Advancing puberty in female sheep: It`s all about fat and muscle

Advancing puberty in female sheep:
It’s all about fat and muscle
by
Cesar Augusto Rosales Nieto
Bachelor of Science in Plant and Animal Science
Master of Science in Animal Science
This thesis is presented for the degree of Doctor of Philosophy of
The University of Western Australia
Faculty of Science
School of Animal Biology
2013
Summary
The reproductive efficiency of the entire sheep flock could be improved if ewe lambs go
through puberty early and produce their first lamb at one year of age, rather than the
more ‘traditional’ two years. The onset of puberty is linked to the attainment of critical
body mass so the aim of the research described in this thesis was to test the general
hypothesis that, in Merino ewe lambs, accelerating the rate of growth and the
accumulation of muscle and fat will advance puberty and improve reproductive
success. A secondary hypothesis tested was that the effects of accelerated growth on
reproduction are mediated by changes in the concentrations of follistatin and leptin.
We studied the statistical relationships between reproductive performance and a
variety of measures of growth and body composition: phenotypic values for depth of
eye muscle (EMD) and depth of fat (FAT) and genotypic values (Australian Sheep
Breeding Values) for similar measures made post-weaning, live weight (PWT) and
depths of eye muscle (PEMD) and depth of fat (PFAT). First oestrus was detected with
vasectomized rams and then entire rams as the ewes progressed from 6 to 10 months
of age.
The general hypothesis was supported by observations in several experiments
involving over 840 Merino ewe lambs over two seasons. Age at first oestrus decreased
with increases in values for PWT. The proportion of ewe lambs that achieved puberty
was positively related to increases in the values for EMD, FAT or PEMD, PFAT or
PWT. Ewe lambs that were heavier at the start of mating were more fertile and had a
higher reproductive rate. Fertility and reproductive rate were positively correlated with
values for EMD, FAT, PWT, PEMD, PFAT.
However, many ewe lambs attained puberty (first oestrus) but then failed to
conceive, so fertility and reproductive rate varied significantly in ewe lambs of similar
live weight at the start of mating, suggesting that the outcome is determined by other
factors. We tested the importance of liveweight change during mating. Females that
weighed 40 kg at the start of the mating period were assigned to dietary treatments that
targeted low (40 kg) or high (45 kg) live weights during mating. Females that were
assigned to target 45 kg were more fertile and had a higher reproductive rate than
females that were targeting 40 kg.
We assessed the roles of some hypothetical endocrine mediators between body
tissues and the reproductive system. The link from adipose tissue is obviously leptin
but muscle hormones associated with reproduction have not been clearly identified.
One possibility is follistatin. We therefore measured blood concentrations of leptin and
follistatin so we could test whether they were related to production traits and
reproductive performance. Leptin concentration was positively correlated with values
i
for EMD, PEMD, FAT, PFAT and PWT, whereas, in general, follistatin concentration
was negatively correlated to the same traits. Leptin concentration was also positively
related to age and live weight at first oestrus, the proportion of females that attained
puberty, and fertility and reproductive rate. There was also evidence that leptin
produced by intramuscular fat plays a role in the onset of puberty. By contrast,
follistatin concentration was negatively related with live weight at first oestrus, fertility
and reproductive rate.
We can conclude that, in ewe lambs mated at about 8 months of age, increasing
live weight during mating will increase fertility and reproductive rate, and this can be
achieved by the use of higher breeding values for accumulation of muscle and fat, and
therefore growth. These relationships, certainly for adipose tissue and perhaps also for
muscle, appear to involve a stimulatory role played by leptin on the reproductive control
centres. The regulatory role of muscle might also be mediated by follistatin, but this
hormone appears to be inhibitory and thus has to be withdrawn to allow for puberty to
proceed and fertility to be expressed.
For the sheepmeat industry, it is clear that strategies for genetic improvement of
body composition, particularly with a view to more rapid muscle (carcase)
accumulation, will very likely also advance puberty and improve the overall
reproduction rate of the flock. The goal of strong reproductive performance in the first
year of life is within reach, as are a major step-up in the lifetime reproductive
performance of each ewe.
ii
Table of contents
Summary
i
Table of contents
iii
Dedicatoria
iv
Acknowledgements
v
Statement of contribution
viii
Publications
ix
General introduction
1
Literature review
5
Chapter 1
Selection for superior growth advances the onset of
19
puberty and increases reproductive performance in ewe
lambs (animal 7, 990 - 997.)
Chapter 2
Ewe lambs with higher breeding values for growth
38
achieve higher reproductive performance when mated
at age 8 months (Theriogenology 80, 427-435)
Chapter 3
Prepubertal growth and muscle and fat accumulation in
58
male and female sheep – relationships with metabolic
hormone concentrations, timing of puberty, and
reproductive performance (Domestic Animal
Endocrinology, in preparation)
Chapter 4
Roles of liveweight change during mating and muscle
76
accumulation on puberty and fertility in ewe lambs
(Animal Production Science, in preparation)
Chapter 5
Relationships among body composition, circulating
92
concentrations of leptin and follistatin, and the onset of
puberty and fertility in female sheep (Journal of Animal
Science, in preparation)
General discussion
111
References (general introduction, literature review and general discussion)
116
iii
Dedicatoria
“Being deeply loved by someone gives you strength, while loving someone deeply
gives you courage” (Lao Tzu)
En esta vida no hay accidentes, todo pasa por algo, cada quien es el arquitecto de su
propio destino! Esa es la historia de mi vida! Esa es la historia de nuestras vidas!
Dependerá de nosotros si somos capaces de descifrar ese acertijo para descubrir
nuestro potencial. Las cosas negativas que en su momento vivimos, nos sirvieron para
salir adelante, fortalecernos y darnos cuenta de que el mundo no termina ahí; que la
vida sigue y que dependerá de nosotros hasta donde queremos llegar. Cada fracaso y
cada logro que hemos vivido han fortalecido la relación, ha fortalecido nuestro
carácter. Gracias al ranger por no conocer al comandante Peña. Gracias a eso, hoy
estamos aquí, hoy culminamos una etapa más en nuestra vida. Una etapa que no
cambiaría por nada en el mundo!
Por eso, tenemos que hacer que cada día cuente como si fuera el último. No
sabemos que nos depara el destino. No sabemos cuánto tiempo más vamos a estar
en este mundo. Hay que vivir y gozar la vida! El ayer ya es historia; el futuro es muy
incierto, pero el día de hoy es un regalo; por eso se llama presente y hay que
disfrutarlo como si fuera el último día (Modificado de Master Oogway).
Mónica, Natalia y Romina, esta va dedicada a ustedes; esto es la culminación
de cuatro años de estudio, de preparación y sacrificios. Ustedes son la razón de mí
existir y por lo cual yo me esfuerzo cada día más. Yo estuve tan inmerso en mi trabajo
que me ausente literalmente 3 años de la casa; sin embargo te tuve a ti Moniquiu y tus
hijas te tuvieron a ti; tuviste la casta y el aplomo de sacar esto adelante; casa, trabajo y
lo más importante a las hijas! Los éxitos y reconocimientos que tuve como estudiante y
los que tendré como profesional, fueron y serán logros para todos nosotros; ya que
somos una familia! Todo esto no hubiera sido posible sin su apoyo! Muchas gracias
por aguantar ese ogro que atacaba de repente, pero más importante, por ese apoyo
incondicional, por sus palabras de aliento que nunca me faltaron.
También va dedicada a los que no estuvieron con nosotros este tiempo, mi
mama, hermanas y sobrinos, sin embargo siempre estuvieron al pendiente de nosotros
y que desde lejos nos daban su apoyo incondicional.
Este tiempo que estuvimos fuera de México y que Australia nos cobijó como
nuestro nuevo hogar siempre tuvimos un ángel que nos cuidó. Tu y yo sabemos bien
quién es; gracias por estar siempre cuidándonos!
Mor’du es por ustedes y para ustedes!!
iv
Acknowledgements
“Choose a job you love, and you will never have to work a day in your life” (Confucius)
“Research is what I'm doing when I don't know what I'm doing” (Wernher von Braun)
“Learn from yesterday, live for today, hope for tomorrow. The important thing is not to
stop questioning” (Albert Einstein)
“No amount of experimentation can ever prove me right; a single experiment can prove
me wrong” (Albert Einstein)
To my supervisors
“The way a team plays as a whole determines its success. You may have the greatest
bunch of individual stars in the world, but if they don't play together, the club won't be
worth a dime” (Babe Ruth)
“I hear and I forget. I see and I remember. I do and I understand” (Confucius)
“If I am walking with two other men, each of them will serve as my teacher. I will pick
out the good points of the one and imitate them and the bad points of the other and
correct them in myself” (Confucius)
Thank you for believing in me, thank you for giving me this opportunity and thank you
for accepted to be my supervisors. It was such a great pleasure to work with you. This
was an enjoyable learning experience for me and your guidance taught me many
valuable lessons through this PhD process that will help me in my career. Thanks for
being so detailed in so many aspects, particularly in our papers. I learned to work
professionally and to become a better listener and a fast learner. It was very
challenging to combine different points of view; three great minds that think so
differently. You were very patient with me and you took your time to explain my tasks
and help me to comprehend the work that I needed to accomplish. Thank you for your
support and those words of encouragement that you had for me every time. All my
achievements as a person/student reflect the dedication and contribution that you had
to me. I was very fortunate to have you all.
Here, I would like to extend my thanks and gratitude to Dominique Blache.
Despite the fact that he was not my supervisor, he acted like one. His door was always
open for me and in countless occasions he responded my questions and solved my
doubts.
v
To my team
“Unity is strength... when there is teamwork and collaboration, wonderful things can be
achieved” (Mattie Stepanek)
“When a team outgrows individual performance and learns team confidence,
excellence becomes a reality” (Joe Paterno)
“Success depends upon previous preparation, and without such preparation there is
sure to be failure” (Confucius)
Jan and Claire: OMG, seriously this would not have been possible without your help.
You are an important part of my achievements as a student. How many sheep we bled;
how many sheep we weighed; how many trips we had to Medina, to Ridgefield, to
Moojepin. In this group, we always had long sessions and interminable work but we
always finished the job! Thank you for your assistance, guidance, patience and
friendship! Thank you for taking care of my experiment while I had a broken leg.
Margaret Blackberry, thank you for your help during my time in the lab – without your
guidance my samples would have been a disaster. As well, I want to extend my
gratitude to Duncan, Sarah, Beth and Ox, who were always keen to help!
Thank you Hamish and Dave Thompson for your hospitality and help during my
experiment in Moojepin.
To my sponsors
“Education is what remains after one has forgotten what one has learned in
school” (Albert Einstein)
Thank you for believing in me. I am fortunate to have had the financial support provided
by CONACyT (the Mexican National Council for Science and Technology), the
Department of Agriculture and Food of Western Australia, the Cooperative Research
Centre for Sheep Industry Innovation and the UWA School of Animal Biology.
vi
To Wendy, Alan, Rose, Graeme, Jackie, Nisha, Mark and Thommo
“Some people come into our lives and quickly go. Some people move our souls to
dance. They awaken us to a new understanding with the passing whisper of their
wisdom. Some people make the sky more beautiful to gaze upon. They stay in our lives
for awhile, leave footprints on our hearts, and we are never, ever the same” (Flavia
Weedn)
Thank you guys for your support during all this time. This would have been a very
different history.
To our Mexican family in Perth
Jior, Aletzin, Mariana, Lalo, los Cucos, Susana, Jorge, Lasha and Luis. As Mexicans,
we always look for coexistance and we were very lucky because we found a family in
you!! For the parties, Christmas, New Year’s we celebrated together! We always had a
dish, a beer and history to share! Thanks to your friendship we were not homesick; our
Mexico was here with you!
To my post grad fellows
Dr Machado, Mikaela, Xixi, Yongjuan, Kels, Stacey, Trina, Sam, Steph, Jo, Kirrin, Joe,
Umar and my CRC mates. Thank you for sharing a cup of tea and enlightening the
room with jokes and comments. Thank you for your friendship! We are the network of
the future; let’s keep in touch!! Thank you Mikaela for being part of the noisy room!!
To myself
“The great man is he who does not lose his child's-heart” (Mencius)
“You have to believe in yourself” (Sun Tzu)
“When you are content to be simply yourself and don't compare or compete, everybody
will respect you” (Lao Tzu)
“Try not to become a man of success, but rather try to become a man of value” (Albert
Einstein)
“Knowing others is wisdom, knowing yourself is Enlightenment” (Lao Tzu)
“If you can't explain it simply, you don't understand it well enough” (Albert Einstein)
“The only source of knowledge is experience. You have to learn the rules of the game.
And then you have to play better than anyone else” (Albert Einstein)
Always be yourself, help others, always remember who you are and from where
you are coming from. Be a good husband and excellent dad for Natalia and
Romina.
vii
Statement of contribution
The work presented in this thesis is the original work of the author. This thesis contains
published work and/or work submitted for publication, some of which has been coauthored. The experimental work, statistical analyses and manuscript preparation was
carried out by the author of this thesis and discussed in extensive detail with the
supervisors Mr Mark Ferguson, Dr Andrew Thompson and Prof Graeme Martin before
submission to the journal. All experimental work required the help of volunteers and Mr
Mark Ferguson, Dr Andrew Thompson, Prof Graeme Martin, Dr Mark Hedger, Ms Jan
Briegel, Ms Claire Macleay, Mr Duncan Wood and Mr Hamish Thompson contributed
significantly to the collection of data, or analyses of data, and are co-authors for some
manuscripts in recognition of their contribution.
Cesar Augusto Rosales Nieto
August 2013
viii
Publications arising from work in this thesis
Journal articles (peer reviewed)
Rosales Nieto C.A., Ferguson M.B., Macleay C.A., Briegel J.R., Martin G.B.,
Thompson A.N. Selection for superior growth advances the onset of puberty and
increases reproductive performance in ewe lambs. animal 7, 990-997.
Chapter 1
C.A. Rosales Nieto, M.B. Ferguson, C.A. Macleay, J.R. Briegel, D.A. Wood, G.B.
Martin, A.N. Thompson. Increasing genetic potential for growth improves the
reproductive performance of ewe lambs. Theriogenology 80, 427-435.
Chapter 2
Conference proceedings
C.A. Rosales Nieto, M.B. Ferguson, C. Macleay, J. Briegel, A. Thompson, G.B.
Martin. 2010. Growth rate, muscling and reproduction in Merino ewe lambs. Animal
Production Science (50) vii.
Chapter 1
C.A. Rosales Nieto, M.B. Ferguson, C. Macleay, J. Briegel, G.B. Martin, A. N.
Thompson. 2011. Selection for superior growth advances the onset of puberty in
Merino ewes. Proceedings of the Association for the Advancement of Animal Breeding
and Genetics, 19, 303-306.
Chapter 1
C.A. Rosales Nieto, M.B. Ferguson, C.A. Macleay, J.R. Briegel, M.P. Hedger, G.B.
Martin, A.N. Thompson. 2012. Follistatin, muscle development, puberty and fertility in
ewe lambs. Proceedings of the Annual Meeting of the European Federation of Animal
Science, 18, 284.
Chapter 5
Media coverage
Early breeding to boost flock sizes. Ag in Focus, Department of Agriculture and Food of
Western
Australia. http://agric.wa.gov.au/objtwr/imported_assets/aboutus/pubns/agif_wa_summ
er_2011-12.pdf
ix
Can joining Merino ewe earlier accelerate the flock rebuild? CRC for Sheep Industry
Innovation. http://www.sheepcrc.org.au/information/news.php
Can joining Merino ewe earlier accelerate the flock
rebuild? http://sj.farmonline.com.au/news/state/livestock/sheep/can-earlier-joiningaccelerate-flock-rebuilding/2539077.aspx
Will early joining help increase flock
number? http://www.therural.com.au/news/local/news/general/will-early-joining-helpincrease-flock-numbers/2538474.aspx
Success with joining lambs earlier. Farming
Ahead. http://www.kondiningroup.com.au/StoryView.asp?StoryID=8682432
Promise in early
joining http://tascountry.realviewtechnologies.com/?iid=62566&startpage=page000002
2
Merino ewes accelerate flock rebuild? http://www.silobreaker.com/merino-ewesaccelerate-flock-rebuild-5_2265658601555099736
Finding the best way to build flock numbers. Esperance Express, general news page
11. Circulation 3,500. Date 02/05/2012.
Research focuses on joining Merino ewes at earlier age. Narromine
News. http://www.narrominenewsonline.com.au/news/local/news/rural/researchfocuses-on-joining-merino-ewes-at-earlier-age/2542161.aspx
Can joining Merino ewe earlier accelerate the flock rebuild? Southern
Weekly http://www.southernweekly.com.au/news/local/news/general/can-joiningmerino-ewes-earlier-accelerate-the-flock-rebuild/2544534.aspx
Joining ewes earlier a possibility. Bombala Times, general news page 12. Date
16/05/2012.
Can joining Merino ewes earlier accelerate the flock rebuild? Border News, general
news page 5. Date 7/05/2012.
Seeking ways to grow flocks. Country News inserts, Shepparton Vic. General news
page 49. Regional circulation 49,616. Date 30/04/2012.
Feeding for better fertility. MLA. Meat and Livestock
x
Australia. http://www.mla.com.au/Publications-tools-and-events/Industrynews/Feeding-for-betterfertility#hp=highlight2&article=Feeding%20for%20better%20fertility
Vale la pena usar el servicio temprano a
corderas? http://www.woolpatagonia.com.ar/noticias/internacionales/notas/2012/mayo/
22/corderas.htm
PhD project sets new direction for sheep breeding research. The UWA Institute of
Agriculture. IOA April 2013.
Early mating defies tradition. The Countryman. June 2013.
xi
General introduction
Australia is the principal producer of export lamb and mutton for the world (Meat
and Livestock Australia, 2012), but the Australian sheep industry cannot meet current
demands for export lamb. Therefore, it is necessary to improve the reproductive
efficiency of the sheep flocks and this has renewed attention on the breeding of young
ewes in their first year of life (Martin, et al. 2009; Ferguson, et al. 2011). Mating ewes
as lambs offers the opportunity to increase the number of lambs on the ground and
therefore exploit potential advantages in sheep breeding and selection programs
through the reduction of generation interval. In addition, it has been demonstrated that
if a ewe lamb can rear lamb(s) successfully to weaning there is the potential to
increase profitability (Young, et al. 2013) and lifetime reproductive performance
(Kenyon, et al. 2011). However, this concept is not well accepted by producers due to a
variety of perceptions: increase on-farm costs, more labour required, poor and variable
reproductive performance, an increase in feed demand, greater live weight and body
condition score targets at a young age, low survival of new-born progeny, tendency for
progeny to be lighter at birth and weaning, and greater death rates of ewe lambs
(reviewed by Kenyon, et al. 2013).
Given these potential constraints, we need specific guidelines for the
reproductive management of young animals so producers can maximize the chances
of success. For example, for young ewes raised under Australian conditions, producers
need to know the live weight, liveweight change, body condition and age at which
puberty is reached.
The timing of puberty is the outcome of dynamic interactions among several
genetic and environmental factors (reviewed by Dýrmundsson 1981) and generally
occurs in ewe lambs when they attain 50 to 70% of their expected mature body mass
(Hafez, 1952; Dýrmundsson 1973). If growth is restricted during early life, young ewes
will remain pre-pubertal until the required proportion of mature body mass is reached
(reviewed by Foster, et al. 1985) so slowly-growing lambs achieve puberty later than
rapidly-growing lambs (Boulanouar, et al. 1995). This relationship between growth rate
and puberty explains the earlier puberty in ewes raised as singles compared to those
raised as twins across a range of breeds (Southam, et al. 1971) – single-reared lambs
grow faster to weaning and can remain heavier until 12 months of age despite postweaning compensatory growth in twin-reared progeny (Thompson, et al. 2011). On the
same basis, we would expect puberty to occur earlier in ewe lambs with higher
breeding values for rapid growth.
After puberty, there are positive correlations between weaning weight, growth
and reproductive performance in young female sheep (Barlow and Hodges, 1976;
1
Gaskins et al., 2005). In addition, fertility and reproductive rate in young ewes are
related to live weight at mating (McGuirk, et al. 1968). Therefore, we would also expect
that ewe lambs with higher growth rates due to better environmental conditions or
higher breeding values for growth would be more fertile and have higher reproductive
rates than ewes with lower growth rates.
However, fertility and reproductive rate vary significantly in ewe lambs of similar
live weight at the start of mating, suggesting that the outcome is determined by other
factors such as liveweight change (LWC). An effect of LWC during mating on
reproductive success could be explained by the ‘acute’ metabolic effect in which shortterm changes in nutrition affect ovarian function, independently of changes in live
weight, or the ‘dynamic’ effect associated with changes in live weight before conception
(reviewed by Scaramuzzi et al. 2006). However, it is not known if the ‘dynamic’ effect
simply reflects weight gains during mating that lead to the ewes being heavier when
mated, or if liveweight change itself has some effect on fertility and reproductive rate in
addition to those associated with correlated changes in absolute live weight.
Regardless of the mechanism, it is expected that improving the nutrition of ewe lambs
so they gain weight during mating will increase their fertility and reproductive rates.
In addition to the effects of growth rate and live weight at mating, body
composition might also be related to the timing of first oestrus, fertility, and reproductive
rate. In mature Merino ewes, fertility and reproductive rate are known to be better for
genotypes with higher breeding values for percentages of muscle and fat that they had
when they were yearlings (Ferguson, et al. 2007; 2010). Furthermore, phenotypic
enhancement of muscle mass and fatness, as assessed through condition score, are
known to increase fertility and reproductive rate in ewes mated to lamb at one or two
years of age (Malau-Aduli, et al. 2007). It is not clear whether this also applies to ewe
lambs and their development of reproductive capability but, overall, it appears that ewe
lambs that accumulate fat and muscle more rapidly will achieve puberty earlier, be
more fertile and have a higher reproductive rate when mated at 8 or 9 months than
their counterparts.
All of the observations cited above show that the metabolic status of the animal
is involved in the regulation of sexual maturation and reproductive performance,
presumably due to the effects of physiological signals from metabolic tissues on the
reproductive axis (review: Martin, et al. 2008). To understand the proposed links
between the rates of muscle and fat accumulation and reproduction in young sheep,
we need to search for physiological links between reproductive control systems and the
muscle and adipose tissues. The metabolites and hormones involved in these
processes include growth hormone (GH), insulin-like growth factor (IGF-I), insulin,
ghrelin, leptin, follistatin, glucose and non-esterified fatty acids (NEFA). The primary
2
candidate from adipose tissue is leptin, a potent regulator of nutrition, metabolism and
reproductive endocrinology (Blache, et al. 2000). The role of leptin in puberty has been
extensively examined in a variety of species, including monogastrics (Ahima, et al.
1997; Cheung, et al. 1997; Kiess, et al. 1998; Barb, et al. 2004; Maciel, et al. 2004) and
ruminants (Foster and Nagatani, 1999; Chilliard, et al. 2005).
With respect to muscle, endocrine factors associated with reproductive
performance have not been clearly identified, but IGF-I and follistatin are possibilities.
IGF-I controls muscle growth (reviewed by Oksbjerg, et al. 2004) and circulating IGF-I
concentration has been associated with the onset of puberty in ewe lambs (Roberts, et
al. 1990). Follistatin plays an important role in muscle growth and development during
fetal development and early neonatal life (McFarlane, et al. 2002). In mice, follistatin
overexpression increases muscle weight and mass (Lee and McPherron 2001; Lee
2007; Gilson, et al. 2009), corroborating the earlier work of Matzuk, et al. (1995) who
had reported retarded growth, reduced muscle mass, bone deficiencies, and early
postnatal death in follistatin-deficient mice. Therefore we would expect follistain
concentrations to be high during muscle growth and development and higher in ewe
lambs selected for rapid muscle accumulation. The role of follistatin in reproduction is
less clear. In the hypothalamic-pituitary axis, it has no effect on GnRH secretory
patterns in sheep (Padmanabhan, et al. 2002) but it inhibits FSH secretion in rodents
(Ueno, et al. 1987). In the ovary, it is synthesized by granulosa cells and helps in the
mechanism of follicular selection during the period leading to ovulation (Michel, et al.
1990; Nakatani, et al. 1991; Xiao, et al. 1992). During ovulation, follistatin seems to
bind to activin and control its actions, an important part of the ovulatory process
(Nakamura, et al. 1990; Newton, et al. 2002). Overall, the importance of follistatin in
reproduction was demonstrated in mice: first, deletion of follistatin in adult granulosa
cells alters follicle and oocyte maturation, reduces fertility and terminates ovarian
activity (Jorgez, et al. 2004); second, deletion of one isoform of follistatin reduces litter
size and leads to early cessation of reproduction (Kimura, et al. 2010). Overall, the
situation is not at all clear, but it seems likely that changes in the circulating
concentration of follistatin will influence reproductive function (review: Knight, 1996).
Therefore, the aim of the research described in this thesis was to test the
general hypothesis that, in Merino ewe lambs, the rate of growth and accumulation of
muscle and fat will positively influence the concentration of follistatin and leptin, thus,
the age and live weight at puberty and the reproductive success. The aim was study in
detail Merino ewes with variation in growth and accretion of muscle and fat and
establishes whether these factors were related to reproductive success at young ages.
The following specific hypotheses were tested:
3
1.
Merino ewe lambs with higher values for the rate of growth, and
accumulation of muscle and fat will be younger at first oestrus and a greater
proportion of them will attain puberty than their counterparts.
2.
Merino ewe lambs with higher values for the rate of growth, and
accumulation of muscle and fat will be heavier at first oestrus and a greater
proportion of them will attain puberty than their counterparts.
3.
Higher value for the rate of growth and accumulation of muscle and fat will
allow a greater proportion of Merino ewe lambs to conceive with a greater
reproductive rate, compared to ewe lambs with lower values for these traits.
4.
The effect of the higher values for the rate of growth and accumulation of
muscle and fat on reproductive performance will be greater in ewe lambs that
are lighter at start of mating, or growing less during mating, compared to ewe
lambs with lower values for the rate of accumulation of muscle and fat.
5.
Differences in the rate of growth and accumulation of muscle and fat in ewe
and ram lambs will lead to differences in the circulating concentrations of
metabolites and metabolic hormones that are linked to the control of the
reproductive system, and will therefore be related to the onset of puberty and
the success of reproduction.
6.
Higher values for the rate of growth and accumulation of muscle and fat will
influence age and live weight at first oestrus, and fertility and reproductive
rate, via effects on the concentrations of leptin and follistatin.
4
Literature Review
The Australian sheep industry is showing an interest in improving carcass and
reproductive traits due to the relative increases in prices for meat and the sustained low
prices for wool. In particular, the ability of the lamb industry to meet projected demand
requires drastic improvements in weaning rates from Merino ewes. Mating replacement
ewes to lamb at age 12-14 months rather than 2 years would significantly increase the
profitability of lamb production systems based on crossbred ewes, but the benefit
gained by early mating depends on the reproduction rate achieved (Young, et al.
2010). This review will describe the Australian sheep industry and its needs, and
outline the external and internal factors that affect the onset of puberty and the
reproductive performance in young females. This topic encompasses the physiology of
adipose and muscle tissue and possible links with the reproductive axis. Finally, there
will be an assessment of the impact of genetic selection using Australian Sheep
Breeding Values (ASBVs) on important economic traits in sheep that are link meat
production to reproduction.
The Australian sheep industry
In 1970, the sheep population in Australia was just under 180 million but, by 2010, it
had decreased to 68 million; recently, it has increased slightly to 73 million (Figure 1).
Most sheep are located in New South Wales (26.8 million), followed by Victoria (15.2
million), Western Australia (14 million), South Australia (11 million), Queensland (3.6
million) and Tasmania (2.3 million). Around 75% of sheep in Australia are Merinos
(90% in WA).
Figure 1. Changes in the Australian sheep population. Australian Bureau of Statistics (2013b).
5
The sheep industry used to be focussed on the production of one commodity,
wool, so, until the last decade, most producers retained castrated males (wethers) as a
significant portion of their flock. With the decline in wool profits, wethers lost value so
producers now keep only female progeny and the males are sold as lamb or exported
(when they are 7-24 months old) to Asia, Africa and North America (Meat and
Livestock Australia, 2012). Producers have also responded to the changing markets by
increasing the production of lambs specifically bred for slaughter, rather than just for
wool production. This has led to a change in the composition of the national flock as
producers crossed Merino ewes with other breeds to produce prime lamb.
Another factor troubling the sheep industry is an increasing concern about
methane emissions (Hegarty 2008). Importantly, with respect to the theme of this
thesis, sheep that are not reproducing are only producing methane, so there is
pressure to reduce the length of infertile periods, thus decreasing ‘emissions intensity’
(units of methane per unit of product).
These needs, to produce more lambs, to produce them faster, and to reduce
emissions intensity, are driving a renewed interest in improving the reproductive
efficiency of the sheep flock. The focal points are ovulation rate (number of eggs
released by an animal that ovulates), fertility at mating, embryo mortality, postnatal
mortality, and delayed puberty.
Reproduction in female sheep
General concepts
The reproductive system is primarily controlled by the activity of gonadotrophinreleasing hormone (GnRH) neurons in the brain. GnRH is synthesized by these
neurons and transported along their axons to the median eminence where it is released
into the hypophyseal portal system. The activity of the GnRH neurons is synchronised
by an unknown mechanism, so the GnRH is release in pulses. The frequency of the
GnRH pulses is the major determinant of reproduction: by increasing pulse frequency,
the brain activates the ovary, and by decreasing pulse frequency it allows ovarian
activity to subside.
The hypophyseal portal system transports the GnRH pulses to the anterior
pituitary gland, where they regulate the synthesis and release of gonadotrophins,
luteinizing hormone (LH) and follicle stimulating hormone (FSH), the two major
hormones that control gonadal activity (Figure 2). The pattern of secretion of LH is also
pulsatile, mimicking the GnRH pulses, but FSH secretion is continuous. The
gonadotrophins are transported to the ovaries where they promote the synthesis and
secretion of inhibin and the sex steroids, primarily oestradiol and progesterone. There
6
are two feedback loops, the first involving the follicle(s) and the pituitary gland, and the
second involving the follicles, the CL, and the brain. Both loops involve synergistic
interactions. High progesterone concentrations produced by the corpus luteum reduce
GnRH pulse frequency, leading to reduced oestradiol production by the follicles. Low
progesterone concentrations during the follicular phase allow an increase in GnRH
pulse frequency, so oestradiol production by the dominant follicle will increase and elicit
oestrus, the LH surge (positive feedback), and ovulation. Oestradiol and inhibin, both of
which are follicular products, will reduce FSH concentrations, thus reducing follicular
growth.
Figure 2. The major endocrine pathways that control ovarian function in the female, illustrating
the pituitary control of ovarian production of inhibin and sex steroids, and the roles of these
hormones in negative feedback.
The balance of the hypothalamic-pituitary-ovarian axis is also influenced by a range of
external factors, most acting at brain level (photoperiod, socio-sexual signals, nutrition),
although metabolic and nutritional factors also act directly on the ovary (Fig 3).
Puberty
During the pre-pubertal phase, when the ewe lamb does not show ovulatory activity,
GnRH/LH pulse frequency is low (Bindon and Turner 1974; Foster, et al. 1975a; 1975b;
Huffman, et al. 1987). The low pulse frequency does not provide the sufficient stimulus
for follicular development, so the young ewe remains in anovulatory state (Foster et al.,
1986). The brain of the lamb is capable of generating the high frequency of GnRH/LH
pulses needed for inducing ovulation, and can do so when the ovaries are removed,
but the hypothalamus is very responsive to negative feedback by oestradiol, even in
the absence of progesterone (Fig. 2), so GnRH secretion is inhibited (Foster, et al.
7
1986). Circulating concentrations of FSH reach the adult range by 10-12 weeks of age
(Fitzgerald and Butler 1982; Foster, et al. 1975a), although a rise in FSH concentration
during the peri-pubertal period has been reported (Ryan, et al. 1991).
Figure 3. Schematic summary of the potential relationships among the endocrine and neural
inputs into the systems that control the reproductive system and mediate the responses to
change in metabolic status. Stimulation of the secretion of IGF-I by growth hormone (GH) acting
on the liver is omitted for clarity. Broken lines indicate hypothetical but unproven pathways.
Modified after Martin, et al. (2010).
There is a gradual 3-4-fold increase in GnRH/LH pulse frequency over the week
before first ovulation (Huffman, et al. 1987), because there is a decrease in the ability
of oestradiol to inhibit GnRH secretion (reviewed by Karsch 1987). The high GnRH
pulse frequency initiates a follicular phase that culminates in ovulation (Foster and
Karsch 1975). The first ovulation is often not accompanied by behavioural oestrus
(known a ‘silent’ ovulation), but the second ovulation usually is (Foote, et al. 1970;
Ryan, et al. 1991). First ovulation and first oestrus thus typically occur within 2 to 3
weeks of each other and several patterns have been reported with respect to the
temporal relationship between them (Ryan, et al. 1991).
The corpus luteum produced by the first ovulation can also be short-lived,
leading to a short luteal phase and a second ovulation 2-8 days after the first (Ryan, et
al. 1991; Baird, 1992). Both the failure of oestrus and the short cycle are caused by the
lack of circulating progesterone in the period leading up to the first ovulation, so the
presence of progesterone during the first normal luteal phase generally guarantees
oestrus (Ryan, et al. 1991; Baird, 1992; Keisler, et al. 1985). Thereafter, progesterone
8
assumes the role of ‘cycle controller’ and determines the interval between ovulations
(Thimonier 1979).
First oestrus is an indication of attained of sexual maturation and occurs in ewe
lambs when they have attained 50-70% of mature body weight (Hafez, 1952;
Dýrmundsson 1973). However, the onset of puberty varies from individual to individual,
from flock to flock within breed, and from location to location, and the timing is affected
by a variety of internal and external factors:
a)
Season of birth can affect age and body weight at puberty (Watson and Gamble
1961; Wiggins, et al. 1970; Hawker and Kennedy 1978);
b)
Local environmental factors, such as environmental temperature, which is closely
associated with light changes in natural conditions, may contribute to the variation
of the onset of puberty. Ewe lambs attain puberty more slowly in tropical regions
than in temperate regions (Moule 1950). Interestingly, the delay of puberty under
extreme temperatures is attributed to a slower growth rate (Hafez, 1964).
c)
Social cues, such as the sudden introduction of mature rams will advance puberty
(Oldham and Grey 1984).
d)
Genetic factors – there are breed differences and variation among individuals
within the same breed in the age and body weight at first oestrus (Hafez, 1952;
Dickerson and Laster 1975; Quirke, et al. 1985; Fogarty, et al. 2007).
The importance of body weight shows the relevance of growth rates. Failure to
grow to the appropriate body size will leads to maintenance of the anovulatory
condition (Hafez, 1952). Puberty is earlier in ewes raised as a single than in those
raised as a twin, across a range of breeds (Southam, et al. 1971). Modification of
growth by manipulation of nutrition will influence age and body weight at puberty. Thus,
severe under-nutrition, or restriction of the intake of protein or energy, delays the onset
of puberty in the ewe lamb (Foster and Olster, 1985; Boulanouar, et al. 1995). It is
arguable that such effects are due to the plane of nutrition per se or due to the growth
rate of the animal as result of the plane of nutrition.
Seasonality
Reproductive seasonality is common to many species that inhabit the non-equatorial
regions of the world. It is an adaptation to the annual cycles in temperature and food
availability and it evolved to ensure birth at the optimal time of the year for lactation and
for the growth of the new-born. Most genotypes of sheep exhibit ovarian activity
primarily between late summer and early winter. During the remainder of the year, they
are anoestrous, as indicated by the almost total absence of sexual behavior. These
changes in the annual cycles can profoundly affect the age at puberty in ewe lambs
9
(Foster, 1981; 1983; Ebling and Foster, 1988). In general, the sexual season will be
longest in sheep from topical regions and shortest in sheep from sub-polar regions
(Hulet, et al. 1974a; 1974b; Chemineau, et al. 1992). Some flocks located in tropical
regions have been reported to show little seasonality (Jackson, et al. 1990;
Chemineau, et al. 2004). Seasonal breeding has a long evolutionary history but is a
major limitation to reproductive efficiency in industrial systems.
Nutrition-reproduction interactions
There is a strong association between nutrition and reproduction, ranging from minor
changes in ovulation rate or frequency to cessation of breeding or extension of the
anoestrus season if nutrition is very restricted. Undernutrition during early stages of
development, either before or after birth, can induce a reduction in the lifetime
reproductive capacity of the female offspring (Gunn, et al. 1995; Rhind, et al. 1998).
Undernutrition disrupts the hypothalamic GnRH pulse generator and thus the secretion
of LH and FSH (Dunn and Moss 1992; Schillo 1992; Mani, et al. 1996) and decreases
the release of LH during the luteal phase of the oestrous cycle in sheep and heifers
(Mackey, et al. 1999; Kiyma, et al. 2004). At the level of the ovary, the outcome is
reduced follicular growth (Bergfeld, et al. 1994), causing atresia in those follicles that
begin development during nutrient restriction (Diskin, et al. 2003), leading to a
reduction in the percentage of females that ovulate or ovulation rate (Killeen 1982;
Abecia, et al. 1997; Mackey, et al. 1999). On the other hand, nutritional
supplementation increases the concentration of progesterone and the amplitude and
frequency of the LH pulses (Imakawa, et al. 1983 and 1986; Rhind, et al. 1985), and
improves ovulation rate (Viñoles, et al. 2005 and 2009).
In addition, dietary restriction will reduce conception rate (reviewed by Abecia,
et al. 2006) and, during pregnancy, compromise the development of the embryo
(Lozano, et al. 2003; Luther, et al. 2007) and reduce lamb birth weight (Warrington, et
al. 1988; Gao, et al. 2007). Brink (1990) suggested that the pregnant ewe that goes
through feed restriction during early pregnancy is able to compensate live weight and
body condition score during the later stages of pregnancy. Unfortunately, nutritional
restriction during pregnancy also has trans-generational consequences, compromising
the lifetime reproductive performance of the progeny (Gunn, et al. 1995; Rae, et al.
2002).
Ruminants can partially or completely compensate for an earlier period of slow
growth or body weight loss due to a low nutritional plane through increased feed intake
(reviewed by Hornick, et al. 2000; Daniel, et al. 2007). The magnitude and nature of
compensatory growth is influenced by factors such as severity of feed restriction, diet
10
type during and after nutrient restriction, duration of nutrient restriction, breed and age
of the animal (Hornick, et al. 1998).
Mating ewe lambs
Mating ewe lambs at, say, 9 months of age rather than the traditional 18 months, is a
practice that has not been widely adopted, but recent studies indicate that the practice
can be successful. Resistance is due to perceptions of cost, poor reproductive
performance and a compromised performance in adult life. Quality of the ovum
(McMillan and McDonald 1985), ovulation rate (Davies and Beck, 1993; Beck, et al.
1996), pregnancy rate (Donald, et al. 1968; Annett and Carson, 2006; Quirke, 1981),
litter sizes at birth (Forrest and Bichard, 1974; Muñoz, et al. 2009) and lamb survival
(Morel, et al. 2010) have all been reported to be lower for ewe lambs than for older
ewes. Other potential disadvantages are: increase in farm costs, more labour required,
poor and variable reproductive performance, an increase in feed demand, greater live
weight and body condition score targets at a young age, decreased survival of newborn
progeny, tendency for progeny to be lighter at birth and weaning, and a greater rate of
deaths of the young ewes (reviewed by Kenyon, et al. 2013).
On the other hand, mating ewes as lambs enables an increase in the number of
lambs on the ground, and reduces the generation interval thus allowing the producer to
exploit potential advantages in sheep breeding and selection programs. Ewe lambs
need to weigh more than 40 kg at start of mating and, if they can rear lamb(s)
successfully to weaning, economic analyses suggest that the strategy will be profitable
if the lamb price is greater than $4/kg carcass weight and more than 60 lambs are
marked per 100 ewes mated (Coop, 1962; Young, et al. 2013). In addition, if the
females are well fed and their body condition is maintained, there is the potential to
increase lifetime reproductive performance (Schoeman, et al. 1995; Kenyon, et al.
2011). Finally, by gaining an extra year of production, successful early mating can lead
to a major reduction (theoretically 16% in a 6-year production life) in methane
emissions intensity in the production system.
Given these potential constraints and risks, producers need specific guidelines
for the reproductive management of young animals so they can maximize the chances
of success. For example, for young Merino ewes under Australian conditions,
producers need to know the optimum combination of live weight, liveweight change,
body condition and age for achieving puberty and maximising fertility.
Growth
Growth can be simply defined as an increase in body mass over time, but it must be
differentiated from development, in which the increase in mass is accompanied by a
11
change in body composition. Body composition is the term used to describe the
percentages of fat, muscle and bone. For the purpose of brevity in this review, we will
exclude bone from the discussion.
The increase of body mass occurs during two specific stages, prenatal
(hyperplasia) and postnatal (hypertrophy), and involves regulated processes of growth
and differentiation that are modulated by a wide variety of factors. During the prenatal
stage, the mass of muscle and fat increases by cellular replication (hyperplasia) to
increase the number of cells; then, during the postnatal stage, those cells increase in
size (hypertrophy). As the animal matures, muscle cells lose their ability to replicate
and grow, but fat cells retain those capabilities and so continue to increase in number
and size (Berg and Butterfield, 1968; reviewed by Robelin, 1986; Azain, 2004). Growth
accelerates during the pre-pubertal stage then decelerates after puberty (Owens, et al.
1993; Robelin, 1986). The rate of change in body composition depends on internal and
external factors such as nutrition, age, gender, mature body size, genetics, and health
(Berg and Butterfield, 1968; Owens, et al. 1993; Nattrass, et al. 2006; Greenwood, et
al. 2006a and 2006b).
Adipose tissue
Adipose tissue is the body’s largest energy reservoir and is essential for regulating and
coordinating energetic and metabolic homeostasis (reviewed by Galic, et al. 2010). As
an energy storage site, adipose tissue enables the organism to adapt to a range of
different metabolic challenges (starvation, stress, periods of excess energy) and it has
also been implicated in endocrinological function through, primarily, the secretion of
leptin, a hormone that is involved in the modulation of puberty and fertility in a variety of
species (reviewed by Foster and Nagatani 1999; Casanueva and Dieguez 1999;
Frühbeck, et al. 2001; Smith, et al. 2002). The discovery of leptin corroborated the
hypothesis of Frisch (1984) that puberty and fertility are linked specifically to the
amount of fat stores.
Adipose tissue growth (adipogenesis) involves the proliferation, differentiation
and conversion of pre-adipocytes into adipocytes (reviewed by Hausman, et al. 2001;
Kokta, et al. 2004; Novakofski 2004; Fernyhough, et al. 2007). This process begins
during embryonic and fetal development (Hausman and Richardson 2004) and
continues during the postnatal stage (Azain, 2004), although the increase in fat mass in
this stage is mainly due to hypertrophy of adipocytes, a process that is prolonged by
over-nutrition (Lemonnier 1972; Faust, et al. 1978; Klyde and Hirsch 1979). Excess
energy is stored in adipocytes, the primary storage site (Azain, 2004; Nafikov and Beitz
2007), first in the abdominal cavity, then intermuscular spaces, subcutaneous regions
and, finally, intramuscular spaces (Vernon, et al. 1981; Pethick, et al. 2005). Variation
12
in the ability to synthesize and store lipids in fatty tissues is closely linked to sex and
genotype and is reflected to the differences in total body fat. Moreover, the rate of
adipose tissue accumulation increases with age in meat animals (Owens, et al. 1993).
The interaction between genotype and nutrition was initially observed by Robelin
(1986) who concluded that fat deposition is more pronounced in the early maturing
animals due to the ability to store more excess of energy.
Adipose tissue is mainly classified as white, the main type, or brown (Cinti
2005). White adipose tissue is distributed throughout the body in subcutaneous regions
and plays a major role in energy storage and in the regulation of activities as diverse as
insulin sensitivity, lipid metabolism, and satiety (Trayhurn and Beattie 2001; Galic, et al.
2010), with flow-on consequences for many other tissues and processes, including
hypothalamus, pancreas, liver, skeletal muscle, kidneys and the immune system
(Frühbeck, et al. 2001). White adipose tissue accumulates rapidly in response to
nutrient availability (Kirtland and Harris 1980). It begins to develop in late gestation
(Vernon 1980; Tang, et al. 2008), increases in mass through puberty and is relatively
steady in maturity (Hirsch and Han 1969).
Brown adipose tissue is located in specific regions and mainly participates in
thermogenesis (reviewed by Rothwell and Stock 1979; Himms-Hagen, 1990; Cannon
and Nedergaard 2004). During late gestation, brown adipose tissue expands primarily
to maintain body temperature upon birth (Frontini and Cinti 2010).
Hormonal links between adipose tissue and reproduction are primarily mediated
by leptin (Fig. 3). Leptin secretion is strongly influenced by the immediate and longterm nutritional status of the animal (Chilliard, et al. 2005) and its major role is
activation of the central satiety networks that suppress appetite (Zhang, et al. 1994). It
provides information to the hypothalamus about the quantity of energy stored in the
adipose tissue and, in response, the neural networks responsible for energy
homeostasis make appropriate changes in energy intake or expenditure to maintain
homeostasis. Leptin concentrations help to predict growth or carcass composition and
determine the body fat content of lambs (Altmann, et al. 2006), beef cattle (Geary, et al.
2003) and pigs (Berg, et al. 2003). Leptin, therefore, is involved in the direct regulation
of adipose tissue metabolism by both inhibiting lipogenesis (Bai, et al. 1996) and
stimulating lipolysis (Frühbeck, et al. 1997).
During growth and development, leptin also provides a signal to the brain
indicating whether metabolic status is adequate for the initiation of reproduction (Fig.
3). It acts directly on hypothalamic sites to enhance the secretion of GnRH and
gonadotrophins (reviewed by Nagatani, et al. 1998; Casanueva and Dieguez 1999;
Clarke and Henry 1999; Chilliard, et al. 2001). During the onset of puberty, leptin
concentration modulates the activity of kisspeptin (Backholer, et al. 2010), a
13
neuropeptide that exerts fundamental control over the pulsatile secretion of GnRH
(Smith, et al. 2006; Navarro, et al. 2007; Smith and Clarke 2007). Several studies have
therefore shown that leptin mediates the effects of body weight and adiposity in
regulating the onset of puberty (Ahima, et al. 1997; Cheung, et al. 1997; Foster and
Nagatani 1999). For example, in immature rats, puberty can be induced by a re-feeding
stimulus for 72 h (Messer and I'Anson 2000) and by administration of recombinant
leptin (Cheung, et al. 1997; Almog, et al. 2001). Likewise, in female mice, leptin
treatment decreased food intake but advanced the onset of puberty (Ahima, et al.
1997; Chehab, et al. 1997) and re-activated the reproductive endocrine system of
males and females in the ob/ob genotype (Barash, et al. 1996). In Merino ewe lambs,
an intra-cerebroventricular infusion of leptin resulted in re-activation of the GnRH-LH
axis after it had been suppressed by fasting (Wójcik-Gladysz, et al. 2009).
Muscle tissue development
Myogenesis, the formation of muscle cells, takes place in two waves during prenatal
development. The first wave is during the initial stage of myoblast fusion and results in
fibres that are primary myoblasts or myofibres (Rehfeldt, et al. 2000). In the second
wave of myoblast proliferation, the primary myofibres serve as a template on which the
secondary muscle fibers develop (Stockdale 1997; Rehfeldt, et al. 2000). In the sheep,
myogenesis takes about 115 d (Greenwood, et al. 1999). The first wave of primary
myofibres occurs in the period leading up to 32-38 d of gestation and the second wave
from 38-62 d (Wilson, et al. 1992).
The number of primary muscle fibres produced during the hyperplasia phase
seems to be an important factor for muscle mass determination and it is regulated
mainly through genetic processes (Dwyer, et al. 1993 and 1994). Secondary muscle
development can be influenced by nutrition and by the addition of growth factors,
particularly IGF-I and GH (Rehfeldt, et al. 1993; Dwyer, et al. 1994; Greenwood, et al.
1999 and 2000). Fetal size (Greenwood, et al. 1999) and litter size (single vs twins)
(McCoard, et al. 2000) have no significant influence on muscle fibre number but, during
a seasonal restraint, the fibre number is differentially affected by season and fetal
number (McCoard, et al. 1997).
Hypertrophy is also markedly affected by the endocrine system, particularly
those that control the uptake and intracellular metabolism of nutrients. Among the
hormones implicated are growth hormone (GH), insulin, insulin-like growth factors
(IGFs), thyroid hormone (TH), transforming growth factor-ß (TGF-ß), glucocorticoids
(GC), insulin and leptin (Etherton 1982; Brameld, et al. 1998; Dauncey, et al. 2004;
Glass 2005; Zeidan, et al. 2005; Velloso, et al. 2008).
14
Promoters and regulators of muscle growth and development
Myostatin (GDF-8) is a growth and differentiating factor that belongs to the
transforming growth factor beta (TGF-ß) super-family and has been identified as an
important inhibitory regulator of muscle development (McPherron, et al. 1997). It is
mainly synthesized in skeletal muscle, although low levels have been detected in
adipose tissue (McPherron, et al. 1997). It appears to regulate both prenatal
myogenesis and postnatal hypertrophy. During myogenesis, it regulates muscle fibre
number by controlling myoblast proliferation and differentiation whereas, during
postnatal stages, it regulates the activation of satellite cells (Thomas, et al. 2000; Lee
and McPherron 2001; McCroskery, et al. 2003).
A mutation in the myostatin gene is responsible for “double muscling” in some
breeds of cattle, such as the Belgian blue (McPherron and Lee 1997; Grobet, et al.
1997) and for enhanced muscle development in sheep (Clop, et al. 2006). In cattle,
myostatin concentration has been reported to differ between sexes within the same
breed and among breeds (Grobet, et al. 1997; Kambadur, et al. 1997; McPherron and
Lee 1997; McMahon, et al. 2003). Blockade of the myostatin signal increases the rate
of muscle accumulation and meat tenderness (Ngapo, et al. 2002), but has a negative
effect on fat depots (Zimmers, et al. 2002), skin, bones and reproductive performance
(poor sexual behaviour, delayed sexual maturity in both sexes, reduced size of the
reproductive tract in females, calving difficulty, poor maternal performance; Arthur
1995).
A promoter of muscle growth and development, and natural regulator of
myostatin, is follistatin (Lee and McPherron 2001). It plays an important role in muscle
growth and development during fetal development and early neonatal life (McFarlane,
et al. 2002). In mice, follistatin overexpression increases muscle weight and mass (Lee
and McPherron 2001; Lee 2007; Gilson, et al. 2009), corroborating the earlier work of
Matzuk, et al. (1995) who had reported retarded growth, reduced muscle mass, bone
deficiencies, and early postnatal death in follistatin-deficient mice. Therefore we would
expect follistatin concentrations to be high during muscle growth and development and
higher in ewe lambs selected for muscle.
Adipose, muscle and reproduction – possible interactions and hormonal links
As outlined above, the link between adipose tissue and reproduction has been clearly
identified, and the adipose hormone, leptin, seems to be a major player. In contrast, the
position is still not clear for links between the muscle tissue and the reproductive axis.
One possibility is follistatin – follistatin had no effect on GnRH secretory pattern
(Padmanabhan, et al. 2002) and its major role is to inhibit FSH secretion (Ueno, et al.
1987). Follistatin is also produced by the ovary although not exclusively and within the
15
ovary, follistatin is synthesized mainly by granulosa cells and helps in the mechanism
of follicular selection (Michel, et al. 1990; Nakatani et al.,1991; Xiao et al 1992). During
ovulation, it seems that the role of follistatin is to bind and control activin actions; activin
which is important in ovulation (Nakamura, et al. 1990; Newton, et al. 2002). However,
the importance of follistatin on the reproductive physiology was demonstrated in mice;
deletion of follistatin in adult granulosa cells was found to altered follicle and oocyte
maturation, leading to reduced fertility and termination of ovarian activity (Jorgez, et al.
2004). Moreover, a deletion of one isoform of follistatin in mice reduced litter size and
early cessation of reproduction (Kimura, et al. 2010). It seems likely that changes in the
circulating concentration of follistatin will influence reproductive function (Fig. 3; review:
Knight, 1996).
An alternative is leptin from muscle – it appears to mediate the interactions
between muscle and fat cells that are important for adipogenesis, myogenesis and
lipogenesis (Boone, et al. 2000; Frühbeck, et al. 2001); It is expressed in muscle
(Wang, et al. 1998), where it induces and modulates hypertrophy (Zeidan, et al. 2005)
and exerts direct effects on glycogen and protein synthesis (Kamohara, et al. 1997; Liu,
et al. 1997; Carbó, et al. 2000), fatty acid oxidation (Muoio, et al. 1997) and basal and
glucose-induced insulin secretion (Emilsson, et al. 1997). Thus, follistatin and leptin
from muscle, as well as leptin from adipose tissue, could be involved in the control of
reproduction.
The genetics of body composition
Clearly, growth and body composition are complex traits that are controlled by different
internal factors that interact with each other as well as with the environment. In recent
times, these interactions have become important for the Australian sheep industry
because a strong interest has developed in genetic selection for fast growing animals
that accumulate more muscle than fat. Almost coincidentally, the selection for these
traits should permit reproduction at a younger age because the onset of puberty
depends on attainment of sufficient body mass and fertility and fecundity are highest in
females that are heavier at the start of the mating period. On the other hand, the
emphasis on accumulation of muscle rather than fat leads to a conundrum:
theoretically, the initiation of puberty depends on the adipose-derived hormone, leptin,
and its effects on the brain centres that control GnRH secretion. We would therefore
expect that animals selected for more accumulation of muscle rather than fat will
produce less leptin, thus delaying the onset of puberty. Evidence for an analagous
stimulatory role for muscle on reproduction, perhaps mediated by follistatin, is scanty
but it nevertheless suggests that animals selected for more accumulation of muscle
might experience an earlier onset of puberty and reproductive success. Support for this
16
hypothesis comes from studies with adult ewes with higher values for yearling muscle
(YEMD) showing that fecundity increased (Ferguson et al., 2007).
Australian Sheep Breeding Values (ASBVs)
The performance of an animal is due to a combination of its genes and the
environment in which it is raised (eg, nutrition, birth type, rear type). To provide a single
language for genetic performance, and to improve the quality, scope and utilization of
sheep genetics in Australia, ASBVs were created by industry research organisations
and are delivered as LAMBPLAN™ for terminal and maternal breeds and
MERINOSELECT™ for Merino and Merino-based breeds. Both LAMBPLAN™ and
MERINOSELECT™ collate and analyse performance values, pedigree information and
relevant environmental and management information from participating breeders. The
outcome delivers ASBVs for a range of traits for growth, carcase, wool, reproduction
and parasite resistance, and it can be measured at four different stages: post-weaning
(7 to 10 months of age), yearling (10 to 13 months of age), hogget (13 to 18 months of
age), and adult (more than 18 months of age) (Brown, et al. 2007).
Genetic selection using sires with high estimated breeding values (EBVs) has
resulted in genotypes that vary in economically important traits such as weight (WT),
fat (FAT), muscle (EMD), fleece quality (clean fleece, CFW; fibre diameter, FD; staple
strength, SS; staple length, SL) number of lambs born (NLB), number of lambs weaned
(NLW), scrotal circumference (SC), and worm egg count (WEC).
Carcass and reproduction traits have recently become a focus of interest in the
Australian sheep industry because of sustained high prices for meat and low prices for
wool. Most emphasis has been placed on selection for growth, depth of ewe muscle
(EMD) and fat (FAT). Clearly, selection based on these traits is likely to change growth
response, carcass composition, carcass lean content and dressing percentage
(Hegarty, et al. 2006; Hopkins, et al. 2007; Gardner, et al. 2010). With the alteration in
body composition, we might expect that, in animals with higher values of ASBV for
rapid growth or improving accumulation of fat and muscle, there would be positive
effects on reproductive outcomes. However, at present, there is little information about
the impact this selection strategy may have on reproductive traits.
Therefore, for the present thesis, ASBVs will be a major tool for testing
hypotheses relating growth and body composition to puberty and reproductive
performance in young Merino ewes, and for testing possible roles for leptin and
follistatin as mediators of those relationships.
Conclusion
There is a wealth of literature demonstrating the importance of metabolic status in the
regulation of sexual maturation and reproductive performance, presumably due to the
17
effects of physiological signals from metabolic tissues on the reproductive axis. The
role of adipose tissue and leptin has been studied extensively, and the action of leptin
on brain centres as a trigger for puberty has strong experimental support. On the other
hand, links between muscle and reproduction, especially in the determination of
puberty, are largely unexplored. Follistatin is one possibility – it plays an important role
in muscle growth and development during fetal and neonatal life and appears to be a
regulator of pituitary and ovarian acitivity, but not GnRH secretion. Overall, the situation
for follistatin is not clear, but the possibility that it will influence reproductive function is
worth testing.
Therefore, based on the literature, we expect that, in Merino ewe lambs, factors
that affect the rate of growth and accumulation of muscle and fat will influence the
concentration of follistatin and leptin, and thus the age and live weight at puberty, as
well as reproductive success.
18
Chapter 1
Selection for superior growth advances the onset of puberty and
increases reproductive performance in ewe lambs
1,2,3
C. A. Rosales Nieto
1,2,4
, M. B. Ferguson
2
2
3
, C. A. Macleay , J. R. Briegel , G. B. Martin and A.
1,2,4
N. Thompson
1
CRC for Sheep Industry Innovation and the University of New England, Armidale, NSW, 2351,
2
Australia; Department of Agriculture and Food of Western Australia, 3 Baron Hay Court,
3
South Perth, Western Australia, 6151, Australia; Institute of Agriculture, University of Western
4
Australia, Crawley, Western Australia, 6009, Australia; School of Veterinary and Biomedical
Sciences, Murdoch University, 90 South Street, Murdoch, Western Australia 6150, Australia
Published in animal 2013; 7 990-997.
19
Abstract
The reproductive efficiency of the entire sheep flock could be improved if ewe lambs go
through puberty early and produce their first lamb at one year of age. The onset of
puberty is linked to the attainment of critical body mass so we tested whether it would
be influenced by genetic selection for growth rate or for rate of accumulation of muscle
or fat. We studied 136 Merino ewe lambs with phenotypic values for depth of eye
muscle (EMD) and fat (FAT) and Australian Sheep Breeding Values at post–weaning
age (200 days) for live weight (PWT), eye muscle depth (PEMD) and fat depth (PFAT).
First oestrus was detected with testosterone-treated wethers and then entire rams as
the ewes progressed from 6–10 months of age. Blood concentrations of leptin and IGFI were measured to test whether they were related to production traits and reproductive
performance (puberty, fertility, reproductive rate). Overall, 97% of the lambs reached
first oestrus at average weight 39.4 ± 0.4 kg (mean ± SEM) and age 219 days (range
163-301). Age at first oestrus decreased with increases in values for PWT (P<0.001)
and concentrations of IGF-I (P<0.05) and leptin (P<0.01). The proportion of ewe lambs
that achieved puberty was positively related with increased in values for EMD (P <
0.01), FAT (P<0.05) or PWT (P<0.01), and 75% of the ewe lambs were pregnant at
average weight 44.7 ± 0.5 kg and age 263 days (range 219-307). Ewe lambs that were
heavier at the start of mating were more fertile (P<0.001) and had a higher
reproductive rate (P<0.001). Fertility and reproductive rate were positively correlated
with increases in values for EMD (P<0.01), FAT (P<0.05), PWT (P<0.01) and leptin
concentration (P<0.01). Fertility, but not reproductive rate, increased as values for
PFAT increased (P<0.05). Leptin concentration increased with increases in values for
EMD (P<0.001), FAT (P<0.001), PWT (P<0.001), PEMD (P<0.05) and PFAT (P<0.05).
Many of these relationships became non-significant after PWT or live weight were
added to the statistical model. We conclude that selection for genetic potential for
growth can accelerate the onset of puberty and increase fertility and reproductive rate
of Merino ewe lambs. The metabolic hormones, IGF-I and leptin, might act as a
physiological link between the growing tissues and the reproductive axis.
Keywords: ewe lambs, phenotypic selection, ASBV, reproductive efficiency
Implications
Genetic selection can improve the rates of growth and muscle development in sheep,
and should also permit reproduction at a younger age because the onset of puberty
depends on attainment of sufficient body mass. Our data support this hypothesis,
suggesting that phenotypic and genetic selection for growth or muscling will improve
the reproductive performance of Merino ewes mated to lamb at one year of age. These
20
findings will inform bio-economic models and promote genetic selection strategies with
a view to improving profitability of sheep production systems by achieving
improvements in reproductive efficiency. The data also suggest a physiological link
between muscle and the reproductive system of female sheep, a possible new
direction in reproductive biology that needs further exploration.
Introduction
International demand for lamb and the need to reduce emissions intensity are
increasing the emphasis on reproductive efficiency sheep flocks and renewing attention
on the breeding of young ewes in their first year of life (Martin et al., 2009; Ferguson et
al., 2011). Puberty, defined as the first spontaneous ovulation (reviewed by Foster et
al., 1985), is the result of dynamic interactions among several genetic and
environmental factors (reviewed by Dýrmundsson, 1981) and is generally reached in
ewe lambs when they attain 50 to 70% of their expected mature body mass (Hafez,
1952). If growth during early life is restricted, young ewes will remain pre-pubertal until
the required proportion of mature body mass is reached (Foster et al., 1985) so rapidlygrowing lambs achieve puberty earlier than slower-growing lambs (Boulanouar et al.,
1995). This relationship between growth rate and puberty explains the earlier puberty in
ewes raised as singles compared to those raised as twins across a range of breeds
(Southam et al., 1971). We would thus expect puberty to be advanced by phenotypic
and genetic selection for enhanced growth rate. This could be achieved through
selection of ewes with high Australian Sheep Breeding Values (ASBVs) for post–
weaning weight (PWT). As live weight at mating is related to fertility and reproductive
rate in young ewes (McGuirk et al., 1968), we would also expect that, at first mating,
ewes with higher growth rates would be more fertile and have higher reproductive rates
than ewes with lower growth.
In addition to the relationships between growth rate and early fertility, selection
strategies that alter the body composition of sheep might also be related to the timing
of first oestrus, fertility, and reproductive rate. In Merino sheep there are positive
genetic correlations between reproduction and growth and between reproduction and
body content of muscle and fat (Huisman and Brown, 2009). The fertility and
reproductive rate of adult Merino ewes is also known to be greater for genotypes with
higher body content of muscle and fat (Ferguson et al., 2007; 2010). Furthermore,
phenotypic enhancement in muscle mass and fatness, as assessed through condition
score, are known to increase fertility and reproductive rate in ewes mated to lamb at
one or two years of age (Malau-Aduli et al., 2007). Overall, it appears that ewe lambs
that accumulate fat and muscle rapidly will achieve puberty earlier, be more fertile and
21
have a higher reproductive rate when mated at 8 or 9 months, than their counterparts
with lower rates of fat and muscle accumulation. Moreover, growth, fatness and the
onset of puberty are all associated with circulating concentrations of IGF-I and leptin
(Roberts et al., 1990; Chilliard et al., 2005), so we would expect reproductive
development to be explained by the secretory patterns of these two metabolic
hormones.
We tested these hypotheses by studying the relationships among phenotypic
values and ASBVs for rates of growth and accumulation of fat and muscle, plasma
concentrations of IGF-I and leptin, the timing of puberty and outcomes for fertility and
reproductive rate in Merino ewe lambs.
Material and Methods
This work was done in accordance with the Australian Code of Practice for the Care
and Use of Animals for Scientific Purposes (7th Edition, 2004) and was approved by
the Animal Ethics Committee of the Department of Agriculture and Food, Western
Australia.
Experimental location and animals
The Merino ewe lambs (n = 136; 94 singles and 42 twins) used in this study were born
on a commercial farm (‘Moojepin’) in August–September 2009 to dams that had been
sourced from two Western Australian ram breeding flocks (‘Merinotech WA’ and
‘Moojepin’) and mated to sires with a wide range in Australian Sheep Breeding Values
(ASBV) for growth, muscle and fat. The ASBV values produced by MERINOSELECT
are the result of collation and analysis of individual performance measures, pedigree
information and relevant environmental and management information of the animals
from participating breeders.
The ewe lambs were transported to Medina Research Station (32.2° S,
115.8° E) where the experiment was conducted from February to June 2010. Live
weight (LW) was recorded weekly and the data were used to generate the average
daily gain (ADG). The depths of the longissimus dorsi muscle and subcutaneous fat at
a point 45 mm from the midline over the twelfth rib were measured using ultrasound
when the ewe lambs were aged 164 (range 134 to 176) and 251 (range 221 to 263)
days. The ultrasound data were used to generate phenotypic values for eye muscle
depth (EMD; range 20 to 33 mm) and C–site fat depth (FAT; range 2 to 8 mm). They
data were also used to generate ASBVs at post–weaning age for weight (PWT; range 0
to 9 kg), depth of eye muscle (PEMD; range 0 to 2.6 mm) and fat (PFAT; range 0 to 1.2
22
mm) by MERINOSELECT (Brown et al., 2007). The ewe lambs were shorn when they
were on average 236 days old.
Animal management and feeding
The ewe lambs were initially run in two management groups in two 20 m x 60 m pens
with ad libitum access to clean water and sheep pellets that were introduced over a 7–
day period. The pellets based on barley, wheat and lupin grains, cereal straw and hay,
canola meal, minerals and vitamins were formulated to provide 11.5 MJ of
metabolisable energy per kg dry matter, 15% protein and minerals to meet their daily
requirements for maximum growth (Macco Feeds, Australia). On February 24 (Day 69), when the ewe lambs were 179 days old (range 149 to 191) and weighed 37 ± 0.4
kg, four Merino wethers (rams castrated before puberty) with harnesses (MatingMark®;
Hamilton, NZ) were introduced to detect the onset of oestrus. The wethers had
received a 2 mL subcutaneous injection of testosterone enanthate (75 mg/mL; Ropel®,
Jurox, NSW) one week before they were placed with the ewe lambs. Every 2 weeks,
the injections were repeated and the crayons on the harnesses were changed. Crayon
marks on the rumps of ewe lambs were recorded three times per week to estimate the
date of standing oestrus. Date of oestrus was deemed as the date the first crayon mark
was recorded and age at this point were deemed to be age at puberty. The closest LW
recorded to the first crayon mark was deemed to be LW at puberty.
The wethers were removed at the end of this “teasing period”, on May 4 (day 0),
when the ewe lambs were 249 (range 219 to 261) days old and weighed 41 ± 0.5 kg.
For the “mating period”, the ewe lambs had received a 1 mL intramuscular injection of
supplement of vitamins (Vit A 500,000 iu; Vit D3 75,000 iu, Vit E 50 iu/mL; Vet ADE®,
Auckland, New Zealand) and were allocated, on the basis of LW and sire, into 8
management groups of 15 and moved into 3 m x 7 m pens where they had ad libitum
access to clean water and the sheep pellets. An experienced single Merino ram was
introduced into each group of ewe lambs. The rams were removed on day 47 and the
ewes remained indoors. Pregnancy and number of fetuses were confirmed by
ultrasound scanning 60 days after the rams were removed. The data from the mating
period were used to generate values for fertility (percentage of pregnant ewes per 100
ewes mated) and reproductive rate (number of fetuses in utero per 100 ewes mated).
The date the ewes lambed was recorded and conception date was deemed to have
occurred 150 days previously.
Blood sampling and immunoassay
Ewe lambs were not fasted and blood was sampled by jugular venipuncture on 5
occasions, when the ewe lambs were on average 199, 227, 248, 269 and 285 days old.
23
Blood was collected into heparinised tubes, placed immediately on ice, and later
centrifuged at 2000 g for 20 min so plasma could be harvested and stored at - 20 ºC
until hormone analysis.
Plasma leptin concentrations were determined by radioimmunoassay in
duplicate 100 μL aliquots as described by Blache et al. (2000). The limit of detection
was 0.06 ng/mL and the intra-assay coefficient of variation was 7.3% at 0.73 ng/mL,
4.4% at 0.84 ng/mL, and 2.4% at 1.61 ng/mL. Plasma concentrations of IGF-I were
measured in duplicate samples using the RIA described by Gluckman et al. (1983).
Interference by binding proteins was minimized by acid–ethanol cryoprecipitation, as
validated for ruminants by Breier et al. (1991). The limit of detection for the assay was
0.05 ng/mL and the intra-assay coefficient of variation was 7% at 0.29 ng/mL and 5.1
% at 2.9 ng/mL.
Statistical analysis
Statistical analysis was aided by SAS version 9.3 (SAS, 2010). Changes in live weight
were analyzed using the linear mixed model procedures allowing repetitive measures
(PROC MIXED), with dam source, dam age (years), birth type and age at start of
mating as fixed effects. FAT, EMD, PWT, PEMD or PFAT were each independently
tested as covariates and dam age was used as a random effect.
The relationships among live weight, PWT, PEMD, PFAT, EMD and FAT were
computed using PROC GLM with the MANOVA option, allowing removal of major fixed
effects. Fixed effects included in the model were dam source, birth type and age at the
day of the muscle and fat scan.
Average daily gain (ADG) during the ‘Teasing’ and ‘Mating’ periods was
determined for each lamb using a cubic smoothing spline approach with transformation
regression model procedures, a method that is appropriate when the response is
nonlinear (TRANSREG). The ADG data were then analyzed using the linear mixed
model procedures (PROC MIXED). Fixed effects in the model were dam source, dam
age (years), birth type and age at start of the ‘Teasing’ or ‘Mating’ periods. FAT, EMD,
PWT, PEMD or PFAT were each independently tested as covariates, and dam age
was used as a random effect.
Age and live weight at first oestrus were also analysed using mixed models
(PROC MIXED) and included dam source, dam age (years), birth type and age as fixed
effects. FAT, EMD, PWT PEMD, PFAT, or concentration of leptin or IGF-I, were each
independently tested as covariates. Dam age was used as a random effect.
Data for puberty (marked or not) and fertility (pregnant or not) were analyzed
using the generalized linear mixed model procedures with a binomial distribution and
logit link function (PROC GLIMMIX). Fixed effects were dam source, dam age, birth
24
type, age and live weight at the start of the period (teasing for puberty; mating for
fertility). Average daily gain (split into teasing for puberty and mating for fertility), FAT,
EMD, PWT, PEMD, PFAT, and leptin or IGF-I concentration, were each independently
tested as covariates. Dam age was used as a random effect. Reproductive rate (dry or
pregnant with single or twins) data was analyzed using the generalized linear mixed
model procedures with a multinomial distribution and logit link function (PROC
GLIMMIX). The same fixed effects, covariates and random effects were used as for the
analysis of fertility.
Data for blood concentrations of leptin and IGF-I were analysed using mixed
models (PROC MIXED) allowing for repeated-measures and included, as fixed effects,
dam source, dam age (years), birth type and age and live weight at start of teasing.
FAT, EMD, PWT, PEMD or PFAT were each independently tested as covariates.
Identification number of the ewe lamb within management group and date of sampling
were used as random effects. Mean metabolite concentrations were analyzed using
analysis of variance (repeated measures), where Factor A was hormone concentration
and Factor B was date at sampling (PROC ANOVA).
All 2-way interactions among the fixed effects were included in each model and
non-significant (P > 0.05) interactions were removed from the final model. The data for
puberty, fertility and reproductive rate are presented as logit values and backtransformed percentages.
Mature LW was considered reached when ewes were more than two years old.
Therefore, to estimate it, individual records from LW and body condition score (BCS)
from the ewe lambs from birth to up to two years of age were used. Mature LW was
predicted at BCS 3 based on these records using linear regression of weight and BCS.
Results
Ewe live weight (LW)
From day –138 to day 55, mean (± SEM) LW increased from 24.2 ± 0.3 to 52.6 ± 0.5
kg (Figure 1). The ADG of the ewe lambs was 90 ± 2.5 g/d during the “teasing” period
and 214 ± 5.3 g/d during the “mating” period. Live weight during the experiment (both
periods) was positively related with increases in values for phenotypic traits (EMD or
FAT; P < 0.001) and ASBVs (PEMD or PFAT; P < 0.001). In general, live weight
increased by 1.4 kg as EMD increased 1 mm, by 3 kg as FAT increased by 1 mm, by
2.2 kg as PEMD increased 1 mm or by 4.4 as PFAT increased 1 mm. The ADG during
the “teasing” period was positively correlated with increases in values for EMD (P <
0.001) and FAT (P < 0.001). The ADG increased 4 g/d as EMD increased 1 mm or by
13 g/d as FAT increased by 1mm. PEMD or PFAT had no effect (P > 0.05) on the ADG
25
during the “teasing” period. None of the variables had an effect on the ADG during the
“mating” period.
The correlations among LW, PWT, EMD, PEMD, FAT and PFAT are shown in
Table 1. Strong relationships were observed between PEMD and PFAT, EMD and FAT
and LW and EMD, whereas other relationships were relatively weak.
Table 1: Correlations (r) among post-weaning phenotypic and genotypic values for weight (LW,
PWT), depth of muscle (EMD, PEMD) and depth of fat (FAT, PFAT) in Merino ewe lambs.
Variable
LW
PWT
EMD
PEMD
FAT
PFAT
LW
1
0.68
0.64
0.28
0.56
0.29
PWT
0.68
1
0.53
0.40
0.39
0.34
EMD
0.64
0.53
1
0.41
0.59
0.34
PEMD
0.28
0.40
0.41
1
0.22
0.76
FAT
0.56
0.39
0.59
0.22
1
0.26
PFAT
0.29
0.34
0.34
0.76
0.26
1
Liveweight (Kg)
60
Single
Twin
55
50
45
40
35
Teasing period
30
-80
-60
-40
-20
Mating period
0
20
40
60
Days of the experiment
Figure 1 Average live weight (± SEM) of single-born (●) or twin-born (○) Merino ewe lambs fed
ad libitum high quality pellet (11.5 MJ metabolizable energy per kg dry matter and 15% protein)
during the experiment. Day 0 is the day when Merino entire rams were introduced.
Live weight and age at puberty
Of the 136 lambs in the flock, 132 (97%) displayed oestrus during the “teasing” or
“mating” periods. The average weight at first oestrus was 39.4 ± 0.5 kg (range 26.9 to
55.1 kg) and the average age at first oestrus was 219 ± 3 days (range 163 to 301
26
days). The proportion of ewe lambs that attained puberty was influenced by both their
age (P < 0.05) and their LW (P < 0.01) at the beginning of the “teasing” period. After
adjustment for effects of LW, there were no effects of ewe source, dam age, birth type
or teasing group on the proportion of ewe lambs that attained puberty (Table 2). The
ADG during the “teasing” period had no effect on the proportion of ewes that reached
puberty. The proportion of ewe lambs that attained puberty was positively related with
increases in values for PWT (P < 0.01; Figure 2), EMD (P < 0.01) and FAT (P < 0.05).
The ASBVs for PEMD or PFAT had no effect on the likelihood of a ewe reaching
puberty (Table 3).
Table 2: Effect of classification variables [Birth type (BT), rear type (RT), ewe source and dam
age] on reproductive performance [age or live weight at first oestrus, puberty, fertility and
reproductive rate (Rep Rate)] and on metabolic hormone concentration (IGF-I or leptin) in
Merino ewe lambs.
Variable
Type
Age at
LW at
Rep
IGF-I
Leptin
(%)
ng/mL
ng/mL
**
NS
NS
81
64.7
1.59
62
61.6
1.51
NS
NS
76
64.6
1.55
95
72
61.6
1.60
*
NS
NS
NS
*
209
38.7
97
77
63.9
1.60
234
40.8
97
72
63.6
1.51
NS
NS
NS
NS
NS
NS
1.5
225
39.1
97
77
61.7
1.56
2.5
210
40.2
100
74
70.1
1.62
3.5
219
40.9
100
89
62.6
1.65
4.5
217
38.8
95
74
62.6
1.50
st
st
Puberty
Fertility
1 oestrus
1 oestrus
Days
Kg
(%)
(%)
NS
NS
NS
NS
Single
218
39.8
96
Twin
222
38.5
100
NS
NS
NS
NS
Single
214
39.5
98
Twin
221
39.2
***
Moojepin
MerinoTech
BT
RT
Ewe source
Dam age (years)
Rate
NS
NS
NS
P-values: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; NS P > 0.05
The average LW and age at the beginning of the “teasing” period was 36.8 ±
0.4 kg and 179 ± 1 days (range 149 to 191 days). Live weight and age at first oestrus
differed with ewe source (P < 0.05; P < 0.001), but the other variables tested had no
effect on either LW or age at first oestrus (P > 0.05). There was no relationship
between the ADG and age at first oestrus (P > 0.05; Table 3). On average, of the
lambs that achieved puberty, twin–born lambs were 1.3 kg lighter and 4 days older
than single lambs at their first oestrus (38.5 vs 39.8 kg and 222 vs 218 days).
27
Live weight at first oestrus was estimated to be 62% of mature body weight.
The average mature body LW was 63.7 ± 0.7 kg (range 49 – 86). Live weight at first
oestrus was positively correlated with increases in values for PFAT (P < 0.01), PWT (P
< 0.001) or EMD (P < 0.001) and it increased by 0.3 kg for each 1 mm of PFAT, by 2.3
kg for each 1 kg increase in PWT and by 1.2 kg for each 1 mm of EMD. Neither PEMD
nor FAT had effect on LW at first oestrus (Table 3).
Ewe lambs with higher values for PEMD (P < 0.05) or PWT (P < 0.001; Figure
3) were younger at first oestrus than ewe lambs with lower values for those traits. Age
at first oestrus decreased by 1 day as PEMD increased 1 mm or by 7 days as PWT
increased 1 kg. PFAT, EMD and FAT had no effect on age at first oestrus (Table 3).
After LW at scanning was included in the statistical model for EMD or FAT or
PWT for PEMD; the effect of EMD and FAT on puberty and PEMD on age at first
oestrus was no longer evident.
Table 3: Effect of phenotype [Eye muscle depth (EMD) and fatness (FAT)], ASBV for postweaning weight (PWT), post-weaning eye muscle depth (PEMD), or post-weaning fatness
(PFAT), or metabolic hormone concentration (IGF-I or leptin) on reproductive performance (age
or live weight at first oestrus, puberty, fertility and reproductive rate) or metabolic hormone
concentration (IGF-I or leptin) in Merino ewe lambs.
Variable
Age at 1
st
st
LW at 1
Puberty
Fertility
IGF-I
Leptin
(%)
ng/mL
ng/mL
NA
NA
NA
NA
**
NA
NA
NA
NA
NA
NA
NS
NS
NS
*
NA
NA
NA
***
***
NS
***
ADG (Teasing)
NS
NA
NS
NA
NA
NA
NA
ADG (Mating)
NA
NA
NA
NS
NS
NA
NA
PWT
***
***
**
**
**
NS
***
*
NS
NS
NS
NS
NS
*
PEMD (+PWT)
NS
NA
NS
NS
NS
NA
NS
PFAT
NS
**
NS
*
NS
NS
*
PFAT (+PWT)
NS
NA
NS
NS
NA
NA
NS
EMD
NS
***
**
**
**
NS
***
EMD ([email protected])
NS
NA
NS
NS
NS
NA
*
FAT
NS
NS
*
*
*
NS
***
FAT ([email protected])
NA
NA
NS
NS
NS
NA
***
*
NS
NS
NS
NS
NA
NA
NS
NA
NS
NS
NS
NA
NA
**
NS
NS
**
**
NA
NA
NS
NA
NS
*
*
NA
NA
oetrus
oestrus
Days
Kg
(%)
(%)
Age (Teasers in)
NA
NS
*
LW (Teasers in)
NS
NA
Age (Rams in)
NA
LW (Rams in)
PEMD
IGF
IGF ([email protected])
Leptin
Leptin ([email protected])
Rep
Rate
P-values: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; NS P > 0.05; NA No applicable
28
Puberty reached (%)
100
90
80
70
0
1
2
3
4
5
6
7
8
9
Post weaning weight (PWT) ASBV (Kg)
Figure 2 Relationships between Australian Sheep Breeding Value for post–weaning weight
(PWT) and the proportion of Merino ewe lambs that achieved puberty by the end of mating
when their average age was 296 days. The dashed lines represent upper and lower 95%
confidence limits.
Age at first oestrus (Days)
250
240
230
220
210
200
190
180
170
1
2
3
4
5
6
7
8
9
Post weaning weight (PWT) ASBV (Kg)
Figure 3 Relationships between Australian Sheep Breeding Value (ASBV) for post-weaning
weight (PWT) and age at first oestrus in Merino ewe lambs. Data from single and twin births
were all retained in the model and the relationship was pooled within the birth-type classes. All
lambs were fed ad libitum with high quality pellets from 6 to 10 months of age. The dashed lines
represent upper and lower 95% confidence limits.
29
Fertility and reproductive rate
A total of 102 out of 136 (75%) ewe lambs were pregnant. Fertility was positively
related to liveweight at start of mating. Ewe lambs that were heavier at the start of
mating were pregnant (P < 0.001; Figure 4). Of those that conceived, the average
weight and age at pregnancy was 44.7 ± 0.5 kg (range 35.3 to 59.2 kg) and 263 ± 2
days (range 219 to 307 days). The ADG during the “mating” period had no effect on
fertility. Fertility differed with birth type (P < 0.05) and mating sire (P < 0.05), but the
rest of the variables had no effect (Table 2). On average, twin–born lambs were 0.6 kg
lighter and 7 days older than single lambs at pregnancy (44.3 vs 44.9 kg and 268 vs
261 days).
The pregnancy rate was positively related with increases in values for PWT (P <
0.01; Figure 5), PFAT (P < 0.05), EMD (P < 0.01), FAT (P < 0.05), but these
relationships, except PWT, were explained by correlated changes in LW and
disappeared when LW or PWT was added to the model. The ASBV for PEMD had no
effect on fertility (Table 3).
Fertility (%)
100
80
60
40
20
30
35
40
45
50
55
Liveweight (Kg)
Figure 4 Relationships between live weight at the start of mating and fertility of Merino ewe
lambs between 6 and 10 months of age. Data from single and twin births were all retained in the
model and the relationship was pooled within the birth-type classes. All lambs were fed ad
libitum with high quality pellets. The dashed lines represent upper and lower 95% confidence
limits.
Of the ewe lambs that were pregnant, 84% were carrying a single lamb and
16% were carrying twins. Reproductive rate differed with birth type (P < 0.01).
Reproductive rate was positively related to LW at start of the mating period (P < 0.001).
On average each extra kg at start of mating was associated with extra 4.8 fetuses per
100 ewes (P < 0.001; Figure 6). Reproductive rate was positively correlated with
30
increases in values for PWT (P < 0.01), EMD (P < 0.01) and FAT (P < 0.05). But, the
effect of EMD or FAT on reproductive rate was no longer evident after LW was added
into the statistical analyses.
Fertility (%)
100
80
60
Single
40
Twin
20
0
1
2
3
4
5
6
7
8
Post weaning weight (PWT) ASBV (Kg)
9
Figure 5 Relationships between Australian Sheep Breeding Value (ASBV) for post–weaning
weight (PWT) and the proportion of single- and twin-born Merino ewe lambs that conceived
between 7 and 10 months of age. All lambs were fed at libitum with high quality pellets. The
dashed lines represent upper and lower 95% confidence limits.
Overall lambs per 100 ewes
180.0
160.0
140.0
120.0
100.0
80.0
60.0
40.0
20.0
35.0
40.0
45.0
Liveweight (Kg)
50.0
Figure 6 Relationships between live weight at start of mating and proportion of overall lambs of
Merino ewe lambs fed ad libitum high quality pellets (15% protein) between 6 and 10 months of
age. The dashed lines represent upper and lower 95% confidence limits.
31
Hormone profiles
As the experiment progressed, circulating concentrations of leptin increased from 1.31
± 0.02 to 1.78 ± 0.01 ng/mL (P < 0.001) and concentrations of IGF-I increased from
39.2 ± 1.3 to 85 ± 2.2 ng/mL (P < 0.001). Leptin concentrations differed with ewe
source (P < 0.05), birth type (P < 0.05) and date of sampling (P < 0.001).
Concentrations of IGF-I differed with date of sampling (P < 0.001), but the other
variables were not related to IGF-I values (Table 2).
The concentrations of IGF-I or leptin were negatively correlated with age at first
oestrus, so first oestrus was advanced as leptin (P < 0.01) and IGF-I (P < 0.05)
concentrations increased. Ewe lambs were younger at first oestrus by 0.2 days as IGFI concentration increased 1 ng/mL or by 28 days as leptin increased 1 ng/mL.
Concentrations of IGF-I and leptin were not related to LW at first oestrus or puberty
(Table 3). The concentration of leptin, but not IGF-I, was positively related to fertility
and to reproductive rate (P < 0.01; Table 3).
Ewe lambs with higher values for PWT (P < 0.001), PEMD (P < 0.05), PFAT (P
< 0.05), EMD (P < 0.001) and FAT (P < 0.001) had a greater leptin concentration than
ewe lambs with lower values. Leptin increased by 0.05 ng/mL as PWT increased 1 kg,
by 0.05 ng/mL as PEMD increased 1 mm, by 0.01 ng/mL as PFAT increased 1 mm, by
0.03 ng/mL as EMD increased 1 mm or by 0.09 ng/mL as FAT increased 1 mm. PEMD
was not associated with leptin concentration. Neither ASBV nor phenotypic values had
effect on IGF-I concentrations (Table 3). The effect of leptin on age at first oestrus and
ASBV was no longer evident once LW was added in the statistical analysis. However,
after adjustment for effects of LW, the effect of leptin concentration on fertility,
reproductive rate, EMD or FAT remained evident.
Discussion
The data support the hypothesis that age and LW at first oestrus, onset of puberty,
fertility and reproductive rate are all influenced by higher values for post-weaning
growth. These observations extend those of Hawker and Kennedy (1978) and Alkass
et al. (1994) who showed that ewes that grew faster reached puberty at a younger age.
In addition, our observations agree with those of Kenyon et al. (2010) who observed
that ewe lambs that attained heavier LW and higher condition score at mating were
more likely to get pregnant and improve the fecundity rate. Decades of research has
shown that we need to provide high quality nutrition to young ewes so they can reach
puberty in a timely manner (Cave et al., 2012) but the present study has also shown
that we can also achieve that aim whilst developing lean carcasses through genetic
selection.
32
The fundamental relationship between body mass and the onset of puberty was
not challenged because the average LW of the lambs that reached puberty was about
62% of their mature LW and thus within the critical 50 to 70% range (Hafez, 1952). On
the other hand, there seems to be a critical LW around 45 kg at the start of mating,
reached earlier in faster growing ewe lambs, where fertility improves. We observed a
linear response between pregnancy rate and LW at the start of mating, over the range
30 to 45 kg, expanding upon previous observations in 18-month-old maiden ewes by
Kleemann and Walker (2005). However, once the 45 kg point had been exceeded, the
response became curvilinear. There was also positive linear effect of LW at start of
mating on reproductive rate, consistent with our observations on mature ewes
(Ferguson et al., 2011), with perhaps greater benefit of additional weight for ewe lambs
than for mature ewes. For ewe lambs, each extra kg was associated with 4.8 extra
fetuses per 100 ewes in contrast with 1.7-2.4 extra fetuses for mature ewes (Ferguson
et al., 2011). This increases the value of reaching the critical live weight at start of
mating for ewe lambs.
High ASBV values for growth can also improve fertility and reproductive rate
because ewe lambs with higher values for PWT achieve the critical percentage of
mature LW earlier and are more suitable for mating at younger ages, as reported
previously (McGuirk et al., 1968). This reflects the positive genetic correlation between
weaning weight and fertility (Barlow and Hodges, 1976). On the other hand, in the
present study, ADG during the “mating” period had no effect on fertility or reproductive
rate, perhaps because the pregnancy rates were already maximal with ADG at high
values (more than 200 g per day). Additionally, the ewes that conceived presumably
did so during the second or third cycle after their first oestrus, as observed by Hare and
Bryant (1985). The remainder of the ewes were detected in oestrus by either teasers or
rams but failed to conceive, perhaps reflecting low quality of ovum or a high incidence
of pre-natal mortality (Quirke, 1981; McMillan and McDonald, 1985). Despite this
problem, it is clear that genetic strategies that increase growth rate will not only
advance puberty but also result in greater fertility and reproductive rate in Merino ewe
lambs.
The data support the hypotheses that age and LW at first oestrus, puberty,
fertility and reproductive rate are all influenced by the rate of accumulation of muscle or
fat. The genetic correlations that we observed among live weight and depths of muscle
and fat were small but positive, whereas the phenotypic correlations observed among
live weight and depth of muscle and fat, also positive, were stronger. However, an
important aspect of the present study is the dissection of effects based on LW, a
passive endpoint, into effects that can be specifically attributed to major, physiologically
active body tissues. Thus, the relationship between onset of puberty and the rate of
33
accumulation of muscle and fat is supported by the existence of endocrine factors from
both tissues that are thought to directly affect the brain processes that control the
initiation of puberty. We found that circulating concentrations of leptin and IGF-I
increased progressively as the experiment progressed and puberty approached,
consistent with previous reports (Roberts et al., 1990; Foster and Nagatani 1999),
suggesting that the two metabolic hormones inform the central nervous system of the
metabolic status of the body, perhaps specifically the accumulation of fat (leptin) and
muscle (IGF-I), and thus permit the triggering of puberty. This role for leptin has been
largely confirmed in ewe lambs, but the question still remains open for IGF-I or any
other endocrine factor associated with muscle.
After puberty, leptin (but not IGF-I) might also explain the relationships between
fertility and reproductive rate and the accumulation of muscle and fat in both young
ewes (present study) and mature Merino ewes (Ferguson et al., 2007; 2010). We found
that the concentration of leptin was associated with fertility and reproductive rate,
consistent with earlier evidence linking leptin to the regulation of fertility (reviewed by,
Smith et al., 2002). Furthermore, ewe lambs with higher phenotypic values for EMD or
FAT or ASBVs for PWT, PEMD or PFAT had greater leptin concentrations than ewe
lambs with lower values for those traits. Interestingly, when LW was added in the
statistical model for EMD and FAT, the effect of these traits on leptin concentration
remained. This might be expected, since animals selected for muscling tend to have
bigger muscles and be bigger and heavier and, since leptin is produced by adipose
tissue, changes in leptin concentration are driven by changes in LW (Blache et al.,
2000). It seems that muscle and fat accumulation modifies circulating leptin, exerting a
positive influence on the reproductive performance of Merino ewe lambs.
Conclusion
Live weight at the start of mating is an important determinant of the reproductive
performance of ewe lambs, and the present study shows that we can address this
limitation in Merino sheep by using genetic strategies for increasing the rates of muscle
and fat accumulation and thus advancing puberty and increasing fertility and
reproductive rate. Our data also suggest that there is a physiological link between
muscle and the reproductive system of female sheep, a novel hypothesis that needs
further investigation. For the sheep industry, these findings should promote genetic
selection strategies that improve profitability by improving reproductive efficiency, and
also help establish modern systems of animal management that will reduce emissions
intensity (Martin et al., 2009).
34
Acknowledgements
The authors wish to thank to Andrew Kennedy and David and Hamish Thompson for
their assistance with collection of pedigree data and Margaret Blackberry for her help
with hormone analyses. Cesar Rosales Nieto was supported by CONACyT (the
Mexican National Council for Science and Technology), the Department of Agriculture
and Food of Western Australia, the Cooperative Research Centre for Sheep Industry
Innovation, and the UWA School of Animal Biology during his doctoral studies.
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37
Chapter 2
Ewe lambs with higher breeding values for growth achieve higher
reproductive performance when mated at age 8 months
a,b,c
C.A. Rosales Nieto
, M.B. Fergusona,b,d,1, C.A. Macleayb, J.R. Briegelb, D.A. Woodb,
G.B. Martinc, A.N. Thompsona,b,d,*
a
CRC for Sheep Industry Innovation and the University of New England, Armidale, New South
Wales, 2351, Australia
b
Department of Agriculture and Food of Western Australia, 3 Baron Hay Court, South Perth,
Western Australia, 6151, Australia
c
UWA Institute of Agriculture, University of Western Australia, Crawley, Western Australia,
6009, Australia
d
School of Veterinary and Biomedical Sciences, Murdoch University, 90 South Street, Murdoch,
Western Australia 6150, Australia
1
Present address: The New Zealand Merino Company Ltd, PO Box 25160, Christchurch 8024,
New Zealand
Published: Theriogenology 2013; 80, 427-435
38
Abstract
We studied the relationships among growth, body composition and reproductive
performance in ewe lambs with known phenotypic values for depth of eye muscle
(EMD) and fat (FAT) and Australian Sheep Breeding Values for post-weaning live
weight (PWT) and depth of eye muscle (PEMD) and fat (PFAT). Vasectomized rams
were placed with 190 Merino ewe lambs, when they were, on average, 157 days old, to
detect first oestrus. The vasectomized rams were replaced with entire rams when the
ewe lambs were, on average, 226 days old. Lambs were weighed every week and
blood was sampled on four occasions for assay of ghrelin, leptin and ßhydroxybutyrate. Almost 90% of the lambs attained puberty during the experiment, at
an average live weight of 41.4 kg and average age of 197 days. Ewe lambs with higher
breeding values for EMD (P < 0.001), FAT (P < 0.01), PWT (P < 0.001), PEMD (P <
0.05) and PFAT (P < 0.05) were more likely to achieve puberty by 251 days of age.
Thirty-six percent of the lambs conceived and, at the estimated date of conception, the
average live weight was 46.9 ± 0.6 kg and average age was 273 days. Fertility,
fecundity and reproductive rate were positively related to PWT (P < 0.05) and thus live
weight at the start of mating (P < 0.001). Reproductive performance was not correlated
with blood concentrations of ghrelin, leptin and ß-hydroxybutyrate. Many ewe lambs
attained puberty, as detected by vasectomised rams, but then failed to become
pregnant after mating with entire rams. Nevertheless, we can conclude that, in ewe
lambs mated at 8 months of age, higher breeding values for accumulation of muscle
and fat, and therefore growth, are strongly positively correlated with reproductive
performance, although the effects of breeding values for growth and responses to live
weight are highly variable.
Keywords: ewe lambs, reproductive performance, phenotypic selection, Australian
sheep breeding value (ASBV)
39
1. Introduction
Puberty in females is defined by the first ovulation because that is when
reproduction becomes possible. In ewe lambs, it is usually achieved when they attain
50-70% of their mature live weight, with age per se being a secondary factor [reviewed
by 1,2]. Because of the importance of live weight, environmental factors that affect the
rate of growth before and after weaning are important determinants of age at puberty
[reviewed by 3]. For example, ewe lambs born and raised as singles reach puberty at a
younger age than those born and raised as twins [4], because single-reared lambs
grow faster to weaning and can remain heavier until 12 months of age despite postweaning compensatory growth in twin-reared progeny [5]. As genetic potential for
growth also affects live weight, it is not surprising that Merino ewe lambs with higher
breeding values for growth are younger at puberty and achieve greater fertility and
reproductive rate when mated at 8-10 months of age [6,7].
In addition to the effect of live weight, we have demonstrated in adult ewes the
relationships of fertility and fecundity with the accumulation of muscle and fat [8,9], and
we have begun extending these studies to ewe lambs. We have shown that, under
animal house conditions, ewe lambs with higher breeding values for muscle and fat
attain puberty earlier and are more likely to conceive than ewe lambs with lower
breeding values [7]. Muscle and fat are genetically correlated with live weight [10] but,
in adult sheep, the relationships between muscle and fat on fertility and reproductive
rate were not related to live weight, in contrast with the situation in ewe lambs [7]. The
potential effect of genetically accelerated fat accumulation on reproduction is likely to
be reduced under conditions of good nutrition [9] and good nutritional conditions were
maintained throughout the previous study of ewe lambs [7]. This might have diminished
the impact of these metabolic tissues on their reproductive performance.
Sexual maturation and reproductive performance are closely linked to the
metabolic status of the animal, due to the effects of physiological signals from
metabolic tissues on the reproductive axis [11]. There are two primary candidate from
adipose tissue: i) leptin, a potent regulator of appetite, metabolism and reproductive
endocrinology [12]; and ii) β-hydroxybutyrate, an indicator of energy status when there
is a relatively high glucose demand [13]. From the gut, a likely candidate is ghrelin, a
hormone that acts as a signal of energy insufficiency and thus generally inhibits the
reproductive axis [14]. We therefore tested the hypotheses that phenotypic and
genotypic values for growth, and for accumulation of fat and muscle, will affect the
timing of puberty and the reproductive performance of ewe lambs mated under
extensive conditions, and that these relationships will be related to circulating
concentrations of leptin, ghrelin and β-hydroxybutyrate.
40
2. Material and Methods
This experiment was undertaken in accordance with the Australian Code of Practice for
the Care and Use of Animals for Scientific Purposes (7th Edition, 2004) and was
approved by the Animal Ethics Committee of the Department of Agriculture and Food,
Western Australia.
2.1. Experimental location and animals
The Merino ewe lambs (n = 190; 135 singles and 55 twins) used in this
experiment were born in June 2010 at the research farm of the University of Western
Australia, near Pingelly in Western Australia (32.2° S, 115.8° E). The dams of the ewe
lambs had been sourced from two ram breeding flocks in Western Australia and the
sires had a range in Australian Sheep Breeding Values for growth, muscle and fat
accumulation. The Australian Sheep Breeding Values produced by MERINOSELECT
are the result of collation and analysis of individual performance values, pedigree
information and relevant environmental and management information of the animals
from participating breeders. Data for birth date, birth weight, birth type and rear type to
weaning were collected for the ewe lambs and, on 1 November 2010, they were
transported to the Medina Research Station (32.2° S, 115.8° E) for the first stage of the
experiment. In late December, they returned to the university farm where they
remained until the end of the experiment (Fig. 1).
The ewe lambs were weighed weekly from the start of ‘teasing’ (the introduction
of vasectomized adult rams) on November 30 (Day -70; Day 0 was to the start of the
mating period with intact rams on February 8) to the end of the experiment (Day +98).
Live weight data were interpolated to estimate the live weight at puberty and the
estimated date of conception and to calculate the average daily gain (ADG). After Day
+ 98, the ewe lambs were weighed and condition scored every three months until two
years of age to predict mature live weight. Mature live weight was predicted as the
conceptus-free live weight at condition score 3 from a linear regression of the live
weight and condition score data for each animal.
The depth of the longissimus dorsi muscle and subcutaneous fat at a point 45
mm from the midline over the twelfth rib was measured using ultrasound when the
average age of the ewe lambs was 167 (range 146–186) and 218 (range 198–228)
days. For both measurements, the range in eye muscle depth (EMD) was 20–33 mm
and the range in C-site fat (FAT) was 2–8 mm. Using MERINOSELECT [15], the
ultrasound data were used to generate estimates of Australian Sheep Breeding Values
at post-weaning age for weight (PWT; range 0–9 kg), depth of eye muscle (PEMD;
range 0.0–2.6 mm) and depth of fat (PFAT; range 0.0–1.2 mm).
41
2.2. Animal management and feeding
At the Medina research station, ewe lambs were allocated on the basis of live
weight among eight groups, each housed in a 6 x 14 m pen where they had ad libitum
access to water and to pellets that were introduced over a 7-day period. The pellets
were based on barley, wheat and lupin grains, cereal straw and hay, canola meal,
minerals and vitamins. They were formulated to provide 11.5 MJ of metabolisable
energy per kilogram of dry matter, 15% protein, and minerals and vitamins for
maximum growth. At the start of ‘teasing’, the ewe lambs were on average 157 days
old (range 136–176) and weighed 36.2 ± 0.3 kg (range 24.8–50.8). A vasectomized
Merino ram with a marking harness (MatingMark®; Hamilton, NZ) was introduced into
each pen to detect the onset of oestrus. On December 29 (Day - 41), the ewe lambs
and vasectomized rams were moved to the University farm (Fig. 1) where each of the
eight groups was allocated to a separate 30 x 120 m field.
The vasectomized rams were removed on February 8 (Day 0) and ewe lambs
were allocated on the basis of their live weight and prospective sire into 8 groups with
23–24 ewe lambs per group. An experienced ram with a marking harness was
introduced into each group to begin “Mating period 1” (Fig. 1) when the ewe lambs
were on average 226 days old (range 206–246) and weighed 42.4 ± 0.3 kg (range
24.3–56.4). The rams were removed after 24 days. One month later, after a pregnancy
scan indicated a low fertility rate, a second set of eight experienced rams were
introduced to all ewe lambs, thus beginning “Mating period 2” (Fig. 1). At this stage, the
ewe lambs were on average 285 days old (range 264–304) and weighed 48.4 ± 0.3 kg
(range 29.0–61.5). These rams were removed 21 days later. Throughout both mating
periods, the lambs had access to clean water and had ad libitum access to oaten hay
(9 MJ/kg and 9% protein) plus lupin grain (13.5 MJ/kg and 32% protein). It was
anticipated that the combination of supplement plus dry pasture would allow the lambs
to gain about 100 g/day.
Crayon marks on the rumps were recorded three times per week at Medina and
once per week at the university farm to estimate the date of their first standing oestrus.
Crayon marks were scored at 1, 2 or 3, with score 1 being one narrow mark on the
middle or the edge of the rump and score 3 as being a single large mark covering the
rump. The date when the first score 2-3 crayon mark was recorded was deemed to be
the age at first oestrus and the closest live weight recorded to that date was deemed to
be live weight at first oestrus. Pregnancy rate and the number of fetuses per ewe were
measured at ultrasound scanning in mid-April and mid-June. The data from both
mating periods were combined and used to generate values for fertility (percentage of
pregnant ewes per 100 ewes mated), fecundity (percentage of pregnant ewes with twin
42
fetuses) and reproductive rate (number of fetuses in utero per 100 ewes mated). The
date the ewes lambed was recorded and conception date was deemed to have
occurred 150 days previously.
Ewe lambs
born
Transport
to Medina
Return to
UWA farm
Jun
2010
Nov
2010
UWA farm
-252
29 Dec
2010
Teasing period
-99 -70
-41
Feb 8–Mar 4
2011
7–29 Apr
2011
Mating period 1
Mating period 2
0
24 58
80
Day of experiment
Fig. 1. A schema describing animal management during the experiment, with Day 0 designated
as the start of the first mating period.
2.3. Blood sampling and assay
Blood (5 ml) was sampled into heparinised tubes by jugular venipuncture when
the ewe lambs were on average 144 (Day - 83), 186 (Day - 39), 227 (Day 0) and 254
(Day + 28) days old. The samples were immediately placed on ice and then centrifuged
at 2000 g for 20 min so plasma could be harvested and stored at –20º C until hormone
analysis. Ghrelin was measured in duplicate 100 μL aliquots of plasma with a doubleantibody radioimmunoassay (RIA) method, as modified by Miller et al. [16]. The limit of
detection was 49 pg/mL and the intra-assay coefficient of variation was 6% at 94
pg/mL, 2.6% at 327 pg/mL, and 1% at 1550 pg/mL. Plasma leptin was measured in
duplicate 100 μL aliquots using the double antibody RIA described by Blache et al.
[17]. The limit of detection was 0.05 ng/mL and the intra-assay coefficient of variation
was 16% at 0.47 ng/mL, 3.3% at 1.10 ng/mL, and 3.6% at 1.79 ng/mL. Concentrations
of ß-hydroxybutyrate were determined on an automated analyser (AU400, Olympus,
Tokyo, Japan) using the RANDOX RANBUT reagents supplied by Perth Scientific.
2.4. Data analysis
Statistical analysis was aid by SAS version 9.3 [18]. Changes in live weight
were analyzed using the linear mixed model procedures allowing repetitive measures
(PROC MIXED), with dam source, dam age (years), birth type and age at start of
mating as fixed effects. FAT, EMD, PWT, PEMD or PFAT were each independently
tested as covariates and the identification number of ewe lamb within sire (father)
group was used as a random effect.
43
The relationships among live weight, PWT, PEMD, PFAT, EMD and FAT were
computed using PROC GLM, with MANOVA option, allowing removal of major fixed
effects. Fixed effects included in the model were dam source, birth type and age at the
day of the muscle and fat scan.
Average daily gain (ADG) during the ‘Teasing’ and ‘Mating’ periods was
determined for each lamb using a cubic smoothing spline approach with transformation
regression model procedures, a method that is appropriate when the response is
nonlinear (TRANSREG). The ADG data were then analyzed using the linear mixed
model procedures (PROC MIXED). Fixed effects in the model were dam source, dam
age (years), birth type and age at start of the ‘Teasing’ or ‘Mating’ periods. FAT, EMD,
PWT, PEMD or PFAT were each independently tested as covariates, and the sire
(father) of the ewe lamb was used as a random effect.
Age and live weight at first oestrus were also analysed using mixed models
(PROC MIXED) and included dam source, dam age (years), birth type and age as fixed
effects. FAT, EMD, PWT PEMD, PFAT or concentration of leptin or ghrelin were each
independently tested as covariates. Sire (father) of ewe lamb was used as a random
effect.
Puberty and fertility data were analyzed using the generalized linear mixed
model procedures with a binomial distribution and logit link function (PROC GLIMMIX).
Fixed effects were dam source, dam age (years), birth type, age and live weight at the
start of the period (teasing for puberty; mating for fertility). Average daily gain (split into
teasing for puberty and mating for fertility), FAT, EMD, PWT, PEMD, PFAT, leptin or
ghrelin concentration (for puberty) or leptin or ß-hydroxybutyrate concentration (for
fertility) were each independently tested as covariates. Sire (father) of ewe lamb was
used as a random effect. Reproductive rate data was analyzed using the generalized
linear mixed model procedures with a multinomial distribution and logit link function
(PROC GLIMMIX). The same fixed effects, covariates and random effects were used
as the as for the analysis of fertility.
Blood metabolite data (leptin, ghrelin, ß-hydroxybutyrate) were analysed using
mixed models (PROC MIXED) allowing for repeated-measures and included, as fixed
effects, dam source, dam age (years), birth type and age and live weight at start of
teasing. FAT, EMD, PWT, PEMD or PFAT were each independently tested as
covariates. Identification number of the ewe lamb within sire (father) group was used as
a random effect. Mean metabolite concentrations were analyzed using analysis of
variance (repeated measures), where Factor A was hormone concentration and Factor
B was date at sampling (PROC ANOVA).
All 2-way interactions among the fixed effects were included in each model and nonsignificant (P > 0.05) interactions were removed from the final model. The data for
44
puberty, fertility and reproductive rate are presented as logit values and backtransformed percentages.
3. Results
3.1. Live weight
Mean (± SEM) live weight increased from 38.2 ± 0.3 kg on Day –70 to 52.6 ±
0.4 kg on Day +98 (Fig. 2) and differed with age at the start of the teasing period (P <
0.001). On average, single-born lambs were heavier than twin-born lambs (37.6 vs
35.6 kg; P < 0.01) and single-reared lambs were heavier than twin-reared lambs (37.5
vs 35.7 kg; P < 0.001).
Live weight (Kg)
55
BS
BS
BS
50
45
40
Mating
period 1
Teasing period
35
-70
-40
0
24
Mating
period 2
58
80 98
Day of experiment
Fig. 2 Average live weight (± SEM) of Merino ewe lambs born as singles (black; n = 135) or
twins (white; n = 55). Day 0 is the day when entire rams were first introduced for the first mating
period. BS: blood sample.
The correlations among live weight, PWT, EMD, PEMD, FAT and PFAT are
shown in Table 1. Medium to strong positive correlations were observed between live
weight and FAT and EMD, PWT and EMD and PEMD and PFAT (Table 1).
During the teasing period, the ADG was 117 ± 2 g and differed with age at the
start of the teasing (P < 0.001), but was not affected by dam age (years) nor dam
source (P > 0.05). Ewe lambs born and reared as twins grew around 10 g/d faster than
ewe lambs born and reared as singles (P = 0.06; Table 2). During the mating period,
45
the ADG was 58 ± 2 g and was affected by birth type (P < 0.05; Table 1), but the other
variables had no effect. Ewe lambs born and reared as a twin grew around 9 g/d faster
than ewe lambs born and reared as a single during this period (Table 2).
Table 1: Correlations (r) among post-weaning phenotypic and genotypic values for weight (LW,
PWT), depth of muscle (EMD, PEMD) and depth of fat (FAT, PFAT) in Merino ewe lambs.
Variable
LW
PWT
EMD
PEMD
FAT
PFAT
LW
1
0.79
0.70
0.24
0.52
0.26
PWT
0.79
1
0.63
0.29
0.33
0.11
EMD
0.70
0.63
1
0.76
0.54
0.52
PEMD
0.24
0.29
0.76
1
0.40
0.67
FAT
0.52
0.33
0.54
0.40
1
0.71
PFAT
0.26
0.11
0.52
0.67
0.71
1
Live weight during the experiment was positively related with EMD, FAT, PWT,
PEMD or PFAT (P < 0.001). Ewe lambs were 1.2 kg heavier as EMD increased by 1
mm, 2.1 kg heavier as FAT increased by 1 mm, 2.0 kg heavier as PWT increased by 1
kg, 1.9 kg heavier as PEMD increased by 1 mm, or 3.1 kg heavier as PFAT increased
by 1 mm. During the teasing period, ADG was positively correlated with EMD (P <
0.001), FAT (P < 0.01), PWT (P < 0.001) and PFAT (P ≤ 0.05), but not PEMD (P >
0.05). The ADG during this period increased by 4.8 g/d as EMD increased by 1 mm, by
7.2 g/d as FAT increased by 1 mm, by 10 g/d as PWT increased by 1 kg or 12.8 g/d as
PFAT increased by 1 mm. During the mating period, ADG was positively correlated
with PWT (P < 0.001) and increased by 5 g/d as PWT increased by 1 kg. It was not
related to EMD, FAT, PEMD or PFAT (P > 0.05).
3.2. Live weight and age at puberty
Of the 190 ewe lambs, 170 (89%) displayed oestrus during the ‘Teasing period’
and ‘Mating period 1’, and the proportion that displayed oestrus was not affected by
dam source, dam age (years), birth type or rear type (P > 0.05). Ewe lambs that were
heavier at the start of the teasing period (P < 0.001) and ewe lambs that grew faster
(high ADG) were more likely to achieve puberty (P < 0.05). Thus, on average, the
proportion of ewe lambs that displayed oestrus increased by 4% with each 20 g/d
increase in ADG during the ‘Teasing period’. As shown in table 2, ewe lambs were
more likely to attain puberty if they had high values for EMD (P < 0.001), FAT (P <
0.01), PWT (P < 0.001; Fig. 3), PEMD (P < 0.05) or PFAT (P < 0.05). After adjustment
for effects of live weight, the relationships with muscle and fat accumulation were no
longer evident.
46
Table 2: Relationships among birth and rear type and the average daily gain (ADG; split into
teasing and mating periods), reproductive performance, and blood leptin concentrations in
Merino ewe lambs. LW = live weight.
Birth type
1
n
2
Mean ±
n
SEM
ADG during teasing
Rear type
1
Mean ±
n
SEM
a
2
Mean ±
n
SEM
a
Mean ±
SEM
a
a
135
114 ± 2
55
123 ± 3
140
114 ± 2
50
124 ± 4
135
56 ± 2
a
55
65 ± 4
b
140
56 ± 2
a
50
64 ± 4
121
198 ± 3
49
196 ± 5
125
199 ± 3
45
193 ± 5
121
42 ± 0.5
49
40 ± 0.7
125
42 ± 0.5
45
40 ± 0.8
Puberty (%)
121
90
49
89
125
89
45
90
Age at pregnancy
49
270 ± 4
19
279 ± 7
50
271 ± 4
18
278 ± 7
49
46.7 ± 0.7
19
47.3 ± 1.1
50
46.7 ± 0.7
18
47.4 ± 1.1
49
36
19
35
50
36
a
18
36
a
18
34
(g/d)
ADG during mating
a
(g/d)
Age at first oestrus
a
a
a
a
(days)
LW at first oestrus
a
c
a
c
(kg)
a
a
a
a
a
a
a
a
(days)
LW at pregnancy
a
a
a
a
(kg)
Fertility (%)
Fecundity (%)
49
a
a
12
19
Reproductive rate
a
Leptin (ng/mL)
1.9 ± 0.02
ab
a
b
37
50
a
a
14
a
a
1.9 ± 0.03
a
ac
a
a
1.9 ± 0.02
values within columns with different superscripts are different (P < 0.05);
a
a
1.9 ± 0.04
values within columns with
different superscripts are different (P < 0.01)
At the first detected oestrus, the average age was 197 ± 2 days (range 147–
290) and the average live weight was 41.4 ± 0.4 kg (range 29.1–57.6), 67% of mature
body weight (estimated to be 61.6 ± 0.4 kg; range 46–79). Live weight at first oestrus
differed with birth type (P < 0.01), rear type (P < 0.05) and age at the start of the
‘Teasing period (P < 0.01), but neither birth type nor rear type were related to age at
first oestrus (P > 0.05; Table 2). On average, ewe lambs born and reared as a single
were heavier by 2 kg (P < 0.05) and older by 6 days at first oestrus than those reared
as a twin (P > 0.05; Table 2).
Live weight at first oestrus was positively related with increases in EMD (P <
0.001), FAT (P < 0.001), PWT (P < 0.001), PEMD (P < 0.01) and PFAT (P < 0.01)
(Table 3). Thus, live weight at first oestrus increased by 1.3 kg as EMD increased by 1
mm, 2.2 kg as FAT increased by 1 mm, 2.3 kg as PWT increased by 1 kg, 2.2 kg as
PEMD increased by 1 mm and 2.6 kg as PFAT increased by 1 mm. Age at first oestrus
was not related to any of these traits (P > 0.05; Table 3).
47
Table 3: Outcomes of statistical analyses of the relationships among live weight (LW) or age at
start of the “teasing” or “mating” periods, average daily gain (ADG) split into “teasing” and total
“mating” periods, phenotypic values for eye muscle depth (EMD) and fatness (FAT), ASBV for
post-weaning weight (PWT), post-weaning eye muscle depth (PEMD), post-weaning fatness
(PFAT), and blood metabolite concentrations, and reproductive performance and leptin
concentration in Merino ewe lambs. Information from both mating periods was combined for
fertility, fecundity and reproductive rate.
Age (1
st
st
LW (1
Puberty
Fertility
Fecundity
Reproductive
Leptin
oestrus)
oestrus)
Days
Kg
%
%
%
%
ng/mL
Age (teasers in)
NA
**
NS
NA
NA
NA
*
LW (Teasers in)
NS
NA
**
NA
NA
NA
***
LW (Rams in)
NA
NA
NA
***
*
***
NA
ADG (Teasing)
NS
NA
*
NA
NA
NA
NA
NA
NA
NA
*
NS
NS
NA
PWT
NS
***
***
***
*
***
***
PEMD
NS
**
*
NS
NS
NS
***
PEMD (+PWT)
NS
NA
NS
NS
NS
NS
*
PFAT
NS
**
*
NS
NS
NS
***
PFAT (+PWT)
NS
NA
NS
NS
NS
NS
***
EMD
NS
***
***
**
*
***
***
NS
NA
NS
NS
NS
NS
***
NS
***
**
*
NS
*
***
NS
NA
NS
NS
NS
NS
***
NS
*
NS
NA
NA
NA
NA
NA
NA
NA
NS
NS
NS
NA
NS
*
*
NS
NS
NS
NA
NS
NA
NS
NS
NS
NS
NA
Variable
ADG (Total
Mating)
EMD (+LW at
scan)
FAT
FAT (+LW at
scan)
Plasma Ghrelin
Plasma ßHydroxybutyrate
Plasma Leptin
Plasma Leptin
(+LW at scan)
Rate
P-values: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; NS P > 0.05; NA, not applicable
48
Puberty reached (%)
100
A
80
60
40
20
0
Fertility (%)
80
B
60
40
20
0
1
2
3
4
5
6
7
8
ASBV for Post-weaning weight (PWT; Kg)
9
Fig. 3. The relationship between the Australian Sheep Breeding Value (ASBV) for post-weaning
weight (PWT) and the proportions of Merino ewe lambs that (A) achieved puberty by 251 days
of age (P < 0.001) and (B) were pregnant after mating at age 7-11 months (P < 0.001). Data
from single and twin births were retained in the model and the relationship was pooled within
the birth-type classes. The broken lines represent upper and lower 95% confidence limits.
3.3. Fertility, fecundity and reproductive rate
A total of 68 of 190 (36%) ewe lambs were pregnant with 80% carrying a single
lamb and 20% carrying twin lambs. Ewe lambs that were heavier at the start of the
mating period were more fertile (P < 0.001; Fig. 4) and fecund (P < 0.01) than lighter
animals. The most significant increases in fertility rate occurred when the ewe lambs
weighed more than 40 kg at start of mating (Fig. 4).
49
Fertility (%)
80
A
60
40
20
0
Reproductive rate (%)
140
B
120
100
80
60
40
20
0
35
40
45
Live weight at start of mating (Kg)
50
Fig. 4. Relationships between live weight at the start of mating and (A) fertility (P < 0.001) and
(B) reproductive rate (number of lambs in utero per 100 ewes mated; P < 0.001) in Merino ewe
lambs mated at 8-11 months of age. Data from both mating periods and are combined. Data
from single and twin birth were retained in the model and the relationship was pooled within the
birth-type classes. The broken lines represent upper and lower 95% confidence limits.
At the estimated date of conception, live weight was 46.9 ± 0.6 kg (range 37.6–58.1)
and age was 273 ± 4 days (range 215–321). The ADG during the “Mating period” was
positively related to fertility (P < 0.05) but not fecundity (P > 0.05). By contrast, birth
type influenced fecundity (P < 0.05) but not fertility (P > 0.05; Table 2). Neither fertility
nor fecundity were influenced by dam age (years), dam source or rear type (P > 0.05).
On average, ewe lambs born and reared as twins were 9 days older and 0.7 kg heavier
at the estimated date of conception than ewe lambs born as singles (P > 0.05; Table
2).
50
Reproductive rate was positively related to live weight at the start of mating (P <
0.001; Fig. 4). The relationship was linear with each extra kg of live weight at the start
of mating associated with an extra 4.5 fetuses per 100 ewes mated (Fig. 4). Birth type,
rear type, dam age and dam source had no effect on reproductive rate (P > 0.05).
Fertility was positively correlated with EMD (P < 0.01), FAT (P < 0.05) and PWT
(P < 0.001; Fig. 3), but not PEMD or PFAT (Table 2). Fecundity was positively related
with EMD (P < 0.05) and PWT (P ≤ 0.05), but was not with FAT, PEMD or PFAT (P >
0.05; Table 3). Reproductive rate was positively correlated with EMD (P < 0.001), FAT
(P < 0.05) and PWT (P < 0.001), but not with PEMD or PFAT (P > 0.05; Table 3). After
adjustment for effects of live weight, the effects of muscle and fat accumulation on
fertility, fecundity and reproductive rate were no longer evident.
3.4. Hormone and metabolite profiles
The concentration of leptin increased gradually between November 2010 and
March 2011 from 2.01 ± 0.02 to 2.12 ± 0.03 ng/mL, the exception being a lower
concentration in the blood sample collected during February 2011 (Day 0; 1.2 ± 0.02
ng/mL) In contrast, ß-hydroxybutyrate concentrations decreased from 0.38 ± 0.009 to
0.33 ± 0.008 mmol/L, the exception being a higher value in the sample collected during
February 2011 (Day 0; 0.59 ± 0.12 mmol/L). Leptin concentration was positively related
to the proportion of ewe lambs that attained puberty (P ≤ 0.05) and live weight at first
oestrus (P < 0.01), whereas ghrelin concentration was negatively related to live weight
at first oestrus (P < 0.05). Leptin and ghrelin concentrations were not related to age at
first oestrus and ghrelin concentration was not related to the proportion of ewe lambs
that attained puberty (P > 0.05). Neither leptin nor ß-hydroxybutyrate concentrations
were associated with fertility, fecundity or reproductive rate (P > 0.05; Table 3). Leptin
values differed with birth type (P < 0.05; Table 1), but not with rear type, dam source or
dam age (P > 0.05). Leptin concentration was positively related to increases in PWT (P
< 0.001), PEMD (P < 0.001), PFAT (P < 0.001), EMD (P < 0.001) and FAT (P < 0.001)
(Table 2). Leptin concentration increased by 0.05 ng/mL as PWT increased 1 kg, 0.24
ng/mL as PEMD increased 1 mm, 0.36 ng/mL as PFAT increased 1 mm, 0.05 ng/mL
as EMD increased 1 mm or 0.11 ng/mL as FAT increased 1 mm. The relationships
between leptin and the accumulation of muscle and fat were still evident when live
weight was included in the statistical analysis. Neither ghrelin nor ß-hydroxybutyrate
concentrations were related to phenotypic or genotypic values for growth, fat or muscle
(P > 0.05).
51
4. Discussion
Most Merino ewe lambs achieved puberty by 250 days of age when their
average live weight was around 41 kg, or about 67% of their estimated mature live
weight, within the range of 50-70% that has previously been considered as the
threshold for spontaneous puberty in sheep [reviewed by 1,2]. Ewe lambs with higher
breeding values for growth were more likely to achieve puberty at around 250 days of
age. They were heavier at the start of teasing and grew faster during the teasing
period, and both of these factors were statistically related to the percentage of ewes
achieving puberty by this age. This observation is consistent with previous reports that
faster growth results in more ewes achieving puberty at a younger age in female sheep
[7, 19]. In the current study, more than 90% of ewe lambs with a PWT greater than 5
achieved puberty by this age, whereas the value was less than 40% for lambs with a
PWT of 1, close to the average for Merino ewes on the National Sheep Genetics
database (www.sheepgenetics.org.au). Early puberty also led to first mating at a
younger age and would allow second mating at 20 months of age and ready integration
with the adult ewe flock. Thus, overall, there are many advantages to selection of
Merino ewe lambs with a high breeding value for growth.
Fertility also improved as breeding value for growth increased, reflecting
previous reports of a positive genetic correlation between weaning weight, rapid growth
and reproductive performance in young female sheep [20, 21]. Ewe lambs with higher
breeding values for growth were heavier at the start of mating, grew faster during
mating and had higher fertility, as previously observed [7, 22]. The relationship
between live weight at the start of mating and fertility rate was linear over the range
40–55 kg. High breeding values for growth and live weight at the start of mating were
also associated with improved fecundity and overall reproductive rate. Higher live
weight at the start of mating may provide a greater benefit for ewe lambs than for
mature ewes, because for each extra kg, there was a gain of 4.5-4.8 extra fetuses per
100 ewe lambs mated, in contrast to only 1.7-2.4 extra fetuses for mature ewes [23].
While the ewe lambs own breeding values for growth include both phenotypic and
genetic components, we have recently studied larger data sets and found that the
breeding value for growth of the sire of Merino ewe lambs is significantly related to the
reproductive performance of their daughters when they mated at 8-10 months of age
(A.N. Thompson, 2013, unpublished data). We are therefore able to conclude that
genetic strategies, as well as nutritional management, can be used improve
reproductive rate in Merino ewe lambs.
In the present study, the lambs that were still pregnant at scanning were
estimated to have conceived about 80 days after achieving puberty, during which time
52
they had gained more than 5 kg in live weight. Depending on cycle length, which vary
from 13–19 d and average 17 d [reviewed by 24], it seems likely that on average they
conceived on their fourth or fifth cycle post-puberty. This deduction, together with the
observation that more 90% of ewe lambs appeared to achieve puberty and yet only
36% were scanned as pregnant, suggests poor conception or a high incidence of
embryo mortality in the present experiment. Hare and Bryant [25] found that the
proportion of ewe lambs with living embryos was greater in those that were mated at
their third oestrus, intermediate in those mated at their second oestrus, and lowest in
those mated at their pubertal oestrus. Ewe lambs born as singles were about 2 kg
heavier, but surprisingly 2 days older, at their first oestrus than their twin-born
counterparts, whereas those born as singles were younger and lighter at the estimated
date of conception. Indeed, ewe lambs that were born as a twin gained 7.7 kg between
puberty and conception compared to 4.6 kg for those born as singles which, despite
some evidence of compensatory growth, indicates that the oestrous cycle from which
pregnancy was maintained was later than for those born as singles. These
observations further highlight the importance of achieving puberty as early as possible
and also suggest that twin-born lambs may benefit from either a delayed mating after
teasing or a longer mating period.
This and other experiments indicate significant variation in the relationship
between live weight at mating and fertility and reproductive rate. Fertility, especially
under field conditions, is always lower and more variable for ewe lambs than for mature
ewes [reviewed by 26], suggesting important effects of unidentified and thus
uncontrolled environmental factors. In the present study, the animals experienced high
environmental temperatures (above 32º C) before and during mating, a known cause of
infertility in Merino ewes [27]. A second possibility is variation in metabolic status, as
described below – we did not limit nutrition but the decline in the trajectory of weight
gain during the week before the start of Mating period 1, followed by a low ADG during
the mating period, suggest that negative energy balance may have prevented the
continuation of ovulation or compromised fertility [reviewed by 28, 29]. This did not
happen in our previous study where the ADG during mating was over 200 g/d and 75%
of the ewe lambs conceived [7]. Further research is needed to test this hypothesis.
The depths of muscle and fat were related to live weight at puberty, and to
fertility, fecundity and reproductive rate. These relationships were revealed when live
weight was excluded in the model. This can be explained by the correlations among
these traits that were observed in this experiment. Obviously, live weight includes
muscle and fat and thus raises questions of interpretation. An important consideration
is that live weight per se is simply mass and so encompasses no physiological or
mechanistic process that would affect the reproductive system. On the other hand,
53
tissues that are metabolically, physiologically and hormonally active, such as muscle
and fat, can become involved in control processes at the brain, pituitary, ovarian or
uterine levels. Moreover, the activities of muscle and fat will be affected by nutrition,
whether or not liveweight responses are detectable. Certainly, these processes could
explain differences in outcomes between ad libitum feeding [7] and limited diets
(present study), but confirmation of these using larger data sets is still required.
For adipose tissue, leptin is the likely endocrine signal to the reproductive
system and it has shown to affect directly the neuroendocrine processes that control
the initiation of puberty in female sheep [reviewed by 30]. The present observations
support this concept because leptin concentration was positively correlated with
increases with FAT, PWT and PFAT. For muscle, however, endocrine links to the
reproductive control centres have not been clearly identified. Interestingly, we found
that leptin concentration was positively correlated with increases with EMD and PEMD,
extending on the observations of Wang et al. [31], and raising the possibility that leptin
from intramuscular fat can play a role in the control of the reproductive axis. This needs
to be tested in further research.
Leptin and ghrelin concentrations were not related to age at first oestrus, but
puberty and live weight at first oestrus was positively correlated with leptin
concentration and negatively with ghrelin concentration. These findings align with the
principle that, under favourable metabolic conditions, we can expect an increase in
leptin concentration and a decline in ghrelin concentration because ghrelin plays a
dominant inhibitory role whereas leptin has a promotable effect on the reproductive
axis [12, reviewed by 14]. The variability in live weight observed during the mating
period was reflected in a decrease in leptin concentration in the sample collected one
week before the mating period. We would expect the physiological factor to be more
responsive than live weight to acute changes in energy balance, so we can surmise
that the ewe lambs were in negative balance at the start of the mating period, and this
is supported by the observation of high ß-hydroxybutyrate levels. These circumstances
could explain the relatively poor reproductive performance in the current experiment
compared to our previous study [7]. Leptin and ß-hydroxybutyrate concentrations were
not associated with fertility, fecundity or reproductive rate. On the other hand, leptin
concentrations were positively associated with higher values for growth, muscle and fat
accumulation, and therefore an improvement in reproductive performance.
4.1. Conclusion
In conclusion, Merino ewe lambs with higher breeding values for growth will
achieve puberty earlier and express higher levels of fertility and fecundity. Live weight
at start of mating plays an important role in the reproductive performance of ewe
54
lambs, and muscle and fat both appear to be physiologically active components of the
body mass because phenotypic or breeding values that increase their rate of
accumulation are associated with improved reproductive performance. At this stage,
the physiological factors in these tissues that are responsible for the reproductive
outcomes are not clear, but it seems feasible that intramuscular fat, and the leptin that
it might produce, could be a determinant of reproductive performance. Further research
is required to determine the ADG values, before and during the mating period, that
would guarantee reproductive success in ewe lambs under extensive conditions.
Acknowledgements
The authors wish to thank to Margaret Blackberry (University of Western Australia) for
her assistance with the hormone analyses, and Kristy Robertson for her assistance in
the care and management of the animals. During his doctoral studies, Cesar Rosales
Nieto was supported by Mexico’s National Council for Science and Technology
(CONACyT), the Department of Agriculture and Food of Western Australia, the
Cooperative Research Centre for Sheep Industry Innovation, and the UWA School of
Animal Biology.
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57
Chapter 3
Prepubertal growth and muscle and fat accumulation in male and female
sheep – relationships with metabolic hormone concentrations, timing of
puberty, and reproductive performance
a,b,c
C.A. Rosales Nieto
, M.B. Ferguson
a,b,d,f
b
e
c,g
, J.R. Briegel , M.P. Hedger , G.B. Martin , A.N.
a,b,d
Thompson
a
CRC for Sheep Industry Innovation and the University of New England, Armidale, NSW, 2351,
Australia
b
Department of Agriculture and Food of Western Australia, South Perth, WA 6151, Australia
c
The UWA Institute of Agriculture, University of Western Australia, Crawley, WA 6009, Australia
d
School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150,
Australia
e
Monash Institute of Medical Research, Monash University, Vic 3168, Australia
f
Present address: The New Zealand Merino Company Ltd, PO Box 25160, Christchurch 8024,
New Zealand
g
Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford OX3 9DU,
UK
58
Abstract
Across a variety of species, metabolic homeostasis is aligned with changes in growth
and body composition, through processes mediated by circulating metabolites and
metabolic hormones, and it is eventually linked to reproductive success. In the present
study, with young Merino sheep, we determined the effects of differences in the rates
of growth and muscle and fat accumulation on the circulating concentrations of
metabolites and metabolic hormones, and the relationships between these factors and
the timing of puberty and ensuing reproductive performance. We used 64 females and
62 males with known phenotypic values for depth of eye muscle (EMD) and fat (FAT)
and known Australian Sheep Breeding Values at post–weaning age (220 days) for live
weight (PWT), depth of eye muscle (PEMD) and depth of fat (PFAT). Blood was
sampled every 20 min for 8 h via jugular cannula and assayed for growth hormone
(GH), insulin-like growth factor I (IGF-I), insulin, leptin, ghrelin, follistatin, glucose and
non-esterified fatty acids (NEFA). In males, the only relationships detected were the
positive effects of PWT on the concentrations of GH, follistatin, and glucose, the
positive effects of FAT and PFAT on IGF-I concentrations (P < 0.01). An unexpected
finding was the negative relationships between testosterone concentrations and
muscle variables (P < 0.001) and PFAT (P < 0.05). In females, the only relationship
detected was the positive effect of EMD on insulin concentrations (P < 0.05).
Reproductive variables were measured only in females. Live weight at first oestrus was
related positively to insulin concentration and negatively to GH concentrations (P <
0.05). No other relationships with reproductive variables were significant. The single
day of sampling appears to have limited the opportunities for detecting the
relationships hypothesized, but those relationships that were detected suggest subtle
differences between the sexes in the effects of changing the rates of growth and
muscle and fat accumulation on metabolic homeostasis, perhaps due to interference
by testosterone.
Introduction
The rates of growth and accumulation of muscle and adipose tissues are controlled by
a variety of regulatory and differentiation processes that are affected by many factors,
including nutrition, age, gender, mature body size, genetics, and health. Most of these
interactions are regulated by the endocrine system (Berg and Butterfield 1968; Owens
et al., 1993; Hocquett et al., 2010). For muscle growth and development, the major
hormones are growth hormone (GH), insulin-like growth factor (IGF-I), insulin, leptin,
and follistatin. In males, the anabolic effects of androgens add an extra dimension to
59
the control of muscle accumulation (Schanbacher et al., 1980; reviewed by Etherton
1982; Spencer 1985; Lee and McPherron 2001; Zeidan et al. 2005; Velloso et al.,
2008). For adipose tissue, the critical factors are leptin and non-esterified fatty acids
(NEFA), both of which are implicated in fat accumulation and mobilization. Importantly,
muscle and adipose tissue are not independent but interact with each other through
glucose/insulin, ghrelin and leptin (Kokta et al., 2004; Patel et al., 2006; Barazzoni et
al., 2007; DeFronzo and Tripathy 2009).
Interactions among these factors inform the central nervous system about the
status of body reserves, to which the metabolic control centres change appetite,
metabolic rate, body temperature and behaviour. In parallel, the brain responds to
information on the status of body reserves to implement strategies for reproduction
(Blache et al., 2007). The relationship between the reproductive and metabolic systems
is mediated, at least in part, by physiological signals from metabolic tissues to the
reproductive control centres and tissues, affecting sexual maturation and subsequent
reproductive performance (review: Martin et al. 2009). In a favourable metabolic
environment, the onset of puberty can be promoted by GH, IGF-I, leptin and
testosterone (Bourguignon 1988; Roberts et al., 1990; Foster and Nagatani, 1999;
Wheaton and Godfrey, 2003) whereas, in an unfavourable metabolic environment,
puberty can be delayed by deficiencies in GH, insulin or glucose and high
concentrations of ghrelin and NEFA (Advis et al., 1981; Richards et al., 1989; Bucholtz
et al., 2000; Tena-Sempere, 2007). Additionally, after puberty, fertility can be
modulated by leptin and perhaps follistatin (Smith et al., 2002; Jorgez et al., 2004;
Kimura et al., 2010).
Therefore, we would expect these blood-borne signals to respond to factors,
both environmental and genetic, that affect reproductive success by changing the rates
of body growth and accumulation of muscle and adipose tissue. We tested this
hypothesis in lambs by measuring the concentrations of GH, IGF-I, insulin, glucose,
leptin, ghrelin, follistatin and NEFA and assessing the relationships with variables that
reflect potential for growth and muscle and fat accumulation. In females, we also
assessed the relationships with the onset of puberty and reproductive performance.
Material and Methods
This work was done in accordance with the Australian Code of Practice for the Care
and Use of Animals for Scientific Purposes (7th Edition, 2004) and was approved by
the Animal Ethics Committees of both the University of Western Australia and the
Department of Agriculture and Food (Western Australia).
60
Experimental location and animal management
Merino lambs (n = 380) were born in June 2010 (Day 0) on the research farm of the
University of Western Australia (32.2° S, 115.8° E). The dams of the lambs had been
sourced from two ram breeding flocks in Western Australian (‘Merinotech WA’ and
‘Moojepin’) and the sires had a wide range in Australian Sheep Breeding Values
(ASBV) for growth, muscle and fat. The Australian Sheep Breeding Values delivered by
MERINOSELECT are the result of collation and analysis of individual performance
values, pedigree information and relevant environmental and management information
of the animals from participating breeders. Data for birth date, birth weight and birth
type were collected for the lambs.
Ewe lambs
In November 2010 (Day 139), 190 ewe lambs were transported to the Medina
Research Station (32.2°S, 115.8°E) where they were allocated among eight groups,
each of equal average live weight upon arrival, and each held in a separate indoor pen
(6 x 7 m). Upon arrival, animals were treated with a broad spectrum oral anthelmintic
(12.5 mL/head; Triton®, Merial Australia, Parramatta, NSW), vaccinated against
clostridial disease, and injected with selenium (1 mL/head; Glanvac™ 6S Vaccine,
Pfizer Australia, West Ryde NSW), vitamin B1 (2 mL/head; Thiamine Hydrochloride
[125mg/mL], Nature Vet, Glenorie, NSW) and vitamins A, D and E (1 mL/head; Vit A
500,000 iu; Vit D3 75,000 iu, Vit E 50 iu/mL; Vet ADE®, Auckland, New Zealand). The
animals had ad libitum access to water and to dietary pellets that were introduced over
a 7-day period. The pellets were based on barley, wheat and lupin grains, cereal straw
and hay, canola meal, minerals and vitamins, and were formulated to provide 11.5 MJ
metabolisable energy per kilogram of matter, 15% protein and minerals, thus meeting
the theoretical daily requirements for maximum growth (Macco Feeds Australia).
On November 30 (Day 168), when ewe lambs were on average 157 days old
(range 136-176) and weighed 36.2 ± 0.3 kg (range 24.8-50.8), a vasectomized Merino
ram with a marking harness (MatingMark®; Hamilton, NZ) was introduced into each
pen to detect the onset of oestrus. The crayons on the harnesses were changed every
two weeks. On 29 of December of 2010 (Day 197), 64 of the ewe lambs were selected
on the basis of sire, birth type and live weight, and were transported to the animal
house at Shenton Park Field Station (31.9 °S, 115.8 °E). Ewe lambs were on average
186 days old (range 165 to 205) and weighed 42.4 ± 0.4 kg (range 34 to 49.5).
Females were placed in individual pens (1.0 x 1.78 x 0.78 m) to which they were
acclimatized for 14 days before blood sampling. Each animal had access to clean
water and an amount of the pellets (described above) that resulted in residues between
100 and 200 g per day (approximately 10%).
61
On January 14 (Day 213), ewe lambs were returned to the university farm and
combined with the remainder of the ewe lamb flock and vasectomized rams (Figure 1)
in a 30 x 120 m plot where they had access to clean water, ad libitum oaten hay and
300 g of lupins/head daily. Crayon marks on the rumps were recorded three times per
week at Medina and once per week on the farm to estimate the date of first standing
oestrus. Crayon marks were scored (1, 2 or 3), with Score 1 being one narrow mark on
the middle or the edge of the rump and Score 3 as being one big wide mark on the
rump. Only marks with Score 2 or 3 were accepted as indicating oestrus. When the first
Score 2-3 crayon mark was recorded, the date and ewe lamb age were noted as an
indication of the onset of puberty. The date was deemed to be age at first oestrus and
the closest LW recorded to that date was deemed to be live weight at first oestrus.
The vasectomized rams were removed on February 8 (Day 238) and ewe
lambs were allocated on the basis of live weight and sire into 8 groups of 24. An
experienced ram with a marking harness (MatingMark®; Hamilton, NZ) was introduced
into each group to begin the “Mating period” (Figure 1) when the ewe lambs were on
average 226 days old (range 206-246) and weighed 42.4 ± 0.3 kg (range 24.3-56.4).
Figure 1. Experimental protocol for ewe and ram lambs.
Animals were fed ad libitum oaten hay and 300 g/head daily of lupin grain. The rams
were removed after 46 days. Pregnancy and the number of fetuses were determined
by ultrasound scanning in mid-April and mid-June and the data was used to generate
values for fertility and reproductive rate.
62
The ewe lambs were weighed every week and these liveweight data were used
to detect the live weight at the onset of puberty and pregnancy. The depths of the
longissimus dorsi muscle and subcutaneous fat at a point 45 mm from the midline over
the twelfth rib were measured using ultrasound by an accredited specialist when the
ewe lambs were on average 167 (range 146-186) and 218 (range 198-228) days old.
Over both measurements, the range in eye muscle depth (EMD) was 20-33 mm and
the range in C-site fat depth (FAT) was 2-8 mm. The ultrasound data combined with
the genetic information of the ewe (dam, sire, birth weight, birth type) were used to
generate ASBVs at post-weaning age for weight (PWT; range 0-9 kg), depth of eye
muscle (PEMD; range 0.0-2.6 mm) and fat (PFAT; range 0.0-1.2 mm) using
MERINOSELECT (Brown et al., 2007).
Ram lambs
On 22 December 2010 (Day 190), ram lambs (n = 190) were transported to Medina
Research station (32.2° S, 115.8° E) where they were allocated among 15 groups of
equal average live weight (on the basis of live weight upon arrival), each held in a
separate indoor pen (6 x 7 m). Upon arrival, animals were also subjected to the health
routine described above for the ewe lambs. The animals had ad libitum access to water
and to sheep pellets (described above) that were introduced over a 7-day period.
On 16 March 2011 (Day 274), 64 ram lambs were selected on the basis of sire,
birth type and live weight and transported to the animal house in Medina research
station. They were 264 days old (range 245 to 282) and 56 ± 0.5 kg (range 46 to 62)
and were placed in individual pens (1.0 x 1.5 x 0.6 m), where they were acclimatized
for 14 days. They had access to clean water and pellets as described for the ewe
lambs. Two ram lambs with very nervous temperament were not subjected to blood
sampling but remained in their pens. After the experiment, the animals returned to the
ram lamb flock at the Medina research station.
Live weight was recorded weekly. The depths of the longissimus dorsi muscle
and subcutaneous fat, at a point 45 mm from the midline over the twelfth rib, were
measured using ultrasound by an accredited specialist when the animals were 211
(range 201-217) days old. Over both measurements, the range in eye muscle depth
(EMD) was 20-31 mm and the range in C-site fat depth (FAT) was 2-6 mm. As for the
ewe lambs, the ultrasound data and genetic information were used to generate ASBVs
at post-weaning age for PWT, PEMD and PFAT (Brown et al., 2007).
Blood sampling and immunoassay
The lambs were fitted with jugular catheters at least 18 h before sample collection.
Feeders were removed 3 hours before sampling began. Blood was sampled every 20
63
min for 8 h and 5mL of every sample was placed into heparinised tubes. In addition, 3
mL of samples 4, 7, 10, 13, 16, 19 and 22 were also placed into flouride/oxalate tubes,
and 2 mL of samples 7, 10, 13 and 16 were placed into EDTA tubes. Samples were
placed immediately on ice and then centrifuged at 2000 g for 20 min so plasma could
be harvested. Samples with heparine and fluoride/oxalate were stored at –20 ºC and
samples in EDTA were immersed in liquid nitrogen then stored at –80°C.
All 25 samples with heparin were assayed for growth hormone (GH) and
subsamples from all samples were pooled for each animal for assay of insulin-like
growth factor–I (IGF-I), insulin, follistatin, leptin, ghrelin. Testosterone was also
assayed for the ram lambs. Samples with EDTA were assayed for non-esterified fatty
acids (NEFA) and those with flouride/oxalate were assayed for glucose.
Growth hormone was assayed in duplicate 100 μL samples by double-antibody
radioimmunoassay (RIA), as described by Boukhliq et al. (1997). The limit of detection
was 0.05 ng/mL and the intra-assay CV was 19% at 1.05 ng/mL, 7.1% at 3.22 ng/mL,
and 2.3% at 7.92 ng/mL.
Insulin was assayed in duplicate 100 μL sample by double-antibody RIA, as
describe by Miller et al. (1995). The limit of detection was 0.04 μU/mL and the intraassay CV was 7% at 1.97 μU /mL, 7.3% at 1.98 μU /mL, and 2.3% at 12.9 μU /mL.
Plasma ghrelin was assayed in duplicate 100 μL samples by a modified doubleantibody RIA, as described by Miller et al. (2009). The limit of detection was 49 pg/mL
and the intra-assay CV was 6% at 94.3 pg/mL, 2.6% at 327 pg/mL, and 1% at 1549.9
pg/mL.
Leptin was assayed in duplicate 100 μL samples by double antibody RIA, as
described by Blache et al. (2000). The limit of detection was 0.05 ng/mL and the intraassay CV was 16% at 0.47 ng/mL, 3.3% at 1.10 ng/mL, and 3.6% at 1.79 ng/mL.
Plasma IGF-I was assayed in duplicate samples using the RIA described by
Gluckman et al. (1983). Interference by binding proteins was minimized by acid–
ethanol cryoprecipitation, as validated for ruminants by Breier et al. (1991). The limit of
detection for the assay was 0.05 ng/mL and the intra-assay coefficient of variation was
7% at 0.29 ng/mL and 5.1 % at 2.9 ng/mL.
Follistatin was measured in duplicate 100 μL samples with an RIA that
measures both free and bound forms, as previously described by Klein et al., (1991).
The assay employs purified heterologous bovine follistatin as standard and uses
iodinated bovine follistatin as tracer, as previously described (Robertson et al., 1987).
The limit of detection was 1.16 ng/mL and the intra- and inter-assay CV were 7.9% and
7.8%, respectively.
Testosterone was measured after extraction in a modified version of an inhouse RIA. All the samples and reagents were diluted in 0.01M Phosphate buffered
64
saline containing 0.1% Gelatin (GPBS) unless otherwise stated. The standards, a stock
solution of testosterone (4-androsten-17ß-ol-3-one; Sigma Chemicals Co. Batch 108T0777) was prepared in ethanol. A substock of 24.1 ng/mL was made by diluting the
stock and stored at -20˚C. Standards were made by serial dilution to the following
concentrations: 0.049, 0.098, 0.187, 0.375, 0.75, 1.51, 3.02, 6.05, 12.1 and 24.2 ng/mL
in ethanol. The antiserum against testosterone-3 BSA (20C-CR2140R) was purchased
from Fitzgerald Industries International, diluted to 1:100 and stored at –20˚C. Crossreactions were 100% with testosterone, 0.5% with androstenedione and 0.01% with
DHEA. The tracer (1,2,6,7-)3H-testosterone (Amersham) with a specific activity of 90
Ci/mM was diluted in GPBS to give 15000 cpm per 100 µL. The assay included one
standard curve and up to 250 unknown samples in duplicate. The standard curve also
included triplicate tubes for total counts (TC) and NSB, 10 replicates of zero standard,
3 replicates of each standard, 6 replicates each of three quality control pools and 3
replicates of the solvent as a blank. On Day 1, 25 µL of unknown plasma and were
extracted with 2 mL AR diethyl ether. The extracts were poured into fresh 10 x 75 mm
glass tubes. The extracts and 25 µL of standards were dried and redissolved in 0.2 mL
with GPBS. The tracer (100 µL) and the antibody (100 µL, diluted 1:50,000) were
subsequently added to all tubes except total counts and NSB which received 0.1 mL
GPBS instead of the antibody. The tubes were mixed and incubated at 4˚C for 48 h.
On Day 3, normal rabbit serum (100 µL, 1:800) was added, followed by 100 µl second
antibody (anti-rabbit serum; 1:60 in GPBS containing 0.1M EDTA). The tubes were
mixed and incubated overnight at 4˚C. On Day 4, 2 mL of 2% polyethylene glycol (PEG
6000) in GPBS was added to all tubes (except TCs). The tubes were centrifuged in a
refrigerated (5˚C) centrifuge at 1500 g for 25 minutes, the supernatant aspirated and
the pellet was redissolved in 500 µL 0.05M HCl. The solution was dispensed into
counting vials and then mixed with 2 mL scintillant (Starcint, Packard Chemicals
Operations). The vials were capped, shaken and left in the dark for two hours before
counting in a liquid scintillation counter (Packard Tri Carb 1500). The limit of detection
was 0.02 ng/mL and the intra-assay CV was 11.6% at 0.28 ng/mL, 14.4% at 2.18
ng/mL, and 14.7% at 0.14 ng/mL.
An automatic analyser (AU400, Olympus, Tokyo, Japan) was used to measure
glucose using the glucose reagent kit supplied by Beckman Coulter (Gladesville NSW,
Australia), and to measure NEFA using the WAKO NEFA kit supplied by Novachem
(Collingwood, Victoria, Australia).
Data analysis
Data for the ewes and rams were analyzed separately using SAS version 9.3 (SAS
Institute Inc., Cary, NC, USA).
65
Live weight and age at first oestrus, measures and ASBVs for body
composition, and mean concentrations for hormones and metabolites, were all
analysed using mixed models (Proc Mixed) and included fixed effects: dam source,
dam age (years), birth-reared type and age at blood sampling. Mean concentrations of
hormones and metabolites were each independently tested as a covariate and sire
(father) of the ewe lambs was used as a random effect.
Puberty and fertility were analyzed using the generalized linear mixed model
procedures with a binomial distribution and logit link function (PROC GLIMMIX). Fixed
effects were dam source, dam age (years) and birth-reared type. Insulin, leptin, IGF-I,
follistatin, ghrelin, mean GH, mean NEFA or mean glucose was each independently
tested as a covariate and sire (father) of the ewe lambs was used as a random effect.
Reproductive rate was analyzed using the generalized linear mixed model procedures
with a multinomial distribution and logit link function (PROC GLIMMIX). The same fixed
effects, covariates and random effects were used as the as the fertility analysis.
Mean hormone concentration was analyzed using the PROC GLM procedure.
Where factor A was hormone concentration and factor B was time of sampling, birthreared type, dam age (years), dam source or reproductive state (puberty attained or
not; pregnant or not; pregnant with single or twins) of the ewe lamb.
The relationships among PWT, PEMD, PFAT, EMD, FAT, GH, IGF-I, insulin,
leptin, ghrelin, follistatin, glucose, NEFA and testosterone were computed using
Pearson’s correlation, which is considered appropriate for parametric measures of
linear relationships between two variables, and Fisher’s transformation which helped to
derive confidence limits using PROC CORR.
All 2-way interactions among the fixed effects were included in each model and
non-significant (P > 0.05) interactions were removed from the final model. The data for
puberty, fertility and reproductive rate are presented as logit values and backtransformed percentages.
Results
Growth
In females, PWT was not related to the concentrations of any of the blood factors
measured. In males, PWT was also not related to concentrations of IGF-I, insulin,
ghrelin, leptin or NEFA, but was strongly positively correlated to the concentrations of
GH and glucose and weakly positively correlated to follistatin (Table 1). For
reproductive function in females (Table 2), a strong positive relationship between PWT
and live weight at first oestrus was observed. Thus, higher values for PWT led to
heavier weights at first oestrus, an increased likelihood of conception, and a higher
66
reproductive rate, compared to lower values for PWT. However, PWT did not affect age
at first oestrus or likelihood of puberty.
Table 1: Correlations (r) among post-weaning phenotypic and genotypic values for weight
(PWT), muscle accumulation (EMD, PEMD) and fat accumulation (FAT, PFAT) and the mean
plasma concentrations of factors involved in the regulation of tissue growth and homeostasis in
male and female lambs. GH: growth hormone (GH). IGF-I: insulin-like growth factor I. NEFA
non-esterified fatty acids.
Variable
Male
PWT
EMD
PEMD
FAT
PFAT
*(0.2375)
ns
ns
ns
ns
IGF-I (r)
ns
ns
ns
**(0.2558)
**(0.2868)
Ghrelin
ns
ns
ns
ns
ns
*(0.0352)
ns
ns
ns
ns
ns
ns
ns
ns
ns
*(0.2423)
ns
ns
ns
ns
Leptin
ns
ns
ns
ns
ns
NEFA
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
IGF-I
ns
ns
ns
ns
ns
Ghrelin
ns
ns
ns
ns
ns
Follistatin
ns
ns
ns
ns
ns
Insulin (r)
ns
*(0.0584)
ns
ns
ns
Glucose
ns
ns
ns
ns
ns
Leptin
ns
ns
ns
ns
ns
NEFA
ns
ns
ns
ns
ns
GH (r)
Follistatin
(r)
Insulin
Glucose (r)
Female GH
* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; ns P > 0.05
Muscle tissue
Except for the weak and positive relationship between insulin and EMD in ram lambs,
EMD and PEMD were not related to the concentrations of and of the blood factors in
either sex (Table 1). With respect to reproduction in females (Table 2), a strong positive
relationship between EMD and PEMD and live weight at first oestrus was observed.
Increases in EMD and PEMD were linked to heavier weight at first oestrus and, for
EMD, an increased likelihood to attain puberty. The effect of EMD on puberty became
evident when live weight was removed from the statistical analysis.
67
Adipose tissue
As for the muscle variables, FAT and PFAT were not related to the concentrations of
and of the blood factors in either sex (Table 1), with one exception – the strong positive
relationships with IGF-I values in males. For reproductive performance in females, the
only significant outcome was the positive relationship between FAT and weight at first
oestrus (Table 2).
Table 2: Correlations (r) among measures of early reproductive performance in female sheep
and post-weaning phenotypic and genotypic values for weight (PWT), muscle accumulation
(EMD, PEMD) and fat accumulation (FAT, PFAT), and the mean plasma concentrations of
factors involved in the regulation of tissue growth and homeostasis. GH: growth hormone (GH).
IGF-I: insulin-like growth factor I. NEFA non-esterified fatty acids.
Variable
Age 1st
Oestrus
LW 1st
Puberty
Fertility
Oestrus
Reproductive
Rate
PWT
ns
***(0.5662)
ns
*
*
EMD
ns
***(0.5355)
*
ns
ns
EMD + LW
ns
na
ns
ns
ns
PEMD
ns
*(0.2838)
ns
ns
ns
PEMD +
ns
na
ns
ns
ns
GH
ns
*(–0.2755)
ns
ns
ns
IGF-I
ns
ns
ns
ns
ns
Ghrelin
ns
ns
ns
ns
ns
Follistatin
ns
ns
ns
ns
ns
Insulin
ns
*(0.1762)
ns
ns
ns
Glucose
ns
ns
ns
ns
ns
FAT
ns
*(0.2727)
ns
ns
ns
PFAT
ns
ns
ns
ns
ns
Leptin
ns
ns
ns
ns
ns
NEFA
ns
ns
ns
ns
ns
PWT
* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; ns P > 0.05; na not applicable
Testosterone
Testosterone concentration was strongly negatively related to values for EMD (P <
0.001; r = –0.3281), PEMD (P < 0.05; r = –0.3011) and FAT (P < 0.05; r = –0.2511).
These effects were significant whether or not live weight/PWT was included in the
68
statistical analyses. Testosterone concentration was not related to changes in PWT or
PFAT.
Blood variables and reproduction
Weight at first oestrus was positively related to the circulating concentrations of insulin
and negatively related to the circulating concentrations of GH. There were no other
significant relationships between blood factors and reproductive performance (Table 2).
Discussion
The results of this study generally suggest that fast-growing lambs that gain muscle
and adipose tissue at higher rates have higher blood concentrations of factors that we
would expect to play a role in those processes (Spencer, 1985; Matzuk et al. 1995;
Florini et al., 1996; Oliver et al., 1993). Interestingly, the clearest relationships, such as
that between adipose tissue and IGF-I, were observed in males rather than females,
where there was also evidence of relationships with testosterone concentration.
Equally interesting, the significant relationships with testosterone concentration were all
negative. Overall, however, we detected very few relationships between the tissue
accumulation and any metabolic homeostatic processes, in either males or females.
With respect to reproductive performance in the females, there was a similar lack of
significant relationships, although increases in muscle accumulation, fat accumulation,
and the concentrations of GH and insulin, were all associated with greater weight at
first oestrus.
The predominance of non-significant relationships suggests that we should
reject our general hypothesis that a greater rate of prepubertal growth, specifically
accumulation of muscle or adipose tissue, would be associated with increased
concentrations of metabolites and metabolic hormones that would, in turn, improve
reproductive performance. However, we did observe several positive relationships with
reproductive success, particularly with post-weaning weight and the eye muscle
variables. This agrees with our other studies (Rosales Nieto et al., 2013a,b,c), where, it
must be noted, far more animals were used. For the present study, we needed to
restrict the number of animals so that the blood sampling protocol was technically
feasible. We also needed to select a time for the measurement of the blood factors
when the hypothetical relationships would be evident. These two compromises in the
design of the study probably limited the likelihood of detecting the critical relationships.
The issue of timing might also explain why we did not observe relationships
between the concentration of GH and phenotypic and genotypic measures of growth,
or between measures of growth and the concentrations of IGF-I and insulin, as
69
established previously (Trenkle and Topel, 1978; Klindt et al., 1985; Spencer 1985;
Florini et al., 1996; Francis et al., 1998). Thus, our data do not refute the idea that
these hormones are actively involved in the regulation of the growth process (Oddy et
al., 1995; Speck et al., 1990). Indeed, we did observe (if only in females) that insulin
concentration was positively related to EMD, corroborating previous reports (review:
Spencer, 1985).
Similarly, we did not observe any relationships between muscle phenotype or
genotype and the concentrations of GH, IGF-I, leptin, glucose or follistatin, despite
strong evidence that these factors are involved in the control of muscle growth and
development (review: Oksbjerg et al., 2004; Etherton 1982; Lee and McPherron 2001;
Zeidan et al., 2005; Velloso, et al. 2008). That said, in a previous study with more
animals and a different sampling protocol, we also failed to detect relationships
between the IGF-I concentration and muscle phenotype or genotype (Rosales Nieto et
al., 2013a), yet we did detect clear positive relationships between leptin concentration
and EMD and PEMD (Rosales Nieto et al., 2013a,b). It is therefore feasible that IGF-I
is not involved in the responses to selection for increased rate of accumulation of
muscle mass. Although ghrelin itself is not involved in the process of growth and
muscle accumulation, it helps to the capture of glucose in the muscle and caused
weight gain, but did not detect any relationship with tissue accumulation (Tschop et al.,
2000; Barazzoni et al., 2007).
On the other hand, the strong relationships between measures of adipose
accumulation (FAT, PFAT) and IGF-I concentrations, observed only in males, were
contrary to the expectation that GH would suppress adipocyte growth (Etherton and
Walton, 1986; Zhao et al., 2011). Interestingly, in female sheep, both young and
mature, that had been selected for leanness or fatness, Francis et al. (1998, 1999)
reported differences in the concentrations of GH and glucose, but not IGF-I or insulin. It
is feasible that the sexes differ in these processes, perhaps due to the role played by
testosterone in males.
In the current experiment, we observed very few relationships between the
plasma concentrations of metabolites and metabolic hormones and measures of
puberty, fertility and prolificacy. Obviously, because of the protocol limitations
mentioned above, this does not imply an absence of cause-effect relationship because
there are many observations, including some from our own studies, indicating the
importance of GH, IGF-I, insulin, leptin and follistatin in the regulation of sexual
maturation and fertility (Roberts et al. 1990; Miller et al., 1995; Bourguignon 1998;
Smith et al., 2002; Jorgez et al. 2004; Chagas et al., 2007; Kimura et al. 2010; Rosales
Nieto et al. 2013a,b). For glucose and NEFA, we would have expected relationships
with reproductive performance only if the animals were not metabolically unstable
70
(Canfield and Butler, 1990; Bucholtz et al. 1996; 2000) and the values that we
observed show that this was not the case.
Conclusion
The single-day sampling protocol used in the present study, despite the power offered
by large numbers of serial samples from a considerable number of animals, did not
reveal convincing relationships that support physiological links between rates of
accumulation of muscle or fat and blood-borne metabolic factors that are thought to be
involved in the onset of reproduction in young sheep. There were, however, indications
that changes in the rates of growth, and accumulation of muscle and fat, affect males
and female differently. The lack of relationships between the other metabolic factors
and reproductive success suggest that we need further study with different
experimental protocols. However, studies of this type are challenging with respect to
compromises in animal number, sampling regime and timing of sampling relative to the
anticipated time of puberty, so the results are not conclusive.
Acknowledgements
The authors wish to thank to all the volunteers for their assistance in the data
collection, Mrs Margaret Blackberry (University of Western Australia) and Ms Susan
Hayward (Monash Institute of Medical Research) for their assistance with the hormone
analyses and Gavin Kearney for his advice in statistical analyses. During his doctoral
studies, Cesar Rosales Nieto was supported by CONACyT (the Mexican National
Council for Science and Technology), the Department of Agriculture and Food of
Western Australia, the Cooperative Research Centre for Sheep Industry Innovation
and the UWA School of Animal Biology.
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Chapter 4
Roles of liveweight change during mating and muscle accumulation on
puberty and fertility in ewe lambs
C.A. Rosales NietoA,B,C, M.B. FergusonA,B, D, F, H. ThompsonE, J.R. BriegelB, C.A. MacleayB,
G.B. MartinC, A.N. ThompsonA,B,D,G
A
CRC for Sheep Industry Innovation and the University of New England, Armidale, NSW
2351, Australia
B
Department of Agriculture and Food of Western Australia, South Perth, WA 6151,
Australia
C
UWA Institute of Agriculture, University of Western Australia, Crawley, WA 6009,
Australia
D
School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150,
Australia
E
Moojepin MPM, Nyabing Rd Katanning, WA 6317, Australia
F
Present address: The New Zealand Merino Company Ltd, PO Box 25160, Christchurch
8024, New Zealand
76
Abstract
We tested the effect of liveweight change (LWC) during mating and muscle and fat
accumulation on puberty and fertility using 481 Merino ewe lambs with a range in
phenotypic values for depth of eye muscle (EMD) and fat (FAT), and a range of Australian
Sheep Breeding Values (ASBV) for post-weaning live weight (PWT), eye muscle depth
(PEMD) and fat (PFAT). From age 4 months, vasectomized rams were placed with the
ewe lambs to detect the onset of puberty. At age 8 months, the vasectomized rams were
replaced by entire rams, and the mating groups were assigned to dietary groups to
designed to target low (LOW = 40 kg, n = 244) or high (HIGH = 45 kg, n = 237) live
weights during mating. The ewe lambs weighed 39.2 ± 0.3 kg for LOW and 39.7 ± 0.3 kg
for HIGH at the start of the mating period and 39.3 ± 0.3 kg (LOW) or 45 ± 0.3 kg (HIGH)
at the end. Together, the vasectomized and entire rams detected first oestrus in 69% of
ewe lambs (average weight 37.8 ± 0.2 kg; average age 232 days, range 177-281). A
greater proportion of ewe lambs with higher values for PWT (P < 0.001) or EMD (P < 0.01)
attained puberty. HIGH females were more fertile (38% vs 7%) and had a higher
reproductive rate (P < 0.001) than LOW females. Fertility and reproductive rate were
positively associated with live weight at the start of mating (P < 0.001), regardless of level
of nutrition during mating, and with higher values for LWC, PWT and EMD (P < 0.001), and
FAT, PEMD and PFAT (P < 0.05). We conclude that increasing live weight during mating,
and the rate of muscle accumulation, will increase fertility and reproductive rate in ewes
mated at age 8 months.
Extra Keywords: Ewe lambs, nutrition, liveweight change, muscle accumulation, fertility
Introduction
Ewe lambs usually achieve puberty when they attain 50-70% of their mature live weight
(Hafez, 1952; Dýrmundsson, 1973). Therefore, important determinants of the timing of
puberty include environmental factors that influence the rate of growth, both pre- and postweaning (reviewed by Foster et al. 1985). Similarly, genetic factors that influence growth
will therefore affect the timing of puberty – thus Merino ewe lambs with higher breeding
values for growth attain puberty earlier and also have a higher reproductive performance
than those with lower breeding values (Rosales Nieto et al. 2013a;b). However, in ewe
lambs of similar live weight at start of mating, fertility and reproductive rate vary
77
significantly within experiments and between years (Rosales Nieto et al. 2013a;b),
suggesting that other factors also determine reproductive success.
Liveweight change (LWC) during mating could contribute to this variation in
reproductive success. We observed 75% pregnancy in Merino ewe lambs that weighed 41
kg at start of mating and gained 200 g/day during mating in one study (Rosales Nieto et al.
2013a), and only 35% in another study where ewes lambs weighed 42 kg at start of mating
and gained only 50 g/day during mating (Rosales Nieto et al. 2013b). An effect of LWC
during mating on reproductive success could be explained by the ‘acute’ metabolic effect
in which short-term changes in nutrition affect ovarian function, independently of changes
in live weight, or the ‘dynamic’ effect associated with changes in live weight before
conception (reviewed by Scaramuzzi et al. 2006). However, it is not known if the ‘dynamic’
effect simply reflects weight gains during mating that lead to the ewes being heavier when
mated, or if liveweight change itself has some effect on fertility and reproductive rate in
addition to those associated with correlated changes in absolute live weight. Regardless of
the mechanism, it is expected that improving the nutrition of ewe lambs so they gain more
weight during mating will increase their fertility and reproductive rates.
In addition to live weight and LWC, differences among genotypes might also affect
the outcome of mating – for both ewe lambs and adult ewes, genotypes with higher
breeding values for muscle and fat have higher fertility and reproductive rate than
genotypes with lower values (Ferguson et al. 2007; 2010; Rosales Nieto et al. 2013a;b). In
the ewe lambs, but not adult ewes, the relationships between the reproductive
performance and body composition in ewe lambs were not evident until live weight was
excluded from the statistical analyses. Therefore, it is likely that the differences in muscle
and fat accumulation would not influence the hypothesised interaction between live weight
at mating and LWC during mating on reproductive performance. In the present study, we
tested whether improving the nutrition of ewe lambs during mating, to permit positive LWC,
would improve their fertility and reproductive rate. We also tested whether the rates of
muscle and fat accumulation are likely to be reflected in the timing of puberty and
reproductive performance.
Material and Methods
This work was done in accordance with the Australian Code of Practice for the Care and
Use of Animals for Scientific Purposes and was approved by the Animal Ethics Committee
of the Department of Agriculture and Food, Western Australia.
78
Experimental location and animals
The Merino ewe lambs (n = 481) used in this study were born at ‘Moojepin’, a farm near
Katanning in Western Australia, during July-August 2010. Their mothers had been mated
to sires with a wide range in Australian Sheep Breeding Values (ASBV) for growth and for
depth of eye muscle and fat. The ASBV values delivered by MERINOSELECT were the
result of collation and analysis of individual performance values, pedigree information and
relevant environmental and management information of the animals from participating
breeders. The ewe lambs were weighed every week from 115 days before mating (Day 115) and to 60 days after start of mating (Day 60) and these data were used to estimate
live weight at puberty, the estimated date of conception, and LWC during the experiment.
The depths of the longissimus dorsi muscle and subcutaneous fat, at a point 45 mm from
the midline over the twelfth rib, were measured using ultrasound when the ewe lambs
were on average 311 days old (range 289 to 318). The range in ewe muscle depth (EMD)
was 16-34 mm and the range in the C-site fat (FAT) was 1-8 mm. The ultrasound data
were used to generate ASBVs at post-weaning age for weight (PWT; range 0-9 kg), depth
of eye muscle (PEMD; range 0-2.6 mm) and fat (PFAT; range 0-1.2 mm) using
MERINOSELECT (Brown et al. 2007).
Animal management and feeding
Ewe lambs were maintained in a 75 ha paddock where they had ad libitum access to clean
water, oaten hay and sheep pellets. The pellets were based on barley, wheat and lupin
grains, cereal straw and hay, canola meal, minerals and vitamins, and had been
formulated to provide 11.5 MJ of metabolisable energy per kg dry matter, and 15% protein
and minerals, sufficient to meet their daily requirements for maximum growth (Macco
Feeds, Australia). A ‘Teasing Period’ was begun when 8 vasectomized Merino rams
bearing marking harnesses (MatingMark®; Hamilton, NZ) were introduced to detect the
onset of oestrus on November 26 (Day –115), when the ewe lambs were 135 days old
(range 112 to141) and weighed 25.3 ± 0.2 kg (range 15 to 40). The crayons were changed
every two weeks and crayon marks were recorded weekly to estimate the date of the first
standing oestrus. Crayon marks were scored, with Score 1 being one narrow mark on the
middle or the edge of the rump and Score 3 being one large, wide mark across the rump
(only Scores 2 or 3 were accepted as indicating standing oestrus). When the first Score 23 crayon mark was recorded, the date and the age of the ewe lamb were noted. The date
79
was deemed to be age at first oestrus and the closest live weight recorded to that date
was deemed to be live weight at first oestrus.
The vasectomized rams were removed on March 22 (Day 0), when lambs were on
average 244 days old (range 224 to 253 days). On Day 0, the ewe lambs were allocated
by birth type and sire to one of two dietary groups that would be fed diets designed to
achieve different target live weights during mating: a) the LOW group (target 40 kg; n =
244) received oat hay ad libitum and 300 g lupin grain per head three times per week, but
no pellets; ii) the HIGH group (target 45 kg; n = 237) received the hay and lupin grain, plus
sheep pellets ad libitum. LOW lambs weighed 39.2 ± 0.3 (range 30 to 53 kg) and HIGH
lambs weighed 39.7 ± 0.3 kg (range 30 to 53 kg) and each dietary group was then
subdivided into 4 groups of 65 on the basis of their prospective sire. Each sub-group was
placed in a separate 1 ha enclosure with access to clean water ad libitum and the
experimental diet. For the ‘Mating Period’, one experienced entire Merino ram bearing a
marking harness was introduced into each group and crayon marks were recorded weekly
and crayons changed bi-weekly. At the end of the Mating Period (Day 28), the rams were
removed and the ewe groups were combined on a common 75 ha paddock. The LOW
lambs weighed 39.3 ± 0.3 (range 29 to 54 kg) and HIGH lambs weighed 45 ± 0.3 kg
(range 33 to 58 kg). The numbers of fetuses were determined by ultrasound scanning 50
days after ram removal and the data were used to calculate fertility (percentage of
pregnant ewes per 100 ewes mated) and reproductive rate (number of fetuses in utero per
100 ewes mated).
Statistical analysis
The data were analysed using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). Ewe
live weight data were analyzed using the linear mixed model procedures allowing repetitive
measures (PROC MIXED) with fixed effects including dam age (years), birth type – reared
type (BTRT) and dietary group. LWC data (subdivided into the ‘Teasing’ and ‘Mating’
periods), FAT, EMD, PWT, PEMD or PFAT were each independently tested as covariates.
Identification number of the ewe within the sire (father) of the ewe lambs was used as a
random effect.
The relationships among live weight, PWT, PEMD, PFAT, EMD and FAT were
computed using PROC GLM with MANOVA option, allowing removal of major fixed effects.
Fixed effects included in the model were dam source, birth type and age at the day of the
muscle and fat scan.
80
For the ‘Teasing’ and ‘Mating’ periods, LWC was determined for each lamb using a
cubic smoothing spline function with the transformation regression model procedures,
deemed appropriate when the response is nonlinear (TRANSREG). LWC data were
analyzed using the linear mixed model procedures (PROC MIXED). Fixed effects in the
model were dam age (years), BTRT and dietary group. FAT, EMD, PWT, PEMD or PFAT
were each independently tested as covariates. The sire (father) of the ewe lambs was
used as a random effect.
Live weight and age at first oestrus were analysed using mixed models (PROC
Mixed) and included: dam age (years), BTRT, live weight at the start of the ‘teasing period’
as fixed effects. LWC during the ‘Teasing Period’, FAT, EMD, PWT, PEMD or PFAT were
each independently tested as covariates. Sire (father) of the ewe lambs was used as a
random effect.
Puberty and fertility data were analyzed using the generalized linear mixed model
procedures with a binomial distribution and logit link function (PROC GLIMMIX). Fixed
effects were dam age (years), BTRT, dietary group for fertility and live weight at the start of
the ‘teasing’ period (for puberty) or the ‘mating period’ (for fertility). LWC (divided into
‘teasing period’ for puberty and ‘mating period’ for fertility), FAT, EMD, PWT, PEMD or
PFAT were each independently tested as covariates. Sire (father) of the ewe lambs was
used as a random effect. Reproductive rate data were analyzed using the generalized
linear mixed model procedures with a multinomial distribution and logit link function (PROC
GLIMMIX). The same fixed effects, covariates and random effects were used as the as for
the analysis of fertility.
All 2-way interactions among the fixed effects were included in each model and
non-significant (P > 0.05) interactions were removed from the final model. The data for
puberty, fertility and reproductive rate are presented as logit values and back-transformed
percentages.
Results
Ewe live weight and liveweight change
As shown in Figure 1, the mean (± SEM) live weight increased gradually from 25.3 ± 0.2
kg on Day –115 to 39.7 ± 0.3 kg on Day 0 when the ewe lambs were allocated to dietary
treatments. During the ‘Mating Period’ (Days 0 to 28), live weight remained stable in the
LOW group (39.3 ± 0.3 kg on Day 28) but increased to 45 ± 0.3 kg in the HIGH group.
Mean live weight differed with BTRT (P < 0.001; 35.6 ± 0.2 for single-single, 34.5 ± 1.3 for
81
twin-single and 31.6 ± 0.5 for twin-twin) and dietary treatment (P < 0.01; 34.5 ± 0.3 kg for
LOW and 35.6 ± 0.3 kg for HIGH). Throughout the experiment, ewe lambs with higher
values for EMD, FAT, PWT, PEMD or PFAT (P < 0.001) were heavier than ewe lambs with
low values.
Fig. 1. Mean (± SEM) live weights of the Merino ewe lambs during the experiment. Data from single
and twin births were retained in the model and the relationship was pooled within the birth-type
classes. On Day 0 (arrow), the vasectomized rams (‘teasers’) were removed and replaced with
SIRE rams, and the animals were allocated to HIGH (○) or LOW diets (●).
During the ‘Teasing Period’, LWC was 123.8 ± 1.2 g day-1 and was not affected by
BTRT (P > 0.05). LWC was higher in ewe lambs with higher values for EMD, FAT, PWT,
PEMD or PFAT than in ewe lambs with lower values (P < 0.001). LWC during the ‘Mating
Period’ was 2.4 ± 4.2 g day-1 for the LOW group and 179.3 ± 3.8 g day-1 (P < 0.001) for the
HIGH group. LWC was higher in ewe lambs with higher values for EMD, FAT, PWT,
PEMD or PFAT than in ewe lambs with lower values (P < 0.001). BTRT had no effect on
LWC during the mating period (P > 0.05).
The correlations among live weight, PWT, EMD, PEMD, FAT and PFAT from each
dietary group are shown in Table 1. The correlations differed slightly between dietary
groups for each trait. A strong positive correlation was observed, in both dietary groups,
between LW and EMD, PWT and EMD and PEMD, EMD and FAT and PFAT and PEMD
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and PFAT. The remaining the correlations were also positive but weak for both dietary
groups (Table 1).
Table 1: Correlations (r) among post-weaning phenotypic and genotypic values for weight (LW,
PWT), depth of muscle (EMD, PEMD) and depth of fat (FAT, PFAT) in Merino ewe lambs from
LOW and HIGH nutritional groups.
Group
Variable
LW
PWT
EMD
PEMD
FAT
PFAT
LOW
LW
1
0.79
0.74
0.41
0.59
0.43
1
0.64
0.64
0.33
0.51
0.30
0.79
1
0.74
0.69
0.54
0.58
0.64
1
0.76
0.72
0.42
0.53
0.74
0.74
1
0.83
0.65
0.68
0.64
0.76
1
0.85
0.56
0.65
0.41
0.69
0.83
1
0.54
0.77
0.33
0.72
0.85
1
0.39
0.72
0.59
0.54
0.65
0.54
1
0.84
0.51
0.42
0.56
0.39
1
0.70
0.43
0.58
0.68
0.77
0.84
1
0.30
0.53
0.65
0.72
0.70
1
HIGH
LOW
PWT
HIGH
LOW
EMD
HIGH
LOW
PEMD
HIGH
LOW
FAT
HIGH
LOW
PFAT
HIGH
Live weight and age at puberty
Of the 481 lambs in the flock, 332 (69%) displayed oestrus at least once during the
combined ‘Teasing’ and ‘Mating’ periods, or had cycled by the end of the Mating Period.
On average, first oestrus was observed at weight 37.8 ± 0.2 kg (range 25.2 to 51.5) and
age 233 days (range 177 to 281). The proportion of ewe lambs that attained puberty by
270 days was influenced by BTRT (P < 0.001). A greater proportion of twin-born and
single-reared ewe lambs (92%) attained puberty than females born as a single and reared
as a single (72%) or born and reared as a twin (48%). The proportion of ewe lambs that
attained puberty by Day 270 was not influenced by LWC during the ‘Teasing Period’ (P >
0.05). As values for PWT (P < 0.01) and EMD (P < 0.01) increased, it became more likely
that puberty would be reached, but this did not apply to FAT, PEMD or PFAT (P > 0.05;
Table 2).
Age and live weight at first oestrus differed with BTRT (P < 0.01). Ewe lambs born
and reared as singles were on average younger (230 days) and heavier (37.6 kg) at first
oestrus than ewe lambs born as twin and reared as single (239 days and 37.2 kg) and ewe
lambs born and reared as twin (244 days and 34.5 kg). LWC during the ‘Teasing Period’
83
did not influence age at first oestrus (P > 0.05; Table 2). Ewe lambs with higher values for
PWT, PFAT, PEMD, FAT or EMD were heavier at first oestrus (P < 0.001).
Ewe lambs with higher values for PWT (P < 0.001) EMD (P ≤ 0.05) and FAT (P <
0.05) were younger at first oestrus (Fig. 2). After live weight at scanning was included in
the statistical model, effects of FAT on age at first oestrus and EMD on puberty and age at
first oestrus were revealed, but not effects of PEMD and PFAT (P > 0.05; Table 2).
Table 2: Relationships among age, live weight, liveweight change (LWC; split into the teasing and
mating periods), phenotypic (Eye muscle depth [EMD] and fatness [FAT]) or ASBV (post weaning
weight [PWT], post weaning eye muscle depth [PEMD], post weaning fatness [PFAT]), and the
reproductive performance (age or live weight at first oestrus, puberty, fertility and reproductive rate)
of Merino ewe lambs mated at age 8 months.
Age at 1st
Oestrus
LW at 1st
Oestrus
Puberty
Fertility
Rep
Rate
Live weight (Rams in)
NA
NA
NA
***
***
LWC (Teasing)
NS
NA
NS
NA
NA
LWC (Mating)
NA
NA
NA
***
***
PWT
***
***
***
***
***
PEMD
NS
***
NS
**
**
PEMD (+ PWT)
NS
NA
NS
NS
NS
PFAT
NS
***
NS
*
*
PFAT (+ PWT)
NS
NA
NS
NS
NS
*
***
**
***
***
NS
NA
NS
*
*
*
***
NS
**
***
NS
NA
NS
NS
*
Variable
EMD
EMD (+ Live weight at scan)
FAT
FAT (+ Live weight at scan)
P-values: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; NS P > 0.05; NA Not applicable
Fertility and reproductive rate
A greater proportion of ewe lambs were pregnant at scanning in the HIGH group (38%;
90/237) than in the LOW group (7%; 18/244) (P < 0.001). Ewe lambs that were heavier at
the start of the mating period were more fertile regardless of dietary group (P < 0.001; Fig.
3).
84
Fig. 2. Relationships between Australian Sheep Breeding Values for post-weaning weight (PWT; P
< 0.001) and age at first oestrus in Merino ewe lambs. The broken lines represent upper and lower
95% confidence limits.
Fig. 3. Relationships between live weight at start of mating, dietary group (LOW, grey lines; HIGH,
black lines) and fertility (P < 0.001) in Merino ewe lambs mated at 8 months of age. The broken
lines represent upper and lower 95% confidence limits.
85
Fertility rate differed with dietary group (P < 0.001; 38% for HIGH vs 7% for LOW),
but not with BTRT. In those pregnant, age at conception was on average 264 d (range
256-280) for the LOW group and 260 d (range 225-280) for the HIGH group, while live
weight was 41.2 ± 1 kg for the LOW group and 43 ± 0.5 kg for the HIGH group. The
likelihood of pregnancy increased with increases in the values for LWC (P < 0.001), PWT
(P < 0.001), PEMD (P < 0.01), PFAT (P < 0.05), EMD (P < 0.001) or FAT (P < 0.01). The
effect of EMD on fertility was still evident after live weight at scanning was included in the
statistical analysis, whereas the effects of the other variables were only significant before
live weight was included (P < 0.05; Fig. 4).
Fig. 4. Fertility in Merino ewe lambs mated at 8 months of age was related Australian Sheep
Breeding Values for eye muscle depth (P < 0.05), and depended on dietary group (LOW, grey lines,
HIGH, black lines). Live weight was retained in the analysis for eye muscle depth. Data from single
and twin births were retained in the model and the relationship was pooled within the birth-type
classes. The broken lines represent upper and lower 95% confidence limits.
In the LOW group, 16/18 (89%) pregnant ewe lambs were carrying a single fetus
while 2/18 (11%) were carrying twins. In the HIGH group, 71/90 (79%) pregnant ewe
lambs were carrying a single and 19/91 (21%) were carrying twins. Reproductive rate was
influenced by dietary group (P < 0.001) but not by BTRT (P > 0.05). Reproductive rate was
positively related to live weight at start of mating, regardless of dietary group (P < 0.001).
Each extra kg from ewe lambs subjected to LOW was associated with 0.4 extra fetuses
per 100 ewes; whereas each extra kg from ewe lambs subjected to HIGH was associated
86
with 3.4 extra fetuses per 100 ewes. Reproductive rate increased with increases in the
values for LWC (P < 0.001), PWT (P < 0.001), PEMD (P < 0.01), PFAT (P < 0.05), EMD (P
< 0.001) and FAT (P < 0.001). The effect of EMD and FAT on reproductive rate was still
evident after live weight at scanning was included in the statistical analysis (P < 0.05), but
the effects of PEMD, PFAT and FAT were not revealed until live weight was excluded from
the statistical analyses. An extra mm of eye muscle depth in the LOW group was
associated with 1.2 extra fetuses per 100 ewes; whereas an extra mm of eye muscle in the
HIGH group was associated with extra 5.5 extra fetuses per 100 ewes (Fig. 5).
Fig. 5. The relationships between reproductive rate and rate of muscle accumulation for the two
dietary groups (P < 0.05; LOW, grey lines; HIGH, black lines) in Merino ewe lambs mated at age 8
months. Data from single and twin births were retained in the model and the relationship was
pooled within the birth-type classes. Live weight was retained in the analysis for eye muscle depth.
The broken lines represent upper and lower 95% confidence limits.
Discussion
Improved nutrition was associated with an increase in fertility and reproductive rate in
ewe lambs. The pregnancy rate in the LOW group, where live weight was maintained at
about 40 kg during mating, was lower than in the HIGH group where live weight increased
from 40 to 45 kg during mating. In the LOW group, the result did not seem to be due to
failure of ovulation because crayon marks were registered during both periods, so
impaired conception is more likely. Supporting our observations, Fletcher et al. (1970)
reported increases in pregnancy and fecundity in maiden ewes offered high levels of
nutrition during the breeding season. However, previous reports are not clear about the
87
effect of an improved nutrition during mating on reproductive performance (Killeen 1967;
Gunn et al. 1992; Lassoued et al. 2004).
The positive linear effect of live weight at start of mating on the reproductive rate was
independent of the dietary group. Each extra kg from ewe lambs subjected to LOW was
associated with 0.4 extra fetuses per 100 ewes. By contrast, each extra kg from ewe
lambs subjected to HIGH was associated with 3.4 extra fetuses per 100 ewes, a value that
is consistent with our previous observation of 4.5-4.8 extra fetuses per 100 ewes (Rosales
Nieto et al. 2013a;b). Overall, it seems likely that reproductive performance may be
improved by reaching key live weights at start of mating, by better nutrition as well as by
selection of animals with higher values for growth (Rosales Nieto et al. 2013a;b).
The effect of improved nutrition was reflected in the LWC and, supporting our
hypothesis, increased LWC during mating was associated with improved reproductive
performance. The reproductive efficiency was higher with LWC above 150 g/d (HIGH) than
with LWC below 5 g day-1 (LOW). This outcome explains contrasting results in two of our
previous studies: in the first, 75% of Merino ewe lambs conceived when they were gaining
200 g day-1 during the mating period (Rosales Nieto et al. 2013a), whereas only 35%
conceived in the second experiment when the ewe lambs gained only 50 g day-1 (Rosales
Nieto et al. 2013b). Interestingly, an increase in LWC above 150 g day-1, as observed in
the HIGH group, was reflected in the total live weight, whereas an increase in LWC below
5 g day-1, as observed in the LOW group, was not. However, an increase in LWC during
the mating period has been reported elsewhere as ineffective –Mulvaney et al. (2010a;b)
did not observe any differences in pregnancy rate in Romney ewe lambs offered medium
or ad libitum diets, or gaining 100 or 200 g day-1, and Adalsteinsson (1979) did not observe
an effect of LWC during mating on fecundity in mature Icelandic ewes. The disagreements
could be due to differences in breed, age and season, or to in dietary treatments. We
nevertheless conclude that a solid gain in live weight during mating is likely to improve
pregnancy in ewe lambs. More research is needed to compare different rates of LWC
during mating and to determine whether there is a threshold in LWC for higher pregnancy
rates.
Supporting our second hypothesis, and our previous observations (Rosales Nieto et al.
2013a,b), ewe lambs with higher values for PWT grew faster, were heavier at first oestrus
and at start of the mating period, and more fertile. It is thus clear that nutritional and
selection of animals with higher values for growth, therefore both, will not only advance
puberty but also will improve the likelihood of pregnancy.
88
With respect to commercial animal management, the important conclusion is that, in
ewe lambs that grow faster, through management or genetics, the first mating would be
earlier and a second mating would be feasible at 20 months of age, permitting integration
with the annual rhythm of the adult ewe flock. Such practices are also likely to reduce the
methane emissions intensity of the production system.
In addition, body composition, particularly the depths of muscle and fat, affects
reproductive performance – EMD, PEMD, FAT and PFAT were strongly related among
themselves, with age and live weight at puberty, and with fertility and reproductive rate. As
we reported previously (Rosales Nieto et al. 2013a;b), these relationships became obvious
when live weight was excluded in the statistical model, although the effect of EMD on
fertility and EMD and FAT on reproductive rate were strong enough to be evident when
live weight was retained. In the present study, high rates of muscle accumulation
increased fertility and reproductive rate, independently of weight gain during the mating
period. Interestingly, we observed that an extra mm of eye muscle depth in ewe lambs was
associated with 1.2 extra fetuses per 100 ewes in the LOW group, and an extra 5.5 extra
fetuses per 100 ewes in the HIGH group. This suggests that 1 mm more eye muscle depth
would be more beneficial than 1 kg more live weight at start of mating for the fertility in ewe
lambs. Overall, the present observations, along with those in our previous studies, strongly
support the existence of a physiological link between muscle tissue and reproductive
function in female sheep.
In the present study, rams marked most ewes during at least one of the two periods, so
reproductive failure appears to be due to failure of conception, not ovulation. Early reports
suggest that fertility remains low when live weight is below 40 kg (Coop, 1962; Killeen,
1972). Our ewe lambs weighed approximately 40 kg at start of mating, but the range within
dietary groups was 30-50 kg. In addition, recent data indicates that the ram:ewe ratio used
in the present experiment could have limited reproductive performance (B Paganoni, 2013,
unpublished data). It is clear that more research is warranted to establish the key live
weight at start of mating and the optimal of ram:ewe ratio.
Conclusion
The rates of fat and muscle accumulation, and thus live weight, at start of the mating
period, strongly affect reproductive performance in ewe lambs. These attributes can be
attained by nutritional management and by selecting fast-growing animals with high values
for fat or muscle accumulation. Moreover, growth must not stop during the mating period –
89
live weight gains above 150 g/d during the mating period seem to be necessary for
adequate pregnancy and reproductive rates.
Acknowledgements
The authors wish to thank to Dave Thompson from Moojepin farm for allowing us to use
his animals and for his help during the experiment. During his doctoral studies, Cesar
Rosales Nieto was supported by CONACyT (the Mexican National Council for Science and
Technology), the Department of Agriculture and Food of Western Australia, the
Cooperative Research Centre for Sheep Industry Innovation, and the UWA School of
Animal Biology.
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Killeen, I (1967) The effects of body weight and level of nutrition before, during, and after
joining on ewe fertility. Australian Journal of Experimental Agriculture 7, 126-136.
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Lassoued, N, Rekik, M, Mahouachi, M, Ben Hamouda, M (2004) The effect of nutrition
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at the start of the breeding period and liveweight gain during the breeding period and
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Mulvaney, FJ, Morris, ST, Kenyon, PR, Morel, PCH, West, DM (2010b) Effect of nutrition
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Rosales Nieto, CA, Ferguson, MB, Macleay, CA, Briegel, JR, Martin, GB, Thompson, AN
(2013a) Selection for superior growth advances the onset of puberty and increases
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Rosales Nieto, CA, Ferguson, MB, Macleay, CA, Briegel, JR, Wood, DA, Martin, GB,
Thompson, AN (2013b) Ewe lambs with higher breeding values for growth achieve
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91
Chapter 5
Relationships among body composition, circulating concentrations of
leptin and follistatin, and the onset of puberty and fertility in female sheep
C.A. Rosales Nieto,*†‡, M.B. Ferguson,*†§3, C. A. Macleay,† J.R. Briegel,† M.P.
Hedger,# G.B. Martin,‡|| A.N. Thompson,*†§2
*
CRC for Sheep Industry Innovation and the University of New England, Armidale, NSW, 2351, Australia
† Department of Agriculture and Food of Western Australia, South Perth, WA, 6151, Australia
‡ The UWA Institute of Agriculture and School of Animal Biology, University of Western Australia, Crawley,
WA, 6009, Australia
§ School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA, 6150, Australia
# Monash Institute of Medical Research, Monash University, Vic, 3168, Australia
|| Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford OX3 9DU, UK
3
Present address: The New Zealand Merino Company Ltd, PO Box 25160, Christchurch 8024, New
Zealand
92
Abstract
The onset of puberty is linked to the attainment of critical body mass, and increases in
body mass imply accumulation of muscle and fat. Therefore, as we attempt to improve
body composition by improving the rate of muscle accumulation, we expect to affect
the onset of puberty and subsequent fertility. There is strong evidence for leptin acting
as the physiological link between adipose tissue and the reproductive axis but, for
muscle, such links have yet to be identified. One possibility is follistatin, a regulator of
muscle growth that also binds activin and has thus been implicated in the control of
reproduction. We therefore studied the relationships, during prepubertal development
in young female sheep, between circulating concentrations of follistatin and leptin and
the rates of growth and accumulation of muscle and fat, and measures of reproductive
performance. In two field experiments, we used 326 females with known phenotypic
values for depth of eye muscle (EMD) and fat (FAT) and known genotypic values for
post-weaning live mass (PWT), eye muscle depth (PEMD) and fat depth (PFAT).
Leptin concentration was positively correlated with values for EMD, PEMD, FAT, PFAT
and PWT (P < 0.001), whereas follistatin concentration was negatively correlated with
values for EMD (P < 0.001), PEMD (P < 0.01), FAT (P < 0.05) and PWT (P < 0.001).
Leptin concentration was also negatively correlated with age and live weight at first
oestrus (P < 0.05), the proportion of females that attained puberty (P ≤ 0.05), fertility (P
< 0.01) and reproductive rate (P < 0.01). By contrast, follistatin concentration was
negatively related to live weight at first oestrus (P < 0.01), fertility (P < 0.01) and
reproductive rate (P < 0.05). These observations suggest that accelerating the
accumulation of muscle and adipose tissues can advance puberty, probably due to the
stimulatory role played by leptin. By contrast, if follistatin mediates inputs from muscle
into the control of reproduction, its role appears to be inhibitory and thus would have to
be withdrawn to allow for puberty to proceed.
Keywords: Ewe lambs, follistatin, leptin, puberty, fertility
93
Introduction
Puberty is usually achieved in female sheep when they attain 50-70% of their mature
body mass and recent data suggests that ewe lambs with higher genetic merit for
growth, or for the accumulation of muscle and fat, attain puberty at a younger age and
are more fertile than those with lower genetic merit (Rosales Nieto et al. 2013a,b,c).
The relationships between reproductive performance and metabolic status are
mediated by physiological signals from metabolic regulatory tissues (review: Martin et
al. 2008). For adipose tissue, the primary signal is leptin (Foster and Nagatani 1999)
but for muscle, endocrine factors associated with reproduction have not been clearly
identified. One possibility is follistatin.
Follistatin is secreted by the sheep ovary (Tisdall et al. 1992), but there is little
variation in circulating concentrations during the oestrous cycle (McFarlane et al.
2002), probably because it is highly expressed in several other tissues, particularly
muscle, where its importance for muscle growth and development has been clearly
demonstrated (Matzuk et al. 1995; Lee and McPherron 2001; Lee 2007; Gilson et al.
2009). With respect to reproduction, it seems to have no effect on the hypothalamic
GnRH secretion in sheep (Padmanabhan et al. 2002) but it appears to act at pituitary
level to inhibit FSH secretion in rodents (Ueno et al. 1987). In mice, deletion of
follistatin in adult granulosa cells reduces fertility and terminates ovarian activity
(Jorgez et al. 2004), and deletion of one isoform of follistatin reduces litter size and
leads to early cessation of reproduction (Kimura et al. 2010). Overall, it appears that
follistatin inhibits pituitary FSH synthesis and also FSH action in the ovary (Knight et al.
2012).
We tested for statistical relationships among the circulating concentrations of
leptin and follistatin, the phenotypic and genotypic values for rates of growth and
accumulation of muscle and adipose tissues, age and body mass at puberty, fertility
and reproductive rate in ewe lambs.
Material and Methods
This experiment was undertaken in accordance with the Australian Code of Practice for
the Care and Use of Animals for Scientific Purposes and was approved by the Animal
Ethics Committee of the Department of Agriculture and Food, Western Australia.
Experimental location and animals
94
Experiment 1 used Merino ewe lambs (n = 136) that were born in August-September
2009 on a commercial farm (‘Moojepin’). They were transported to Medina Research
Station (32.2° S, 115.8° E) where the experiment was conducted from February to
June 2010. Experiment 2 used Merino ewe lambs (n = 190) that were born in June
2010 on the research farm (‘Ridgefield’) of the University of Western Australia (32.2° S,
115.8° E). In November 2010, they were also transported to the Medina Research
Station (32.2° S, 115.8° E) for the first stage of the experiment and, in late December,
they returned to ‘Ridgefield’ where they remained until the end of the experiment. In
both experiments, the dams of the experimental animals had been sourced from two
ram breeding flocks in Western Australian and the sires had a wide range in Australian
Sheep Breeding Values (ASBV) for growth, muscle and fat. The values for ASBV
delivered by MERINOSELECT are the result of collation and and analysis of individual
performance values, pedigree information and relevant environmental and
management information of the animals from participating breeders. Data were
collected for birth date, birth weight, birth type (single or twin) and rear type to weaning
(single or twin). The ewe lambs were weighed every week and the data were used to
calculate the average daily gain (ADG) and to estimate the live body mass at puberty
and the date of conception. The depths of the longissimus dorsi muscle and
subcutaneous fat at a point 45 mm from the midline over the twelfth rib were measured
using ultrasound when the ewe lambs were aged 164 (range 134 to 176) and 251
(range 221 to 263) days for Experiment 1, and 167 (range 146 to 186) and 218 (range
198 to 228) days for Experiment 2. For both measurements for each experiment, the
range in eye muscle depth (EMD) was 20-33 mm and the range in C-site fat (FAT) was
2-8 mm. Using MERINOSELECT (Brown et al. 2007), the ultrasound data were used to
generate estimates of Australian Sheep Breeding Values at post-weaning age for
weight (PWT; range 0–9 kg), depth of eye muscle (PEMD; range 0.0–2.6 mm) and
depth of fat (PFAT; range 0.0–1.2 mm).
Animal management and feeding
The ewe lambs from Experiment 1 were initially managed as two groups in two 20 m x
60 m pens. The ewe lambs from Experiment 2 were allocated on the basis of live
weight to eight groups and housed in separate pens (6 x 14 m) at Medina research
station. The animals from both experiments had ad libitum access to water and pellets
that were introduced over a 7-day period. The pellets were based on barley, wheat and
lupin grains, cereal straw and hay, canola meal, minerals and vitamins. They were
formulated to provide 11.5 MJ of metabolisable energy per kilogram of dry matter, 15%
protein, and sufficient minerals and vitamins for maximum growth.
95
On February 24 (Day - 69), when the ewe lambs from Experiment 1 were 179
days old (range 149 to 191) and weighed 37 ± 0.4 kg, four Merino wethers (rams
castrated before puberty) with harnesses (MatingMark®; Hamilton, NZ) were
introduced to detect the onset of oestrus (“pre-mating period”). The wethers had
received a 2 mL subcutaneous injection of testosterone enanthate (75 mg/mL; Ropel®,
Jurox, NSW) one week before they were placed with the ewe lambs. Every 2 weeks,
the injections were repeated and the crayons on the harnesses were changed.
For Experiment 2, the “pre-mating period” commenced on November 30 (Day 70), when ewe lambs were on average 157 days old (range 136 to 176) and weighed
36.2 ± 0.3 kg (range 24.8 to 50.8). A vasectomized Merino ram with a marking harness
(MatingMark®; Hamilton, NZ) was introduced into each pen to detect the onset of
oestrus. On December 29 (Day -41), ewe lambs and vasectomized rams were moved
back to ‘Ridgefield’. Each pen/group was allocated to a separate 30 x 120 m plot, with
access to clean water, ad libitum oaten hay (9 Mj/kg and 9% protein) plus lupin grain
(13.5 Mj/kg and 32% protein). It was anticipated that the combination of supplement
plus dry pasture would allow the lambs to gain approximately 100 g/day.
The wethers from Experiment 1 were removed on May 4 (day 0), when the ewe
lambs were 249 (range 219 to 261) days old and weighed 41 ± 0.5 kg. The ewe lambs
received a 1 mL intramuscular injection of supplement of vitamins (Vitamin A 500,000
iu; Vitamin D3 75,000 iu; Vitamin E 50 iu/mL; Vet ADE®, Auckland, New Zealand). For
the “mating period”, they were allocated, on the basis of body mass and sire, into 8
management groups of 15 and moved into 3 m x 7 m pens where they had ad libitum
access to clean water and the sheep pellets. A single, experienced Merino ram was
introduced into each group of ewe lambs. The rams were removed on Day 47 and the
ewes remained indoors.
The vasectomized rams from Experiment 2 were removed on February 8 (Day
0) and ewe lambs were allocated on the basis of their body mass and sire into 8
groups. An experienced ram with a marking harness was introduced into each group to
begin the “mating period” when the ewe lambs were on average 226 days old (range
206 to 246) and weighed 42.4 ± 0.3 kg (range 24.3 to 56.4). The rams were removed
after 45 days.
Crayon marks on ewe rumps were recorded three times per week at Medina
and once per week at Ridgefield to estimate the date of first standing oestrus. Crayon
marks were scored (1, 2 or 3), with Score 1 being one narrow mark on the middle or
the edge of the rump and Score 3 as being a single large mark covering the rump. The
date when the first Score 2-3 crayon mark was recorded was used to estimate age at
first oestrus and the closest live weight recorded to that date was deemed to be live
weight at first oestrus. Pregnancy rate and the number of fetuses per ewe were
96
confirmed by ultrasound scanning for each experiment 60 d after rams were removed.
The data from each experiment separately was used to generate values for fertility
(percentage of pregnant ewes per 100 ewes mated) and reproductive rate (number if
fetuses in utero per 100 ewes mated).
Blood sampling and immunoassay
For both experiments, 5 ml blood was sampled by jugular venipuncture on 4 occasions,
without fasting, when the ewe lambs were on average 199, 227, 248, and 269 days old
(Experiment 1) or 144, 186, 227 and 254 days old (Experiment 2). The samples were
placed immediately on ice, and then centrifuged at 2000 g for 20 min so plasma could
be harvested and stored at -20º C until hormone analysis. Plasma concentration of
total follistatin was measured in duplicate 100 μL samples by RIA using purified
heterologous bovine follistatin as standard and iodinated bovine follistatin as tracer, as
previously described (Robertson et al. 1987; Klein et al. 1991). The limit of detection
was 1.16 ng/mL and the intra- and inter-assay CVs were 7.9% and 7.8%. Plasma leptin
concentrations were determined by RIA in duplicate 100 μL aliquots, as described by
Blache et al. (2000). For Experiment 1, the limit of detection was 0.06 ng/mL and the
intra-assay CVs were 7.3% at 0.73 ng/mL, 4.4% at 0.84 ng/mL, and 2.4% at 1.61
ng/mL. For Experiment 2, the limit of detection was 0.05 ng/mL and the intra-assay
CVs were 16% at 0.47 ng/mL, 3.3% at 1.10 ng/mL, and 3.6% at 1.79 ng/mL.
Data analysis
The data were analysed using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA).
Ewe body mass during the experiment was analyzed using the linear mixed model
procedures allowing repetitive measures (PROC MIXED) and included dam source,
dam age (years) and birth type as fixed effects. Follistatin or leptin concentration was
each independently tested as covariate and sire (father) of the ewe lambs was used as
a random effect.
Average daily gain (ADG) during the experiments was determined for each
lamb using a spline approach with the regression model procedures, which is
appropriate when the response is nonlinear (TRANSREG). ADG was analyzed using
the linear mixed model procedures (PROC MIXED). Fixed effects in the model were
dam source, dam age (years), birth type and age at start of the experiments. Follistatin
or leptin concentration was each independently tested as a covariate and sire (father)
of the ewe lambs was used as a random effect.
97
The correlations among body mass, follistatin, leptin, PWT, PEMD, PFAT, EMD
and FAT were computed using PROC GLM with MANOVA option, allowing removal of
major fixed effects. Fixed effects included in the model were dam source, birth type and
age at the day of the muscle and fat scan.
Age and live weight at first oestrus were analysed using mixed models (PROC
Mixed) and included: dam source, dam age (years), birth type and age as fixed effects.
Concentration of follistatin or leptin was each independently tested as a covariate. Sire
(father) of the ewe lambs was used as a random effect.
Puberty and fertility data were analyzed using the generalized linear mixed
model procedures with a binomial distribution and logit link function (PROC GLIMMIX).
Fixed effects were dam source, dam age (years), birth type, age and body mass at the
sampling date. Concentration of follistatin or leptin was each independently tested as a
covariate. Sire (father) of the ewe lambs was used as a random effect. Reproductive
rate data was analyzed using the generalized linear mixed model procedures with a
multinomial distribution and logit link function (PROC GLIMMIX). The same fixed
effects, covariates and random effects were used as for the fertility analysis.
Average body mass, PWT, EMD, PEMD, FAT and PFAT were analysed using
mixed models (PROC MIXED) and included the fixed effects: dam source, dam age
(years), and birth-reared type. Average hormone concentration (leptin or total follistatin)
was each independently tested as a covariate. Sire (father) of the ewe lambs was used
as a random effect.
Hormone concentration (follistatin, leptin) was analysed using mixed models
(PROC MIXED) allowing for repeated-measures, and included the fixed effects: dam
source, dam age (years), birth-reared type and age and body mass at start of teasing.
FAT, EMD, PWT, PEMD or PFAT were each independently tested as a covariate. Sire
(father) of the ewe lambs was used as a random effect. Mean hormone concentration
was analyzed using analysis of variance model procedures, where Factor A was
hormone concentration and Factor B was date at sampling (PROC ANOVA).
All 2-way interactions among the fixed effects were included in each model and
non-significant (P > 0.05) interactions were removed from the final model. The data for
puberty, fertility and reproductive rate are presented as logit values and backtransformed percentages.
Results
Body mass, leptin and follistatin
In Experiment 1, mean body mass increased from around 37 kg on Day –75 to around
54 kg on Day 57 (Figure 1A), with an ADG of 144 ± 2.4 g. There was a clear set-back
98
in growth between Days -20 and +20, around the time of introduction of fertile males.
Mean leptin concentration increased from 1.3 ng mL-1 on Day –50 to 1.7 ng mL-1 on
Day +20, (P < 0.001; Figure 1B), although the progressive increase was also
interrupted around Day 0 at the time of the arrest in weight gain (upper panel). By
contrast, total follistatin concentration decreased gradually from 3.1 ng mL-1 on Day –
50 to 2.7 ng mL-1 on Day 0 (P < 0.001), after which it did not change (Figure 1B).
In Experiment 2, body mass increased from 37 kg on Day -75 to 48 kg on Day
59 (Figure 1C). On several occasions, growth was briefly negative (eg, Days –50,–12,
–8, +24) so the overall ADG (69 ± 4.7 g) was about half that observed in Experiment 1.
In Experiment 2, Leptin concentrations remained around 2.0 to 2.2 ng mL-1 except on
Day 0 (Figure 1D) when there was a temporary but major decline (P < 0.001) that
appeared to be associated with a brief arrest in growth. By contrast, total follistatin
concentration decreased markedly from 5 ng mL-1 on Day -80 to around 3.5 ng mL-1 on
Day 0 (P < 0.001; Figure 1D), before rising again when growth appeared to stop
(Figure 1C).
Body mass (Kg)
55
C
B
D
50
45
40
35
5.5
2.4
5.0
2.2
4.5
2.0
4.0
1.8
3.5
1.6
3.0
1.4
2.5
2.0
-80
Leptin (ng/mL-1)
Follistatin (ng/mL-1)
A
1.2
Pre-mating period
-60
-40
-20
Pre-mating period
Mating period
0
Days of the experiment
20
40
60 -80
-60
-40
-20
Mating period
0
20
40
1.0
60
Days of the experiment
Figure 1. Changes in body mass in Experiment 1 (A) and Experiment 2 (C) and circulating
concentrations of follistatin (○) and leptin (●) in the Merino ewe lambs in Experiment 1 (B) and
Experiment 2 (D). Day 0 is the day fertile Merino rams were introduced. Values are mean ±
SEM.
Leptin concentration was strongly positively correlated with body mass in both
experiments (P < 0.001; Figure 2A, B). By contrast, for follistatin concentration, the
99
relationship was significant but weak in Experiment 1 (P < 0.01; Figure 2A) and not
significant in Experiment 2 (Figure 2B).
Figure 2. Correlation between body mass and circulating concentrations of follistatin (○ grey
lines) and leptin (● black lines) in Merino ewe lambs in Experiment 1 (A; P < 0.01 for follistatin
and P < 0.001 for leptin) and Experiment 2 (B; P > 0.05 for follistatin and P < 0.001 for leptin).
Growth, muscle and fat
The correlations among live weight, PWT, EMD, PEMD, FAT and PFAT are shown in
Table 1 for both experiments. In both experiments, strong positive relationships were
observed between EMD and live weight and PWT, live weight and FAT, and PEMD
and PFAT; the remaining relationships were also positive but relatively weak (Table 1).
100
Table 1: Correlations (r) among post-weaning phenotypic and genotypic values for weight (LW,
PWT), depth of muscle (EMD, PEMD) and depth of fat (FAT, PFAT) in Merino ewe lambs from
Experiments 1 and 2.
Experiment
1
2
1
2
1
2
1
2
1
2
1
2
Variable
LW
PWT
EMD
PEMD
FAT
PFAT
LW
PWT
EMD
PEMD
FAT
PFAT
1
0.68
0.64
0.28
0.56
0.29
1
0.79
0.70
0.24
0.52
0.26
0.68
1
0.53
0.40
0.39
0.34
0.79
1
0.63
0.29
0.33
0.11
0.64
0.53
1
0.41
0.59
0.34
0.70
0.63
1
0.76
0.54
0.52
0.28
0.40
0.41
1
0.22
0.76
0.24
0.29
0.76
1
0.40
0.67
0.56
0.39
0.59
0.22
1
0.26
0.52
0.33
0.54
0.40
1
0.71
0.29
0.34
0.34
0.76
0.26
1
0.26
0.11
0.52
0.67
0.71
1
Leptin concentration was strongly and favourable related to PWT, EMD, FAT
and PFAT in both experiments. The correlation with PEMD was also significant in both
experiments, but strong and favourable in Experiment 2 and relatively weak and
favourable in Experiment 1. By contrast, where the relationships with follistatin
concentration were significant, they were all negative. In Experiment 1, the
relationships were strong for PWT, EMD, PEMD and FAT but, in Experiment 2, there
were only two significant correlations, with EMD and PEMD, and both were relatively
weak and favourable (Table 2).
Potential effects of muscle accumulation on leptin and follistatin concentrations
are of particular interest, so the relationships with EMD are explored in Figure 3. For
leptin, the relationships are relatively robust in both experiments whereas, for follistatin,
the relationships are also significant but explain only 2-10% of the variation.
101
Table 2. Correlations (r) among post-weaning phenotypic and genotypic values for weight
(PWT), depth of muscle (EMD, PEMD) and depth of fat (FAT, PFAT) and the circulating
concentrations of leptin and total follistatin in Merino ewe lambs. Information has been provided
for analyses with or without live weight (LW, PWT) included in the statistical model.
Experiment 1
Experiment 2
Variable
Leptin
Follistatin
Leptin
Follistatin
PWT (r)
*** (0.46)
*** (-0.28)
*** (0.27)
NS
EMD (r)
*** (0.47)
*** (-0.32)
*** (0.55)
* (-0.15)
EMD + LW
*
**
***
*
PEMD (r)
* (0.19)
** (-0.27)
*** (0.53)
** (-0.18)
PEMD + PWT
NS
*
***
**
FAT (r)
*** (0.41)
** (-0.23)
*** (0.47)
NS
FAT + LW
***
NS
***
NS
PFAT (r)
*** (0.26)
NS
*** (0.45)
NS
PFAT + LW
NS
NS
***
NS
Puberty
As shown in Table 3, leptin concentration was positively associated with the proportion
of ewe lambs than attained puberty for Experiment 2 (P ≤ 0.05), but the relationship
was not significant for Experiment 1 (P = 0.08). Age at first oestrus was weakly
negatively correlated with leptin concentration in Experiment 1 (P < 0.01), but the
association was not significant in Experiment 2 (P > 0.05). Live weight at first oestrus
was weakly positively correlated with leptin concentration for Experiment 2 (P < 0.05),
but not Experiment 1. These effects of leptin concentration only were evident before
live weight was included in the statistical analysis (Table 3). Follistatin concentration
was not related to the proportion of ewe lambs entering puberty or to age at first
oestrus. There was a weak and significant negative relationship with live weight at first
oestrus, but only in Experiment 1 (Table 3).
Fertility and reproductive rate after puberty
As shown in Table 2, for Experiment 1, leptin concentration was positively correlated
with the proportion of ewe lambs that became pregnant (P < 0.01) and with
reproductive rate (P < 0.01). By contrast, follistatin concentration was negatively
correlated with the proportion of ewe lambs that became pregnant (P < 0.01) and with
reproductive rate (P < 0.05). The effect of leptin on fertility and reproductive rate was
evident whether live weight was included or not in the statistical analyses, but the
effects of follistatin on fertility and reproductive rate were only significant when live
weight was not included.
102
The strong but contrasting relationships observed in Experiment 1 between
fertility and leptin, and between fertility and follistatin, are illustrated in Figure 4. For
Experiment 2, neither leptin nor follistatin concentration were related to the proportion
of ewe lambs that became pregnant or with reproductive rate (Table 3).
Leptin (ng/mL-1)
Follistatin (ng/mL-1)
8
y = 0.0458x + 0.2496
R² = 0.1967
A
y = -0.1633x + 7.3099
R² = 0.0957
7
2.8
2.4
6
5
2
4
1.6
3
1.2
2
1
0.8
8
y = 0.0571x + 0.3721
R² = 0.2484
B
y = -0.0924x + 6.2031
R² = 0.0226
7
2.8
2.4
6
5
2
4
1.6
3
1.2
2
1
0.8
17
Figure
21
25
29
Depth of Eye muscle (EMD; mm)
33
3.
Correlation analysis for the effect of depth of eye muscle (EMD) on the concentrations of
follistatin (○ grey lines) and leptin (● black lines) in Merino ewe lambs from Experiment 1 (A; P <
0.05 for leptin and P < 0.01 for follistatin) and Experiment 2 (B; P < 0.001 for leptin and P < 0.05
for follistatin).
103
Table 3. The correlations (r) between the hormone concentrations (follistatin and leptin) and the
advent of puberty, the age and live weight at first oestrus, and reproductive performance
(fertility, reproductive rate) in Merino ewe lambs mated at 8-9 months of age. Information has
been provided for analyses with or without live weight (LW) included in the statistical model
except for fertility where both outcomes are provided.
Experiment 1
Experiment 2
Variable
Leptin
Follistatin
Leptin
Follistatin
Puberty (%)
NS
NS
*
NS
Age at first oestrus (days) (r)
** (-0.21)
NS
NS
NS
Age at first oestrus + LW
NS
NS
NS
NS
LW at first oestrus (kg) (r)
NS
* (-0.17)
* (0.19)
NS
Fertility (%)
**
**
NS
NS
Fertility + LW
*
NS
NS
NS
Reproductive Rate (%)
**
*
NS
NS
Reproductive rate + LW
*
NS
NS
NS
Discussion
In these field studies with Merino ewe lambs, circulating concentrations of leptin
increased progressively as the animals grew and puberty approached, consistent with
previous reports (Foster and Nagatani 1999). The most consistent relationships, across
both experiments, showed the dependence of circulating leptin concentrations on the
rate of growth and the rates of muscle and fat accumulation. Thereafter, leptin
concentration was itself positively related to age and live weight at first oestrus, as well
as to the success of puberty and fertility, and reproductive rate, observations that are
consistent with previous studies (reviewed by Smith et al. 2002). Overall, our
observations support the idea that increases in the rates of accumulation of adipose
tissue, perhaps acting through the leptin that it produces, will inform to the brain
centres that control reproduction about the body composition of the animal and
advance the onset of puberty.
Follistatin concentrations, by contrast, decreased as the animals grew and the
amount of muscle increased. Interestingly, the concentration of total follistatin was
similarly low at the onsets of puberty and conception in both Experiments. Moreover,
whenever there were significant relationships with measures of reproductive function,
they were negative. These observations are all consistent with the suggestion that, in
young female sheep, follistatin delays puberty and reduces fertility and reproductive
rate. If circulating follistatin acts as a physiological signal between muscle tissue and
104
the reproductive axis, it appears to be an inhibitory factor that needs to be reduced
before reproduction can proceed.
1
1.5
Leptin (ng/mL-1)
2
2.5
3
3.5
100
Fertility (%)
80
60
40
20
0
1
2
3
4
5
6
Follistatin (ng/mL-1)
7
8
Figure 4. Effect of leptin (black line) and follistatin (grey line) concentration on fertility in the
Merino ewe lambs from Experiment 1. The broken lines represent upper and lower 95%
confidence limits (both relationships: P < 0.01).
There were inconsistencies among the observations from these two field
studies. For example, the dependency of leptin concentration on the rates of growth
and the rates of muscle and fat accumulation significant in both experiments, but
relationships were stronger in Experiment 2 than in Experiment 1, particularly for
phenotypic and genotypic measures of muscle accumulation (EMD, PEMD). For
follistatin concentration, on the other hand, relationships with the rates of growth and of
muscle and fat accumulation were weaker for Experiment 1 than for Experiment 2. Ewe
lambs from Experiment 2 were heavier and had higher circulating concentrations of
both hormones than the ewe lambs from Experiment 1 but the average growth rate was
lower for Experiment 2 than for Experiment 1. Our interpretation is differences between
the experiments is largely explained by variation in growth rate during the mating
period, a variable that is difficult to control with field studies in an extensive
management system. It is clear that, under these conditions, external factors affected
the secretion of leptin and follistatin, and the reproductive performance of ewe lambs.
The relationships described above were revealed because we used large numbers of
animals.
Interestingly, we observed significant relationships between muscle
accumulation and leptin concentration and between muscle accumulation and
reproductive performance. Leptin is thought to be produced primarily by adipose tissue,
105
but the large variation in circulating leptin concentrations at similar levels of adiposity
implies roles for factors other than fat mass (Flier 1997). Our observations suggest that
intramuscular adipose tissue might also be a biologically significant source of leptin, a
concept supported by other findings: the leptin gene is expressed in muscle; leptin
induces muscular hypertrophy and regulates energy expenditure and fat oxidation in
muscle; and, as muscle mass increases, the concentration of intramuscular fat
increases (Gong et al. 1997; Muoio et al. 1997; Wang et al. 1998; Zeidan et al. 2005;
Zhong et al. 2011). It is therefore possible that the leptin produced in muscle,
particularly in intramuscular fat, might work together in parallel with that from adipose
tissue to inform the brain about metabolic reserves.
We hypothesized that follistain concentration would be high during muscle
growth and development, and therefore higher in ewe lambs selected for rapid muscle
accumulation. We have rejected these hypotheses because all of the relationships that
we observed were negative. Our observations were made under field conditions so is
possible that day-to-day variations in food intake and weight gain affected follistatin
secretion (review: Silanikove 2000). Indeed, Phillips et al. (1998) reported that
restriction or reduced food intake affected circulating concentrations of follistatin in
Romney ewe lambs. On the other hand, we also need to explore the relationship
between myostatin and follistatin. Myostatin is an important inhibitory regulator of
muscle development (McPherron et al. 1997; Thomas et al. 2000; Lee and McPherron
2001) and a mutation in the myostatin gene and increases in the production of growth
factors, including follistatin, are responsible for enhanced muscle development in
sheep (Clop et al. 2006). However, in animals selected for accelerated muscle
accumulation, but less specialized for meat (eg Merino), the ratio of follistatin to
myostatin is probably more important in determining muscle mass (McPherron et al.
1997). To date, there have been no studies of the processes that underpin muscle
accumulation in Merino sheep that have been selected for rapid muscle accumulation.
With respect to reproduction, the decline in total follistatin concentration at the
approach of puberty and conception reflects previous observations (Foster et al. 2000;
McFarlane et al. 2002). Among the variety of effects that follistatin has been reported to
have on the reproductive axis, our observations are consistent with its role in blocking
the activins – at pituitary level, reducing FSH synthesis and at ovarian level reducing
the actions of FSH on granulosa cells. Under these circumstances, withdrawal of
follistatin as mature body mass is achieved would aid the progress of puberty and the
maximization of fecundity.
In conclusion, selection for rapid accumulation of muscle or fat will advance
puberty and improve reproductive performance in sheep, apparently because the
changes in tissue accumulation affect the circulating concentrations of leptin and
106
follistatin. These hypotheses have been generated from correlations with large
numbers of animals in field studies, and now need to be tested in intervention
experiments.
Acknowledgements
The authors wish to thank to Mrs Margaret Blackberry (University of Western Australia)
and Ms Susan Hayward (Monash Institute of Medical Research) for their assistance
with the hormone analyses and Gavin Kearney for his advice in statistical analyses.
During his doctoral studies, Cesar Rosales Nieto was supported by CONACyT (the
Mexican National Council for Science and Technology), the Department of Agriculture
and Food of Western Australia, the Cooperative Research Centre for Sheep Industry
Innovation and the UWA School of Animal Biology.
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110
General Discussion
The aim of the studies in this thesis was to test the general hypothesis that increases in
the rate of growth and, specifically, the rates accumulation of muscle and adipose
tissues, during pre-pubertal life, will advance puberty and improve subsequent
reproductive success. As a secondary consideration, it was expected that this
connection between accelerated growth and early puberty would be mediated by
changes in the circulating concentrations of the hormones produced by the key tissues,
follistatin from muscle and leptin from adipose tissue.
The general hypothesis was supported by observations in several experiments
involving over 840 Merino ewe lambs over two seasons. We found that ewe lambs with
higher phenotypic or genotypic (breeding) values for growth were more likely to attain
puberty at a younger age, and then conceive, than their counterparts with low potential
for growth. This was strongly linked to the fact that lambs selected for rapid growth
reached the ‘critical weight’ for puberty earlier in life. The relationships between
accelerated growth and puberty were observed initially in the study described in
Chapter 1 and later corroborated by the studies described in Chapters 2 and 4. These
findings extend on those by Hawker and Kennedy (1978) and Alkass, et al. (1994) who
showed that ewes that grew faster reached puberty at a younger age. In addition to
puberty, we found that the fertility rates could be improved with higher genetic values
for growth (Chapters 1, 2 and 4), supporting previous reports of a positive genetic
correlation between weaning weight, rapid growth and reproductive performance in
young female sheep (Barlow and Hodges 1976; Adalsteinsson 1979; Gaskins, et al.
2005). Ewe lambs with greater potential for growth were heavier at the start of mating
and grew faster during mating, and both of these factors were associated with an
increase in fertility.
Most of the ewe lambs in the experiments described here achieved puberty
when they had achieved 50-70% of estimated mature body weight, a threshold for
spontaneous puberty in sheep that was established long ago (Hafez, 1952;
Dýrmundsson, 1973). Our data reinforce the observation that fast growth does not limit
the ability of ewe lambs to attain the critical percentage of mature live weight,
suggesting that there is significant scope for selecting animals for higher growth
potential with positive outcomes for reproductive efficiency as well as meat production.
This strategy should enable the mating of Merino ewe lambs at a younger age and with
better fertility. Reaching puberty earlier also increases the likelihood that, when these
ewe lambs mate for a second time at 20 months of age, their reproductive cycle can be
integrated with that of the adult ewe flock.
111
In Chapters 1, 2 and 4, we confirmed that live weight at the start of mating plays
an important role in determining fertility and reproductive rate, expanding upon
previous observations in 18-month-old maiden ewes by Kleemann and Walker (2005).
We found a linear response between pregnancy rate and live weight at the start of
mating, over the ranges of 30 to 45 kg (Chapter 1) and 40 to 55 kg (Chapter 2). There
was also a linear response in reproductive rate to increases in live weight at start of
mating. Each extra kg from ewe lambs at the start of mating was associated with 4.5
(Chapter 1) or 4.8 (Chapter 2) extra fetuses per 100 ewes. The range of 40-45 kg
seems narrow but it covers major differences in performance for Merino ewe lambs. In
Chapter 4, for example, we found that the pregnancy rate was lower in the group that
started with a body weight of 40 kg and only maintained it during mating, than in the
group that started at 40 but grew to 45 kg during mating. Each extra kg in the lambs
that maintained low weight was associated with 0.4 extra fetuses per 100 ewes;
whereas each extra kg in ewe lambs gained weight was associated with 3.4 extra
fetuses per 100 ewes. Moreover, from comparison with other studies in our laboratory
(Ferguson, et al. 2011), it seems that improving live weight at the start of mating will
provide a greater benefit for ewe lambs (3.4-4.8 extra fetuses per 100 ewe lambs) than
for mature ewes (1.7-2.4 extra fetuses per 100 mature ewes). This gain in reproductive
performance in ewe lambs, by ensuring that they reach the key live weight at start of
mating, can be achieved through nutritional management and probably also through
genetic selection for fast growth.
Comparison of the experiments in this thesis suggests that conception rate is
affected by live weight change (LWC) during mating. In the study described in Chapter
1, 75% of Merino ewe lambs conceived with an LWC of 200 g/day during the mating
period whereas, in Chapter 2, only 35% conceived with an LWC of 50 g/day during
mating. This is supported by the study described in Chapter 4 for the LWC range of 5
to 150 g/d. The literature is controversial for this topic, but our data support the view
that a positive LWC above 150 g/d during the mating period is necessary for
maximizing pregnancy success.
An important aspect of the studies in this thesis was the dissection of effects
based on total body mass into effects that can be specifically attributed to major,
physiologically active tissues. Compared to lambs with low phenotypic and genotypic
values for fat and muscle, lambs with high values were younger and heavier at first
oestrus, conceived more readily, and had a higher reproductive rate. These
observations apply regardless of whether the ewe lambs were fed ad libitum during
mating (Chapter 1) or fed restricted diets during mating (Chapter 2). In many cases,
these relationships did not become evident until live weight was removed from the
statistical model, thus allowing us to focus on biologically active constituents of body
112
mass. In Chapter 4, on the other hand, the relationship with adiposity potential and age
at first oestrus, and the relationship between muscling potential and fertility and
reproductive rate, were still evident when live weight was retained in the statistical
model. These observations extend on those for mature Merino ewes by Ferguson, et
al. (2007), who reported a positive correlation between fecundity and the rate of muscle
accumulation. We therefore support the well-established concept that adipose tissue is
an important determinant of puberty and add a novel dimension of great interest – a
potential link between muscle accumulation and reproductive function.
The final step in our attempts to understand the relationships between growth or
body composition and puberty and reproductive performance was an assessment of
potential physiological links that might be involved. We focused on metabolic factors
that were likely to be affected by rates of growth and muscle and fat accumulation, and
that have been implicated in the regulation of sexual maturation and reproductive
performance (review: Martin, et al. 2008). The primary candidate from adipose tissue is
leptin, a potent regulator of appetite and metabolism that has shown to directly affect
the brain processes that control the initiation of puberty and modulate fertility (reviews:
Foster and Nagatani 1999; Blache, et al. 2000; Smith, et al. 2002). With respect to
muscle, however, where endocrine factors associated with reproductive performance
have not been clearly identified, the literature guided us to IGF-I and follistatin.
IGF-I is important in skeletal muscle growth and development (review:
Oksbjerg, et al. 2004; Duan, et al. 2010) and increases on IGF-I concentration during
prepubertal growth have been associated with the onset of puberty in female sheep
(Roberts, et al. 1990). Follistatin is secreted by the ovary, but there is little variation in
circulating concentrations during the oestrous cycle so other tissues, such as muscle,
have become the focus of attention (McFarlane, et al. 2002). In mice, follistatin is
important for muscle growth and development, and overexpression increases muscle
weight and mass (Matzuk, et al. 1995; Lee and McPherron 2001; Lee 2007; Gilson, et
al. 2009). The importance of follistatin in reproduction seems to be clear for mice: first,
deletion of follistatin in adult granulosa cells reduces fertility and terminates ovarian
activity (Jorgez, et al. 2004); second, deletion of one isoform of follistatin reduces litter
size and leads to early cessation of reproduction (Kimura, et al. 2010).
In Chapter 1, we found that the circulating concentrations of leptin and IGF-I
increased progressively as live weight increased and as puberty approached,
confirming previous reports (Roberts, et al. 1990; Foster and Nagatani 1999). This
suggests that these two metabolic hormones inform the central nervous system of the
metabolic status of the body, perhaps specifically the accumulation of fat (leptin) and
muscle (IGF-I), and thus lead to the triggering of puberty. After puberty, the
concentration of leptin was associated with fertility and reproductive rate, consistent
113
with earlier observations (reviewed by Smith, et al. 2002). In Chapter 2, we observed
an increase in leptin concentration as puberty approached, but the relationship
between leptin and puberty or fertility was not statistically significant. The concentration
of ghrelin was also measured in this chapter, but it was not associated with any aspect
of reproductive rate. However, in Chapter 3, we did not detect relationships between
the concentrations of metabolic factors (GH, IGF-I, insulin, glucose, NEFA, leptin,
ghrelin, follistatin) and the rates of growth, muscle or fat accumulation. Furthermore, in
ewe lambs, the concentration of these metabolites and metabolic hormones did not
seem to be linked to reproductive performance. However, studies of this type are
challenging with respect to compromises in animal number, sampling regime and
timing of sampling relative to the anticipated time of puberty, so the results are not
conclusive.
Finally, in Chapter 5, we attempted to separate the potential effects of adipose
and muscle tissues. We observed that the most consistent relationships showed the
dependence of circulating leptin concentrations on the rate of growth and the rates of
muscle and fat accumulation. Thereafter, leptin concentration was itself positively
related to age and live weight at first oestrus, as well as to the success of puberty and
fertility, and reproductive rate, observations that are consistent with previous studies
(review: Foster and Nagatani, 1999; Smith, et al. 2002). On the other hand, we found
that follistatin concentrations decreased as the animals grew and the amount of muscle
increased. Notably, the concentration of total follistatin was similar at the onsets of
puberty and conception. Moreover, whenever there were significant relationships
between follistatin concentrations and measures of reproductive function, they were
negative. Taken together, these observations are consistent with the suggestion that, in
young female sheep, follistatin delays puberty and reduces fertility and reproductive
rate. Our observations support the idea that increases in the rates of accumulation of
adipose tissue, perhaps acting through the leptin that it produces, will inform to the
brain centres that control reproduction about the body composition of the animal and
advance the onset of puberty. However, by contrast, if circulating follistatin is acting as
a mechanistic link between muscle tissue and the reproductive axis, it appears to be an
inhibitor that needs to be removed before reproduction can proceed.
Another possible explanation of the relationship between muscle accumulation
and reproduction could be leptin. The dogma states that leptin is produced mostly by
the major white adipose deposits and observations in this thesis support that view –
leptin concentrations increased in parallel with adipose stores and were strongly
correlated with live weight and body composition (Chapters 1 and 2). However, we also
observed positive correlations between leptin concentrations and the two measures of
muscle development, PEMD and EMD. Indeed, there is large variation in circulating
114
leptin concentrations at similar levels of adiposity so factors other than simple fat mass
must be involved (Flier, 1997). Observations in Chapters 1, 2 and 5 are consistent with
the possibility that intramuscular adipose tissue is a biologically significant source of
leptin. This concept is supported by other findings: the leptin gene is expressed in
muscle; leptin induces muscular hypertrophy and regulates energy expenditure and fat
oxidation in muscle; and, as muscle mass increases, the amount of intramuscular fat
increases (Gong, et al. 1997; Muoio, et al. 1997; Wang, et al. 1998; Zeidan, et al. 2005;
Zhong, et al. 2011). It is therefore possible that the leptin produced in intramuscular fat
works together in parallel with that from adipose tissue to inform the brain about
metabolic reserves.
Conclusion
The basic process underlying these studies was a search for statistical relationships to
support a priori hypotheses that were based on known physiological processes in the
control of the reproductive system, especially those controlling the timing of puberty
and early reproductive performance. Overwhelmingly, the observations support the
general hypothesis that increases in the rate of growth and, specifically, the rates
accumulation of muscle and adipose tissues, during pre-pubertal life, will advance
puberty and improve subsequent reproductive success. With respect to the
physiological connections between accelerated growth and early puberty, the roles of
the circulating concentrations of the hormones produced by the key tissues are less
clear. There are very positive signs, particularly for leptin and follistatin, but conclusive
evidence awaits experiments with alternative sampling protocols and intervention
treatments.
Finally, the studies in this thesis provide a very solid foundation on which to
develop industry strategies for genetic improvement of body composition, particularly
with a view to more rapid muscle (carcase) development. The advantages of such
strategies for the meat industry are obvious but it seems very likely that increasing the
rate of muscle accumulation will also advance puberty and improve the overall
reproduction rate of the flock. The goal of strong reproductive performance in the first
year of life is within reach, as are a major step-up in the lifetime reproductive
performance of each ewe, simply through an extra year of lamb production. Finally,
young ewes will not waste a year of productive life whilst only producing methane, so
early fertility will contribute to a major reduction in emissions intensity. We therefore
have three reasons to continue to pursue this line of investigation.
115
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