Status paper
on
CYTOGENETIC AND REPRODUCTIVE
PROBLEMS IN FARM ANIMALS
(Pre-Screening of Breedable but not Calvable Animals)
Dr. B.R. Yadav
Dr. R.K. Tonk
Dr. A.K. Srivastava
Livestock Genome Analysis Laboratory
Dairy Cattle Breeding Division
National Dairy Research Institute
(Indian Council of Agricultural Research)
Karnal – 132 001 (Haryana)
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Published by:
Prof. (Dr.) A. K. Srivastava
Director & Vice-Chancellor,
NDRI, Karnal-132 001 (Haryana)
NDRI Publication No:
112/2013
Reviewed and Edited by:
Dr. B.R. Yadav
Dr. R.K. Tonk
Dr. A.K. Srivastava
(Status paper presented in a Brainstorming Session on 15.01.2014 under the Aegis of NADS-NDRI, Karnal)
National Dairy Research Institute
Deemed University
(Indian Council of Agricultural Research)
Karnal – 132 001 (Haryana)
www.ndri.res.in
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
In modern era of fast industrialization, both in either developed or developing countries maintenance
of animals is becoming quite costly in the world. Many of the agricultural produce are reaching to industries
as raw material for finer products. Besides a large number of conventional fodder plants, grains or ingredients
are processed for value addition for human needs. Thus, there is increasing trend in competition for feed and
fodder between man and animals. Since ancient period maintenance of farm animal in India is subsistence of
life, however, in coming future, it is to shift towards and organized sector. The profitability of dairy enterprise
depends on the overall production of animals maintained in herd. Livestock farming is one of the most
important activities of agriculture industry in India, which contributes largest share in agriculture GDP. There
is constant need and increased need of milk, meat and fibres.
Inherent vital instinct exists in all the living beings for their self-propagation. During the long process
of evolution vast variety of life forms have developed. All these have been classified in different kingdoms,
phyla, classes, families, species, breeds, strains, races etc. All the living beings have their specific mechanism
of reproduction. The mechanisms start from simple binary fission to complex sexual system of individual
preferences. In case of mammalian species sexual dimorphism occurs and males and females produce
specific games. The fertilization is internal and females have suitable anatomical and physiological systems
to carry the conceptus to full development and give birth to progeny. The progeny passes through all the
phases of life cycle to give rise the next generation. However, many of the livestock species particularly large
ruminants have long generation interval, duration of maturity and age of first calving. Hence need of early
age and stage prediction of fertility status of domestic animals. It may be both males and females.
All the living beings have their specific genetic makeup known as genome. The term genome is the
combination of gene and chromosome. The genes control all the traits and activities of individuals in a
sequential manner from zygote to full development and subsequently performance. Genes are inherited
from parents and transmitted to next progenies. Though, genes have many alternate forms, alleles, which
1
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
cause variation among individuals in populations. Genes are composed of DNA (the genetic material), which
is consisted of nucleotides. There are three billion pairs of nucleotides in man and same number is reported
in other mammalian species including livestock species. These base pairs are arranged in the genome is
such an order that these can be read like a book, ponder the information and concepts that it contains. The
DNA molecule is packaged into thread-like structures called chromosomes. Each chromosome carries one
strand of DNA tightly coiled and folded many times to fit in the pellicle. Chromosomes were called long back
as vehicles of heredity.
Chromosomes are nuclear components with well-defined organization, morphology, special structure,
individual identity and functions. These are capable of self-replication and maintaining morphologic and
physiologic properties through successive cell divisions. Chromosomes are not visible in the nucleus of cells
and not even under a microscope, while the cell is not in division phase (during interphase). Nevertheless,
the DNA that makes chromosomes first gets duplicated and begins to become more tightly packed as cell
advances towards division, as condensation increases the chromosomes start becoming visible under a
microscope. Chromosomes become well recognizable and identifiable during the metaphase stage of the
cell division. All the details about chromosomes have been explored during cell divisions by cytogeneticists.
Chromosomes are differentiated mainly on the basis of either morphology or functions. Morphological
differences are due to a constriction point called the centromere, which divides the chromosome into two
sections, or ‘arms’. The short arm of the chromosome is labelled as the ‘p’ and the long as the ‘q’. The location
of the centromere on each chromosome gives it a characteristic shape, and can be used to help describe the
location of specific genes. Thus four types of chromosomes are found in higher eukaryotes viz. metacentric
(centromere in the middle or both arms of equal length), submetacentric (centromere towards one end
or both are of unequal length), acrocentrics (centromere close one end) and telocentric (centromere
just on one end). However, last type of chromosomes is rare found in normal configurations. All the four
National Dairy Research Institute
2
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
types of chromosomes found in different species and their breeds and strains. The mainly found different
morphological types of chromosomes are shown in the Figure -1.
Metacentric (Sheep)
Submetacentric (Buffalo)
Acrocentric (Cattle)
Fig. 1: Different types of chromosomes (1st Row of karyotypes of respective species)
Functionally chromosomes are of two types: Autosomes and sex chromosomes. In all the mammalian
species females are homogametic, produce all the gametes of one type only, that is each with one haploid
set of autosomes and one X-chromosome (1n=Autosomes + X). Males are heterogametic, which produce
two types of gametes in equal number (autosomes + X or autosomes +Y). The diploid condition is regained
after fertilization (2n= autosomes+ XX or autosomes+ XY) in females and males, respectively.
Every species has its characteristic constant chromosome number and morphology.
However,
sometimes deviations in chromosomes either numerical or structural do occur, which lead to impairment
in life activities. All these are genetic faults, and if apparent at birth are known as congenital defects or also
known as birth defects. However, are incurable or non-correctable, hence the surviving carriers need to be
identified early and further breeding of their parents is to be accordingly decided. During second half of the
previous century a large number of genetic defects have been encountered, explored, monitored, and even
sometimes reduced in man and other mammalian species including farm and dairy animals.
Chromosomal abnormality if present in a bull is likely to spread quickly in the population with the
use of AI or even natural service (currently at limited places). The earliest one such example is on record
(Gustavsson, 1969). A Robertsonian translocation involving chromosomes 1 and 29 got spread in 14%
3
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
of the general population of Swedish Red and White breed. The abnormally could be traced to one bull
several generations back. The carrier bulls and cows of the abnormality show fertility problems and reduced
production. Subsequently this abnormality (1/29 Robertsonian translocation) has been report in most of
the breeds of taurus and in few of the zebu breeds of cattle. Besides this (1/29), Robertsonian translocation
involving other chromosomes has also been reported in various breeds of cattle.
Currently in pursuit of high productivity the best sires of farm animal species with improved frozen
semen technology and artificial insemination have been used extensively to produce over million progeny
and grand progeny worldwide even at different distant places. This has been particular for wide use of
proven meritorious bulls to obtain fast genetic gain in reproductive and productive performance. These
days there is emphasis on multiplication of superior germplasm (both bulls and dams).
In animal herds sometimes there is an increase in average relationships, inbreeding and the likelihood
that rare defected genes suddenly become common in a population. Though, sometimes due to mutations
and other reasons de novo anomalies also do arise. Besides in the era of industrialization the cost of rearing
and maintenance has also gone quite high. Genetically normal calves in standard management practices
grow well, attain puberty, enter breeding cycle and become replacement in the herd. However, carriers of
genetic anomalies depending upon the severity of the defect go out of programme starting from a day to
years with one or the other reason or mode of disposal.
In the last quarter of the previous century the ARTs (Assisted Reproductive Technologies: ET, MOET,
IVF, Cloning etc.) have made it possible to multiply the female partner of many of the species of human
interest and need. Nevertheless genetic defects are quite damaging and cause substantial economic
losses. Breeders keep evaluating various genetic abnormalities in animals from time to time in most of the
developed countries and in developing countries gross level screening and evaluation breeding record of
parents is carried out in the selection of bulls in organized farms. However, in unorganized farms or farmers
National Dairy Research Institute
4
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
level such cases go undetected. The various anomalies encountered in livestock animals are stunted
growth, late maturity, anoestrus, repeat breeding etc. in females and poor quality semen, poor libido and
high return rates of mates are quite common in males of most of the dairy breeds and other related species.
Intersexuality and freemartinism are also encountered quite often in cattle, buffaloes and occasionally
in goats. However, the goats provide useful mechanism to understand the cause and effect relation of
anomalies and morphological features.
In addition to gross anomalies, the number of hidden problems and particularly the subfertility, cause
higher economic losses. The breeders keep such animal often for long period or spend on their treatment
with hope to use them as replacement. In standard management practices the body weight gain, attainment
of puberty and age of first calving monitored regularly. Each species and breeds there in have a set range of
parameters in terms of reproduction and production. The deviations in such parameters are considered as
genetic defect. However, many genotypic traits do not express in phenotype and remains hidden in animals
and populations.
In characterization of animals there are two main approaches: Top down and bottom up. In first
approach the animals are examined on the basis of their phenotypes and morphological features, which are
usually, species and breed specific. In case of anomalies grossly visible apparent deviations are compared
with breed description. Adult animals with reproductive problems are examined par rectum and deviations
are identified, which are given treatment or culled. Sometimes the advanced tests viz. hormonal evaluation,
karyotyping, genetic profiling (DNA typing) etc. are applied. The second approach is genotyping starts at
the bottom of the genetic bases viz. sequencing of DNA, gene identification, allele mining, chromosome
evaluation, and association with the performance of the animals.
The revelation of association of chromosomal anomalies with the reproductive defects and can be a
tool in culling or exclusion of problem breeding animals early in the age and stage animal maintenance
5
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
programme, and thus the losses of rearing of these animals in future can be prevented. In the first stage
animals are examined, observed and detected for reproductive status. However, many of these tests
become feasible only after puberty or attainment of maturity. These days various diagnostic methods are
available and being developed, though the cost and expertise is required. Chromosome identification for
their number and structure is quite efficient and less costly. Various methods and techniques are available
to identify chromosomal configuration and their structural features.
The conventional methods are useful in detection of gross numerical and structural anomalies and
have limitations as minor deviations in the rearrangement of chromosomes are not detected. These small
variations hamper in meiosis and in the formation of gametes and hence affect fertility. However, advances
in Molecular Cytogenetics have offered opportunities to detection of small fragments.
Presently a number of techniques are available to locate DNA sequences within cells or chromosomes
to be visualized by hybridization with a label specific DNA probe. These are collectively called in situ
hybridization. Fluorescent in situ hybridization (FISH) has a large number of applications in molecular biology
and medical science, including gene mapping, diagnosis of chromosomal abnormalities, and studies of
cellular structure and function. Chromosomes in three-dimensionally preserved nuclei can be "painted"
using FISH. A set of DNA probes derived from a single chromosome type can be used to delineate an entire
region of that chromosome by in situ hybridization. The direct visualization of specific chromosomes by
hybridized fluorescent Whole Chromosome Probes has led to the term “Chromosome Painting” where the
whole chromosome specific probes are referred to as paints. Probes are available in different sizes and
complexity. They are chromosome specific and labelled with different colours. Using fluorescent-labelled
DNA probes one can selectively paint a chromosome while the non-painted chromosomes are stained
with a different colour using suitable counter stains. In livestock animals FISH can be used for diagnosis
of inherited chromosomal aberrations, prenatal and postnatal diagnosis of carriers of genetic disease,
National Dairy Research Institute
6
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
diagnosis of infectious disease, viral and bacterial disease, tumour cytogenetic diagnosis, and detection of
aberrant gene expression.
All the developed and particularly the dairy oriented countries have as per policy chromosomescreening programmes. All the male calves selected (reserved) and reared for bulls are karyotyped at
initial stage, before their use in AI programme, and the carriers of any abnormality are culled. This practice
is gradually being adopted in developing countries, particularly in India, however, is limited to research
organizations and for academic programmes only. Though is all the teaching curricula, cytogenetic courses
have been included in PG programmes, however, hands on and practical work is deficient.
The present paper is description of the chromosome profile of major domestic animal species, the
problems encountered, rationality of cytogenetic investigations and findings of screening of several breeds
of cattle, buffaloes, goats and a few other species in this Institute.
Chromosome Profile of Farm Animals
Chromosomes are specific structures in the nuclei of all the living cells, each of these consists of a single
molecule of DNA bonded to various proteins and that carries the genes determining heredity. During the
process of cell duplication, these strands coil up and condense into much thicker structures that are easily
viewed under a microscope. Chromosomes occur in pairs in all of the nucleated cells in mammals except the
gametes, which carry one of each chromosome. In the evaluation chromosomes are arranged in an orderly
fashion to produce a karyotype. The karyotype represents the metaphase chromosomes characteristic for
an individual, breed or species. The graphical representation of the karyotype is the idiogram, which is a
drawing or photograph of the chromosomes of a particular cell. It contains information on the length of the
chromosomes, morphological characteristics such as location of the centromere, secondary constriction,
etc.
7
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
In animals the cytogenetic investigations started in early 1960s and by late 1970s, the diploid number
was well explored in majority of the known living species. The basic chromosome number and gross
morphology of autosomes and sex chromosomes were explicitly deciphered. During these years and in the
decade of 1980s structural details of chromosomes were analysed and by turn of the century their anatomy
has been well understood. In the current period the functional details and their applications in diagnosis of
diseases, defects and alterations are being studied.
Chromosomes of domestic animals are generally very difficult to (study) identify correctly in
conventional staining because all the autosomes (cattle, goat), or most of them (river buffalo and sheep) or
some of them (horse, donkey) are acrocentrics, which in karyotype construction are arranged in decreasing
order of size. So the acrocentric autosomes are hard to distinguish from one another in conventional Giemsa
staining. The rapid growth of knowledge on human chromosomes in several laboratories, and advances in
technical methods, have given several systems, which have made feasible to name and arranged uniformly
the chromosomes as karyotypes. The information about chromosomes configuration of the major species
of domestic mammalian species of farm animals is given table-1 & 2. Several investigators across the world
have shown similar configuration in all the breeds within respective species. Thus the karyotypes of each
species (cattle, buffalo, goats and sheep) are shown in Figure-2 and briefly described below under separate
heading:
Cattle
In cattle there are two well-known species: Bos indicus (humped Zebu) and Bos taurus (humpless
Taurus). These are well differentiated breeds in each species. Though inter-breeding occurs among all the
breeds within and between species, and the progeny are fertile. The diploid chromosome number in all
the cattle breeds is 60. Among these 58 are autosomes and one pair of sex chromosomes, which in males
is XY and females as XX. All the autosomes are acrocentric in decreasing order of size; X-chromosome is
National Dairy Research Institute
8
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
submetacentric comparable to first pair of autosomes in size, while Y- chromosome is among the smaller
autosomes.
The diploid number in cattle is 60 (2n=60)
Female 2n=60, XX
Male 2n=60, XY
Autosomes 58, all acrocentric
Sex chromosomes 2 (XX female or XY male)
X is submetacentric and
Y is small submetacentric (in Bos taurus) while smallest acrocentric (in Bos indicus) in all the
breeds (however, identified with C-banding in indicus breeds).
a
b
c
d
Fig.2: Morphological details in karyotypes of males of (a) Cattle (inset zebu – Y chromosome),
(b) Buffalo, (c) Goat and (d) Sheep
9
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Buffalo
Normal Riverine buffalo has 50 chromosomes in each diploid cell. Among these 5 pairs are submetacentric
and 19 pairs are acrocentric autosomes, and one pair of acrocentric sex chromosomes (XX in females and
XY in male). The X chromosome is the longest, while Y chromosome is between 18-19 pair (identified only
with banding techniques).
The diploid number in Riverine Buffalo is 50 (2n=50).
Female 2n = 50, XX
Male 2n = 50, XY
Autosomes = 48, first five pairs submetacentric rest nineteen pairs acrocentric
Sex chromosomes = 2 (XX female or XY male)
X is largest acrocentric and
Y is small acrocentrics, between 19th and 22nd pair of autosomes (identified only with
banding particularly with C- banding techniques).
Goat
The diploid chromosome number in goat is 60. The complement consists of 59 acrocentric chromosomes,
including the X- chromosome and a submetacentric Y chromosome, which is the smallest element.
The diploid number in goat is 60 (2n=60).
Female 2n=60, XX
Male 2n=60, XY
Autosomes 58, acrocentric
Sex chromosomes 2 (XX female or XY male)
X is second largest acrocentric and Y is smallest submetcentric
National Dairy Research Institute
10
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Sheep
The diploid number in sheep is 54. The complement consists of six metacentric, 48
acrocentric chromosomes, including the X- chromosome, and a submetacentric Y chromosome,
which is the smallest in size.
The diploid number in sheep is 54 (2Wn = 54).
Female 2n = 54, XX
Male 2n = 54, XY
Autosomes 52, metacentric 6 and acrocentric 46
Sex chromosomes 2 (XX female or XY male)
X is second largest acrocentric and Y is smallest submetacentric
Chromosome Configuration in Other Domestic Animal Species
In additions to cattle, buffalo and goats, the other major species viz. sheep, yak, mithun, pig, horse,
donkey, camel are reared in organized farm conditions under ICAR institutes, State Veterinary/ Agricultural
Universities and Livestock Farms and or by livestock breeders/ farmers. Investigations on chromosomes in
all these species are in progress. Anomalies have also been reported, which are like other livestock species
inflict reproductive and other problems. Chromosome profile of these species is also shown in table-1. The
screening of all the breeds and species for early diagnosis and remedial measures is essential like dairy
animals.
India has rich resources of flora and fauna, besides in domestic conditions many species are maintained
in reserved forests, safaris and captivity in zoos. Experimental animals are also maintained in research
institutes. Usually population size at such places is small and hence inbreeding increases leading to various
genetical, reproductive and survival problems. Thus it is essential to evaluate their chromosome profile from
time to monitor anomalies. The normal chromosome complement of such species is shown in table-2.
11
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Table-1: Chromosome number and morphological details in different species of
domestic farm animals and man
Sr. Species (common name)
2n
No.
Autosomes
Sex chromosomes
No. of
Sm/m
No. of
acrocentrics
X-
Y-
1.
Bubalus bubalis (River buffalo)
50
10
38
A
A
2.
Bubalus bubalis (Swamp buffalo)
48
10
36
A
A
3.
Bos indicus (Zebu cattle)
60
-
58
Sm
A
4.
Bos taurus (Exotic cattle)
60
-
58
Sm
Sm
5.
Bos grunniens (Yak)
60
-
58
Sm
Sm
6.
Bas frontalis (Mithun)
58
2
54
Sm
Sm
7.
Capra hircus (Goat)
60
-
58
A
m
8.
Ovis aries (Sheep)
54
6
46
A
m
74
10
62
m
m
74
10
62
m
m
11. Sus scrofa (Domestic Pig)
38
24
12
Sm
m
12. Sus scrofa (European wild Pig)
36
26
8
Sm
m
13. Equus caballus (Horse)
64
26
36
Sm
m
14. Equus asinus (Donkey)
62
48
12
Sm
m
15. Mule (Hinny)
63
37
24
Sm
m
16. Mule (Pony)
63
37
24
Sm
m
17. Homo sapiens (Man)
46
34
10
Sm
A
Camelus dromedaries (Single humped
Camel)
Camelus bacterianus (double humped
10.
Camel)
9.
2n= Diploid number; SM= Submetacentric chromosome; M= Metacentric chromosome;
A= Acrocentric chromosome
National Dairy Research Institute
12
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Table-2: Chromosome number and morphological details in different species of semidomestic, experimental and major wild animal species in captivity in India
Sr.
No.
Species (common name)
Autosomes
Sex chromosomes
No. of No. of acroXSm/m
centrics
46
12
32
Sm
56
12
42
Sm
38
34
2
Sm
38
32
4
Sm
38
32
4
Sm
38
32
4
Sm
78
76
Sm
78
76
Sm
74
76
Sm
36
32
2
Sm
40
20
18
Sm
40
20
18
Sm
Male:7 & Female:6
Sm (X,t-3)
68
2
64
A
30
26
2
Sm
58
12
44
A
68
2
64
A
56
14
40
A
82
Subtelo=80
Sm
2n
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
13
Boselaphus tragocamelus (Nilgai)
Elephas maximus (Indian elephant)
Acinonyx jubatus (Cheetah)
Panthra tigris (Tiger)
Panthera leo (Lion)
Felis domestica (Domestic cat)
Canis familiaris (Dog)
Canis lupus (wolf )
Ursus thibetanus (Asiatic Black Bear)
Funplus vulpes (Common fox)
Crocutacrocuta (Spotted hyena)
Hyaena brunnea (Brown hyena)
Muntiacus muntjak (Indian muntjac)
Dama dama (Fallow deer)
Antilope cervicapra (Black buck)
Cervus unicolour niger (Indian Sambar deer)
Cervus elaphus (Red deer)
Cervus duvauceli (Barasingha)
Rhinoceros unicornis (Great Indian onehorned Rhinoceros)
Ceratotherium simum (White Rhinoceros)
Mus musculus (Mouse)
Rattus rattus (Black Rat)
Rattus norvegicus (Rat)
Cavia porcellus (Guinea Pig)
Ocryctolagus cuniculus (Rabbit)
Lepus nigricollis) (Indian hare)
84
40
38
42
64
44
48
Subtelo:18
Acro: 64
38
22
14
22
18
Subtelo=62
34
8
?
?
Sm
A
A
A
Sm
Sm
Sm
YA
m
A
Sm
Sm
Sm
Sm
Sm
Sm
A
A
A
m
Sm
Sm
A
m
m
m
A
A
A
A
A
A
A
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Genetic and Hereditary Defects in Animals
Genetic defects have been encountered, explored, monitored, and sometimes reduced in man and other
mammalian species including farm animals. In recent generations, the best sires in animals have been used
to produce even over million progeny and grand progeny. However, this increases in average relationships,
inbreeding and the likelihood that the genes responsible of rare defects can suddenly become common in
a population. Genetic abnormalities are quite damaging and sometimes cause substantial economic losses.
In most of the developed countries evaluation of various genetic abnormalities in animals and particularly
males is carried out prior to their selection as replacement of herd. In developing countries gross level
screening in organized farms is carried out of bulls during their selection, however, in unorganized farms or
farmers level such cases go undetected.
The various anomalies encountered in livestock animals of most of the dairy breeds and other related
species. In females these are stunted growth, late maturity, anoestrus, irregular heat cycle, repeat breeding,
abortions, ovarian hypoplasia, smooth and non-functional ovaries etc. While in bulls these could be
crytorchidism, poor libido, poor quality semen or non-freezability, high return rate of mates or abortions in
mates are quite common. Intersexuality, hermaphroditism, pseudohermaphroditism and freemartinism are
also encountered in cattle, buffalo, goats and sheep. The goats and sheep provide a useful mechanism to
understand the cause and effect relation between anomalies and morphological features.
Reasons or Causes of Genetical Defects
Genetic disorders are diseases of defects that are caused by abnormalities in the DNA of individuals,
which reflect in their performance. Abnormalities can range from a small mutation in a single base in the
gene to the addition or subtraction of an entire chromosome or set of chromosomes.
National Dairy Research Institute
14
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
The main genetic defects fall in three categories, viz. (i) lethal, (ii) semi-lethal and (iii) balanced. The first
type of cases (lethal) either do not get implanted, so animal turns out to be of irregular heat cycle, or the
foetus die during embryonic development. In the second category progeny (semi-lethal) the offspring die
usually in perinatal stage or survive infrequently for longer duration though with reduced life expectancy
and performance. In the balanced types of genetic defects, the carriers are born and behave like normal
individuals; however, condition is more deleterious economically. The carrier does not have the genetic
disease related to the abnormal gene. A carrier can pass this abnormal gene to its progeny. For many genetic
disorders, carrier testing can help determine likelihood that offspring will have the disease: Sometimes these
carriers produce gametes and progeny in the initial age of breeding, however, later is age become sterile.
Nutritional Deficiency and Reproductive Defects
During gestation and initial growth period insufficient nutrition causes bone structure malformations
and susceptibility to disease. Majority of birth defects are the direct result of a lack of essential nutrients
needed for proper development of foetus and growing progeny. Such birth defects are not hereditary in
the sense of genetics, but are hereditary in the sense of an inherited nutrient deficiency, which lead to
impairment of a particular organ or system or multiple effects. However, carriers of genetic errors fail to
reproduce even with high feeding or best management condition.
In normal calves feeding of colostrum at birth and high plan of ration leads to fast growth and early
maturity. Deficiencies of various trace minerals, inadequate vitamin supplementation, energy- protein
imbalances and excessive proteins are considered as contributors to infertility and poor reproductive
performance. Implications of feeding programmes have been shown as the cause of many breeding
problems in dairy cows. Energy intake may be the most important nutritional factor affecting reproduction
on most dairy farms. Inadequate energy intake in heifers and early lactation cows reduces reproductive
performance. Excessive energy intake during late lactation and the dry period can cause ‘fat cow’ problems
which lower reproductive efficiency in next lactation.
15
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
In animal husbandry programmes normally 50 to 70 per cent of input costs are associated
with feed; manipulating nutrition can make operations more profitable. Cows need to be in good
enough condition to resume oestrous cycles after parturition and overcome general infertility,
anoestrus, short oestrous cycles, and uterine involution just to maintain a yearly calving interval.
Thus nutrition plays an important role in reproduction, particularly in most cases of severe nutritional
deficiencies have been observed as one of the possible causes of reproductive problems. Heifers,
if are fed inadequate amounts of energy, take longer duration to reach sexual maturity, in such
conditions even they stop cycling.
Hormonal Imbalance and Reproductive Defects
In animals development, growth, physiology, behaviour reproduction and lactation are regulated by a
number of hormones, the chemical messages of their body that are secreted by the specialised ductless or
endocrine glands directly into the blood stream. The blood carries these chemical messengers to the parts
of the body where they produce their effects, directly or indirectly also in various aspects of reproduction.
The secretion of these hormones is essential for the maintenance of a proper internal environment to ensure
successful reproduction. The hormones involved in reproduction may be divided into two groups: (a) primary
and (b) secondary hormones of reproduction. The primary hormones are: follicle stimulating hormone (FSH),
lutenizing hormone (LH), (ICSH), oxytocin, testosterone, estradiol, progesterone, chorionicgonadotrophin
etc. While secondary hormones include: Somatotropin releasing hormone (STH), thyroid stimulating
hormone (TSH), adrenocorticotropic hormone (ACTH), vasopressin (antidiuretic horomne) (ADR), thyroxine,
aldosterone, 17-OH corticoids (cortisone) (cortisol) (corticosterone), insulin, parathormone etc. The primary
hormones are directly involved in various aspects of reproduction, such as spermatogenesis, oogenesis, and
ovulation. The secondary hormones are needed for the normal functioning of the organisms which makes
it possible for reproduction to occur.
National Dairy Research Institute
16
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Sexual reproduction is a complex phenomenon; it involves two sexes and a series of intriguing process
in higher animals. Thus during gamete formation and development in both males and females; several
events take place in germinal layers, gonadogenesis, organosis etc. however, some errors do occur though
fortunately very rarely. Imbalance in hormonal profile is one of the leading causes of infertility, particularly
in females. The inability to ovulate and regulate hormone levels cause a production of too much or too less
of one particular hormone. Usually hormonal imbalance is manifested by symptoms that can be detected
allowing for treatment. These symptoms include anoestrous, repeat breeding, irregular heat cycles,
abortions and sometimes excessive weight gain or loss. In animals quite often observed as stunted growth
in both sexes or bullish appearance in females.
Treatments are available for restoration of deficiency of hormones or their level in the body with external
sources treatment. Though, treatment of sterility with hormones is not always successful. Sometimes failures
can be due to the fact that the exact amounts of various hormones necessary for normal reproduction are not
known, and the use of hormones have necessity been largely on a trial and error basis. Some conditions are
beneficial in improvement of reproductive performance, however, with appropriate hormones treatment.
Environmental and Chemical Factors for Reproductive Failures
Environmental factors influence directly physiological and pathological state of animals, and indirectly
to reproduction and then production. One or more of these factors in sub optimal level, affects both the
calving interval and culling rate due to reproductive failure, which can decrease profitability of animal
breeders. Stress perception of females is also alleged as one of the causes of infertility, impaired foetal
development, late pregnancy complications or abortions.
Monitoring temperature and relative humidity influence the reproductive efficiency of dairy cows. The
temperature-humidity index (THI) is a widely used tool to assess the impact of heat stress on dairy cows.
17
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
The effects of heat stress become apparent on first conception rate, calving to conception interval and
calving interval. Thus in planning layout of an organized breeding farm various aspects are kept in mind,
which include suitable housing system to protect from high and low temperature, movement of air and
commingling of animals.
Inadvertent exposure to chemicals in the environment might predispose birth defects and reproductive
failure in both males and females. Every year millions of chemicals are handled and various types of products
are produced worldwide. Chemicals are present at the workplaces, in the environment (from air pollution
and from carelessly disposed materials that contaminate the land, water supply, or food chain), and food
additives. He said that exposure to these chemicals and other low-level environmental contaminants such
as phthalates, polychlorinated biphenyls (PCBs), dioxins and pesticides might subtly undermine the ability
to reproduce.
Infertility and Sterility
During the selection of young calves or kids for replacement in herd the various criteria used include
the pedigree record, growth rates, body parameters, and other trivial features, and the question of fertility.
In organized herds or field conditions breeders keep observations on growing young stock to proper
growth, maturity and attainment of puberty. The well fed, healthy, individuals (males and females) enter
in breeding programmes as the standard criteria and age of the specific breed and species. However, still
many fail due to infertility. Many of the infertility problems are due to congenital causes often inherited.
These include developmental anomalies of the ovaries, oviducts, uterus, cervix, vagina and vulva. Some
have morphological implications and others a functional significance. Common morphological conditions
include ovarian (gonadal) hypoplasia and aplasia, anomalies of the tubular genitalia, hermaphroditism,
freemartinism, arrested development of the Mullerian ducts and even double cervix. Some of these authors
have observed and others well documented in literature.
National Dairy Research Institute
18
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Sterility is the inability of a sexually mature individual to produce offspring. Sterility is synonymous with
infertility, although the term ‘sterility’ is ordinarily used in botany and horticulture and the term ‘infertility’ is
usually applied to man and animals. Sterility may develop as a result of the impairment of meiosis and the
malformation of sexual cells (gametes), the suppression of the reproductive process, the death of fertilized
eggs (zygotes), or the abnormal development of reproductive organs. It may be hereditary (genetic), or it
may be caused by external factors, or be induced artificially by sterilization.
Sterile animals are of little significance if an appropriate culling programme is practiced. Such cases of
females and males have limitations in development and appearance, which are well identified at early age
either due to lack of masculine features in males or feminine in females or vice versa and are culled from
herd without much expenditure on them. Though the birth of infertile calves is disadvantage as deprivation
of progeny; however, less economical loss as are removed early from herds.
Sub-fertility or Semi-sterility
Subfertility means decreased fertility or a decreased chance of attainment of conception, but not a
complete inability in getting pregnancy. A common definition of sub- and infertility is very important for
the appropriate management of infertility. Subfertility generally describes any form of reduced fertility
with prolonged time of non-conception. Infertility may be used synonymously with sterility with only
sporadically occurring spontaneous pregnancies. The major factor affecting the individual spontaneous
pregnancy prospect is the time of non-conception, which determines the grading of subfertility.
Primary infertility is where the females have not conceived before, while Secondary infertility describes
the situation where there has been a previous conception, though not necessarily resulting in the birth of live
progeny. It is to some extent, failing to conceive is normal. Common causes of subfertility include ovulatory
disorders, tubal disease, peritoneal adhesions, endometriosis, uterine abnormalities, abnormalities of sperm
19
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
and advancing female age. Such infertility cases remain unexplained after thorough physical evaluation in
about 5-10% of cases, which are considered as genetic defects.
Different farms have different management practices and this may have an impact on fertility after AI.
Reproductive planning (intervals between calving, season, age of first mating, AI technique, etc.) and animal
handling (feeding, health, preparation of AI lots, etc.) have a great effect on fertility results. Thus, in order to
improve fertility results, handling conditions on farms must be improved, along with more widespread use
of AI techniques.
These types of animals are more un-economical as the decision of culling gets delayed and expenditure
keeps increasing with hopes of conception. The observations show that in females at an early age ovulation
takes place, which results in progeny; however, subsequently most of the remaining oocytes get atrophied
and animal turns out anoestrous.
Congenital – Birth Defects
Congenital abnormalities are manifestation of features or differences that are present in new
born contrary to normal offspring. These are also known as birth defects, which also express as physical
deformities. Birth defects present at birth might be result of a genetic mutation or some other non-genetic
factors. Often the causes of birth defects are less understood including cleft lip and palate, clubfoot, spina
bifida, water on the brain (hydrocephalus), diabetes mellitus, and some heart defects. Sometimes several
clinically recognizable deviated features are observed in individual, which syndrome together concurrently.
Congenital gross defects usually cause abortion, however, sometimes present at time of birth. They
are uncommon but do occur in most of the breeds of cattle. Such defects can be abnormalities in skeleton,
body form, and body functions. Abnormalities may result from genetic or environmental causes. Once the
National Dairy Research Institute
20
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
environment is the cause, adjustments can reduce further economic losses. However, genetic (inherited)
causes are much more complex and difficult to correct.
The calves born with gross phenotypic deviation from the standard features of the breed and species
are known as abnormal. The defects of the body that are present at the time of birth are called congenital
defects. Such anomalies might involve whole body or some part of the body or an organ system. The
individuals with multiple deformities are called as monsters, the malformation due teratogenic effects of
environmental biohazards or genetic reasons. Some congenital defects can be minor and resolve with
growth, while others might prevent normal development and function or even cause premature death.
Though certain defects may be quite obvious at birth, many may remain hidden for months, years, and
some even lifelong. Congenital defects can occur for no known reason, can be inherited, can be caused by
environmental factors, or can be a combination of any of these. Congenital causes of infertility are often
inherited.
In females the congenital defects include developmental abnormalities of the ovaries, oviducts, uterus,
cervix, vagina and vulva. Some are lethal; a few have a morphological significance and others a functional
significance. Common morphological conditions include ovarian (gonadal) hypoplasia and aplasia,
anomalies of the tubular genitalia, hermaphroditism, freemartinism, arrested development of the Mullerian
ducts and double cervix. All the carriers turn out sterile and non-curable.
Congenital defects in males have also been observed; the main defects include gross anatomical
deformities of one or the other organ visible in morphology. Some influence genital system viz. hypospadia
and cryptorchidism; however, many of the reproductive anomalies come to knowledge in advancement of
age or on attainment of maturity. In males (bulls), the high return rate of mates when a large percentage
of females do not conceive, the first thought is generally a bull problem. In human males the occurrence of
congenital defects has been shown higher than females.
21
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
There is no way to prevent all birth defects. However, if in some pedigree there is history of birth defects,
then screening of parents (dam and sire) becomes necessary before further breeding. There are methods of
diagnosis of abnormal foetuses in the event of pregnancy during early developmental stages, and if carrier
their termination. Some abnormalities can be corrected with surgery. Experimental procedures have been
used successfully in correcting other defects. The risk of birth defects can be reduced and with the advent
of ‘gene therapy’ approach may come to salvage of some of the defects, however, majority of the congenital
defects may remain incurable.
Syndromes in Livestock
The word syndrome (Greek word meaning ‘run together’) however, is represented by a number of
symptoms without an identifiable cause. Occasionally in mammalian species atypical calves are born,
with different organs system affected showing a variety of symptoms appeared together but without
a known cause. The syndromes (calves) are born with a group of symptoms that collectively indicate
or characterize a disease, disorder, or other abnormal condition. Some of the well-known examples
include baldy calf, brachygnathia, bulldog (achondroplasia) contractural arachnodactyly (CA), also
known as “fawn-calf” syndrome (FCS), cleft palate or cleft lip, rigid joints (arthrogryposis), hairlessness
(hypotrichosis), dwarfism and many others. The offspring with malformation of one or more organs
systems are usually called monsters.
Chromosome anomalies have been found in the carrier animals as cause of the deformities in many of
the syndromes, however, still cause of others remain unknown (idiopathic). Most of the monsters and many
of the syndromes are born either dead or die within few hours or days of parturition. In some of the calves
survive for longer duration.
National Dairy Research Institute
22
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Reproductive Problems Frequently Encountered in Mammalian Species
The reproductive problems are caused by one of the various factors including environmental, nutritional,
hormonal imbalance or genetical or a combination of two or all. Most congenital defects in animals are
due to environmental factors, however, some are due to genetic reasons. Reproductive efficiency is a
major contributor to profitability of dairy herds. Dairymen must then be concerned with its improvement.
Early identification and diagnosis of disorder for achieving good reproductive efficiency in a dairy herd
is necessary. Different types of reproductive disorders are found in females and males of each species of
farm animals. In females such disorders are: anoestrous, repeat breeding, irregular heat cycle, abortions etc.
while in males are cryptorchidism, poor libido, poor quality or abnormal semen (azoospermia, oligospermia,
asthenospermia), poor freezability and high post-thaw mortality etc. The description of such anomalies is
briefly described in this section.
Reproductive Problems Encountered in Females
In animal breeding programme the female partner gives birth to the offspring, which becomes
replacement for the next generation. There is a cascade of anatomical organ system, physiological and
developmental activities responsible for the successful birth of normal progeny. However, if somewhere
some error occurs the process gets hampered.
Rectal Palpation-Observations of Reproductive Tract and Ovaries
The mature females with suspected ailments or breeding problems during rectal palpation reveal
different conditions of the reproductive tract and ovaries. The main deviations of the tract include: infantile
or rudimentary or under development tract and organs. The ovarian deformities include: size quite
small, smooth, and non-functional. The individual of some particular breed or species shows ambiguous
23
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
appearance of whole body or some organ and are likely to have stunted growth or bullish features much
different to normal. These animals usually have deformities of their reproductive tract, organs or ovaries.
In females quite often observed condition is ovarian aplasia/ hypoplasia, which is due to the absence or
failure of development of the ovaries. Ovarian failure exists in a variety of forms and has a variety of causes.
Extreme peculiar deformities include one or both ovaries missing (ovarian agenesis), ovarian hypoplasiaone or both ovaries are small, functionless and composed of largely undifferentiated parenchyma. Oocytes
and follicles are virtually absent.
Abnormalities of the uterine tubes, uterus and cervix include segmental aplasia of the paramesonephric
ducts; developmental defects of the paramesonephric (Müllerian) ducts lead to a wide range of anomalies of
the vagina, cervix and uterus. In uterus unicornis situation one horn either left or right is fund present, while
the other absent. Sometimes horns are comprised as flat band of tissues with no lumen and a blind residual.
Occasionally uterus and horns are found filled with fluid could be clear, hydrosalpingitis, haematosalpingitis
etc. One such example is White heifer disease, hereditary problems due to high inbreeding and leads to
infertility.
All the animals with deformities of reproductive system and organs, which might be due to different
reasons, however many of these reveal various chromosome anomalies. There is limitation about rectal
palpation as it is feasible on after attainment of a certain age, however, chromosome evaluation can be
carried out soon after birth.
Anoestrous
Anoestrous is considered a problem when cows are not seen in heat. The true anoestrous condition
can be ruled out by palpating the ovaries. The condition of anoestrous in animals has been shown under
the control of various factors. On the basis of the effects of these factors the problem can be divided in
several categories: Congenital anoestrous, seasonal and nutritional.
National Dairy Research Institute
24
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Congenital anoestrous: Numerical and structural chromosomal abnormalities considered to be
significant and contributing to anoestrous condition. The numerical such as trisomy-X, monosomy- X, sex
chromosome chimaerism, autosomal trisomy have been observed responsible for this condition. Some
structural congenital defects have also been reported to be responsible for the condition such as ovarian
aplasia (a rare condition when one or both ovaries are absent), ovarian hypoplasia (one or both ovaries are
smaller than normal and inactive). Both the latter conditions have been reported controlled through the
recessive trait in the Swedish Highland breed of cattle as is caused by a single autosomal recessive gene
with incomplete penetration.
Seasonal anoestrous: There are some evidences that daylight length (photoperiod) influences fertility
in Brahman breeds, though there is no seasonal anoestrous in some species.
Nutritional anestrous: Nutritional deficiency has been reported to cause the anestrous in some breeds
of the beef cattle. In the postpartum cow, lactation causes a negative energy balance. This is even more so
in first calf heifers are affected mostly by nutritional anestrous. Suckling by the calf inhibits cyclic activity in
the cow by decreasing LH release. Deficiency of vitamins and minerals are often suspected in infertility and
anestrous. Copper requirements are 10 ppm and less than this often cause anestrous. A cobalt deficiency
might cause a delayed first estrus and irregular heats. Magnesium requirements are 40 ppm and less than
this could cause anestrous or irregular heats. Ovarian atrophy is caused from nutritional problems and is
most often seen in high production dairy cows.
Various infectious agents like bacteria, virus, protozoa and fungi have also been reported to affect the
normal oestrus cycle of the animal and many times the animal become anoestrus. This type of animal is also
known as pathologic anoestrous.
25
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Cystic ovarian disease or follicular cysts: It is one of the quite frequently diagnosed gynaecological
findings in dairy cattle. It is a large follicular structure (>2.5 cm) on one or both ovaries in the absence
of a corpus luteum. Cyst formation in the ovaries is common during early lactation and is diagnosed in
about 4% of all dairy cows milked by hand or machines, however, much rare in cows with suckling calves.
Clinically, it is most frequently characterized by anoestrous. The most widely accepted hypothesis is that
cystic ovarian disease results from a neuroendocrine imbalance involving the hypothalamic-hypophysealgonadal axis. The causes of cystic ovaries are not well established but there is evidence to suggest that the
condition is more common in daughters of cows that have had cystic ovaries. One of the possible causes of
this imbalance is an insufficient feeding of the high-producing cow, resulting in ketosis. Energy deficit and
low insulin concentrations might limit the responsiveness of the ovary to gonadotrophin stimulation. Fatty
liver syndrome, acidosis and phytoestrogens in forage have been associated with cystic ovaries.
The genetic background of cystic ovarian disease has also been investigated. Higher incidence of
anoestrous due to inactive ovaries in buffalo than in cow has been reported. Various hormonal treatments
like GnRH, oestrogen and progesterone either alone or in combination have been tried with variable success.
Repeat Breeding
In breeding of cattle females the mean number of services per conception of 1.6 has been reported
as normal, though some animals need 4-5 service. However, occasionally some heifer even cow after 4-5
services fail to conceive and become repeater. Repeat breeding is reproductive failure or non-conception
in the heifer, which can be equally attributable to both fertilization failure and early embryonic loss prior to
day 8 of gestation. The early loss of embryos usually does not have any effect on interval to next oestrus and
these females return to oestrus at regular intervals.
National Dairy Research Institute
26
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Repeat breeders or those females that return to oestrus after bull exposure for 45-60 days or following
multiple inseminations have been reported quite often in most of the breeds of cattle and other farm animal
species. However, these females have high rates of anovulation, fertilization failure, anatomical defects,
fewer normal embryos, increased chromosomal abnormalities, and lower progesterone levels on day 6 of
gestation. Infertility has been linked to females where persistent follicles grow larger and ovulate later. In
these females, estradiol concentrations are elevated, embryo development is retarded, and lower numbers
of embryos are formed.
Repeat breeding is a substantial problem in livestock species breeding leading to large economic loss
for the dairy producers due to more inseminations, increased calving interval and increased culling rates.
Repeat breeding has been defined as failure to conceive from three or more regularly spaced services in the
absence of detectable abnormalities. Repeat breeder cows usually appear physically and physiologically
normal on examination, however, might have problems either with embryonic losses after conception
or poor embryo recognition, environmental stress, nutritional imbalance or other management factors,
which might have adverse effect on their fertility. Embryonic loss, failure by the embryo to attach itself onto
uterine wall, is considered an important cause to poor conception rates. The main causes of embryonic
losses are also post calving complications that prevent the uterus from recovering and recognising the
embryo. Similarly, if a healthy cow is served too soon after calving, the uterus might not have recovered
sufficiently to recognise the embryo. It is suggested that stress in animals leads to poor conception rates.
The other possibilities are also the condition of endometritis, cystic ovaries or other pathological reasons,
such as herd infection with BVDV or leptospirosis as reasons for non-conception
It has also been proposed that early embryonic loss should be regarded as normal due to an early
elimination of unfit genotypes. During the last 50 years repeat breeding has been the object of several
investigations. There are different opinions among scientists about the cause of repeat breeding. A number
27
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
of experiments have reported a higher proportion of embryos with deviated morphology collected from
repeat breeding animals as compared to control animals suggesting an increased embryonic death rate
as the cause of repeat breeding. Higher progesterone levels during oestrous in repeat breeding heifers as
compared to control animals have been reported, indicating hormonal deviations as one possible cause
of repeat breeding. There are suggestions that physiological alterations linked to individual animals as a
possible cause of repeat breeding. On the other hand, other investigators have reported normal pregnancy
rates in repeat breeders when an additional insemination was performed under controlled conditions
suggesting management and environmental imperfects as the most important factors for the repeat
breeding syndrome. A third theory has also been proposed claiming the repeat breeding phenomenon as
solely the result of a probability distribution since the same proportion of animals might be pregnant after
each AI and there might always be a number of not pregnant individuals after a number of AIs.
Several studies have been conducted on cytogenetic point of view on repeat breeding
animals, which show the chromosome anomalies associated with fertility disturbances. A higher
frequency of a chromosomal abnormality than in the normal population leads poor conception in
females.
Irregular Heat Cycle
In the literature the average length of the normal sexual cycle in all the animal species is fixed, which
cattle and buffaloes is observed to be about 21 and 24 days, respectively. However, a great variation in the
length of the sexual cycles is one of the problems in animal reproduction. Cycles of short duration are few,
while those of longer duration are common. The causes of long sexual cycles could be various disturbances,
such as silent or not detected oestrus with a hidden ovulation, delayed ovulation, fertilization/implantation
failure or embryonic death. All the embryonic losses during earlier stages of implantation or development
delay the return female to oestrus at regular interval.
National Dairy Research Institute
28
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
In chromosomal translocation carriers a reduction of fertility occurs due to the formation of genetically
unbalanced gametes whose frequency varies widely depending on whether the carrier is the cow or bull, and
on the type of translocation. The gametes with an unbalanced karyotype result in chromosomally abnormal
embryos. These chromosomally unbalanced embryos usually do not survive for a complete gestation and
result early embryonic death and animal again entered in oestrous cycle with irregular time period.
In buffalo sometimes heat cycles occur with a length of about 70 days and even extremely long cycles
of 91 to 138 days. Due to the great number of long sexual cycles, the mean length, including both normal
and abnormal cycles, could be much longer than 21 and 24 days for cattle and buffalo. The influence of the
level of nutrition and season on the sexual cycle rhythm of the length of the sexual cycle has been shown
significantly longer for the underfed group.
Abortions
Abortion in farm animals is usually defined as a loss of the foetus between the ages of 42 days to 260
days. Pregnancies lost before 42 days are generally referred to as early embryonic deaths, whereas a calf
that is born dead between 260 days and full term is defined a stillbirth. In well managed herds it remains
between 3 to 5% abortions per year and is often considered normal. However, beyond 5% it becomes
alarming and efforts are made to find the cause and take remedial measure.
Many reasons have been postulated to explain the high rate of embryo wastage, including an increased
frequency of genetic abnormalities, several factors individually or collectively can cause the abortion in
animals. While infectious agents such as bacterial protozoal, viral are perhaps the most frequently thought
of cause of abortion, there are other factors which might cause a proportion of pregnancies to terminate
with an abortion.
29
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Genetic abnormalities, adverse environmental conditions, nutritional deficiency, toxic agents,
reproductive organ anomaly and hormonal imbalance are the some important factors which play an
important role in abortion. Occurrence of chromosomal abnormalities in embryos might get introduced
during its development or might be transmitted through its previous generation. Chromosomal
abnormalities constitute a major cause of embryonic loss and abortions in mammals. These abnormalities,
which might not cause a change in the outward appearance of the foetus, could result in abortion because
of the growing foetus inability to develop properly in the uterus. Abortions and stillbirths negatively impact
the annual cow production rate.
Dystocia and Stillbirths
Dystocia and stillbirth are related terms; however, are not the same traits. Approximately 50% of stillborn
calves are born without difficulty. Dystocia is difficult or abnormal labour or delivery. From the Greek ‘dys’
meaning ‘difficult, painful, disordered, abnormal’ and ‘tokos’ meaning ‘birth’. Dystocia or difficult calving is
stipulated that the heifer or cow is unable to calve without assistance. Stillbirth is defined as birth in which
an animal is born dead or dies during or within 24 hours’ time after parturition and after at least 260 days of
gestation. If the dead calf is delivered earlier than 215 days it is categorised as an abortion.
In most of the dairy farm two of the major problems are calving complications and stillborn calves.
Among calving complication dystocia is the difficult birth, typically caused by a large or awkwardly
positioned foetus, by smallness of the maternal pelvis, or by failure of the uterus and cervix to contract
and expand normally: the incidence of dystocia or difficult calving or disordered abnormal calf or monster.
Dystocia is the term used to describe difficult birth and causes significant economic loss to both the beef
and dairy industries due to the death of the calf or dam.
National Dairy Research Institute
30
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Dystocia is one of the leading causes of calf crop loss, resulting from a disproportionate size of the
dam and foetus at parturition. Dystocia and calf mortality occur more frequently in heifers than cows. Calf
mortality also increases with severity of dystocia and is observed higher in males than female calves and large
versus small calves. Dams with larger pelvic areas tend to have lower incidences of dystocia. The factors that
have the greatest impact therefore are pre-calving pelvic area and sex of the calf. Most dystocia problems
arise at conception with breeding of females with small pelvic diameters, since these females will still have
a relatively small pelvic size at calving. Foetal mal-presentation also may cause calf loss but accounts for less
than 1% of all losses. Finally, females that experience dystocia are more likely to have rebreeding problems.
The causes of dystocia can be many: either dam or calf or management practices ranging from breeding
genetics and nutrition to the cow or heifer during delivery. Quite often genetic syndromes, Malformed
(teratogenesis) or monster calves are do born and their birth is not normal. Other diseases (e.g. Milk Fever)
where there is a decrease in calcium which decrease muscle tone causing the cow to become too weak
to push out the calf, or uterine torsion where the cervix is twisted. Dystocia, of the many factors affecting
calf survival, is the most important and usually results in death of calves and cows, production losses in
both dam and calf, and delayed reproduction rates. In addition to death losses and veterinary and drug
costs, dystocia often causes other less noticeable, losses. These losses include lower rate of body weight
gain in surviving calves, higher number of days open in dams, lower conception rates, and decreased milk
production in dairy cows.
Stillbirths and calving difficulties are caused by genetic and/or environmental factors. The genotype of
the calf as direct effect and that of the dam as maternal effect contribute to the risk of both traits. Further
factors are the birth weight and sex of calf, parity and age of dam as well as season of calving. Causes of
stillbirths are not related with dystocia are infections (e.g. BVD), insufficient placenta development, metabolic
disorders of the dam, and congenital malformations of the calf. Chromosome anomalies have been found in
many of the congenital defective or stillborn calves. Hence there is always a need of chromosome screening
of such cases to know the cause and effect.
31
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Diseases Inflicting Reproduction Problems
Diseases cause reproductive failure; and are generally manifested in abortions, early embryonic death,
calves born weak or dead, and calves that die soon after birth. The diseases affect the reproductive systems
in abnormal hormone production by the ovaries or the testes or by other endocrine glands, such as the
pituitary, thyroid, or adrenals. Such diseases can also be caused by genetic or congenital abnormalities,
infections, tumours, or disorders of unknown cause. The most common reproductive diseases in cattle are
brucellosis (Bang’s disease); leptospirosis; infectious bovine rhinotracheitis (IBR) and bovine virus diarrhea
(BVD) complexes; vibriosis; and trichomoniasis. Infected animals usually are not dying; in most cases,
especially in males, they do not even appear ill. Some animals never show symptoms of the disease, yet
remain a major threat to the rest of the herd because they carry disease organisms.
In literature on some of the beef herd 50% of all reproductive losses been shown as result of infectious
diseases. These diseases often result from the introduction of opportunistic organisms at times of stress.
Disease may occur systemically or only in particular organs such as the vagina or uterus. Metritis or
inflammation of the uterus often occurs after calving. It occurs frequently following dystocia and retained
placenta. Fertility in these females is often reduced due to infection damaging the uterus.
True venerealdiseases are those that are spread during breeding. The most important diseases for beef
producers are infectious agents that cause premature termination of gestation. Bovine Viral Diarrhoea (BVD)
is a viral infection that is the leading cause of most cases of abortion. Infectious Bovine Rhinotracheitis
(IBR) is also caused by a virus that induces high rates of abortion in unvaccinated females. Leptospirosis is a
bacterial agent, which typically causes abortion in late gestation. Trichomoniasis is a venereal disease caused
by aprotozoan, that results in infertility and sometimes induces abortion. Using bulls and semen tested to
be free of this disease is the best prevention. Camphylobacter infection is a venereal disease caused by
a bacterium that is ingested and causes infertility and abortion. Failure to properly vaccinate animals for
many of these organisms may predispose the breeding herd to disease.
National Dairy Research Institute
32
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Among the reproductive system diseases, each has its own specific causes and symptoms, which are
inflicted either by a bacterium, virus, fungus or other organism. Specific reproductive diseases are often
symptoms of other diseases and disorders, or have multiple, or unknown causes making them difficult to
classify. Some infections are easily detectable, treatable and can be cured, some are more difficult, and some
are even non curable. Many times infected animals become barren for rest of the life.
Reproductive Problems Encountered in Males (Bulls)
A systematic and intensive multistage selection process of the males is practiced in bull mother farms,
commercial and institutional herds. The selected males are subjected to a general clinical examination, an
examination of the reproductive organs, a semen examination and freezability test and an assessment of
serving ability. These tests are done routinely at the time of collection of progeny test doses of semen. The
fertility performance of each bull is recorded from conception rates based on pregnancy diagnosis and the
first service. Depending upon the breeding goals level of selection and culling is adopted. The selection
and ranking of bulls is usually done on the basis for their genetic value and different attributes of estimated
breeding value (EBV).
In standard programmes the optimum serving capacity and seminal profile are indispensable
parameters for evaluation of breeding soundness of dairy bulls. However, a wide variation in the male
reproductive parameters is observed within and between the breeds of different species of dairy animals.
The production traits in animals are mainly genetic in nature; however, reproduction parameters are also
more influenced by environment. One of the notable observations in dairy males also has been shown as
high incidence of subfertility, semen production ability and simultaneously the low freezable.
The reproductive performance of males (bulls) is dependent on various aspects including physical
condition and health, structure of genital organs and system, libido, output of sperm cells and their
33
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
morphology, count of spermatozoa in semen, movement of sperm cells, mating ability, age etc. Fertility of
bulls can be affected by numerous reasons including lameness, leading to inability or reluctance to work,
hereditary defects and diseases. Besides if their hoofs are not regularly trimmed or have digital dermatitis,
cause problem in breeding. Regular body condition scoring is also essential to monitor dietary requirements
and in addition to routine herd health policies (i.e. application of fly repellents in summer, vaccination
against disease etc.). However, even if a bull does become ill, veterinary attention should be given as soon as
possible to try to reduce the impact on his health, welfare and also potential or ability to serve cows. Sperm
production is also adversely affected by diseases, especially pyrexia, with effects taking several months
to resolve due to the duration of sperm maturation in the male reproductive tract. Vaccination and other
treatments also adversely effect on the quality and amount of semen.
The reproductive efficiency is a complex phenomenon controlled by both genetic and non-genetic
factors, the non- genetic factors being climate, nutrition, and level of management. In breeding farms and
filed conditions often encountered problems of bulls, which mainly includes: poor libido, cryptorchidism,
poor semen profile, low freezability of semen and high post thaw mortality of spermatozoa. All these are
described briefly as below:
Poor Libido
Libido is the desire or sex drive of the male (bull) to mate. However, the willingness to breed remains
highly variable among bulls and can have a major impact on animal breeding programmes. Mounting and
thrusting behaviour, sniffing of the genital region and flehmen reaction (curling of the upper lip of the male
in response to detecting sexual readiness of the female) are well established common behaviours of normal
farm animals. This behaviour is regulated by the release of testosterone, produced by specialized cells in
the testes. In some species or breeds bulls show libido almost continuously once they reach puberty. In
others, there is a marked decline in libido during the non-breeding season or the problem is associated with
confinement. Underfed or excessively fat males have been observed with reduced libido. The desire to mate
National Dairy Research Institute
34
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
also decreases with age and disease conditions, such as arthritis. The polluted environment in which most
of us live has also created havoc with male fertility.
Low libido can result from physical or structural problems such as foot rot, lameness, or poor hind-leg
conformation, which may prevent the bull from mounting or seeking out oestrus females. Reproductive
problems involving a bruised, swollen, or deviated penis are painful and may discourage the bull from
attempting to breed. Low libido in newly introduced bulls may be related to the type of rearing environment
and the social interaction and dominance hierarchy with other bulls in the herd.
Evaluation of libido may include frequency of mounting in a time period, time to mount from
introduction of a female, and delay period between successive mountings.
Mostly poor libido problem is behavioural and management related, however, genetical reasons have
also been reported. Ejaculation is rare, and hence fertility rate usually remains undiagnosed. Management
usually eliminates such animals quite early. However, it is highly essential to diagnose the underlying cause
of the problems. Some males have inherently poor libido due to genetic anomalies. This phenomenon has
also been shown in twin born (chimeric bulls- XX/XY complement of chromosomes.
Cryptorchidism
During the early stage of foetal development of males the formation of testicles begins with the
immigration of primordial germ cells into testicular cords along the genital ridge in the abdomen of the
early embryo. Subsequently testicles gradually descend into the scrotum, usually in the last part (months)
of gestation. In order to function normally, the testicles need to be slightly cooler than normal body
temperature. However, in some unusual cases both or one testis fails to move into the scrotum (the bag of
skin that hangs below the penis) before the birth. This condition is known as cryptorchidism that is absence
35
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
of one or both testes from the scrotum, with either palpable or unpalpable testicles. The disorder depending
on the location of testicles is of three types: (i) Abdominal or intra-abdominal - the testicle remain inside the
abdomen, generally near the upper opening of the inguinal canal and unpalpable. (ii) Inguinal - the testicle
has moved into the inguinal canal, but has not moved down enough to be detected by touch (unpalpable).
(iii) Atrophic or absent - an atrophic testicle is very small, while an absent one was never formed (missing)
cases of unpalpable testicles.
Cryptorchidism comes in the category of testicular dysgenesis syndrome (TDS), which includes a
concept that poor semen quality, testes cancer, undescended testes and hypospadias. All these symptoms
come under the testicular dysgenesis syndrome (TDS), which is increasingly becoming papparent due to
adverse environmental influences.
Cryptorchidism usually occurs as a result disturbance in steroid hormone metabolism, possibly through a
perturbed hypothalamic–pituitary–gonadal axis. Disturbance may be genetic, or extrinsic through endocrine
disruptors. Recently, the role of insulin‐like factor‐3 (INSL3; alternatively called relaxin‐like factor) has been
highlighted through the cryptorchid phenotype of mice where genes for either INSL3 or its receptor have been
ablated. The origin of TDS is presumed to occur either through the influence of genetic mutations (e.g. X0/XY
mosaicism or point mutations), or through endocrine disruption at the time of sex determination. This can
then lead to disturbed Sertoli and/or Leydig cell function. If the former, then germ cell defects can arise, e.g.
reduced semen quality; if the latter, then there would as a result be at least an androgen deficiency, and hence
a disturbance of androgen‐dependent differentiation of the external genitalia (cryptorchidism, hypospadia,
micropenis). Nevertheless, cryptorchidism is often found as a unique symptom.
Typically, cryptorchidism is detected at birth or shortly thereafter. In such cases fertility rates are
low and infertility is a major health problem. TDS is the result of disruption of embryonal programming
and gonadal development during foetal life. An endocrine disrupter hypothesis to explain the adverse
National Dairy Research Institute
36
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
trends has been proposed. Cryptorchidism causes severe spermatogenic arrest. Often affected individuals
produce fewer than normal numbers of sperm, with an increased percentage of abnormal sperm. Thus
low sperm counts, poor sperm quality and decreased fertility are more likely in cases with undescended
testicles. A large number of congenital malformation syndromes including cryptorchids the genetics are
not fully understood. Nevertheless, the general perception has been that cryptorchidism is a single disease
with moderate heritability, incomplete penetrance, expressed only in males (sex specific expression), and
concentrated by inbreeding or minimized by culling affected males and all siblings. Currently it is accepted
that cryptorchidism has many causes including genetic, epigenetic, and environmental components.
However, chromosome evaluation can give some insights, and molecular makers and DNA sequences might
unravel the real reason.
Cryptorchidism appears to be rare in bovine bulls; however, a planned study on cryptorchids is still
lacking and is considered an inherited trait. Animals with bilaterally undescended testes are generally
infertile and unable to father young. Animals with unilaterally undescended testes are generally fertile and
able to father young. This is because they have one normally-descended scrotal testicle.
Scrotal Circumference
In animal breeding farms reproductive efficiency is a major determinant of cow-calf profitability. Mainly
depends upon the calves and milk produced by the cows, however, cow in heat to conceive depends mostly
on the number of normal sperm cells deposited in her reproductive tract. Thus fertility of the male is a major
contributor to overall reproductive performance. Predicting the fertility of bulls is an area of research that
has been active for years and on-going activity. Research and experience have identified a number of factors
that influence bull fertility. Breeding soundness examination (BSE) is of utmost importance. Usually involves
physical and reproductive system examination of bulls. (i). Physical examination: Usually starts at the first
stage of selection of male calves for bulls and then examination in a systematic way for any problem that
37
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
would hamper ability to impregnate cows. (ii). Reproductive organs and tract examination: The reproductive
tract consists of the scrotum, testicles, penis, prepuce and their associated structures. These structures can
be examined externally both visually and by manual palpation.
Measurement of Scrotal Circumference (SC): Measuring scrotal circumference is a crucial part of the
BSE. Scrotal circumference has been determined to be the measurement that best predicts the output of
sperm cells of bulls. The measurement technique involves the use of a circular tape. This measure is useful as
there is a correlation between the scrotal circumference and the volume of semen-producing tissue that the
bull possesses. Since SC increases with the age and weight of the bull the circumference must be interpreted
in relation with the age of bull.
Scrotal circumference as has been considered one of the best predictors of bull fertility, however,
during measurement of the testicles proper procedure and care is essential for the measurement to be
accurate. It is also important to use a circular tape and be closed tightly, on the scrotum for measurements
to be consistent. There has been formalized the procedure by the Society for Theriogenology (the study of
reproduction in domestic animals) (SFT), whose members have standardized the bull evaluation
Defects in male accessory glands (MAGs): The main glands include ampullary, vesicular, prostate,
bulbourethral, and urethral glands. The products of these glands have different types of activities, which are
to nourish and activate the spermatozoa, to clear the urethral tract prior to ejaculation, serve as the vehicle of
transport of the spermatozoa in the female tract, and to plug the female tract after placement of spermatozoa to
help ensure fertilization. These glands are usually branched tubular or branched tubuloalveolar; however, vary
in their organization and distribution in different species. Secretions of male accessory glands contain a variety
of bioactive molecules. During mating, transfer of these molecules with semen exert wide-ranging effects on
female reproductive activity and improve the chances of siring offspring. Seminal proteins are critical for fertility.
These proteins can activate sperm or enhance sperm storage within the female, and can improve the chance to
National Dairy Research Institute
38
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
fertilize eggs. However, any defect in accessory gland or deviations in production of seminal secretion causes
reduction siring ability of bulls. Genetic and molecular evaluation is essential in such cases.
Semen Quality
Semen is a white or grey liquid occasionally appear yellowish. Pink or red semen suggests that blood is
present. Semen is discharged from the urethra (tube in the penis) on ejaculation. Semen is consisted of cells
(normally sperms) and fluid part rich in fructose (a sugar) that fuels sperm as they travel through the female
reproductive tract. The major volume of semen consists of secretions and contains alkaline substances
that neutralize the acidic environment of the vaginal canal to make it a less hostile environment for the
sperm. Sperms are the male reproductive cells produced by the male reproductive organs (testicles) while
the fluid is produced by accessory glands viz. the prostate, bulbourethral glands, and the seminal vesicles.
Sperm normally develops to full maturity within testes of males from puberty onward. Sperm capability
to fertilize an egg is an absolute requirement to achieve pregnancy. An abnormality in such production or
maturation can result in male infertility. Spermatozoa continuously change and develop from their origins
as somatic cells until their destination as highly specialized cells capable of fertilization. The final stages of
spermatozoon development are induced by the immediate environment of the oocyte and its zona.
The purpose of semen is purely for reproduction, as a vehicle to carry the spermatozoa into the female
reproductive tract. Although ejaculation of semen accompanies orgasm and sexual pleasure, erection and
orgasm are controlled by separate mechanisms and semen emission Semen consists of the secretions of
several glands but only 5 per cent comes from the testicles.
Semen prior to freezing or use is evaluated as per standard guidelines immediately just after collection
from the bull. There occurs variation in volume of semen donated by different bulls. However, count of
spermatozoa per millilitre of normal semen discharge is species specific. Usually, each millilitre of semen
39
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
contains millions of spermatozoa (sperm). Among these (spermatozoa) at least 60 per cent should have a
normal shape and show normal forward movement (motility). Evaluation includes two main parameters viz.
morphology of sperm cells and their motility.
Sub-fertile semen ejaculates are characterized by gross evaluation for low volume, off colour or odour
and microscopic evaluation for low concentrations. Sperm morphology basically describes the percentage
of sperm that look normal. Common causes of abnormal morphology include varicocele, drugs, heat,
infrequent ejaculation, age of the bull and frequency of ejaculation or collection.
In order to assess the potential for the reproductive success of a bull, a systematic approach to bull
evaluation has been developed. This evaluation involves an assessment, performed in as objective a manner
as possible, providing for the prediction of bull fertility. This procedure is termed the Breeding Soundness
Examination (BSE) and has been formalized by the Society for Theriogenology (the study of reproduction
in domestic animals) (SFT), whose members have standardized the bull evaluation. It has been that there
are substantial differences in fertility among individual bulls. Compensable sperm quality attributes are
those that affect the ability of a sperm to access and fertilize an ovum. Therefore, measurements of motility,
viability, and membrane function are commonly used to estimate compensable sperm quality.
Poor Quality Semen
Semen must be evaluated as soon as possible after collection, because changes in temperature,
exposure to light, and exposure to any type of chemicals, lubricants etc. can change sperm motility and
adversely affect fertility. Motility should be examined as soon as possible, as motility is the most influenced
parameter in the semen analysis. Use a wooden stick to handle semen, because a wooden stick is thermoneutral and will not cold-shock the sperm cells. Morphology is usually examined with an eosin-nigrosin
(Society for Theriogenology) stain (background stain) to highlight the cells.
National Dairy Research Institute
40
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Sperm concentration, quantity and sperm motility are considered important variables influencing
fertility. However, aspermia is absence of semen (zero volume ejaculate) is also observed. Individual sperm
or mass motility score or both are commonly used as criteria for ejaculate rejection in most AI centres. The
count of sperm cells in semen: viz. azoospermia (absence of sperm in the ejaculate), oligozoospermia (low
numbers of sperm), asthenozoospermia (poor sperm motility), teratozoospermia (abnormal morphology of
sperm than usual), necrozoospermia (all sperm in the ejaculate are dead) and leucospermia (a high level of
white blood cells in semen) etc. are good indicators of bull potential.
Cryopreservation of Semen
Semen cryopreservation implies storage of spermatozoa at subzero (i.e. frozen) temperatures.
Cryopreservation of semen continues to be one of the most frequently employed technique for use in
modern animal production. Maintaining the highest fertilizing potential of semen through the freeze/thaw
process requires the best available conditions for cryopreservation. Regardless of the successful research
to improve cryopreservation, some spermatozoa fail to survive freezing and thawing. The use of artificial
insemination to accomplish this goal has become an integral part of the dairy industry. Bull sperm indefinitely
through cryopreservation, frozen -thawed sperm are even more fragile and shorter -lived than freshly
ejaculated sperm. The success of cryopreservation depends upon many factors, viz. interactions between
cryoprotectant, type of extender, cooling rate, thawing rate and packaging, as well as the individual animal
variation.
Poor Freezability of Semen
The success of AI depends up on the delivery of an adequate number of viable, fertile spermatozoa to the
site of fertilization at the proper stage of oestrous. Freshly ejaculated sperms have a limited lifespan, which
gets reduced with the procedures of cryopreservation. The efforts to improve post -thaw survival of bull
sperm always get prime importance; however, semen will remain a perishable product, thus a compromise
41
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
in the procedures to make it available for AI with suitable number and condition. All the precaution during
semen processing and handling in the AI programmes through thawing and insemination on the farm is the
best method to improve fertility of frozen semen.
The technology of semen collection to cryopreservation and subsequently use in AI is well standard,
however, not equally successful in all the livestock species and even breeds, particularly in crossbreeds.
Such problem of poor freezing has been shown in bulls of KF and other crosses of cattle.
Post-thaw Motility
The procedure for thawing semen is equally important and potentially just as lethal as the freezing
process. Straws of bull semen are thawed rapidly, either in a warm water or keeping in pocket. The number of
viable sperms in a straw is directly related to the processing and handling from collection through thawing.
The effects of cryopreservation on sperm function and fertility have been widely studied, particularly
in bovine. Various sperm organelles have been known to be affected due to the detrimental effects of
cryopreservation. Cooling is a major stressor, as a result of which membrane bound phospholipids reorient
themselves into a different configuration that disrupt membrane function and permeability. Induction of
premature acrosomal reaction, altered mitochondrial function, reduction of motility and failure of chromatin
decondensation, all of which influence the viability and fertility of the sperm cells have been reported by
different investigators.
The stress response shown by spermatozoa as a reaction to a drop in temperature is referred as cold
shock. Generally, cold shock damage manifests itself as a decline in cell metabolism, altered membrane
permeability, loss of intracellular components, irreversible loss of motility of spermatozoa and an increase in
the number of dead spermatozoa. However, even with the most up to date procedure cryopreservation still
National Dairy Research Institute
42
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
causes detrimental effect on sperm compartments and their function. There has been considerable concern
worldwide about possible semen quality deterioration during freezing.
Chromosomal abnormalities, protamine status, and perhaps abnormalities of mRNA are believed
to be uncompensable defects, as these sperm would be expected to initiate fertilization but not sustain
development. Furthermore, laboratory processing such as flow-sorting sperm for sex selection can increase
the number of uncompensable sperm defects.
Cost of Rearing of Animals from Calf Stage to their Conception-Calving
In farm animals reproductive defects are usually expressed at puberty of later in life (3-5 years of age),
which lead to failure in conception, abortion or birth of abnormal calf. All these also increase treatment costs.
Reproduction can be negatively affected in many ways. Viral and bacterial diseases can cause abortions, and
dystocia may cause the death of calf or dam and reduce reproductive performance of the dam and also loss
of the calf. Congenital or poor growth or delay in attainment of puberty or reproductive problems or failure
in breeding, diseases and abnormal conditions result in economic losses.
Infertility, regardless of cause, is a major reason for culling animals. Accurate quantification of these
costs is straightforward when parameters are set and data analyses are confined to a few individual
operations. However, on the other hand, national cost estimates based on parameter coefficients from
several production studies and disease experiments are difficult to calculate because research goals, study
methodology, and quantification criteria differ greatly from study to study.
In breeding herds replacement of animals, costs from drug, labour, and veterinary service. Cows and
heifers that do not become pregnant to first service contribute largely unknown but substantial costs to
both dairy and beef industries. Three-fourths of the cost for reproductive diseases and conditions can be
43
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
attributed to female infertility and dystocia and the failure to produce a healthy calf that will survive the first
24 h of life. There is no such study in Indian conditions or India, nevertheless, cost conversion of European
observations is reasonable for our herds in organisations or field conditions.
Methodology for Chromosome Preparation and Evaluation
During the 1960s many newer approaches of genetics and cytogenetics were developed. Chromosome
profile were identified in most of the species, breeds and types of animals in the coming two decades and
subsequently molecular technologies were added to the armoury of scientists for animal characterization
in 1980s. Presently the cytogenetical and molecular markers are utilized for the characterization of animals
and breeds, diagnosis of diseases and causes of anatomical, physiological and reproductive defects.
In normal animals identification of correct number and gross morphological features of chromosomes
can be carried out by conventional staining with Giemsa dye. However, for delineation of structural details
of chromosomes and particularly of mutated chromosomes various banding techniques are applied.
1. Preparation of Chromosomes for Evaluation
Chromosomes are dynamic genetic entities, continuously changing in appearance and carrying out
inherent activities. For their physical examination the highly dividing cells are arrested at metaphase stage
where they can be counted, measured and evaluated for various features. Such a stage can be obtained
in vivo or in vitro growing cells. The later approach is routinely followed in laboratories for obtaining
chromosome preparations for cytogenetical investigations.
1.1 Procedure
Short-term whole blood culture method is routinely used for obtaining chromosome preparations.
The technique involves the steps: peripheral blood drawn from the jugular vein of individuals is cultured
National Dairy Research Institute
44
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
in sterilized synthetic TC media* supplemented with adult cattle serum (~10%). Whole blood cultures (0.5
ml blood in 5ml medium) are established and grown at 37.5±0.50 for 72 hours. Subsequently metaphases
are arrested with colchicine (1.3µg / 5ml culture) and cells are harvested. The cultured cells are treated with
hypotonic salt solution (0.075M KCl) for 6-8 minutes and later fixed in methanol: acetic acid (3:1 ratio). The
cells are washes at least three times; every time adding 5ml fixative, centrifuged at 1000 rpm, supernatant
is discarded and packed cells are used further. In the end a milky white suspension is left in the centrifuge
tubes. Chromosome preparations are made by flame-drying (for conventional) or air-drying (for banding)
methods by dropping 2-3 drops of cell suspension from 1-2 feet height on clean glass slides.
*There are a large number of TC media, however, TC199, Hams F10 or RPMI 1640 are preferred for
chromosomes studies. In preparation of liquid media, recommended amount of powered medium is added
to sterilized GDW to which are added various other constituents viz. pokeweed mitogen (0.20mg/100ml),
streptomycin (100µg per culture) and penicillin (100 units per culture). Then the pH is adjusted by adding
sodium bicarbonate (4.46 %) drop wise till the desired level of 7.2 – 7.4 is attained. The medium is filtered
through 0.2µ filters under vacuum. Subsequently 15-20 % adult cattle serum is added to make the medium
complete. Precautions: All the glassware and plasticware used should be sterilized and all the steps are
carried out in sterile conditions (in a Laminar-flow hood).
1.2 Chromosome Slide Preparations and their Staining (Conventional Method)
The chromosome preparations are made on glass slides and are stained with Giemsa dye. Normally
the staining is carried out in 2% Giemsa dye at pH 6.8 (without filtration). The slides are dried, dipped in
xylene for a few minutes, mounted with cover glass and examined under X1000 magnification. However, if
preparations are to be made permanent for longer storage and examination, dried keeping in an incubator
at 37oC for at least 10 minutes. Dried slides are kept in a jar filled with xylene for 5-15 minutes for clearing.
A few drops (2-3) of DPX are placed on the slide, and then a cover glass dipped in xylene is placed gently
avoiding entry of air-bubbles. Still if some air bubbles come, are removed with a gentle pressure on the
45
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
cover glass. The slides are kept overnight in an incubator. Subsequently slides are cleaned to remove excess
DPX, examined or stored for evaluation.
Giemsa staining gives crystal violet colour to the chromosomes. The chromosomes appear solid rod
like structures with their general morphological features of shape and size (Fig. 1). This staining is useful in
the study of normal and gross anomalies in number and morphology, gaps and breaks etc. This method of
evaluation is conventional; to evaluate fine details or homology identification banding techniques are used.
1.3 Identification and Characterization of Chromosomes
Every species has its characteristic constant number and morphology of their chromosomes. However,
sometimes deviations occur in the number of morphology of chromosomes. Hence different types techniques
revealing such patterns have been developed to correctly identify normal and abnormal number or structure
of chromosomes. These techniques help in identification of whole or part of chromosomes, their homology
and structural details revealing a certain level of genome organization. The term chromosome banding
refers to these processes of producing light and dark patterns on chromosomes. Bands are lengthwise
variations in staining properties along a chromosome. Band patterns can be produced with a variety of ways
on chromosomes that show the structural differentiation along their length, which are uniformly stained
with specific dyes depending upon the region to be explored.
However, certain banding patterns are used or recommended for specific purposes of revelation details,
identification and characterization of chromosomes. The various types of band patterns named after the
technique by which they are revealed or structure shown are described briefly in table- 3 below:
National Dairy Research Institute
46
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Table-3: Different Banding Techniques Routinely Used, their Characteristics and Applications
Name of the banding Characteristics of patterns
technique
Q-banding
Fluorescing bands visible after staining with
(Quinacrine)
quinacrine mustard or similar compounds, reveal
light and dark bands at alternate positions
G-banding (Giemsa)
Revealed by Giemsa staining and additional
techniques, chromosome segments take up the dye.
Q and G bands are identical (structurally) except
staining procedure).
R-banding (Reverse of Stained after controlled denaturation by heat. The
Q- and G-banding)
bands at the reverse (vice-versa) position of Q or G
bands, the light regions takes dark, while dark take
light stain.
C-banding
These are localized in pericentric regions of
(constitutive
centromere of chromosomes. Single armed
heterochromatin)
(acrocentrics) take usually dark stain irrespective of
autosomes or sex chromosomes. Bi-armed show a
thin band in livestock species
NOR-staining
The bands appear at the site rich in genes for 18s and
(Nucleolar organizer
28s rRNA
regions)
Purpose / utility /
Application
Precise identification of
specific chromosomes
SCE-staining (Sister
chromatid exchange)
Identification of mutations
and mutagens
FISH (Fluorescent in
situ hybridization)
Applied for the detection of different cycles of
replications and exchanges of segments between the
chromatids of the same chromosome
Fluorescent probes bind to sites of the chromosome
with which they show a high degree of sequence
complementarity
Precise identification of
specific chromosomes
Precise identification of
specific chromosomes
Precise identification sex
chromosomes
Identification of
chromosomes involved in
re-generation of nucleolus
Precise identification
of chromosomes and
exchange of segments
In the routine work of screening of chromosomes and their gross anomalies three commonly used
techniques i.e. C-, G- and R-banding are described here.
47
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
1.4
Chromosomes Abnormalities and Reproductive Problems
Cytogenetic studies have revealed chromosomal abnormalities animals. The role of chromosomal
abnormalities have been found as a cause of reproductive problems, early mortality of embryos and
congenital birth defects, poor semen quality. Different types of anomalies reported in farm animals are
summarized in Box-1 and in figures 3-14.
Box -1: Chromosomal Abnormalities Encountered in Farm Animal
Variation in chromosomes
I. Numerical
Euploidy
Aneuploidy
Monoploidy – 1n
Nullisomy
Diploidy
- 2n
Trisomy
Triploidy
- 3n
Tetrasomy
Polyploidy
Sex chromosomes
a). Missing XO
b). Extra
XXY or XXX
1). Deletion / deficiency
2). Duplication
3). Inversion
4). Translocation
(Haploidy)Monosomy
Auto-
II. Structural
5). Iso-chromosome
6). Ring chromosomes
7). Dicentric
8). Chimaerism
i). Zygotic
Allo-
ii). Post-zygotic
iii). Twin chimaerism
9). Mosaicism
i). Developmental
ii). Proliferative
10). Endoreduplication
11). Pulverization
12). Gaps & Breaks
National Dairy Research Institute
48
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
a
b


Fig. 3: Trisomy – X Chromosome in buffalo (a) Giemsa stained (b) C-banded
a
b
Fig. 4: Monosomy – X Chromosome in buffalo (a) Giemsa stained (b) C-banded
49
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
a
b
Fig. 5: Pericentric inversion of Chromosome-4 in buffalo (a) Giemsa stained (b) G-banded
a
b
Fig. 6: Autosomal trisomy of Chromosome-5 in buffalo (a) Giemsa stained (b) G-banded
National Dairy Research Institute
50
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
a
b
Fig. 7: Mosaicism (XO / X del-X) in cattle (a) 59,XO (b) 60,Xdel-X
Fig. 8: In goat 59, XO karyotype
51
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Fig. 9: Giemsa stained, C- and R-banded X- chromosomes (showing fragile and late replicating-X) in Murrah buffaloes
National Dairy Research Institute
52
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
a
b
c
Fig. 10: Translocation in cattle 60, XY t(1;5) chromosomes (a) Giemsa stained, (b) C-banded and (c) R-banded
karyotypes
53
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
Fig. 11: Metaphases showing fragile X- chromosomes (a) Cattle and (b) buffalo
National Dairy Research Institute
54
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
X
Y
X
X
Fig. 12: Two metaphases (Giemsa stained) showing XY (left side) and XX (right side) (arrow marked) chromosome
complements of Karan Fries cattle freemartin
X
X
Y
Fig. 13: Two metaphases (Giemsa stained) showing XY (left side) and XX (right side) (arrow marked) chromosome
complements of buffalo freemartin
X
Y
X
Fig. 14: Two metaphases (Giemsa stained) showing XY (left side) and XX (right side) (arrow marked) chromosome
complements of goat freemartin
55
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
1.5
Applications of Chromosome Investigations
Chromosome studies are contributing greatly in understanding biological processes and to the field
of medicine. While these studies are progressing fast in man, the progress is slow with regard to animals of
economic importance. The major applications in farm animals are enumerated as follows:
a)
Precise identification and cataloguing of karyotypes in animals of different species.
b)
Understanding karyological evolution, within and between genera and families of livestock species
including strains and breeds.
c)
Chromosomal studies on inter-species crosses such as horse/donkey, yak/cattle, cattle/Mithun,
Swamp buffalo/River buffalo, wild pig/domestic pig, sheep/goat etc.
d)
Sterility and breeding problems in species hybrids in F1 or further generations.
e)
Understanding the structure and function of observed abnormal chromosomes.
f)
Chromosomal abnormalities as causative agents of anatomical, clinical, pathological, reproductive
and genetical defects and syndromes.
g)
Elucidating the mechanisms of determination, differentiation and development of sex genital
organs and reproductive system of problematic animals.
h)
Location and the order of various genes on chromosomes.
i)
Prenatal diagnosis and genetic disorders.
j)
Understanding mechanisms of gene action.
k)
Sexing of embryos before transplantation, etc.
l)
Evaluation of sperms and early embryos produced after semen sorting (separation of X- and
Y-bearing spermatozoa).
National Dairy Research Institute
56
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
m)
All the progenies produced using ARTs must be screened early in the age
n)
Breeding bulls can spread anomalies far and wide in large populations, hence must be screened
before selection and use.
Chromosome Abnormalities Discovered for the First Time at NDRI
57
1.
Trisomy X chromosome (51, XXX) in buffalo
2.
Monosomy of X chromosome (49, XO) in buffalo
3.
Pericentric inversion (4th autosome) in buffalo
4.
Chimaerism (50, XX / 50, XY) in buffalo
5.
Mosaicism of different types of chromosomes anomalies
6.
Autosomal trisomy in buffalo
7.
Autosomal trisomy (27th autosome) in a Sahiwal cattle calf
8.
Secondary constriction in buffalo
9.
Dicentric chromosomes were observed in cattle
10.
Deletion of chromatin of X- chromosome in cattle
11.
Two cell lines: 59, XO / 60, X del (X) in cattle
12.
Sterility in bulls born as co-twins to male or female
13.
Sterility due to endoreduplications in cattle
14.
Endoreduplications, pulverization and isochromosomes in cattle, buffalo and goats
15.
Autosomal translocation and its transmission to progeny in Sahiwal
16.
Fragile sites and their association with reproductive problems
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
The Following Chromosomal Abnormalities and other Findings were Confirmation
of those Reported in Literature or an Extension of the Knowledge:
1.
A female cattle with 60, XY (male complement)
2.
Cytogenetic evidence of occurrence of Swamp buffaloes in India
3.
Monosomy of X chromosome in cattle
4.
Turners' stigmata in goat
5.
Freemartinism in goat
6.
Intersexuality in goats
7.
Pseudohermaphroditism in male goat
8.
Hypospadiac condition in goat
9.
Change of chimaerism with age in cattle
10.
Parallelism of sex chromosomes chimaerism in heterosexual twins of cattle and buffalo
11.
Cases of fertile females born co-twin with males in cattle
12.
Forced inversion of Y chromosome in cattle
13.
Freemartinism in different breeds of cattle and buffalo
14.
Heteromorphism of sex chromosomes in cattle and buffaloes leading to sterility
15.
High frequency of polyploidy in late maturity and old age animals
16.
High frequency of gaps / breaks in chromosomes of some reproductively deficient cattle and buffaloes
17.
Sterility due to fragility of X chromosome in different breeds of cattle and buffalo.
18. Sex chromosomes chimaerism (XX/XY) was observed in quadruplet calves, which revealed their
development in the same pouch of foetal membranes in uterus and hence blood vascular anastomosis
occurred.
National Dairy Research Institute
58
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
19.
The exhibition of chimaerism and characteristics of external genitalia are well-founded indicators that
all the female calves will turn out to be freemartins.
20.
The male calf with physical indicators and chimaerism of sex chromosomes will not be a good bull for
breeding programme.
21.
Impression of presence of testicles in female calves, is very rare, however, indicator of hermaphroditism.
Highlights of Other Findings of Ndri are as Follows:
1.Standard karyotypes, relative lengths and idiograms of 16 cattle, 7 buffaloes, 8 goats (Pure and cross breeds)
2.
Chromosome studies of 18 different species including domestic and livestock
3.
Identification of Y chromosome position in the karyotype of buffalo
4.
Identification of late replicating X-chromosome in cattle, buffalo and goat breeds
5.
Random distribution of sex chromosomes in the metaphase plates of cattle and buffalo
6.
The long duration investigations on reproductively problematic have revealed that about 8 % animals carry
gross chromosomal abnormalities.
Recommendations Emerged from Studies
•
Chromosome preparations be made and examined conveniently by short-term whole blood (lymphocyte)
culture technique.
•
Chromosomes must be examined at an early age and stage of life and bearers of anomalies be culled, to
save rearing cost on such animals.
•
Bulls before their use in A.I. or inclusion in progeny testing programme should be screened for chromosomes.
Secondly bull calves born as co-twin with male or female should not be selected for breeding as are usually
sub-fertile or infertile.
59
National Dairy Research Institute
CYTOGENETIC AND REPRODUCTIVE PROBLEMS IN FARM ANIMALS
•
Animals with reproductive problems carry higher percentages of abnormalities of chromosomes, hence for
the start of medication, must be karyotyped.
•
In mating plans of goats one of the parents must be horned, for this a record must be kept at time of
dehorning, as in dominant homozygous condition polledness leads to intersexuality.
•
•
Oocyte chromosomes can be studied to assess their fertilization potential.
Cells from early in vitro produced or manipulated embryos must be used for identification of their genetical
defects or chromosome anomalies.
•
•
These days ther is emphasis on ARTs, however, the progeny must be karyotypes at an early age.
In ET programmes only one embryo is to be placed in the recipient to avoid twinning, and in such case
failure in conception is better than production of twin calves.
EPILOGUE
Reproductive success depends on a large number of carefully orchestrated biological cascade of
events that must occur in a specifically time sequence. The interference with one or more of the sequences
or events results in total reproductive failure or a subtle reduction in reproductive potential. Thus many
factors must be considered when reproductive failure occurs. These can be genetical, nutritional, hormonal,
environmental or managemental errors. The major emphasis is for cytogenetical screening at early age and
stage of animals, which are to be used as replacement of breeding population. During breeding stage a
large number of animals are encountered with reproductive problems and up to this age farmer / owner
has spent time and resources in their feeding and health coverage. Besides usually a heavy expenditure is
also incurred on the treatment and breeding efforts of such animals. Hence so as to save the rearing cost on
sterile individuals, all the calves must be screened for gross anomalies.

National Dairy Research Institute


60