Animal Reproduction Science 62 Ž2000. 143–172
www.elsevier.comrlocateranireprosci
Storage of boar semen
L.A. Johnson a,) , K.F. Weitze b, P. Fiser c , W.M.C. Maxwell d
a
c
Germplasm and Gamete Physiology Laboratory, Agricultural Research SerÕices,
US Department of Agriculture, BeltsÕille, MD 20705, USA
b
Institut fur
Hochschule HannoÕer, HannoÕer, Germany
¨ Reproduktionsmedizin, Tierarztlichen
¨
Centre for Food and Animal Research, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada
d
Department of Animal Science, UniÕersity of Sydney, Sydney, NSW 2006, Australia
Abstract
The problems, aspects and methods of liquid storage and freeze–thawing of boar semen are
discussed and a review is given on examination of spermatozoa by the recent flourescent staining
methods. Published by Elsevier Science B.V.
Keywords: Liquid and frozen semen; Cold shock; Diluent; Cryoprotectant; Thawing of semen; Staining of
sperm
1. Introduction
The first attempts on artificial insemination ŽAI. of pigs were made in 1926–1927 by
Ivanov, which were continued between 1930 and 1936 by Milovanov et al. Žcited by
Serdiuk, 1970.. The early diluents Žglucose–sulphate and glucose–tartrate. for boar
semen were proposed by Milovanov in the years of 1931–1933 ŽMilovanov, 1962..
Since these earliest studies of semen storage in the pig, it has been understood that only
a portion of spermatozoa in the original semen sample survives preservation of any type.
It is also well known that survival of cells is much greater after liquid than frozen
storage of semen. As temperature declines, there is an inevitable reduction in the
proportion of spermatozoa that maintain normal membrane integrity, ultrastructure and
biochemical components.
Utilisation of preserved semen for AI in pigs has increased approximately threefold in
the past 15 years. More than 99% of the estimated 19 million inseminations conducted
worldwide are made with semen that has been extended in the liquid state and used on
)
Corresponding author.
0378-4320r00r$ - see front matter. Published by Elsevier Science B.V.
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L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
the same day, or stored at 15–208C for 1 to 5 days. Eighty-five percent of all
inseminations are conducted on the day of collection or on the following day. Virtually
all AI with liquid-stored semen is used for market hog production. Frozen boar semen
has been available commercially since 1975 both in pellet form and in straws. However,
less than 1% of all inseminations are made using frozen–thawed semen, most often after
export from one country to another and primarily for the purpose of upgrading the
genetic base in a particular country or herd. Countries differ in the percentage of sows
and gilts that are inseminated: in some only 5% of their breeding females, while in
others, mainly in Europe, nearly 90% of the females are inseminated in a particular
country.
From 1975 through 1990, increases in the use of liquid-stored boar semen grew with
astounding proportions in Europe. Since 1990, however, the growth has been primarily
in North and South America where it is still continuing at the time of writing. This
increase in pig AI, particularly in the Western Hemisphere, is due to improved
technology of semen processing, increased demand for higher quality pork, increased
profitability from high quality carcasses and improved transportation systems, making
the liquid-stored semen available on a same or next day basis, virtually anywhere in the
world. It should also be noted that a greater proportion of pig producers are innovative
and willing to try new technology. The United States has experienced substantial growth
in pig AI in the last 5 years, moving from about 5% of the sow population inseminated
in 1993 to an estimated 45–50% in 1998–1999.
Three international conferences over the past 15 years have been devoted to improvement of methods, providing information and stimulation of research on storage of boar
semen. For more complete details on various aspects related to storage of boar semen
and AI, readers are advised to consult the proceedings from the First, Second and Third
International Conferences on Boar Semen Preservation ŽJohnson and Larsson, 1985;
Johnson and Rath, 1991; Rath et al., 1996.. This chapter will not be devoted to an
extensive and detailed examination of the various critical aspects of liquid and frozen
storage of boar semen. Rather it will cover highlights of the advances made in the two
semen storage methods and also give an update on the in vitro evaluation of semen and
refer the reader to relevant protocols available in the literature.
2. Storage of boar semen in liquid state
Two main factors influence sperm cell function after ejaculation and during in vitro
storage: the temperature at which the semen is collected and stored after dilution, and
the conditions of the suspension medium.
2.1. Cold shock
It is known that boar spermatozoa are very susceptible to cold shock. This occurs
when freshly ejaculated boar spermatozoa are cooled quickly from body temperature to
temperatures below 158C, which results in a loss of viability of an increasing number of
spermatozoa, especially when cooling is quickly continued to 1–28C.
L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
145
When prediluted semen samples are held above 158C for several hours, the spermatozoa acquire a gradual resistance to cold shock ŽPursel et al., 1973.. Rapid cooling from
358C to 158C of freshly diluted semen leads to a significant loss of motility, whereas
cooling down large volumes Ž100 ml. slowly in 108C steps enhances cooling resistance.
Preincubation for at least 24 h before a possible decrease of temperature below 158C
enhances cold shock resistance, an effect which deserves consideration in routine work
ŽWeber, 1989..
The changes within the incubated cells that render them resistant to cold shock are
not entirely known. As recently reviewed by Watson Ž1996., it is believed that cold
shock may be related to the lipid composition of the membrane bilayer affecting the
fluidity of the plasma membrane. As the temperature is lowered, restriction of lateral
movement of membrane phospholipids could result in a transition from a fluid to a gel
phase. Due to the different transition temperatures for different membrane lipids, phase
separations may occur, whereby proteins become irreversibly clustered ŽDe Leeuw et al.,
1990.. These authors concluded that the response of a particular cell membrane to low
temperature is strongly related to the detailed membrane composition of that cell.
Observed differences in the cold-induced structural changes between cold-resistant bull
and cold-sensitive boar spermatozoa could be explained by the differences in membrane
composition. Since the fatty acid composition of phospholipid determines phase behaviour, the most striking differences in the type of phospholipids between boar and bull
spermatozoa, that is a lower percentage of phosphatidylcholine and the higher percentage of phosphatidylethanolamine and sphingomyelin in boar, make it difficult to assess
the stability of boar sperm membranes on the basis of differences in phospholipid
composition.
Another factor which influences the thermotrophic behaviour of membranes is the
percentage of cholesterol. As the cholesterolrphospholipid ratio in boar spermatozoa is
very low, and as cholesterol is distributed asymmetrically, it is present more in the outer
than the inner monolayer of the membrane. This combination could render the inner
monolayer especially vulnerable to cold shock. Cold-induced reorganization of membrane particles, although partially reversible, could influence membrane function in a
number of ways. These include an increase of permeability Žleakage of cations and
enzymes., a reduction in enzyme activity and diffusion controlled membrane processes,
and changes in lateral motion in channels ŽDe Leeuw et al., 1990..
Dilution and cooling render the boar sperm membrane more permeable ŽOrtman and
Rodriguez-Martinez, 1994.. Watson Ž1996. emphasized that phase events and concomitant entrance of free calcium ions from the environment into the cell could stimulate
calcium dependant processes associated with capacitation and consequently render the
cell membrane more fusagenic, so that the usual mechanism of capacitation may be
by-passed. More insight is needed into the mechanisms responsible for cold-induced
membrane changes and the consequences to the preservational state of the spermatozoa.
Important in a practical sense is that cold shock sensitivity of the boar spermatozoa
seems to change in a time and temperature-dependent manner. Factors which are
believed to play a role are maturation state, seminal plasma, dilution and ageing during
incubation. As the maturation of spermatozoa cannot be influenced when ejaculated
semen is used, and seminal plasma seems not to play an essential role ŽKotzias-Bandeira
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L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
et al., 1997., research on temperature dependent medium-membrane interactions during
incubation and storage could increase knowledge on how to prevent or reduce cooling
changes ŽPetrounkina et al., 1997..
2.2. Dilution effect
Spermatozoa are diluted with seminal fluids from the accessory glands at ejaculation
and their motility is retained for a few hours. To extend their survival in vitro, it is
necessary to reduce the metabolic activity by chemical inhibitors or by lowering the
temperature, which also requires dilution.
In addition to a prolonged lifespan, mammalian spermatozoa respond to dilution by
an initial increase of activity, followed by a loss of motility and an increase in
membrane damage. With excessive dilution, especially when using pure electrolyte
media, there is considerable loss of cell viability. No definite explanations have been
offered for this so-called dilution effect, but Watson Ž1995. considers it to indicate cell
injury, as a consequence of loss of intracellular components andror dilution of a
protective agent in seminal fluid. A reduction in the concentration of important seminal
plasma components may contribute to the effect, which can be eliminated by the
addition of albumin. Furthermore, minimal additions of millimolar concentrations of Kq
may also assist to maintain the motility of spermatozoa, as a high concentration of Kq
in the seminal fluid is essential for cell viability ŽHarrison et al., 1978..
Harrison et al. Ž1982. proposed that the dilution effect is due to the absence of
proteinaceous motility stimulants from seminal plasma and showed that serum albumin
could stimulate motility in a reversable manner. When spermatozoa were washed in the
absence of albumin, an increased tendency to stick to glass surfaces indicated surface
membrane changes. Waberski et al. Ž1989. found that bovine serum albumin ŽBSA.
stimulated the motility of spermatozoa during a six day storage test. Buhr Ž1990., on the
other hand, used BSA in an attempt to overcome the fluidity of the membrane, and
therefore the deleterious effect of lysophospholipids and free fatty acids, produced by
cooling. It was assumed that changes in membrane lipid fluidity could affect trans-membrane movement of Ca2q, which is essential in the capacitation process but deleterious
during storage. However, BSA induced only a temporary decrease in membrane fluidity
which was difficult to interpret. Nevertheless, BSA improved fertility when the semen
was stored between 3 and 5 days ŽWaberski et al., 1994a..
2.3. Diluents
The requirements of a storage medium have been established on several occasions
and were reviewed by Watson Ž1990.. The important factors are the pH, ionic strength,
type of ions and osmotic pressure of the medium. Anti-microbial substances are also
commonly included in diluents.
In freshly ejaculated boar semen the pH varies between 7.2 and 7.5, and below this
the motility and metabolism of spermatozoa are reduced gradually. The high glucose
content of most diluents for boar semen causes a considerable reduction of intracellular
pH below 6.0. This intracellular acidosis obviously enables the cells to survive storage
L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
147
of some days at ambient temperature. The glycolytic metabolism of boar spermatozoa is
very weak in comparison to other species, and since diluted boar semen is placed
routinely in plastic tubes with little or no access to oxygen, this situation causes a
reduction of motility even at relatively high temperatures.
The ionic strength of the diluent seems not to be of primary importance in diluents
for boar semen where the osmolality is maintained by non-ionic components, such as
glucose. This may explain the importance of surface-bound proteins which are more
readily solubilized in high ionic strength media ŽWatson, 1995.. The ions in media for
fresh semen are introduced as sodium bicarbonate andror sodium citrate and, in certain
diluents, potassium chloride. The former compounds are used as buffers, the latter in
low concentration Žminimum 4 mM Kq. to maintain the Naq–Kq pump of the cells in
preventing intracellular Kq-exhaustion and a loss of motility ŽAlvarez and Storey, 1982..
Bicarbonate, on the other hand, is known to cause changes in membrane lipid architecture within a few minutes of exposure, which initiates final membrane destabilization as
an important step towards capacitation ŽHarrison, 1996..
Newer diluents are based on zwitterionic organic bufferŽs., especially TES and
HEPES ŽCrabo et al., 1972, Weitze, 1990., which capture heavy metals and control pH.
Boar spermatozoa tolerate a relative wide range of osmolality between 240 and 380
mosM, but it seems that isotonic or slightly hypertonic media offer better preservation of
fertilising capacity than hypertonic diluents ŽWeitze, 1990.. One of the extenders in this
group, Androhep is used extensively ŽTable 1..
As boar semen has to be stored in a liquid state at ambient temperature above
15–188C, a number of extenders have been developed which decrease the metabolic
activity of spermatozoa using an environment high in CO 2 . Different modifications of
glucose–sodium citrate media were mainly used. The IllinoisVariable Temperature
ŽIVT. diluent, developed in the 1950s for storage of bull semen at ambient temperature,
contains a glucose–citrate–bicarbonate–egg yolk solution gassed with CO 2 and was
used in a modified form for boar semen by du Mesnil du Buisson and Dauzier Ž1959..
The medium, supplemented with potassium chloride, is still used as a ‘‘post-diluter’’ in
the two step extension system ŽStahr
¨ and Nehring, 1997.. The composition of several
extenders is provided in Table 1.
Ethylenediamine-tetra-acetic acid ŽEDTA., as a chelating substance, captures divalent
metal ions, especially Caqq, and is believed to limit their movement across the plasma
membrane ŽWatson, 1990., preventing the initiation of capacitation and the acrosome
reaction. The inclusion of EDTA in diluents was an important step towards widespread
use of liquid-stored semen in pig AI ŽKiev extender, Plisko, 1965.. Several modifications have been made to the Kiev extender in an attempt to increase storage time and
fertility, but the results have been unequivocal.
At the present time one of the most widely used extenders is the Beltsville-TS ŽBTS.,
developed by Pursel and Johnson Ž1975. for thawing boar spermatozoa frozen in the
pellet form, and later adapted for liquid storage ŽJohnson et al., 1988.. This extender
contains a low concentration of potassium and is believed to play a role in maintaining
the intracellular concentration of this ion at physiological levels during storage.
The Zorlesco diluent is a relatively complex medium Žcontaining tris buffer, citric
acid, BSA and cysteine, in addition to glucose and EDTA. ŽGottardi et al. 1980. and the
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L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
Table 1
Composition of extenders used commercially for liquid storage of boar semen Žall extenders are also
supplemented with antibiotics
Ingredient Žgrl.
Extenders
BTS c
KIEV d
37.0
1.25
6.0
1.25
0.75
60.0
3.7
3.7
1.2
IVT e
3.0
24.3
2.4
0.4
ANDROHEP b
11.5
2.3
11.7
1.25
26.0
2.4
8.0
1.2
f
¨
SCHONOW
I
Glucose
EDTA
Sodium–citrate
Sodium–bicarb
Potassium chloride
TRIS
HEPES
Citric acid
Cysteine
BSA
ZORLESCO a
40.0
2.0
3.7
1.2
II
3.0
18.0
2.1
0.4
6.5
9.0
0.05 g
4.1
0.1
5.0
2.5
a
Gottardi et al. Ž1980..
Weitze Ž1990..
c
Pursel and Johnson Ž1975. and Johnson and Garner Ž1984..
d
Plisko Ž1965..
e
Du Mesnil du Buisson and Dauzier Ž1959..
f
Stahr
¨ and Nehring Ž1997..
g
Acetylcysteine.
b
Androhep diluent ŽWeitze, 1990., contains HEPES and BSA. The latter extender is used
for up to 5 days of semen storage in routine AI and has therefore been called a
long-term extender. Liquid semen extension is straightforward in that ingredients can be
weighed out and diluted with high quality water.
The use of sulfhydral-group bearing substances ŽSchonow
I, Zorlesco. is believed to
¨
stabilize sperm membranes and inhibit capacitation. Dilution with the Schonow
I
¨
medium gives better viability of spermatozoa after storage, but is more laborious as the
diluted semen should be further extended with the Schonow
II diluent portion before
¨
insemination. Thus, the simple one step-methods are favoured commercially, especially
when the semen is stored for less than 50 h ŽStahr
¨ and Nehring, 1997..
To prevent the growth of microflora in the stored semen, the diluents are supplemented with antibiotics. Currently, up to 300 mg gentamycin or 1.0 g neomycinsulfate
are added to 1 l of diluent, replacing penicillin and streptomycin which still have some
importance in supressing leptospira. The legislative ruling in the European Community
prescribes the use of an effective combination of antibiotics, in particular against
leptospira and mycoplasma, which must produce an effect at least equivalent to
combinations of 500 IUrml penicillin, 500 IUrml streptomycin, 150 mgrml lincomycin and 300 mgrml spectinomycin.
2.4. Duration of storage
Structural and functional changes to spermatozoa connected with liquid storage of
boar semen resemble a natural ageing process and may be determined by the conditions
L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
149
and length of storage. Ageing of spermatozoa occurs during storage in vitro and after
insemination in vivo when the fertile sperm population awaits ovulation to be released
from the lower part of the isthmus. This means that the success of fertilisation is
influenced by two periods of ageing: the in vitro storage for a period of up to 5 days and
in vivo ageing, which increases when the interval between AI and ovulation is extended
over 12 to 24 h ŽWaberski et al., 1994b; Soede et al., 1995a..
From a practical standpoint, a decrease in fertilising ability during storage cannot be
prevented, even when so-called ‘‘long-term’’ media are used. Nevertheless, special
media components, such as BSA and type of buffer, seem to reduce storage-dependent
ageing processes and, therefore, a decrease in fertilising capacity. Increased number of
spermatozoa per dose, the presence of seminal plasma and a low dilution rate seem to
reduce the loss of fertility associated with storage. In this context, clear differences in
fertility between boars must be considered, which can only partly be related to
differences in the quality of spermatozoa such as motility and morphology ŽWeitze,
1990..
The ageing-related functional changes within different compartments of the spermatozoon, such as the mitochondria, flagellum, acrosome and plasma membrane, are poorly
understood. The reduction of motility which occurs during storage has long been the
main parameter used to judge the decrease of fertilising ability. The loss of ATP and
cAMP, as well as reduced calcium uptake, are characteristic of decreased motility. An
important indicator of storage-related membrane damage is a change in membrane
permeability, such as increased permeability to stains and a release of intracellular
substances. In relation to this permeability, a distinction must be made between the
acrosomal and plasma membranes of spermatozoa. A loss of net negative charge also
gives an indication of the ageing processes. This could be caused by lipid peroxidation,
due to the relatively high content of unsaturated fatty acids in the phosospholipids of the
boar sperm membrane, through which the lipid phase of the membrane could be
damaged. These changes are balanced by the protective action of the seminal antioxidants superoxide dismutase and glutathione peroxidase ŽPetzoldt and Nehring, 1988..
As revealed in AI experiments monitored by sonographic ovulation detection,
inseminations with liquid semen performed within 2 days after collection did not reduce
fertility levels with increasing intervals between AI and ovulation ŽWeitze et al., 1989..
However, when semen stored for 48 to 87 h was used, the fertilisation rate decreased
even when ovulation occurred between 12 and 24 h after insemination. The dependence
of fertility results on time of ovulation became more striking when inseminations were
performed with semen stored for longer than 87 h, suggesting a combined effect of in
vitro and in vivo ageing of spermatozoa ŽWaberski et al., 1994b.. From the results of
further insemination experiments in which ovulation was monitored, the authors concluded that apart from slight differences between media for storage of boar semen, the
decrease in fertilising capacity due to in vitro ageing cannot be prevented, even during
the first day of storage. Improvement of the AI procedure, with special emphasis on the
time of insemination relative to the time of ovulation, as well as the selection of boars
with high quality semen, are still essential to obtain satisfactory results with liquid stored
boar semen. In conclusion, ageing of spermatozoa after ejaculation and liquid storage
seems to be a physiological process which cannot be avoided completely by preservation
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L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
measures, so additional care must be taken on timing of insemination relative to
ovulation to maintain satisfactory fertility.
2.5. Physiological relationships
On the basis that epididymal spermatozoa can be successfully used for insemination
in a number of species, including pigs, it could be assumed that seminal plasma from the
accessory glands is of minor importance. However, today there is no doubt that seminal
plasma has important regulatory functions in the processes before penetration of oocytes
by the spermatozoa. These functions are based on direct interactions between seminal
plasma and the spermatozoa and refer to nutrition, protection, regulation of motility,
capacitation, gamete recognition and binding. Indirect effects influencing the physiology
of spermatozoa are directed to the female genital tract, as seminal plasma is known to
enhance uterine contraction, relax the tubal isthmus and modulate immune responses in
the uterus. It is believed that these effects together ensure the survival, maturation and
transport of the spermatozoa in the female genital tract and are involved in the binding
of spermatozoa to the zona pellucida ŽWaberski, 1996..
Extension of semen for AI results in a quick and considerable dilution of the seminal
plasma shortly after ejaculation, so that only a small proportion of the original seminal
fluid is still present in an insemination dose. Normally, dilution does not surpass a 1:10
ratio. Either this low concentration of seminal plasma is sufficient to ensure the survival,
maturation and transport of the spermatozoa, or the initial ejaculatory contact between
seminal plasma and spermatozoa before dilution enables them to survive and arrive at
the place of fertilisation with retained fertilising capacity. When seminal plasma or
extender was introduced into the uterus before the semen, significant differences in egg
fertilisation rates and numbers of accessory spermatozoa in the zona pellucida were
obtained ŽWillmen et al., 1989.. Using an inseminate dose of 2 = 10 9 spermatozoa, a
pre-infusion of seminal plasma increased the percentage of embryos from 84.7% to
95.8% and resulted in an increase of the number of accessory spermatozoa from 8.0 to
74.7 per zona. This beneficial effect of seminal plasma on fertilisation rate and
accessory sperm number was attributed to enhanced cell transport to the site of
fertilisation. In addition, other mechanisms aimed directly at the spermatozoa, which
increase their fertilising competence, could be responsible for the higher numbers of
spermatozoa in the zona pellucida of the day 3–5 embryos ŽRath et al., 1989, Waberski
et al., 1994b..
2.6. Assessment of quality and quantity of spermatozoa
The fertilising competence of the inseminate is often estimated in terms of the quality
Žmotility, morphology, functional integrity, microbial contamination. and quantity of
spermatozoa Žthe number of high quality spermatozoa per dose.. The effects of motiltiy,
morphology and number of spermatozoa are discussed here.
2.6.1. Motility of spermatozoa
Progressive motility of spermatozoa is an indicator of both unimpaired metabolism
and intactness of membranes. Estimation of motility, therefore, has fundamental importance in the daily quality control of the semen. The percentage of motile spermatozoa,
L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
151
furthermore, is used to calculate the required degree of dilution and to estimate the
number of ‘‘intact’’ spermatozoa per insemination dose. Regular motility checks after
dilution and during the holding period give information concerning the preservability of
the semen of each boar and of its individual peculiarities. As boar spermatozoa show a
higher percentage of circular movement than those from other species, it is recommended to estimate the different forms of motility, including proportions of progressive
spermatozoa. Such estimates, undertaken by phase contrast microscopy within 20–30
min after dilution, cannot be integrated easily into the production processes. Stored
semen should be examined daily, with motility values above 60% considered satisfactory.
2.6.2. Morphology of spermatozoa
Boars used for commercial AI are selected to a certain degree on the basis of a low
incidence of morphologically abnormal spermatozoa, so that statistical calculations
concerning their correlation with fertility are not very informative. Nevertheless, the
variance between the percentages of proximal and distal cytoplasmic droplets on
spermatozoa in the ejaculates is sufficiently large to establish a statistical relationship
with fertility. A number of studies have also shown a significant negative correlation
between the percentage of spermatozoa with cytoplasmic droplets and farrowing rate
and number of live born piglets ŽZeuner, 1992.. Since cytoplasmic droplets generally
represent the most frequent morphological alteration, Waberski et al. Ž1994a. investigated the effect of proximal and distal droplets on fertility in a field experiment. They
found a negative correlation between the proportion of distal droplets and fertility, which
contradicts the assumption that these droplets depress fertility less than proximal
droplets ŽHurtgen, 1984.. The retention of cytoplasmic droplets is probably a primary
defect, originating in the testis and hindering the physiological migration of the droplet
in the epididymis. Waberski et al. Ž1994a. concluded that the presence of cytoplasmic
droplets is a serious morphological defect, which is of particular importance when
semen stored for long-term is used for AI and might be associated with lowered
resistance of spermatozoa against in vitro ageing. They recommend that the total
percentage of cytoplasmic droplets in ejaculates used for AI should not exceed 15%,
especially when stored semen is used.
A complete morphological examination is recommended when boars are introduced
into the AI centre and during subsequent regular routine examinations. In addition to the
incidence of cytoplasmic droplets, the percentage of other morphological alterations
should not exceed 20%. The use of a phase-contrast microscope and glutaraldehyde–BTS
or formol–citrate fixed wet preparations is recommended, since they avoid the artifacts
associated with dried smears ŽPursel et al., 1972..
2.6.3. Number of spermatozoa
The number of spermatozoa plays an important role in fertilisation, because fertility
improves with increasing number of spermatozoa delivered up to a certain threshold.
The threshold varies with semen quality ŽSaacke et al., 1991., and differences in fertility
between semen treatments Ži.e. extender, age of semen. can only be revealed when
numbers of spermatozoa in the inseminate are below threshold values ŽSaacke et al.,
1994..
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L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
In the commercial situation, the aim is to maintain spermatozoa in a viable state for a
variable period of time in order to fertilise a high proportion of ova with a minimum
sperm dose ŽReed, 1985.. Fullfilling all of these requirements has not been achieved by
any available boar semen extender when storage time exceeds 3 days. A number of field
trials using semen stored for longer periods in various diluents have yielded reduced
fertility Žreviewed by Weitze, 1990.. However, doubling of the sperm dose to 6 = 10 9
on the 4th day of storage ŽJohnson et al., 1988., as well as the use of boars with good
semen quality ŽMartin Rillo, 1984. can prevent the fertility decline of stored semen. This
option usually is not practicable for commercial use, but gives valuable insight into the
interaction of the quality and quantity of spermatozoa. When the number of spermatozoa
in the inseminate was reduced from 2 to 0.5 = 10 9 , a significant reduction of fertility
from 90.2% to 58.3% was reported ŽWillmen et al., 1991.. Moreover, pretreatment of
the uterus with seminal plasma compared with saline before insemination of 0.5 = 10 9
spermatozoa per gilt gave no improvement in fertility. Considering the low number of
spermatozoa used, both accessory spermatozoa counts and fertilisation rates were
surprisingly high in the two groups. This was attributed to optimum AI conditions, such
as winter season, close boar contact, skillful animal handling and detection of oestrus,
and to high quality of ejaculated semen. A reduction in the inseminate dose to 0.3 = 10 9
spermatozoa resulted in a decrease of normal embryo development rate to 60%. This
was due to a higher percentage of degenerated embryos, and confirms the assumption of
Saacke et al. Ž1994. that low numbers of spermatozoa at the site of fertilisation reduces
competition among them and thereby allows fertilisation by less competent spermatozoa.
Thus, less competent spermatozoa are capable of fertilisation, but are unable to sustain
normal embryonic development. The observation that normal and degenerated embryos
had similar numbers of accessory spermatozoa supports the hypothesis that less competent spermatozoa have normal binding and penetration ability ŽWaberski et al., 1996..
Thus, quantitative and qualitative deficiencies in semen might lead to different degrees
of fertilising incompetence of the sperm population in the female genital tract, both
resulting in low fertility.
2.7. Fertility results after insemination of liquid-stored semen
As mentioned earlier, there are a number of extenders used for dilution of boar
semen. Some have been in use at one or more locations for 30 years. Quite frequently
the results of fertility trials that initiated the use of the extender in the first place are not
applicable in current practice. However, as a general rule one can expect farrowing rates
ranging from 65% to 70% if the semen is used in the first 2 days after collection and a
reduction down to about 50% with 5-day-old semen. The litter size may also be affected
by longer storage times. Several references covering various extenders over the past 10
years can be consulted for specific information on fertility ŽJohnson et al., 1988;
Johnson and Rath, 1991; Rath et al., 1996; Johnson, 1998..
2.7.1. Commercial use of liquid-stored boar semen
Pig producers have adapted the AI technology to their specific needs, some by
collecting semen and inseminating females on their own farms, which is frequently
L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
153
referred to as do-it-yourself AI ŽDIYAI.. Other countries adopted extensive networks of
AI Centres with services set up to deliver the semen. The delivery systems have worked
well to establish AI. However, as AI has grown in use in the last 10 years there has been
a decrease in the use of outside inseminators and a preponderance of DIYAI. The latter
has been encouraged by the increased use of AI for crossbred pig production. High
animal health and disease prevention standards also encourage the use of DIYAI, as
producers are able to close their farms to outsiders. For example, increased DIYAI was
used for insemination in the Netherlands during the 1997 swine fever outbreak.
Individual collection facilities have been used in the United States for many years,
where many production units are large and can easily manage their own collection and
insemination. However, the use of semen delivered from local AI Centers is also
increasing in the USA. There is an increasing number of regional AI centers, where
technical help can provide a consistent semen quality standards and levels of expertise in
collection and dilution of semen. Many of these AI centres are organized along a
cooperative structure.
Little variability exists worldwide in the methods of packaging liquid semen. The
standard procedure involves collection of the whole ejaculate, a modified whole
ejaculate or the sperm rich fraction. Dilution varies considerably in practice. However,
dilution to about 3 = 10 9 in 80 ml is recommended. Where collection is done on the
farm, producers will frequently inseminate higher doses of spermatozoa, since they tend
to use all the semen collected on any given day, rather than store it. Furthermore, there
is considerable mixing of semen Žheterosperm. from different boars, which encourages
the use of more semen than is actually needed.
2.7.2. Life span of gametes in the female genital tract
The fertile life span of oocytes and spermatozoa in the female genital tract is limited.
Therefore, the timing of insemination relative to ovulation is crucial for achieving high
fertility. Sub-optimum insemination time before ovulation leads to low conception rates
andror litter sizes, either by a failure of senescent spermatozoa to fertilise all released
eggs, or by fertilisation of oocytes with less competent spermatozoa, resulting in
increased embryonic mortality.
The fertile life span of gametes in the female genital tract should be viewed as the
period of time during which the ability to undergo normal fertilisation and give rise to a
viable embryo is retained ŽHunter, 1988.. This information can be reliably obtained only
from insemination trials where the time of ovulation is known, which can be assessed
now by sonographic monitoring of the ovaries ŽWeitze et al., 1989.. Recent studies
using sonographic detection of ovulation in spontaneously cycling and ovulating sows
show differing ‘optimum’ insemination times relative to ovulation where fertilisation
rates were at a maximum ŽTable 2..
The viable life span of spermatozoa in the female genital tract is not regarded as a
constant figure. Variations are due to heterogeneity of the sperm population and the
condition of the female genital tract, for example the composition of the fluids in the
tract ŽHunter, 1988.. Variations found in insemination trials depend on season, farm
managment, skill of the inseminator and quality and number of spermatozoa. Improvement of conditions can result in increased fertilisation even with low numbers of
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Table 2
Time of insemination in pigs Žafter Soede and Kemp, 1997.
Optimum time range
Žhours before ovulation.
Dose of sperm Ž=10 9 .
Ovulation assessment
Gilts
Dziuk Ž1970.
Hunter Ž1967.
Waberski et al. Ž1994a.
Waberski et al. Ž1994b.
12 Ž6–18.
6–8
0–12
0–24
10–15% of an ejacul.
80–120 ml semen
2
2
40 h after hCG
41–42 h after hCG
ultrasound every 4 h
ultrasound every 12 h
Sows
Soede et al. Ž1995a.
Soede et al. Ž1995b.
Nissen Ž1995.
0–24
y8–24
y4–28
3
3
2
ultrasound every 4 h
ultrasound every 4 h
ultrasound every 6 h
AuthorŽs.
spermatozoa at extended AI to ovulation intervals ŽWaberski and Weitze, 1996..
However, in commercial practice, where many factors cannot be controlled, the fertile
life span of spermatozoa in the female genital tract should not be overestimated. The
time interval between two inseminations and between the last insemination and ovulation, respectively, should not exceed 12 to 18 h. In vitro storage of semen for a
considerable length of time before insemination reduces the viability of spermatozoa in
vivo and requires, therefore, a more proper timing of AI compared to that with fresh
semen. The relationship of in vitro ageing and subsequent in vivo ageing of spermatozoa
with fertilisation rates and numbers of accessory spermatozoa in the zona pellucida has
been described by Waberski et al. Ž1994b..
2.7.3. Insemination in relation to oÕulation
Ovulated oocytes have a limited life span in the female genital tract. Although
successful fertilisation of pig oocytes has been observed with inseminations performed 4
h ŽWaberski et al., 1994a; Nissen, 1995. or even 8 h ŽHunter, 1967; Soede et al., 1995a.
after ovulation, there is evidence that farrowing rates are reduced due to ageing of
oocytes compared with insemination before or close to ovulation. It is therefore
recommended that insemination should be performed not later than 2 h after ovulation in
order to obtain the maximum number of normally developed oocytes ŽHunter, 1967..
In current AI practice, the majority of sows receive the first insemination before
ovulation which occurs late in oestrus ŽTable 3.. In a recent study, in which sows were
first inseminated 24 to 0 h before ovulation, a second insemination 0 to 5 h after
ovulation did not influence the percentage of normally developed embryos ŽSoede et al.,
1995b.. This confirms that shortly after fertilisation an effective block against polyspermy
is established even when high numbers of spermatozoa subsequently reach the site of
fertilisation. However, insemination in late oestrus, after ovulation, when blood progesterone levels rise up to 10 ngrml, might affect fertilisation rates due to a decreased
resistance of the endometrium to infectious agents ŽDe Winter et al., 1996.. Thus, the
effect of multiple inseminations during oestrus on fertilisation remains questionable
ŽWaberski and Weitze, 1996..
L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
155
Table 3
Oestrus and ovulation in sows after weaning in different herds
Significant correlation between weaning–onset of oestrus and onset of oestrus–ovulation intervals
Herd 1: Hulsenberg
Research Station, Germany; ns 483 sows ŽWeitze et al., 1994.
¨
Herd 2: Wageningen University, Netherlands; ns151 sows ŽSoede et al., 1995a.
Herd 3: Commercial Farm, Denmark; ns143 sows ŽNissen et al., 1997.
Oestrus intervals
Weaning–oestrus Žh.
Duration of oestrus Žh.
Ovulation intervals
Oestrus onset–ovulation Žh.
Time of ovulation in oestrus Ž%. a
Herd 1
Herd 2
Herd 3
Mean"sd
Range
Mean"sd
Range
Mean"sd
Range
124"94
60"15
67–240
33–153
93"18
50"13
65–153
24–88
92"13
60"14
64–134
30–89
45"13
71
19–120
35–100
35"8
72"15
10–58
39–133
42"11
71"14
17–68
a
Median time of ovulation for herd from beginning of oestrus, where 0%s beginning of oestrus and
100%send of oestrus. Determined by ultrasound.
2.7.4. Insemination strategy adapted to oestrus behaÕiour
The interval from onset of oestrus to ovulation in the sow may vary from 19 to 120 h
ŽWeitze et al., 1994.. However, a significant relationship has been reported between the
onset of oestrus after weaning, the duration of oestrus and the time of ovulation ŽTable
3., which suggests that an adaptation of the insemination strategy to oestrous behaviour
of individul animals may be necessary. Sows can have early, regular and late oestrus
after weaning. Those showing early oestrus after weaning usually have a long oestrous
period, in contrast to sows having late oestrus after weaning which is of short duration.
Sows with early oestrus should be inseminated later than those showing late oestrus,
which should receive insemination immediately after detection of oestrus ŽWeitze et al.,
1994..
Further knowledge on the influence of factors, like farm managment, season and
breed on the oestrous pattern and ovulation time of individual sows would allow the use
of a more precise strategy for AI ŽWaberski and Weitze, 1996..
3. Storage of boar semen in frozen state
Boar semen differs in several aspects from the semen of other domestic animals. It is
produced in large volumes and is extremely vulnerable to cold shock or sudden cooling
immediately after collection. These and other characteristics of boar semen require
special consideration in the design of freezing protocols ŽPursel and Johnson, 1971,
1975; Paquignon and Courot, 1976; Larsson et al., 1977; Westendorf et al., 1975;
Larsson, 1978..
It is not the intention of this chapter to describe in detail the freezing procedures
developed in past decades and used with varying success. Reviews on the subject have
been published by Einarsson Ž1973., Graham et al. Ž1978., Watson Ž1979., Johnson
Ž1985. and Bwanga Ž1991.. Here the freezing methods will be discussed in general,
focusing on the factors in the freezing protocol which could be readily modified and
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thus influence directly the success of frozen storage. In addition to the reviews
mentioned above, the proceedings of the international conferences on boar semen
preservation provide further detailed information on the subject.
Successful freezing of boar semen depends on an understanding of the factors and
their interactions which influence the capacity of spermatozoa to survive freezing and
thawing. The factors may be classified into two categories: Ž1. internal or fixed factors,
such as the inherent characteristics of spermatozoa, and differences between boars and
ejaculates, and Ž2. external factors, such as composition of diluents, type and concentration of cryoprotective agents, rates of dilution and of cooling, equilibration, and method
of freezing and thawing of semen.
The external, unlike the internal, factors can be manipulated or modified in order to
‘‘optimize’’ the freezing protocol. In any protocol, rapid cooling of spermatozoa from
body temperature to temperatures close to freezing irreversibly reduces viability of
spermatozoa. Under such conditions, spermatozoa lose their membrane integrity and
motility with irreversible reduction of carbohydrate metabolism ŽWatson, 1981; Watson
and Morris, 1987.. Rapid cooling also results in release of intracellular enzymes ŽPursel
et al., 1970; Pursel and Johnson, 1974. and lipids ŽDarin-Bennett et al., 1973. and a
redistribution of ions ŽHood et al., 1970.. The magnitude of the cold shock depends on
the rate of cooling and on the final temperature to which semen is cooled ŽMorris and
Watson, 1984.. For more details on cold shock, see the section on liquid storage.
Boar spermatozoa may acquire resistance to cooling simply by incubation at ambient
temperature for several hours ŽPursel et al., 1973.. The effect is temperature dependant
and the environment of seminal plasma is not essential, but the viability of spermatozoa
is improved when incubated in its seminal plasma. After 2–7 h incubation most
spermatozoa develop resistance to cooling ŽPursel et al., 1973; Butler and Roberts,
1975..
Egg yolk, which provides protection against cold shock to spermatozoa of different
domestic animals, does not give the same level of protection to boar spermatozoa
ŽBenson et al., 1967.. However, its protective effect can be improved by adding Orvus
Es Paste ŽOEP. to the extender ŽGraham et al., 1971; Pursel et al., 1978.. OEP Žnow
known as Equex Stm; Nova Chemical, Scituate, MA., a synthetic detergent based on
sodium and triethanolamine lauryl sulphate, has been included in most egg yolk-containing diluents for freezing of boar semen ŽPursel and Johnson, 1975; Westendorf et al.,
1975.. It has been suggested that OEP gives protection by modifying the egg yolk
constituents of the diluent ŽPursel et al., 1978; Strzezek et al., 1984., as it has protective
qualities only when used in combination with egg yolk.
Some other compounds reported to alleviate cooling injury are the antioxidants
ditret-butyl-kresol ŽDTBK., echinochrome Žan extract of sea urchins. ŽGolyshev, 1985.
and butylated hydroxytoluene ŽBHT; Graham and Hammerstedt, 1992.. The longevity of
boar spermatozoa was increased by the presence of low concentrations of BHT ŽBamba
and Cran, 1992.. However, BHT was effective only when spermatozoa were subjected
to a slower rate of cooling to 58C.
Based on the results of other species ŽFiser and Fairfull, 1984. and also reported for
cold shocked pig spermatozoa, the damage by cooling seems to be independent of the
freezing damage, that is the spermatozoa which survive cold shock regardless of its
L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
157
severity, have the same chance to withstand freezing and thawing as those not subjected
to cold shock. However, it should be born in mind that the proportion of spermatozoa
surviving a severe cold shock is low.
3.1. Diluents and cryoprotectants
During the past three decades, many diluents have been elaborated, most for a
particular processing protocol, so that generally diluents are not transferable from one
method to another ŽOsinowo and Salamon, 1976a,b.. In general the diluents are
composed of sugars, proteins and lipoproteins, buffers, additives and cryoprotective
agents, and may be divided, based on their constituents, into two categories: Ž1. diluents
without buffers, such as egg yolk–glucose ŽBaier, 1962; Polge et al., 1970., egg
yolk–lactose, ŽRichter et al., 1975; Westendorf et al., 1975., egg yolk–saccharose–
EDTA, Mg and Ca salts ŽMilovanov et al., 1974., and Ž2. diluents with buffers such as
glycine–phosphate and glucose–phosphate ŽIida and Adachi, 1966., egg yolk–glucose–
citrate ŽSerdiuk, 1970., egg yolk–glucose–citrate–EDTA–potassium–unitol–urea
ŽShapiev et al., 1976., Beltsville F3 ŽBF3; Pursel and Johnson, 1971., Beltsville F5
ŽBF5; Pursel and Johnson, 1975., Tes-tris-fructose–citrate–egg yolk ŽTEST; Graham et
al., 1971., Tes-NaK–glucose–egg yolk ŽCrabo and Einarsson, 1971; Larsson et al.,
1977., Tris-fructose–EDTA–egg yolk ŽSalamon and Visser, 1973. Tris-glucose–
EDTA–egg yolk ŽPark et al., 1977..
Glycerolated diluent portions are usually added when the partially diluted semen has
been cooled to 58C ŽAlmlid and Johnson, 1988.. Although the composition of diluents
varies, most are characterised by a low concentration of glycerol and short equilibration
time ŽWilmut et al., 1973; Pursel and Johnson, 1975; Westendorf et al., 1975.. Almlid
and Johnson Ž1988. exposed diluted semen to glycerol from 0.5 to 75 min and observed
no difference in the respective cryosurvival. The semen–glycerol equilibration time was
re-visited recently ŽFiser and Fairfull, 1996. for semen packaged in 0.5 ml straws and
protected with various glycerol levels Ž0–6%., with surprising results. Longer times of
exposure at 58C were beneficial, with 4 h equilibration producing the highest percentage
of motile spermatozoa with a normal apical ridge. As semen with no glycerol benefited
from the exposure to 58C similar to glycerolated semen, it seems likely that the holding
time at 58C is solely responsible for changes in sperm membranes and may render them
less susceptible to freezing damage. It seems unnecessary for boar semen to be
equilibrated with glycerol, but only exposed to a temperature of 58C. In this case,
glycerol can be added any time before freezing, as glycerol penetrates rapidly into
spermatozoa ŽWilmut and Polge, 1974; Almlid and Johnson, 1988..
Many cryoprotective agents have been tested, but none have proved better for
preserving boar spermatozoa than glycerol ŽWatson, 1995.. From the other compounds
tested, only exythriol, xylitol, adonitol, acetamide and DMSO, in relatively low concentrations, improved the post-thawing motility of boar spermatozoa, but the proportion of
sperm with intact acrosomes decreased ŽPaquignon, 1985.. Thus, glycerol has been used
extensively for protection of boar spermatozoa, and in general it is the cryopreservative
of choice for the semen preservation of most mammals. However, boar spermatozoa
show greater sensitivity than spermatozoa of other domestic animals to glycerol levels
adequate for optimum cryopreservation ŽAlmlid and Johnson, 1988..
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L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
The concentration of glycerol required for maximum survival of spermatozoa is
determined by several considerations. One of them is cooling velocity Žfreezing rate..
When static liquid nitrogen vapour and dry ice were used for freezing, the volume and
the geometry of the semen sample influenced the rate of freezing and the glycerol
concentration used. However, it is generally accepted that only relatively low concentrations of glycerol may be used in order to obtain satisfactory post-thaw survival ŽPursel
and Johnson, 1975; Westendorf et al., 1975; Paquignon and Courot, 1975; Scheid et al.,
1980.. Levels of glycerol necessary for adequate cryoprotection ŽMazur, 1985. were
reported to be detrimental to fertilisation by boar spermatozoa ŽCrabo and Einarsson,
1971; Wilmut and Polge, 1974. and resulted in damaged acrosomal membranes and
altered membrane permeability ŽWatson, 1981..
3.2. Rate of cooling and its interaction with the concentration of glycerol
At present, two freezing methods developed in the mid-1970s, are commercially used
for boar semen: the Beltsville and Westendorf methods. In the former method, ŽPursel
and Johnson, 1975. the sperm-rich fractions of the ejaculates are held for 2 h after
collection in the presence of seminal plasma and then, after centrifugation, cooled about
3 h, the semen is pelleted on dry ice Ž0.15–0.20 mlrpellet..
In the Westendorf method ŽWestendorf et al., 1975., which has been modified for
commercial use ŽAlmlid and Johnson, 1988; Almlid and Hofmo, 1996. the semen is
diluted Ž1:2, semen:diluent and cooled. in Hulsenberg
8 diluent and subsequently frozen
¨
in maxi-straws.
The method of Visser and Salamon Ž1979., modified by Maxwell and Salamon
Ž1979., also merits attention. The sperm rich fraction of the ejaculate is not held prior to
processing over a several hour period; after processing and cooling, the semen is
pelleted on dry ice. The method has not been used commercially.
The above packaging systems Žpellets and maxi-straws. allowed studies on the effect
of glycerol concentration on cryosurvival of boar spermatozoa, but the investigations of
the effect of freezing and thawing rates were limited. The geometry of the maxi-straw,
with its large gradient of temperatures, did not allow objective assessment of fast
cooling rates, while the pellet allows only slight modification of freezing rates by
changing its volume to surface ratio, as the surface of dry ice is of constant temperature
ŽFiser and Langford, 1980.. Weitze et al. Ž1987. reported that the boar semen packaged
in maxi-straws froze 3.75 times faster at the periphery than in the centre, resulting in
significantly lower survival compared to semen in straws of smaller diameter.
The control of cooling and warming rates can be improved by using packages with a
larger surface to volume ratio ŽWeitze et al., 1988., which will allow faster rates and
minimise the temperature range within the package ŽFiser and Fairfull, 1990.. Therefore,
new designs of semen packaging have been developed, such as 2 ml ‘‘flat straws’’
ŽWeitze et al., 1988; Ewert, 1988. or 5 ml plastic bags ŽRodriguez-Martinez et al.,
1996.. Neither of these systems are currently in use.
The optimum concentration of glycerol required for best protection of spermatozoa is
determined by several factors. One of them is cooling velocity. When a sperm
suspension is cooled below the freezing point of the solution, the spermatozoa and the
L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
159
diluent became increasingly supercooled until ice starts to form. The contents of the
spermatozoa remain unfrozen and supercooled as the membrane system blocks the
expansion of the ice. As the temperature decreases, supercooled cellular water, in
response to the increased differential osmotic pressure, diffuses out of the cell and
freezes. The rate of exosmosis is influenced by fixed cellular factors, in particular by the
permeability of the cell membrane to water and glycerol at specific temperatures, the
cell volume and the volume to surface ratio. The external factors that can be manipulated to regulate exosmosis are freezing rate and the concentration of the cryoprotectant.
If the freezing rate is slow enough, the spermatozoa will be capable of losing their
freezable water by exosmosis and thereby avoid supercooling; they dehydrate and no
internal ice forms. If the spermatozoa are frozen rapidly, the intracellular water does not
leave the cell before it freezes. According to the two factor theory of Mazur et al.
Ž1972., damage to the cell can result from two principal causes: dehydration at
suboptimum rates, also known as the solution effect, involving exposures of cells to high
concentrations of solutes, pH changes and solute precipitation ŽLovelock, 1953., and by
intracellular ice formation when the semen is frozen too rapidly. The optimum freezing
rate usually represents a compromise between these two extremes ŽMazur, 1970..
However, most studies on freezing of boar semen have used a fixed freezing protocol
and thus only the effect of glycerol concentration was evident ŽPursel et al., 1972;
Scheid et al., 1980; Almlid and Johnson, 1988..
The importance of the interaction between glycerol concentration ŽAlmlid and
Johnson, 1988. and cooling velocity has been recognised in the case of boar spermatozoa by Fiser and Fairfull Ž1990.. The authors demonstrated an inverted U-shape survival
curve for boar spermatozoa frozen over a range of cooling rates from 18C to 15008Crmin.
The interaction between glycerol concentration and cooling velocity in relation to the
percentage of motile spermatozoa indicates that the optimum cooling velocities shift to
higher values with the decreasing glycerol concentration. At each glycerol level, the
spermatozoa tolerate a range of cooling velocities without appreciable changes in
survival. It was also shown that optimum glycerol concentration varied with the
parameter investigated. Thus for motility, the best glycerol concentrations were 3–4%,
whereas for acrosomal integrity ŽNAR., the optimum concentration was 0–1%. Contrary
to glycerol concentration, the best cooling velocity for both parameters Žmotility and
NAR. was 308Crmin. Therefore, for preservation of both components of spermatozoa
function the authors recommended a compromise with 3% glycerol and cooling rate of
308Crmin. These conditions are similar to those recommended by Almlid and Johnson
Ž1988. in a study on the effect of glycerol concentration on the viability of spermatozoa
after freezing of semen packaged in 1.3 ml straws at 208Crmin. The latter authors also
reported acrosomes damaged by concentrations of glycerol as low as 1–2%. These
observations contribute to the growing body of evidence that the two factor theory of
cryoinjury applies also to boar spermatozoa ŽWatson, 1990, 1995..
A relationship between concentration of cryoprotectant and cooling rate exists for
most cell types ŽMazur, 1985.. The gradual decrease in motility and acrosome integrity
of boar spermatozoa as the glycerol concentration exceeds 6% may be due to the
chemical toxicity of glycerol, or to osmotic shock by rapid dilution of the glycerol at
thawing ŽBamba and Cran, 1988.. Most studies on membrane permeability have been
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L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
conducted with simple models, such as red blood cells ŽLeibo, 1976.. However, the
spermatozoon has a more complicated structure than the erythrocyte, comprising a
several geometries and different regions. Therefore, the optimum conditions for one part
of the cell may be unsuitable for other regions, or damage to some parts may have
greater consequences than to others.
The lipid composition and the organisation of sperm membranes changes during rapid
cooling ŽBuhr et al., 1994. resulting in increased membrane permeability ŽOrtman and
Rodriguez-Martinez, 1994.. There has always been a suspicion, and now there is
growing evidence, that frozen–thawed spermatozoa display characteristics of capacitated
spermatozoa ŽWatson, 1996.. This has also been noted when spermatozoa have been
subjected to other types of insult, such as cell sorting in the presence of a fluorescent
stain ŽJohnson, 1991.. Maxwell et al. Ž1997. have shown that there are significantly
more acrosome reacted spermatozoa after, than before the sorting procedure.
3.3. Induced ice nucleation and its effect on boar spermatozoa
The lack of induced ice nucleation Žseeding. in semen processing procedures often
has been criticised by cryobiologists. This technique could eliminate the damage from
the excessive supercooling, crystallisation of cellular water and growth of ice crystals by
aggregation and fusion after spontaneous nucleation, particularly in straws cooled
slowly. Without external seeding the semen sample will supercool well below its
freezing point to y158C ŽWatson, 1995.. Nevertheless, the elimination or minimization
of supercooling by induced nucleation at temperatures slightly below the freezing point
of the sperm suspension did not improve the cryosurvival of boar spermatozoa ŽFiser et
al., 1991.. The induced nucleation had no effect on post-thaw survival of spermatozoa
frozen at optimum rates and only slight improvement in acrosomal maintenance was
observed in boar spermatozoa frozen at suboptimum rates. This slight improvement is
similar to that reported by Pursel and Park Ž1985. for boar semen frozen in maxi-straws
which, due to their geometry, cannot accommodate optimum Žhigher. cooling rates.
There is no practical value in this improvement, because of the overriding effect of
suboptimum cooling rate which decreases the motility and acrosome integrity of
spermatozoa to approximately half of the respective values for semen cooled at the
optimum rate, regardless of induced nucleation. The reasons that the survival of boar
spermatozoa in particular, and of mammalian spermatozoa in general, does not appear to
be affected by seeding to the degree observed for mammalian embryos ŽWhittingham,
1980. may be due to differences in their sensitivity to glycerol and cooling rate.
Moreover, glycerol levels used for freezing of spermatozoa ŽWatson, 1995. are considerable lower than those adopted for embryos ŽWhittingham, 1980; Wilmut, 1986.. Thus,
embryos in 0.25 ml straws would be supercooled to lower subzero temperatures than
would spermatozoa. The increased rate of cooling after spontaneous nucleation is
tolerated quite well by spermatozoa which require higher cooling rates ŽFiser and
Fairfull, 1990. for best survival, but it is lethal to embryos ŽLeibo et al., 1978. in which
it causes intracellular freezing due to the insufficient removal of intracellular water by
exosmosis ŽMazur, 1985.. Nevertheless, it should be kept in mind that ‘‘live spermatozoa’’ at y1968C can be easily killed or damaged by the incorrect thawing Žwarming.
procedure.
L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
161
3.4. Thawing of semen
The rate of thawing through the critical temperature range is an important factor
affecting survival of spermatozoa. Various methods of thawing boar semen have been
described and are summarised in the review by Bwanga Ž1991.. Boar semen has been
frozen mostly as pellets on dry ice, but more recently in maxi-straws using static liquid
nitrogen vapour. Survival of spermatozoa after rapid thawing of pellets in preheated
diluent was superior to that obtained with spermatozoa packaged in maxi-straws in
which, because of their diameter, a fast thawing rate could not be achieved effectively
ŽSalamon et al. 1973; Weitze et al., 1987.. The effectiveness of thawing rate depends on
the original rate of freezing ŽMazur, 1985.. This interaction, which greatly influences the
survival of any mammalian spermatozoa, was assessed for boar semen by Fiser et al.
Ž1993., who recognised the limitations of packaging in pellets or maxi-staws, and
employed 0.5 ml plastic straws cooled by a flow of liquid nitrogen vapour, using a
programmable freezing system, to control freezing rate. The rate of thawing resulted
from exposure of straws to air or water of various temperatures for a specific time. In
boar semen frozen at the optimum rate ŽFiser and Fairfull, 1990., the motility and
acrosome integrity of spermatozoa improved with increasing thawing rate; similarly,
faster thawing rates were beneficial for pellets ŽSalamon et al., 1973; Pursel and
Johnson, 1976.. This is consistent with the idea that cryoinjury after rapid freezing is
caused predominantly by the regrowth of minute ice crystals during slow thawing and
that a high warming velocity is essential for cryosurvival ŽMazur, 1985.. The effect of
thawing rate on the acrosome is influenced by the glycerol concentration, higher
concentrations being more damaging to acrosome integrity ŽAlmlid and Johnson, 1988..
It should be noted that only a small proportion of spermatozoa frozen at suboptimum
rates survive after slow or rapid thawing. This suggests that boar spermatozoa are
damaged critically by slow cooling and only a limited rescue can be achieved by the
‘‘optimum’’ thawing rate. Effective freezing requires the optimum combination of all
three variables Žglycerol concentration, cooling and warming velocities.. Considering the
maintenance of motility and acrosome integrity, the optimum represents a compromise:
3% glycerol, freezing rate Žin the range between 08C and y508C. of 308Crmin, and a
thawing rate of 12008Crmin.
The final temperature of rapid thawing seems to be of importance for boar spermatozoa. Bamba and Cran Ž1985. reported that fast warming of boar semen in the
temperature range between 58C and 378C damaged acrosome membranes, resulting in
undulation of the outer membrane with its invagination and the development of tubular
networks within the acrosome. However, no release of enzymes or decrease of motility
was observed. The magnitude of this so-called ‘‘warm shock’’ is influenced by the
temperature range, suggesting that a phase change in the membrane lipids may occur
ŽBamba and Cran, 1988..
3.5. Fertility results of frozen–thawed boar semen
Attempts to obtain pregnancies with frozen–thawed semen before 1970 generally
failed. Polge et al. Ž1970., using surgical insemination into the oviducts, explicitly
demonstrated the fertilising capacity of frozen–thawed boar spermatozoa. In 1971,
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farrowings were reported after cervical insemination with thawed semen ŽCrabo and
Einarsson, 1971; Graham et al, 1971; Pursel and Johnson, 1975.. At the present time,
the indications are that frozen semen may give up to 50% farrowing rate and about
seven pigs per litter. This average was borne out by a study conducted on 36 farms in
the Netherlands in 1978, where the average farrowing rate and litter size from single
frozen–thawed Žpellet method. and fresh semen inseminations respectively, were: 47%
and 7.4 pigs per litter and 79% and 10.6 pigs per litter ŽJohnson et al., 1981.. The study
also showed a significant breed effect, with better ‘‘freezing’’ results produced by Dutch
Large White than Dutch Landrace semen. In a review of nearly all the published fertility
results conducted with frozen semen from 1970 to 1985, the average farrowing rates
Žlitter sizes. were 55% Ž8.3. and 58% Ž9.0. for the pellet and straw methods, respectively
ŽJohnson, 1985.. Results of commercial use of frozen boar semen over a five year period
in Norway indicate a 48% farrowing rate with 10.4 pigs per litter ŽAlmlid and Hofmo,
1996., using the modified maxi-straw method of freezing and double inseminations. In
order to improve the farrowing rates and litter sizes with frozen semen, it is necessary to
inseminate as close to ovulation as possible. Current commercial practice utilises double
inseminations: the first about 30 h after detection of oestrus and the second 10 to 12 h
later.
Frozen semen is not used generally for production of pigs sold for slaughter because
of the lower farrowing rate and litter size obtained after insemination with frozen–thawed
compared with fresh semen ŽJohnson et al., 1981; Johnson, 1985.. This limits the
progress toward more intensive use of AI with frozen–thawed semen worldwide.
Nevertheless, frozen semen is the vehicle for international transport of genetic material.
The world’s pig population is being consistently upgraded through the utilisation of
frozen semen, while slaughter pig production is improved locally through the use of
liquid-stored semen. However, the usefulness of frozen-stored semen from boars of high
genetic merit for repopulation after natural disasters, such as serious disease outbreaks,
cannot be underestimated.
4. Examination of spermatozoa after flourescent staining methods
Fertilising potential is directly related to the functional capacity of spermatozoa.
Researchers have sought for generations to use in vitro technologies to measure the
fertilising ability of spermatozoa. Examination of motility and acrosome integrity are
still the current standards for assessment of boar spermatozoa in vitro, nothwithstanding
the knowledge that boar spermatozoa that survive freezing can be motile but do not
necesarily fertilise ŽPolge, 1956.. However, new technologies have come to the fore
which utilise fluorescent dyes that bind to various regions of the cell to demonstrate
particular functional characteristics of spermatozoa. The technologies that could give
greater reliability to the estimation of the fertilising capacity of spermatozoa are
described below.
4.1. Assessment by microscopy after staining with fluorescent stains
Fluorescent staining of boar spermatozoa to determine viability of large numbers of
cells was adapted to flow cytometry by several laboratories in the 1980s ŽResli et al.,
L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
163
1983; Johnson and Garner, 1984; Garner et al., 1986.. Resli used fluorescein diacetate
ŽFDA., others used 6-carboxy fluorescein diacetate ŽCFDA. or more recently 6-carboxy
methyl fluorescein diacetate ŽCMFDA; Molecular Probes, Eugene, OR.. The latter two
stains tend to be more stable than the original FDA. In this system, CFDA is converted
by esterase enzymes in the live spermatozoa to a non-permeant fluorescent compound
that is retained in the plasma membrane of living spermatozoa. When used along with
propidium iodide ŽPI., CFDA or CMFDA is an effective indicator of viability of fresh
andror for frozen–thawed spermatozoa. Almlid and Johnson Ž1988. found these
combined stains with flow cytometry useful for monitoring membrane damage in
frozen–thawed boar spermatozoa during evaluation of various freezing rates. There are
normally three populations: live-green positive, dead-red positive and a third population
which are stained with both and represent dying spermatozoa. This process can be used
effectively with flow cytometry where 10,000 spermatozoa may easily be evaluated in
less than a minute. It can be used also for microscope examination. This staining system
has the disadvantage that it is time dependent when samples must be run at a set time.
4.2. Assessment of Õiability by flow cytometry after staining with SYBR-14 and PI
A newly developed membrane-permeant nuclear stain was released for testing in
1993 by Molecular Probes. The stain, known as SYBR-14 and sold in kit form with PI
by the developer as Fertilight, stains the DNA of living cells. The PI stain is used as a
counterstain since it penetrates the plasma membranes of dead spermatozoa in several
species ŽGarner and Johnson, 1995.. SYBR-14rPI works effectively as a liverdead
stain for either fluorescence microscopy or flow cytometry. Flow cytometry is the
preferred method since large numbers of spermatozoa can be analysed. The usefulness
of the stain has been demonstrated for several species ŽGarner et al., 1994; Garner and
Johnson, 1995.. Fig. 1 illustrates the strong correlation between spermatozoa stained for
SYBR-14 Žliving. and those spermatozoa stained by PI Ždead.. In boars, the live stained
cells represents 80 to 90% of the sperm population. Fresh ram spermatozoa, on the other
hand, tend to have a lower percentage of SYBR-14 staining Ž49%. than fresh boar
spermatozoa. SYBR-14 staining provides a new tool for measuring the viability of
spermatozoa based on the condition of the DNA within the nucleus and the apparent
membrane potential of the spermatozoa. Its use in combination with PI is useful for
determining within a population those spermatozoa that are live as well as dead. The
advantages of the method are its relative rapidity and that the levels of both dyes
ŽSYBR-14 and PI. can be measured in the same sample.
4.3. Assessment by microscopy after staining with exclusion dye
Fixing and subsequent staining of spermatozoa with fluorescent dye has proven
effective according to the report of De Leeuw et al. Ž1991.. The report described the use
of Hoechst 33258 to stain spermatozoa after they had been fixed with glutaraldehyde.
Hoechst 33258 stains dead or damaged spermatozoa, since the stain will not penetrate
the membranes of living cells. A count of the proportion of dead cells can be obtained
after treatment of spermatozoa with fixative Žgenerally 2% glutaraldehyde. and stain Ž10
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Fig. 1.
mg per ml Hoechst 33258 final concentration. as a wet mount and then counting about
200 cells per slide under a phase contrast microscope equipped with epi-fluorescence.
This method can also be adapted to flow cytometry. However, flow cytometric analysis
requires a laser that operates in the ultraviolet light range which is not a standard feature
on the smaller analytical machines.
4.4. Assessment by microscopy or flow cytometry after staining with CTC or FITC-PSA
The fluorescent antibiotic chlortetracycline ŽCTC. and the fluorescent lectin ŽFITCconjugated agglutinin derived from Pisum satiÕum, FITC-PSA. have been utilised in
studies to identify the degree of destabilisation of the sperm membrane ŽMaxwell and
Johnson, 1997a,b.. The CTC assay, first described for mouse spermatozoa by Saling and
Storey Ž1979., is based on the transfer of neutral and uncomplexed CTC across the cell
membranes. The CTC enters intracellular compartments containing high levels of free
calcium, ionizes to an anion and binds to the calcium becoming more fluorescent as a
result ŽTsien, 1989.. This CTC–Caq
2 complex preferentially binds to hydrophobic
regions of the cell membrane resulting in a pattern of membrane staining characteristic
of various transitional phases of destabilisation, indicated in the literature by the letters F
Žuncapacitated, acrosome intact., B Žcapacitated, acrosome intact. and AR Žacrosome
reacted.. A flow-cytometric assay for capacitation has also been developed based on
CTC-fluorescence ŽMaxwell and Johnson, 1997a.. The CTC assay based on fluorescence microscopy requires the manual counting of relatively low numbers of cells, and
their subjective classification according to the F, B and AR patterns. This has potential
disadvantages such as lack of precision due to the number of cells assessed, and the
subjective nature of the assessment. The flow cytometric approach allows rapid counting
L.A. Johnson et al.r Animal Reproduction Science 62 (2000) 143–172
165
of large numbers of cells Ž10,000. based upon the intensity of their fluorescence alone.
Although this lacks the precise delineation of specific fluorescence categories, it does
allow for a progressive increase in fluorescence, which might be associated with
membrane destabilisation in the process of capacitation.
The integrity of sperm membranes can be assessed also by using FITC-PSA
ŽMaxwell and Johnson, 1997b., which is reported to bind specifically to the acrosomal
contents ŽCross et al., 1986.. Graham et al. Ž1990. established that fluorescently labelled
PSA could be used in flow cytometry to assess the percentage of cells with or without
intact acrosomes based on comparisons with naphthol yellowrerythrosin B, and an
assessment of the percentage of cells with intact acrosomes when the acrosome reaction
was induced with lysophosphatidylchloine. In order to detect changes taking place
specifically in the live sperm population, Maxwell and Johnson Ž1997b. stained the
spermatozoa with PI, and the PI-positive cells were excluded from the estimate of
acrosome intact Žlow. and reacted Žhigh FITC fluorescence.. The proportion of acrosome
reacted spermatozoa estimated by FITC-PSA fluorescence tended to be higher than by
the CTC method Žcompare Fig. 2a and c., but as yet the acrosome status has not been
confirmed in these spermatozoa by microscopy. Fig. 2 shows the membrane integrity of
fresh spermatozoa after incubation Ž388C, 4 h. and after cooling to 58C at 0.258Crmin
as assessed by Ža. microscopy after CTC staining, Žb. by flow cytometry after staining
with CTC or Žc. with FITC-PSA. These treatments increased the percentage of B pattern
Žcapacitated. cells and of spermatozoa with high CTC fluorescence in comparison with
fresh, uncapacitated ŽF pattern. cells. Freezing and thawing and flow cytometric sorting
of boar spermatozoa into X and Y populations also causes a capacitation type response
ŽJohnson, 1991; Maxwell and Johnson, 1997b.. In other experiments, Maxwell and
Johnson Ž1997a. observed percentages of F Žuncapacitated. and B Žcapacitated. pattern
cells of 84.0 vs. 37.6% and 8.0 vs. 44.8% for fresh vs. flow-sorted spermatozoa,
respectively. Whether these membrane changes after flow cytometry may be considered
akin to normal capacitation still remains to be proven. Satisfactory cleavage has been
achieved after in vitro fertilisation of in vivo matured oocytes without further incubation
of boar spermatozoa sorted for sex containing a significant proportion Ž55%. of B- and
AR-pattern spermatozoa as assessed after staining with CTC ŽMaxwell et al., 1999..
Fig. 2.
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The tests of membrane integrity described above may provide valuable information in
the assessment of commercial semen processing methods.
4.5. Concluding remarks on semen eÕaluation
Assessment of the functional capacity of spermatozoa in vitro is of particular interest
to the AI industry in order to select sires with good fertility. Evaluation of semen
generally has been based on microscopic examination of motility and morphology of
spermatozoa. Frequently, eosin stain has been used to determine the proportions of live
and dead spermatozoa, while giemsa stain has often been used for morphological
evaluation. These methods, along with visual inspection of the semen, are useful but
their repeatability is questionable. Fluorescent staining improves microscopical evaluation of spermatozoa, but its major drawback is that only 100 or 200 spermatozoa can be
evaluated per sample. The advent of flow cytometry for spermatozoa analysis in the
1980s, and the development of new stains in recent years, has led to more accurate
means for assessing the viability of spermatozoa. However, finding one indirect test to
evaluate the fertilising potential of spermatozoa continues to be elusive. The use of
several tests, in combination, such as motility, acrosome integrity and a fluorescent
assessment of membrane integrity of spermatozoa, would add significant credibility to
evaluation of semen in the laboratory.
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