ARTIF

FICIAL INSEMI
I INATIO
ON AND INFERT
TILITY
M
MANAGGEMENT IN DA
AIRY AN
NIMALSS

Editors:

Drr. Anuj Kum

mar & Dr. Sarvjeet
S Yaddav

P
Published by:

U.P. Deen dayal Upaddhyaya Pashhu Chikitsa Vigyan Vishwa Vidyaalaya Evam
G Anusanddhan Sansthhan, Mathurra-281001 (U.P.)
Go- ( INDIIA

Finaancial Supp
port:

Uttar Pradeesh Livestoock Develop

pment Boardd (UPLDB))
 ARTIFICIAL INSEMINATION AND INFERTILITY

MANAGEMENT IN DAIRY ANIMALS

Course Director - Dr. Sarvjeet Yadav

Course Co-ordinator - Dr. Anuj Kumar

CORE FACULTY

NAME DESIGNATION DEPARTMENT

Dr. Atul Saxena Professor & Head Veterinary Gynaecology

Dr. Sarvjeet Yadav Professor & Head Veterinary Physiology

Dr. Vijay Singh Associate Professor Veterinary Gynaecology

Dr. Vinod Kumar Associate Professor Animal Nutrition

Dr. Brijesh Yadav Assistant Professor Veterinary Physiology

Dr. Anuj Kumar Assistant Professor Veterinary Gynaecology

Dr. Mukul Anand Assistant Professor Veterinary Physiology

Dr. Dilep Swain Assistant Professor Veterinary Physiology

Dr. Shankar Singh Assistant Professor Veterinary Medicine

Dr. Yajuvendra Singh Assistant Professor Livestock Production and

Management

Dr. Vikas Sachan Assistant Professor Veterinary Gynaecology

U.P. Deen dayal Upadhyaya Pashu Chikitsa Vigyan Vishwa Vidyalaya Evam Go-
Anusandhan Sansthan, Mathura-281001 (U.P.) INDIA
 CONTENTS

S. Title Faculty Page

No.
1 Management of anoestrus in cattle and buffalo Vikas Sachan 1

2 Principal and technique of semen cryopreservation Brijesh Yadav 3

3 Recent advances in treatment of repeat breeding syndrome in Vijay Singh 7

bovine
4 Handling and upkeep of artificial insemination and semen freezing Mukul Anand 11
instruments
5 Use of ultrasonography in bovine reproduction Anuj Kumar 15

6 Assessment of optimum time for A.I. and artificial insemination Vijay Singh 22
technique

7 Role of nutrients in animal reproduction Vinod Kumar 28

8 Semen handling at artificial insemination centre Sarvjeet Yadav 37

9 Nutritional management of peri parturient animals Shanker Singh 48

10 Evaluation of frozen semen to optimize the conception rate in Dilip Swain 57

bovine
11 Current concept in management of post-partum uterine infection Anuj Kumar 64
in cattle and buffalo
12 Impact of herd management on reproductive efficiency in bovine Yajuvendra 68
Singh
 Management of anoestrus in cattle and buffalo
Vikas Sachan

"Anoestrus is a broad term that indicates the lack of oestrus expression at an

expected time". It is most common cause of infertility. It is one of the most common
cause of the infertility. At field level anoestrous is the very old and common problem in
cattle and buffaloes which is still responsible for the huge economic wastage of the
animal owner. It is very necessary to know the cause and the remedy of this harmful
condition.
It can be categorized on the basis of corpus luteum (CL) and on the basis of stage
of the animal. Anoestrous with the presence of functional corpus luteum termed as
functional anoestrus or apparent anoestrus. It may be due to pregnancy (physiological
persistent CL), pyometra, mummified foetus, macerated foetus (Pathological persistent
CL) and also in the condition of silent heat or weak heat. Condition of persistent CL or
cyclic CL can be diagnosed by ovarian palpation at the interval of 10 days. Weak or
silent estrous and unobserved estrous are the false anoestrous conditions. Cause of weak
or silent estrous is due to lack of estrogen, advanced age, arthritis, Poor nutrition,
Seasonal stress and suckling. Unobserved heat is due to short estrous or managemental
problems. Anoestrous with the absence of functional corpus luteum termed as true
anoestrus. In this condition the ovaries are quiescent, inactive and do not have any
functional CL. It may be due to insufficient release of gonadotropins or failure of the
ovaries to respond. This may be due to malnutrition (Lack of energy and protein,
deficiency of P, Co, Fe, Cu, I, Mn and Vit. A), lactational stress (negative energy
balance), seasonal stress, chronic wasting disease, Senility etc. Condition of persistent
CL or cyclic CL can be diagnosed by ovarian palpation at the interval of 10 days.
Anoestrous can be of different modes according to the different stage of the
animal as prepubertal anoestrus, postpartum anoestrus and post service anoestrus.
Conditions characterized with abnormal reproductive tract such as Freemartins,
hermaphrodites, segmental aplasia of paramesonephric duct and ovarian hypoplasia etc
are the causes of prepubertal anoestrous. Due to undernutrition there may be delayed
puberty. It is to be noted that a heifer must attain approximately two-third of its adult size
before the attainment of puberty. Debilitating disease such as chronic pneumonia may
also lead to the prepubertal anoestrous. Postpartum anoestrus may be due to the
physiological anoestrus for 2-3 weeks after parturition, lactational stress, nutritional
effects such as negative energy balance and deficiency of micro nutrients. Uterine
diseases such as RFM, metritis, pyometra and Chronic debilitating diseases are also the
cause of the postpartum anoestrus. Pregnancy, Pyometra, luteal cyst, Silent oestrus are
the reasons of Post service anoestrus.
The act of suckling stimulates the prolactin secretion which increase the period of
anoestrus. It has been shown that suckling decreases LH secretion. This prevents the first
postpartum ovulation and return of cyc1icity. Plasma cortisol level increases in suckled
cow which depresses LH secretion and sensitivity of pituitary gland to GnRH. Severity
of anoestrus can be measured by per-rectal examination. The cow with very small,
inactive ovaries, which are devoid of any significant structures (i.e. no palpable follicles
or luteal tissue) are considered to be in greater depth of anoestrus than those large ovaries
containing palpable follicles.
The range of the causes of anoestrous are very large but it is necessary to
diagnose them. Treatment of anoestrus can be done according to its cause.
Managemental practices should be improved first as proper management for exercise,
elimination of stress factors and nutritional management. Deworming (e.g. Albendazole,

1 
 
Fenbendazole etc) once and mineral mixture supplement for one month should be at
priority to provide the required minerals to the body without any loss due to
endoparasites and ectoparasites. Gentle ovarian massage and painting of external os with
Lugol's iodine on alternate day three times. Application of lugol’s Iodine on the cervix
causes local irritation and results in reflex stimulation of anterior pituitary for secretion
of gonadotrophins and thus, cyclicity starts. If the above conventional treatments fail,
then herbal heat inducer drug can be used for example Prajana, Sajani, Fertikit etc.
Prajana Forte 2 capsule daily for three days orally. If she does not come in heat then
repeat on 10th, 11th and 12th days. Clomiphene citrate (Fertivet, Clofert etc.) one tablet
for five days orally. One tablet should be dissolved in 500 ml of water. Just before
drenching the medicine, 125 ml10 % Sodium bicarbonate or 1 % Copper sulphate should
be drenched which closes rumeno-reticular groove so that the medicine directly goes to
abomasum. Phosphorus injection (Tonophosphan) 10 ml. intramuscularly for three days
may also be given.
Hormonal therapy should be tried at last. Administration of GnRH analogues as
receptal (Buserelin 20 mg) 5 ml- I/M can be given but in the cows and heifers which are
in deep anoestrus, the administration of a single injection of GnRH may be ineffective in
stimulation of ovulation. Therefore, second injection of GnRH 10 days later is necessary.
This effect may involve a long-term stimulation of FSH secretion, which initiates
follicular growth or development. This therapy works better after correcting the
nutritional deficiency or systemic disease. The calf removal for about 2 days improves
the ovulatory response by GnRH. The most potent gonadotrophic drug that is available
for use in cattle is equine chorionic gonadotropin (eCG). It can be used to stimulate
ovarian activity and can induce follicular growth and oestrus within 2-5 days. Dose 1500
- 3000 IU I/M or I/V. When eCG or FSH is administered at higher dose, it causes
superovulation rather than initiating normal ovarian activity so not recommended.
Therefore, it is not advised to inseminate in the induced oestrus but inseminate in next
normal oestrus. Progesterone therapy is also used to treat anoestrous as it mimics the
luteal phase and sudden withdrawal of the progesterone induces heat. Intramuscular oily
preparation of progesterone injection (Duraprogen) does not give good result because it
should be given daily for several days and also concentration does not decline suddenly
at the end of treatment. Intravaginal route of administration of progesterone gives the
most favourable result. PRID or CIDR device is placed in anoestrous cows for 7-14 days.
Most cows show oestrus within few days of their removal. After removal of the device,
sudden decline of progesterone occurs unlike duraprogen. Subcutaneous ear implant of
norgestomet also gives good result. Estrogens easily induce behavioural oestrus but no
follicular growth or ovulation occurs. For treating silent heat injection of anyone PGF2
analogue when active CL remain present and do double AI after 48-72 hrs.
 
 
* * * * * * * 
   

2 
 
 Principles and techniques of bovine semen cryopreservation
Brijesh Yadav, Anuj Kumar and Sarvajeet Yadav

Spermatozoa were the first mammalian cells to be cryopreserved successfully.

Since then, many methods have been developed for various types of cells, tissues and
organs. Increased understanding of the causes of cryo-injury has continually helped to
improve cryopreservation methods. Research into fundamental cryobiology has provided
the basis for new cryopreservation methods such as vitrification and addition of different
additives. The two most commonly used cryopreservation methods for animal
germplasm are slow-freezing and vitrification. These are quite different methods, but
relate to the same physico-chemical relationships. The differences between the two can
be explained by first describing what happens during slow freezing.

Slow Freezing
In slow-freezing, cells in a medium are cooled to below freezing point. At some
stage, ice masses containing pure crystalline water will form. What remains between the
growing ice masses is the so-called unfrozen fraction, in which all cells and all solutes
are confined. The concentrations of sugars, salts and cryoprotectant increases, while the
volume of the unfrozen fraction decreases. The increase in osmotic strength in
extracellular matrix causes an efflux of water from the cells. Slow cooling minimizes the
chance of intracellular ice formation. As cooling continues, the viscosity of the unfrozen
fraction ultimately becomes too high for any further crystallization. The remaining
unfrozen fraction turns into an amorphous solid that contains no ice crystals.

Cold shock during slow freezing

Cells may be damaged by very rapid cooling (cold shock) or be damaged by low
temperature. Behaviour and function of membrane lipids and proteins may be affected by
temperature. For example, membrane lipids may solidify at non-physiological
temperatures, which can alter their function and cryocapacitation of the production of
reactive oxygen species begins. Decreasing the temperature may cause an imbalance in
cellular processes because the rate of one process may be affected more strongly than
that of another.

Supercooling
In slow-freezing methods cells are brought into a suitable freezing medium and
cooling is continued below the freezing point of the medium. Spontaneous ice nucleation
will occur after the solution is supercooled to a temperature between -5 and -15 °C.
Thereafter, ice will grow rapidly in all directions, and the release of the latent heat of
fusion which will cause the sample to warm up abruptly until the freezing/melting
temperature of the solution (i.e. of the remaining unfrozen fraction) is reached. At this
point, the ice formation will stop, or will proceed at a rate governed by the rate at which
the heat of fusion is transported from the sample. Finally, the sample can “catch up”
again with the lower temperature in the freezing apparatus. From a practical perspective,
this means that the cells undergoing cryopreservation in a typical semen straw have to
withstand a series of large and abrupt temperature changes.

3 
 
Conditions in the unfrozen fraction
Cells are faced with very high concentrations of solutes in the unfrozen fraction.
Dehydration and high salt concentration may result in loss of stability in the membranes
or denaturation of proteins. Moreover, high salt concentrations may cause extracellular
salts to enter the cells, a process known as “solute loading”. The fast efflux of water
causes a rapid decrease in the volume of the cells to approximately 50 percent of their
original volume. This leads to structural deformation of the cells. Further mechanical
stress may be caused by cells being confined in very narrow channels of unfrozen
solution and squeezed between growing masses of ice.

Influence of cryoprotectants
Sugars can be used as non-electrolyte solutes, but they will only affect the
extracellular salt concentration. Moreover, high concentrations of impermeable solutes
impose osmotic stress on the cells already before freezing. This is much less the case
when a membrane permeable solute, such as glycerol, is used rather than a non-
permeable solute. When cells are brought into a hypertonic glycerol medium, water will
leave the cells because of the osmotic pressure difference. However, at the same time,
glycerol will enter the cells. After a short period of equilibration, the cells will have
regained their original volume. The osmotic stress imposed by a hypertonic glycerol
solution is therefore much smaller than that imposed by a hypertonic sugar solution.
Hence, glycerol can be used at greater concentrations than sugars without damaging the
cells. A substantial initial glycerol concentration in the medium means that part of the
extracellular and intracellular water is replaced by the glycerol. Hence, the amount of ice
formed is lower, the unfrozen fraction remains larger, the degree of shrinkage of the cells
is limited, and the electrolyte concentration in the unfrozen solution and in the cells will
be relatively small. The mechanisms through which other membrane permeable
substances, such as ethylene glycol and dimethyl-sulfoxide (DMSO), provide
cryoprotection are similar to those involving glycerol.
There are additional mechanisms through which polyols, such as like glycerol
and several sugars provide cryoprotection. These substances can stabilize lipid
membranes by hydrogen bonding with the polar head groups of membrane lipids which
is especially important under severely dehydrated conditions. In addition, these
substances may affect the mechanical properties of the unfrozen fraction, especially its
viscosity and glass-forming tendency. The degree to which cells shrink and re-swell after
addition of a membrane-permeable cryoprotectant depends on the concentration of the
cryoprotectant and the relative permeability of the membrane to water and to the
cryoprotectant. For instance, bull sperm shrink very little when brought into a freezing
medium with glycerol whereas bovine embryos react much more strongly. Upon
thawing, removal of the cryoprotectant has the opposite effect on cells: they first swell
and then they shrink again. This may lead to damage if the cells expand too much.
Damage due to over-swelling of cells can be prevented by stepwise removal of the
cryoprotectant.

Influence of cooling rate

Ice growth is a rapid process, but transport of water through the cell membrane is
relatively slow, because the membrane acts as a resistance barrier. Therefore, as cooling
and extracellular ice growth continue, the liquid water of the unfrozen fraction remains
very close to equilibrium with the ice, but the intracellular water lags behind. This means
that the water concentration (i.e. the chemical potential of water) is too high for
thermodynamic equilibrium, and there may be a risk of intracellular ice formation. The

4 
 
optimal cooling rate falls in a range that is neither too fast nor too slow. When cells are
cooled very slowly, the intracellular water lags behind only a little, and the risk of
intracellular ice formation is minimal. However, it also means that the dehydration of the
cells is maximal, which is not desired. At higher cooling rates, intracellular dehydration,
intracellular solute concentration and shrinkage of the cells is less excessive. Moreover,
the cells are exposed to the unfavourable conditions for a shorter period of time.
However, when cooling rates are increased too much, the dehydration may not be fast
enough to prevent intracellular ice nucleation. Fast-cooling damage can also be caused
by other factors. For instance, it has been proposed that rapid water flow through
membrane pores could lead to an uneven distribution of pressure on the membrane. Fast-
cooling damage could also result from the very sudden changes in size, shape and
ultrastructure, caused by the rapid efflux of water.

Interactions of cooling rate with thawing rate and cryoprotectant concentration

The optimal cooling rate may depend on various other factors, such as the
cryoprotectant concentration and the thawing rate. It has been observed in semen from a
number of species that the combination of fast cooling and slow thawing is particularly
damaging to the cells. If intracellular ice nucleation occurs at a low temperature and
cooling proceeds rapidly, it may be that the cytoplasm turns into glass before the
intracellular ice crystals grow to a significant size, thus causing only sublethal, or no
damage. During slow thawing, the small crystals can grow and subsequently damage the
cells. In addition, cells may be damaged by extracellular restructuring of ice masses, a
process known as “recrystallization”.

Programmable and non-programmable freezers

Biological material can either be frozen using quite simple, non-programmable,
freezers or using more sophisticated, programmable, freezers. Although programmable
freezers are more expensive, they do not necessarily yield more satisfactory results,
especially for experienced technicians and cryobiologists. Therefore, the choice between
programmable and non-programmable systems will depend on the financial resources
available and the experience of the technicians. In some cases, even the most
experienced technicians prefer the operating simplicity of programmable models. In most
programmable freezers, the straws or vials are cooled by cold nitrogen vapour. The
temperature inside the cooling chamber can be accurately controlled and the time course
of the temperature can be programmed. However, the time course of temperature inside
the straws may be different due to the generation of heat of fusion.
In non-programmable freezers, the straws may be cooled by being exposed to vapour (or
a cold surface) at a constant low temperature. An example of a simple system is the
freezing of straws placed on a rack in a Styrofoam box partially filled with liquid
nitrogen without ventilation. The height of the straws above the liquid nitrogen
determines the rate of heat exchange. Alternatively, straws can be placed on a piece of
Styrofoam that floats on the liquid nitrogen. The thickness of the Styrofoam piece
determines the rate of heat exchange.

Vitrification
The term “vitrification” refers to any process resulting in “glass formation”, the
transformation from a liquid to a solid in the absence of crystallization. According to this
definition, cells that are properly slow frozen become “vitrified”. If, in slow-cooling
methods, cells ultimately become vitrified, how do so-called vitrification methods differ?
Vitrification methods involve the use of a medium that has a very high solute

5 
 
concentration to begin with. Thus, ice cannot form in any part of the sample. As no ice
forms, cooling does not have to be slow. In fact, it may be beneficial to cool very rapidly.
The vitrified state and the associated physico-chemical conditions obtained using
vitrification methods, are to some extent similar to those obtained by slow cooling, but
the way of reaching this point is quite different.

Cryoprotective agents in vitrification

In vitrification methods, cells or tissues are brought into a medium that has a very
high concentration of cryoprotective agents, also known as cryoprotectants. If the
concentration of solutes is high enough, vitrification solutions will solidify to a glass
without any risk of intracellular or extracellular ice formation during cooling or
warming, independently of the cooling and warming rates used. However, the very high
concentrations of cryoprotective agent needed for vitrification may cause damage due to
abrupt osmotic changes, extremely low water potential or chemical toxicity.

Freeze drying
Storage of freeze-dried biological material is extremely cost efficient, as no
expensive and bulky liquid nitrogen containers are necessary. Furthermore, it is safe. The
material may be stored at ambient temperature and, unlike cryogenic storage; there is no
risk of equipment malfunction or of personal injury from liquid nitrogen. On the negative
side, however, freeze drying generally reduces cell viability. Therefore, standard
insemination procedures generally cannot be used for freeze-dried sperm. However,
freeze-dried sperm have been successfully used to produce live offspring using ICSI in
mice and rabbits.
 
 
* * * * * * * 
   

6 
 
 Recent advances in treatment of repeat breeding syndrome in bovine
Vijay Singh

Repeat breeder:
The repeat breeding cow / buffalo is one that has normal or near normal estrous
cycles and estrus periods and has been bred 3 or more times by a fertile bull or semen,
yet failed to conceive. These animals show normal genitalia and clear oestral genital
discharge.
Incidence: In India, incidence of repeat breeding is 5.5 to 33.33 % in cattle and 6 to 30
% in buffaloes.
All the causes of repeat breeding will lead to either failure of fertilization or early
embryonic mortality:
I-Failure of Fertilization
A Female
1. Abnormal ovulations: Failure of ovulation, delayed ovulation.
2. Obstruction of the oviduct: It is a serious condition since ovum fails to pass through
after ovulation and as a result fertilization does not take place in bilateral blockage.
When fluid accumulates in the blocked tube then tube in the course of palpation gives an
impression of a thickened Zig- Zag appearance on the finger tip. Sometime a palpable
adhesion of fallopian tube to nearby structure may be felt. In addition to rectal palpation
10 ml of 0.1% phenol red dye is injected into one uterine horn at a time. The urine
collected within three hours of dye injection if color appears red, the tube is patent and
yellow colour urine indicates that fallopian tube is blocked.
The patency of tube is established by
1. Chromohydrotubation
2. Hydrotubaton
3. Air insufflation method

3. Inability of ova to get fertilized is due to aging of ova, defective ova and short
life of ova.
B-Male
Inability to fertilize is due to defective sperm and aging of sperm.
4. Management deficiencies:
Inadequate estrous detection, resulting in:
(i) Improper timing of insemination in relation to the onset of standing estrus.
(ii) Buffaloes/Cows being inseminated that have not actually been in estrus.
II- Early embryonic mortality
It has been estimated that about 40% of embryos are lost in bovine, and most of
these losses occurs between days 8 and 16 of pregnancy. Normally embryonic mortality
is from 25 to 45% and it up to 65% in Repeat Breeder
Early embryo loss (EEL): Embryonic loss is from fertilization to day 27 of Gestation
Period (20.5 to 46.3%).
Late embryo loss (LEL): Embryonic loss is from day 28 to 42 of Gestation Period (3 to
14%).
Fetal loss: It is after day 42 of Gestation Period.
Factors affecting early embryonic mortality:
1. Genetic Factors: Genetic abnormalities, expression of lethal genes, abnormal
chromosomal number, meiotic errors and polyspermy. Most genotypic abnormalities will
cause the death of the embryo within the First two weeks of pregnancy. Lethal gene will

7 
 
be expressed which causes the death of the embryo within the rst ve days of
pregnancy. Abnormal chromosome number that results in abnormal growth of the
embryo and usually death occurs within the First trimester of gestation. Causes of
abnormal chromosome numbers include polyspermy (when more than one sperm
fertilizes an egg), and meiotic errors within the gametes or developing embryo. Higher
incidences of polyspermy can occur when artificial inseminations occur nearer the time
of ovulation as opposed to the optimal 12 h after the onset of estrus.
2. Hormonal deficiency or excess: Progesterone (P4) vs. Conception rate and
embryonic
mortality
• Lower P4 causes decrease in conception rate and increase in early embryonic
mortality.
Mechanism of reduced luteal function
• Deficiencies in the maturational processes within the Pre Ovulatory Follicles and
/ or inadequacies of ovulatory stimulus.
• Short coming in the support of the Corpus Luteum (C.L.) once they have formed.
• Premature activation of the luteolytic process.
Persistent follicle
• P4 treatment more than 7 days causes ovulation of large persistent dominant
follicle, decreases fertility/ increases repeat breeding. C.L. is normal from such
follicle.
• Uterine function is also normal.
• Decrease fertility is due to premature nuclear maturation of oocytes (36 vs. 91%).
• Such oocytes are fertilizable but did not reach the 16 cell stage.
Effect of Lactation
High milk producing animals have high metabolic rate which will increase P4
metabolism and it will lead to decrease in Conception rate.
Influence of Follicular Development
Increase Estradiol (E2) from large follicle during day 14 to 17 after breeding will
compromise embryo survival through interference with Maternal Recognition of
Pregnancy (MRP) and luteal maintenance.

Effect of Proestrus
Inadequate Proestrus will lead to decrease exposure to E2 and it will decrease P4
receptors in uterus leading to increase in PGF2 secretion. Thus, conception rate
is decreased.
• In GPG protocol ovulation of follicles <11mm were resulted in reduced
conception rate because P4 rose at slower rate from such follicles.
• Embryos might have been expected to be less advanced and produced less IFN-
Tau.
• The reducing the Proestrus length induces subsequent short luteal phase and
reduces pregnancy rates.
• Diameter of Pre-ovulatory Follicle correlated with diameter of C.L. and
pregnancy in buffaloes
3. Hostile uterine environment: It may be due to endometritis, mild or severe. Animal
should be treated with antibiotics.
4. Nutritional causes: Balanced ration should be fed to animals. Body score of animal
should be above 3. Under nutrition lead to delayed ovulation, anovulatory condition,
embryonic death and even death of fetus.

8 
 
Treatment
1. Supplementation of nutrition including minerals, trace elements and vitamins
2. Sexual rest
3. Examination of the animal for one or two cycles for ovulation, endometritis,
estrus cycle length, duration of estrus etc.
4. If suspected for endometritis
Treatment of endometritis with mild antiseptic douches or physiological saline
douches
Treatment of endometritis with antibiotics based on ABST
Treatment with PGF2alpha ( a luteolytic dose) during mid luteal phase
Combinations of antibiotic and PGF2alpha
E.D.T.A. + Tris can be used along with antibiotics.
Selenium and vitamin E - 30 to 50 mg selenium and 650 I.U. of vitamin E
intramuscularly.
Per acetic acid: 30 to 50 ml of 0.2 % of intrauterine infusion is effective. This is
less irritant than the lugol’s iodine
Bacterial lipopolysaccharides or bacterial free filtrate: Intrauterine infusion of
100 mg of E. coli polysaccharide is effective in cow with mild to moderate
infections. Infusion of 100 ml of bacterial free filtrate is also effective
5. If anovulations or delayed ovulation:
a) By Inj. hCG ( Chorulon) at the dose rate of 1500 – 3000 I.U. I/M or I/V
b) Inj. of GnRH ( Receptal) at 10 – 20 µg of GnRH analogue (2.5 ml to 5 ml of
Receptal) I/M or I/V
6. If corpus luteum insufficiency or Progesterone deficiency after breeding:
a) Administer Progesterone 3-5 days after service and continued for a period of 2
weeks. Progesterone can be administered either by Inj (like Duraprogen) or silastic ear
implants (Crester) or (PRID) etc.
b) Administration of hCG at the dose rate of 1500 - 4500 I.U. after 48 hrs of service or
an day 4 or 5 after service to promote growth and development of corpus luteum and its
production of progesterone
By Inj. GnRH prior to Artificial Insemination
a) 10 - 20 µg of GnRH analogue is injected I/M on 12 - 14 day of previous cycle and
insemination at detected estrus
b). By injecting GnRH post A.I. period (mid luteal)
c). By injecting GnRH on 0 day and mid luteal phase

7. Insulin injection:
• Long acting bovine insulin Injected S/C @ 0.2 IU/Kg at day 8,9 & 10th of
oestrous cycle
• Increased conception rate (24%) due to better follicular growth, oocyte quality
and early embryonic development.
8. Management Intervention:
1) Synchronization of ovulation with insemination
2) Double Artificial Insemination
3) Improving heat detection and correct time of insemination
4) Improving A.I. technique.
5) Check and improve semen processing.
6) Education of Veterinarians in handling of frozen semen, semen shippers and A.I.
equipment.

9 
 
 7) Adaptation of proper procedures in thawing of semen, loading of semen in the
gun etc.
8) A.I. is done immediately, after thawing of semen.
9) Strict hygienic procedures should be adopted in A.I.
10) Deposition of semen at correct site in the cow.
11) Give rest for the animal before and after artificial insemination in the hospital
under shade.
12) Advised the owner to keep the cow on the day of insemination at cool climate
without exposing to high ambient temperature and excitement.
13) Examination of semen quality at Artificial Insemination centers.
14) Maintenance of LN2 level in the containers.
15) Screening of cows and breeding bulls for infectious diseases.
16) Culling of cow which is repeating each year for several years and those cows
which are permanently sterile.
 
 
* * * * * * * 
   

10 
 
 Handling and upkeep of artificial insemination
and semen freezing instruments
Mukul Anand

Assisted reproductive technology in recent years has progressed at tremendous pace. Semen
cryopreservation and Artificial insemination are two remarkable innovations in field of assisted
reproductive technology that have contributed to genetic improvement through planned breeding
programme. These two techniques compliment each other and if performed with necessary
precautions, hygiene and standard protocol results in successful conception in farm animals. The
complete process for successful conception involves following steps-
1- Semen collection
2- Semen cryopreservation
3- Semen storage tank management
4- Artificial Insemination (A.I)
All these processes are complex and need sound knowledge and expertize to be
performed successfully. Handling and upkeeping of the instrument is an important component
that needs to be taken into consideration for execution of process. Following measures should be
taken at each step for optimum outcome in terms of successful Artificial Insemination and better
conception rate.

1- Semen collection
It is the first step in process is semen collection. It is the process of semen collection from
male animal using a dummy/cow. The success of semen cryopreservation and Artificial
Insemination largely depends upon semen collection. The process involves clean, hygienic
semen collection as this step forms the basis of future semen quality and influences post thaw
semen quality. The semen collection is performed using artificial vagina which has following
parts-
(i) An outer heavy rubber cylinder,
(ii) Inner sleeves of rubber,
(iii) The semen receiving cone,
(iv) Semen collecting vial made of glass or plastic.
Artificial Vagina (AV) and semen collection
Before using artificial Vagina for semen collection all the parts should be washed and
sterilized properly, and assembled as artificial vagina. The rubber liner is inserted into the
hose inverting both ends back by folding back from either side opening, or fastening with
rubber bands. Now the space between the hard rubber hose and inner rubber liner forms a
water tight compartment. The water jacket of the artificial vagina is filled with hot water at a
temperature of 45°C (113°F) by opening the nostle. The temperature of the water to be taken
depends upon the liking of the bull, and season of the year. The graduated semen collection
tube is fixed to the narrow end of the artificial vagina hose, and fastened by a rubber band.
The modern artificial vagina types are also provided with an airscrew along with the water
screw, which can be used for blowing the air between the two layers to create the desired
pressure The inner side of the rubber liner on the anterior side of the artificial vagina is
lubricated with sterile jelly to a length of 3 to 4 inches. The cow or dummy is secured in
service create. The artificial vagina assembled is held at 45° angle from the direction of penis
as the thrust is in that angle. The artificial vagina is held with the left hand by a right handed
person and when the bull mounts the cow, the sheath of the bull should be graphed by the
operator, directing the gland penis into the artificial vagina, and then the bull gives a thrust to
ejaculate. After the bull dismounts, the artificial vagina is taken off from penis and the air
vent is opened to release the pressure from the jacket. Water from the jacket is also drained
by opening the nostle. This allows the ejaculate to flow from the cone to the semen collection
tube. The semen collection tube is detached from the cone, plugged with cotton wool, and
taken to the laboratory for examination.

11 
 
Precaution during semen collection-
1- The temperature of the artificial vagina is to be checked, at each collection, and it should
resemble natural vagina at mounting time.
2- The operator should evince care so as not to touch the exposed past of the penis.
Touching the penis will cause shyness and bull dismounts.
3- The rubber cone and the semen collection tube should be protected from external
contamination or heat by covering with an insulation bag with zip.
4- Prior to collection all these parts are cleaned, sterilised and assembled into artificial
vagina. A sterile condition of the apparatus ensures disease control.
5- Prior to collection, bulls are usually allowed to become excited by bringing the bull to
cows or other dummy.
6- It is free from the extraneous secretions.
7- Avoid contamination of the semen from water, lubricating jelly or other harmful
substances.
8- Avoid cold shock by providing adequate protection for the collection tube and funnel.
9- The preputial hair should be trimmed to 2 cm length. Long preputial hair cause adhering
of the dung to it , spoiling the ejaculate. If closely trimmed it will cause irritation leading
to frequent masturbation by the bulls.
10- The semen collector shall wash his hand before every collection with mild disinfectant
and use separate napkin for drying hands or shall use the disposable hand gloves.
11- Immediately after semen collection the AV shall be kept immersed in a tub with neutral
detergent lotion.
12- Use of lubricant shall be avoided. If it is extremely essential to use lubricant, separate
sterilized glass rods shall be used for smearing K- Y Jelly on each AV.
13- The entry of visitors/staff/ laborers’ shall be strictly prohibited in the collection arena at
the time of semen collection and inside the semen laboratory.
14- Semen stations must follow the norm of two ejaculates per collection and two
collections per bull per week for taking annually at least 90 collections and 180
ejaculates from each adult bull.

2. Semen cryopreservation
Semen cryopreservation (commonly called sperm banking) is a procedure to preserve sperm
cells at a very low temperature (-1960C) for long period of time. The process starts just after
semen collection and involves semen evaluation, semen dilution, equilibration and straw filling,
deep freezing and storage of semen straws in liquid nitrogen. All these procedures should be
carried out in one room fully air conditioned and maintaining 20 to 22°c. Before starting the
laboratory procedures, all the parts of equipment handled during processing shall be cleaned with
70% alcohol. The semen extender shall be prepared in laminar flow station with the help of
standard chemicals. The chemicals used for preparation of extender shall be either G.R. or A.R.
quality, coming from reputed firms. The eggs used for the preparation of egg yolk extender shall
be from known poultry farm and shall be fresh. Only fresh semen extender shall be used, the pH
of the leftover extender will change on storage and the extender then may not remain suitable for
extending semen. The combination of antibiotics in the extender should be such that can control
contaminants, like mycoplasma and other ubiquitous organisms. This can be effectively done by
a combination of Tylosin, Lincospectin and Gentamycin, alternatively crystalline penicillin,
Streptomycin can be used. The laboratory shall be fumigated twice a week e.g if the laboratory is
fumigated with 5% formaldehyde on Wednesday, then on Saturday it should be fumigated with
12 % formaldehyde for half an hour to two hours. The laboratory should be wet mopped twice a
day before starting and after finishing the work with disinfectant having gluteryldehyde like
Lysol. The Laminar flow stations should be cleaned with 70% alcohol and should be tested for
DOP for every six months and should have annual maintenance contract. The cold handling
cabinets should also be cleaned with 70% alcohol and should maintain 4 to 5°c temperature for
equilibration. The washing and sterilization of glassware and rubber ware should be carried out
in separate rooms. There shall be a separate room for AV preparation and washing and separate
room for glassware washing and sterilization.

12 
 
3. Semen Tank Management
The semen storage tank is a large vacuum-sealed metal bottle with an extremely efficient
insulation system. Because of the vacuum bottle construction, the temperature can be maintained
at – 1960C (liquid nitrogen temperature) as long as two inches of liquid nitrogen is present. The
maintenance of liquid nitrogen temperatures in the inner chamber is due to high quality solid
insulation material and vacuum in the outer chamber. Although the newer tanks are better
insulated, they are still susceptible to damage from mishandling. The inner chamber containing
liquid nitrogen is actually suspended from the outer shell by the neck tube. Abnormal stress on
the neck tube caused by sudden jarring or excessive swinging motion could crack the tube and
result in vacuum loss. Since vacuum is the major insulation component of the tank, a loss of
vacuum causes an increase in temperature within the inner chamber and a rapid evaporation of
nitrogen. Accumulation of frost at the top of the tank indicates a rapid evaporation of liquid
nitrogen. Semen must be kept at temperatures well below critical temperatures where the
recrystallization of ice begins to occur. Thermal injury to sperm is permanent and cannot be
corrected by returning semen to liquid nitrogen. The following semen handling practices are
recommended to minimize thermal damage:
Handling Semen within the Tank
1. Transfer of semen between tanks must be coordinated and rapid. Two people should be
involved, and tanks should be arranged side by side. If possible, fill the tanks with
nitrogen before transfer. Raise canisters only to a level necessary to locate the appropriate
rack of semen.
2. Develop a semen inventory system and mount it on the wall above the tank. It is best to
keep semen from one bull on each rack. Such systems help avoid unnecessary searching
and exposure of semen to dangerously high temperatures within the neck region.
3. Prepare to thaw semen by raising the canister into the lower portion of the neck where the
desired rack of semen can be grasped.
4. Use tweezers to transfer the straw to the thaw bath. Quickly lower the rack of semen and
canister into the tank body.
5. Routinely monitor nitrogen levels and keep a record of nitrogen loss. Remember, even
new tanks can have defects and fail.
6. Store the semen in an area with good light but out of direct sunlight. Observe the tank
daily. Once a tank fails, nitrogen is lost very rapidly. Plan to have an alternative semen
tank available in case of a failure.
7. Keep the tank elevated above the concrete floor or other wet and poorly ventilated
surfaces. Corrosion of the outer shell will shorten the functional life of the tank and
possibly cause failure.
8. Store only the amount of semen needed for six months.

4- Artificial Insemination (using A.I gun)

The primary objective of proper semen handling is to optimize conception by preserving
sperm fertility until insemination has taken place. A technician’s goals include minimizing the
time of exposure of semen to extreme fluctuations in temperature and direct sunlight (ultraviolet
light destroys semen) and preventing contamination with manure, water, detergents, and other
substances. When preparing the A.I. gun, the straw of semen should be removed from the goblet
with plastic tweezers – not your fingertips. The straw should be shaken after removing it from
the tank to eliminate any drops of nitrogen at the end of the cotton plug. This will prevent the
plug bursting off when it is put in the water bath. Accuracy should be maintained by regularly
checking temperatures and calibrating thawing thermometer. After the thawing straw, it should
be dry it off with a clean towel and checked for information printed on the outside to verify the
bull’s identity. Semen should be used within 15 minutes of thawing. In cold weather, gun should
be warmed by rubbing it with your hands. The sealed end of straw should be cut at a 90-degree
angle about ¼ inch from the lab seal. If the straw is not cut squarely, the plastic sheath may not
seal tightly against the straw and some semen will then back flow between the sheath and the
straw rather than going inside the cow. A sterile plastic sheath should be placed over the gun and
then sealed. The end should be wrapped in a paper towel to prevent exposure to the sun and to

13 
 
maintain sanitation. Sheath protectors can also be useful. After loading the gun, the region of the
vulva should be cleaned to prevent contaminating the vagina and uterus. After loading the end of
the gun should be palpated with finger just outside the cervix. Be sure the gun is passing through
the cervix and there should be no stretching the vagina. When the tip of the insemination gun
passes through the front ring of the cervix, it is in the uterine body. The location should be
checked by placing index finger in front of the cervix where tip of the gun should be felt. The
cervix and uterus should be picked up properly and semen should be deposited in the body of the
uterus. While insemination the gun should be inserted into the cow upward at a 30-degree angle
to avoid entering the bladder.

Precautions while performing Artificial Insemination

1- Thawing temperature and thaw time for frozen semen should be monitored properly.
Thaw semen straws in warm water (95–98°F) for a minimum of 45 seconds.
2- Always check the ware temperature in the thawing device below pulling a frozen straw
from the liquid nitrogen tank.
3- Always keep the canister below the frost line when Always keep the canister below the
frost line when locating a straw of semen. Avoid lifting the canister too high or too long
during this process.
4- Once a straw is thawed, it is recommended that the semen be deposited into the cow
within 15 minutes. Always wipe the straw completely dry with a clean paper towel
before loading it into the inseminating syringe.
5- Plunger should not pulled back before loading insemination gun. Always pull back the
plunger approximately 6 inches before loading a straw into a semen gun. Use a slow,
gentle motion to depress the plunger on the inseminating gun. Complete depression
should be accomplished in no shorter than 5 second.
6- Always lock the split plastic sheath into place on the inseminating gun with an o-ring.
Otherwise, the sheath will slip, leading to improper semen placement during
insemination of the cow.

Thus the success of semen cryopreservation and artificial insemination involves careful
handling of equipment’s and adopting hygienic measures at each step of the standard protocol.

 
* * * * * * * 
   

14 
 
 Use of ultrasonography in management of bovine reproduction
Anuj Kumar

1. INTRODUCTION
In bovine practice, ultrasonography has become an important diagnostic tool for
evaluating the female reproductive system. With the help of ultrasound technology, it is
possible to view the entire reproductive system in a non-invasive manner. The quality of
the images depends above all on the user's understanding of the interactions between the
ultrasound wave and the organ tissue, as well as proper use of the instrument's controls.
The objective of this manuscript is to understand some basic principles of
ultrasonography, to present its principal uses in bovine reproduction, and the economic
benefits of early pregnancy diagnosis.

2. PRINCIPLES OF ULTRASONOGRAPHY
2.1 Images description
The image on the ultrasound screen represents a fine section of an organ, grossly
resembling a weakly-magnified histological cut. The probe therefore simulates the
passage of a knife, slicing through an organ tissue from top to bottom. Ultrasonography
thus presents a flattened two-dimensional image of a finely-cut section of tissue; whereas
radiography is a two-dimensional superimposed view of the entire thickness of an animal
or of a limb under observation. Ultrasound images are rapidly renewed and images are
superimposed one over the other as the probe moves across a tissue surface. The rapid
succession of tissue section views gives the impression that the structures are moving
like an animated cartoon. When interpreting sectional views of an organ on the screen, it
is essential to have a good appreciation of the three-dimensional shape of the organ in
space.
2.2 Transducer or probe characteristics
The probe is the most delicate component of the ultrasound machine. In the
veterinary practices, usual probes have a frequency of 3.5, 5.0 or 7.5 MHz. The ability of
the instrument to distinguish between two structures located very close together along
the axis of the ultrasound beam is called its axial resolution. The axial resolution is best
when the groups of waves emitted have a short wavelength. Since the number of cycles
in each group of waves is set according to instrument design, the only way to shorten
their length is to use a probe with a higher frequency. For example, the group of waves
produced by a 7.5 MHz probe will be shorter than one produced by a 3.5 MHz probe and
will provide better axial resolution.
There are two types of probes: linear and sectorial (Figure 1). In bovine
reproduction, linear probes are preferred for transrectal examinations of the ovaries and
uterus. This probe has a set row of crystals that are selected electronically to form a
rectangular image. The linear probe provides good resolution for tissues located close to
the probe. Sectorial probes have one or several crystals whose position produces a beam
in the shape of a pie slice. The advantage of the sectorial probe is that it doesn't require a
large surface of contact, and it scans a greater overall surface. The disadvantage is that
the visual field and the lateral resolution (i.e. the ability of a system to differentiate
between two adjacent structures) are more restricted close to the probe. The sectorial
probe is ideal for viewing the small ruminant fetus by transabdominal ultrasound
imaging and for ultrasound-guided transvaginal aspiration of bovine follicles.

15 
 
 Figure 1. Type of probe

The details contained in an ultrasound image (resolution) as well as the depth of

the tissue observed depend on the frequency and the focalization of the scanning beam.
With a lower frequency, tissue penetration will be deep, but the resolution will be lower.
A higher frequency enables better resolution, but beam attenuation will be greater and it
will not penetrate the tissue as deeply.
2.3 Images description
The description of ultrasound images is based on an evaluation of the shape,
contour, size, and position of the structure being studied, as well as its echogenicity,
which depends on the amplitude of the echoes received. An echogenic structure reflects
the majority of soundwaves back to the probe and thus appears from white to different
shades of grey on the screen. An anechogenic structure does not produce echoes; instead,
it transmits the waves on to more deeply situated tissues. An example of an anechogenic
structure is fluid filled cavity like follicular fluid, urinary bladder which appears black on
the screen. The terms hypoechogenic and hyperechogenic indicate respectively a
decrease and an increase in relative echogenicity in comparison with the surrounding
tissue, whereas the term isoechogenic is used to describe similar echogenicity with the
surrounding tissue.

3. ULTRASOUND APPLICATIONS
3.1 Ovary and uterus
With the use of ultrasound since 1984, enormous progress has been made in our
understanding of folliculogenesis and the development of the bovine corpus luteum.
Ultrasound enables us to explain the dynamics of follicular growth in follicles greater

16 
 
than 1 mm in diameter throughout estrous cycle. The same pattern of growth repeats
itself approximately every seven days. It starts with the growth of a cohort of many
follicles, known as a follicular wave, which includes a follicle that will pursue its growth
and will be called dominant while the other follicles of the cohort will regress by atresia.
The most common pathological conditions of the ovaries in bovine practice are cystic
ovaries (follicular and luteal cysts). These two types of cysts can be differentiated by
ultrasonography and treated accordingly. Increase growth of a non-ovulating follicle may
lead to development of a follicular or luteal cyst. An ovary is usually considered cystic
when it contains a hollow structure greater than 25 mm that persists for more than 10
days. A follicular cyst can be differentiated from a luteal cyst by its thin wall and
uniformly anechogenic follicular fluid.

Ovary with follicles

Corpus luteum

Follicular cyst

The two most frequently observed uterine pathologies in ultrasound diagnosis

are, by decreasing order of prevalence, the metritis complex (pyometra-endometritis) and
mucometra. In the case of endometritis-pyometra, the contents of the uterus may vary in
appearance as anechogenic fluid in black, to the presence of echogenic material floating
in a black background, to a purulent exudate that is echogenic in appearance and similar
to the surrounding tissue, or isoechogenic. The characteristic of mucometra resembles a
pregnancy, except for the fact that there is no embryo/foetus or foetal membranes.

17 
 
 Pyometra

Non-pregnant uterus

3.2 Pregnancy diagnosis

3.2.1 Stage of gestation period
Diagnosis of pregnancy in cows is an important part of management of infertile
cow in dairy herds. The pregnancy examination is considered essential and economical
for livestock owners. Transrectal palpation has been used for the past 50 years to
diagnose pregnancy. This diagnosis can be made after 30 to 35 days in adult cows and
slightly earlier in heifers. However, it is important to note that the accuracy of early
diagnosis by palpation varies according to the skill and experience of the veterinarian.
Transrectal palpation in an early phase of pregnancy can be harmfull by causing
embryonic death as a result of improper handling.

30 days pregnancy

Embryonic death is one of the most important factors associated with dairy cow
infertility. It is defined as the loss of an embryo between the moment of fertilization and
the first stages of differentiation, at around day 45 of pregnancy. The majority of
embryonic deaths occur before day 25. However, the period between days 25 and 45 is
critical for the fixation of the embryonic membranes to the uterine epithelium and
corresponds to the period when early pregnancy examinations are performed on dairy

18 
 
cows. Ultrasonography is less harmfull and more accurate than transrectal palpation for
diagnosing pregnancy in dairy cows before day 35.
3.2.2 Accuracy of the method
Diagnosis of pregnancy by ultrasonography can be made as early as day 25 after
insemination. An evaluation of this diagnostic method has demonstrated excellent
sensitivity (detection of pregnancy when the animal is truly gravid): greater than 95% at
day 26 post-insemination. Ultrasonography's greatest advantage for the clinician is to
diagnose non-pregnant animal as early as possible after insemination. The accuracy
increases to 98 and 100% if the pregnancy diagnosis is performed respectively after day
30 and day 31 post insemination. This excellent characteristic enables veterinarians for
management or treatment of non-pregnant animals as quickly as possible.
The accuracy of early pregnancy diagnosis using ultrasonography depends upon
many factors. Out of these factors most important is the period of pregnancy when the
reproductive examination is performed. Theoretically, with advancement of pregnancy,
the more space the amniotic vesicle and the embryo will occupy within the uterus and the
easier they will be to visualize. Other factor is the position of the uterus in the pelvic
canal. There are more false negatives between days 24 and 33 after insemination when
the uterus is located at the cranial part of the pelvic bone. It appears that a complete
exam of the reproductive tract is more difficult when the uterus is located in this
position. The cow's parity also has an impact on diagnostic accuracy, which has proven
to be inversely proportional to the age of the cow being examined. Diagnostic accuracy
is also influenced by the type of instrument and probe used. In bovine reproduction,
where it is important to evaluate ovarian and uterine structures in detail, we prefer to use
linear probes that produce frequencies of 5 to 7.5 MHz; these give a better resolution
compared to probes with lower frequencies.
3.2.3 Embryo’s viability characteristics
During pregnancy diagnosis, it is also important to observe the embryo's viability
by paying attention to the fetal heartbeat. It is generally visible at the centre of the
embryo starting at day 25 of pregnancy, appearing as a twinkling light with a frequency
that varies between 140 and 160 beats per minute.
Another important sign of normal embryonic development is the appearance of
the umbilical cord between the uterus and the embryo between days 40 and 45 of
pregnancy. Starting on day 45, we may also observe the first movements of the fetus.
Finally, the presence of a homogeneous, anechogenic liquid in sufficient quantities
within the amnios is another way of confirming that viable fetal development is
progressing normally. When the presence of many echogenic particles are noted within
the uterine liquid, the veterinarian should question the viability of the embryo and
wonder if embryonic death has occurred. At this time, it is important to review the
various signs of embryonic vitality, such as the heartbeat, fetal movement as well as its
length in relation to the stage of pregnancy. The presence of a large amount of debris
within the amnionic fluid is generally the result of embryonic degeneration. On occasion,
it is also possible to observe a dead but nearly intact embryo. However, upon further
examination, the embryo's contours may appear irregular and small particles may be
found in the immediate surroundings.
3.3 FOETAL SEXING
The ultrasound diagnosis of the foetal sexing involves three steps: locating and
identifying the fetus, verifying fetal viability and determining fetal sex. The clinician
first locates the fetus in the gravid uterus. With a complete examination of the two
uterine horns the user will be able to determine the number of fetuses present. During
examination, the aspect of the amniotic fluid and the presence of a fetal heartbeat

19 
 
observed to confirm viability. Now the clinician will determine the sex of the fetus in
following ways-
3.3.1 Male fetus
At around day 50, the genital tubercle migrates cranially along the median line
towards the umbilicus. This migration gradually increases the distance separating the
genital tubercle from the tail of the fetus. At around day 58, the genital tubercle reaches
its final destination, slightly caudal to the umbilicus. Following this migration, the
genital swellings are now in a caudal position in relation to the genital tubercle and have
fused together near the median line between days 65 and 70 in the male fetus, we note a
change in the appearance of the genital tubercle. The two-lobed structure observed
earlier in the pregnancy gives way to a four-lobed structure following a view of the
genital tubercle and the urogenital folds. In the male, the genital tubercle is the origin of
the penis, the urogenital folds form the prepuce and the genital swellings form the
scrotum. The migration of the testicles toward the scrotum occurs later and is generally
complete by day 140 of pregnancy.
3.3.2 Female fetus
Starting on day 40, as for the male, a vertical lengthening of the genital tubercle
and the presence of the urogenital folds can be detected. Between days 50 and 58, the
genital tubercle migrates from its initial position between the hind limbs towards the anal
region which is in the opposite direction from that of the male. In the female, the genital
tubercle is located under the tail and the genital swellings undergo progressive atrophy
starting on day 50, and eventually disappear completely. The genital tubercle is the
origin of the clitoris and the urogenital folds form the vulvar labia.
3.3.3 Stage of gestation period for fetal sexing
Ultrasound fetal sexing examination can be done between days 58 and 100 of
pregnancy, but best time period is between days 60 and 70. Even though the genital
tubercle is already visible at day 45, it doesn't reach its definite position until day 58.
Migration may occur more rapidly in certain animals, sometimes enabling definitive
diagnosis at day 54 or 55. Clinician ability to establish a diagnosis at the end of the first
trimester of pregnancy (90 to 100 days) will depend on the position of the uterus within
the pelvic/abdominal cavity and the possibility to reach the gravid uterine horn with the
probe. A uterus deep in the abdominal cavity will make the diagnosis more difficult.
3.4 Diagnosis of twin/multiple foetuses
Diagnosis of multiple fetuses is generally performed by the veterinarian at the
same time as sexing the fetus, after day 57 of pregnancy. By embryo transfer technique,
it has become possible to implant two embryos in a recipient. If the presence of twins is
confirmed before day 55 of pregnancy, it is then important to do another ultrasound
exam after day 60 to determine the sex of the foetuses. If the fetuses are male and
female, the owner will then have the opportunity to choose whether to maintain the
pregnancy and run the risk of obtaining a freemartin heifer or to terminate the pregnancy.
3.5 Abnormal development of fetus
During the ultrasound examination for pregnancy, and especially when
determining the sex of the fetus, it is very important to ensure that the fetus is viable and
its appearance is normal. It is important to recognize the fetal defects like siamese twins;
schistosomus reflexus; amorphous globosus. Certain acquired or congenital defects may
be noted during the ultrasound exam in an older foetus. Among these, it is worthwhile to
mention hydrocephalus, fetal ascites and pericardial effusion. These conditions have a
very poor prognosis for fetal survival. Before proposing the termination of the pregnancy
on the cows presenting these anomalies, it is essential for the veterinarian to do a careful

20 
 
ultrasonographic examination using more than one view and to repeat the scanning more
than once to confirm his definitive diagnosis.
4. CONCLUSION
The diagnosis of reproductive characteristics can be acquired by examining
hundered to thousands of cow by per-rectal examination during a period of time. On the
other hand veterinarians who are less experienced in transrectal examination can improve
their manual mistake by ultrasonography, along with offering a more accurate and
extensive diagnostic service. The use of this diagnostic tool increases the value of a
veterinary clinic's services. The use of ultrasound technology is economically
advantageous for dairy producers and veterinarians.

*******
   

21 
 
 Assessment of optimum time for artificial insemination technique in
bovine
Vijay Singh

INTRODUCTION
y Technique entails deposition of semen in cervix of animal at the appropriate
time during later part of the heat period in order to give best chance for viable
spermatozoa to meet the ovum for successful fertilization
y It is most important that female inseminated at proper stage of cycle i.e. shortly
before ovulation.
Advantages
• Genetic Improvement
• Wide spread availability of genetically superior sires
• Disease Control
• Injured studs available
• Increased use of sires
• Reduced danger from studs
• Cost - relatively cheap
Disadvantages:
• Estrus detection must be good
• Handle and care for semen
• Record Keeping
• Time involved - Restraining and inseminating cow
• Training required to handle semen and breed cow
Check points before insemination:
y If heifer – weight > 250 kg cattle & >300 kg buffalo
y No abnormal discharge
y Non pregnant
y Gap - 60 days post partum in normal calving
90 days post-partum in abnormal calving
(Dystocia, ROP, abortion etc.)
y Type of semen used (Liquid/Cryopreserved).

22 
 
Insemination technique in cattle:

y A)- Loading of A.I. gun

y Ready all necessary instruments before opening liquid nitrogen container.
y Raise desired canister to the neck of container , keeping the straws in goblet as
deep as possible
y Take out desired straw as quickly as possible and shake them in air and put
immediately in water bath for thawing.
y Thaw at 37°C for 30 sec.
y Remove straw from water bath and wipe out all water from outer surface of
straw.
y Hold straw vertically with factory seal end towards downward side and shake air
bubble from middle of straw to lab seal end.
y Withdraw piston of gun and place straw in barrel of A.I. gun.
y Factory seal end should be inside gun and lab seal end should face outside.
y Cut straw at right angle.
y Fix A.I. sheath over A.I. gun from its broad end.
Recto vaginal or cervical fixation method:
y A.I. in cattle is done in standing animals.
y Wear sleeve and lubricate it with soft soap and water.
y Insert hand inside rectum in cone shape manner.
y Remove dung and care should be taken to prevent ballooning.
y Locate cervix and palpate entire genitalia.
y Wipe exterior of vulva and vulvar lips with clean cotton
y Grasp cervix with palm and fingers, palpate external end of cervix with thumb.
y Pull vulvar lips apart and A.I. gun is passed at an angle of 45° through vagina.
y In case vaginal folds creates problems and put hindrance for passing A.I. gun,
cervix may be pushed forward in order to abolish vaginal folds.
y Manipulate A.I. gun so as it strikes thumb placed over external end of cervix and
then A.I. gun is passed into cervix.
y Intra uterine inseminations are more efficient than intra cervical. Insemination

23 
 
 A.I. with liquid semen:

A.I. in cattle and buffaloes is done in standing animal.

Sterilized catheter of 40 to 42 cm in length, outside diameter of 5-6mm,
inside diameter of 1mm is fitted to a clean and dry plastic syringe (2-5ml)
with rubber connector is required.
After sucking some air (0.5 -1.0 ml) about 1ml semen is sucked in the pipette.
A.I. is done with recto-vaginal method.

Semen deposition site:

Proper placement of A.I. gun at internal end of cervix:

24 
 
 Semen deposition at internal os:

Improper placement of A.I. gun in Rt. Uterine horn:

25 
 
 Improper semen deposition in Rt. Uterine horn:

Insemination technique in buffalo:

y Same as cattle
y Difficulty in detecting heats.

26 
 
 y More sensitive to touch and vaginal contractibility
y In India - 80 % of total no of estrous periods recorded in buffaloes were from
October to March and only 20% from April to September.
y High temperature and humidity coincide period of lower sexual activity.
y Oestrous cycle length: Average- 21 days
y Duration of oestrus: Less than 24 hours varies as wide as 4-64 hours. (mostly 24
hours)
y Ovulation Time: 15-48 hour after end of oestrus. (mostly 15-18 hours)

 
 
* * * * * * * 
   

27 
 
 Role of nutrients in animal reproduction
Vinod Kumar

Introduction
Animals need optimal amount of energy, protein, minerals, vitamins and water
for optimum lactation, growth and reproduction. Imbalance or deficiencies of any
above nutrient may lead to adverse affects on reproductive performance and health of
the animal. Insufficient energy in diet or a negative energy balance is considered the
most common cause of nutritional infertility. Protein insufficiency is related with a
delay in the onset of puberty and an increase in days open. Rations of high
producing cow containing more than 18% crude protein may lead to lower
reproductive performance due to excessive ruminal degradable protein, causing
excessive ammonia and urea levels in the bloodstream servicing the ovaries.
Minerals act as structural components of organs and tissues, as cofactors or activators
of enzyme, hormone systems, as constituents of body fluids and tissues, and as
regulators of cell replication and differentiation. Mineral deficiencies, imbalances
and toxicity of certain mineral elements may cause reproductive disorders as
minerals play an important role in health and reproduction of the livestock. Vitamin
A deficiency has long been known to affect reproductive function in cattle.
Periovulatory -carotene and vitamin A supplementation improves follicular growth
and development of the corpora lutea in cows with fertility impairment. Dietary
supplementation of vitamin E and selenium may reduce the incidence of retained
placenta and mastitis by improving immune response. Calcium and phosphorus
deficiencies affect reproduction in cattle, and vitamin D may directly affect
reproductive function in addition to its role in calcium and phosphorus metabolism.
The active vitamin D hormone also contributes to immune, reproductive, and
mammary physiology. Dietary manipulation of a number of other vitamins and
minerals also influences reproductive function.

Nutritional factors affecting reproduction

Energy: Energy is an important nutritional factor related to poor reproductive

function in animals. Short and Adams prioritized the metabolic use of available
energy in ruminants ranking each physiological state in order of importance, as
follows: 1) basal metabolism, 2) activity, 3) growth, 4) energy reserves, 5)
pregnancy, 6) lactation, 7) additional energy reserves, 8) estrous cycles and initiation
of pregnancy, and 9) excess energy reserves. Energy content in diet of Indian animals
is measured as total digestible nutrient (kg) or metabolizable energy or net energy.
On an average for a crossbred animal a daily maintenance requirement of energy for
lactating animal is about 30 g TDN and 107.0 Kcal ME/W0.75kg and milk production
requirement for per kg 4% FCM, energy requirement are 436 g TDN and 1082 Kcal
ME. Out of which, about 45% energy is partitioned for maintenance and rest 55% for
milk production. The maintenance requirement lactating Indian cattle as TDN was
39.5 g/kg W0.75 and for milk production and weight gain were 332 g/kg FCM and
1.78 g/g body weight gain, respectively. In tropical condition low quality fodders
predominates feeding regimen of lactating animal as a result deficiency of energy in
diet leads to adverse effect on health and production. Negative energy balance during
early lactation in dairy cows leads to an altered metabolic state that has major effects
on the production of IGF family members. Low IGF-I concentrations are associated

28 
 
 with poor fertility. Metabolites and metabolic hormones that change in relation to
negative energy balance, insulin-like growth factor-I (IGF-I) is strongly associated
with the calving to conception interval and pregnancy outcome and their effect on
oocyte, ovary, uterus and oviduct effects embryo development, contributing to the
high rates of embryonic mortality in dairy cows.

Protein: A deficiency or excess of dietary protein has adverse effect in reproduction.

Over-feeding of protein has been associated with decreased pregnancy rates in
female dairy cows and buffaloes. The exposure to high levels of ammonia or urea
may impair maturation of oocyte and subsequent fertilization or maturation of
developing embryos. Overfeeding protein during the breeding season and early
gestation, particularly if the rumen receives an inadequate supply of energy may be
associated with decreased fertility. This decrease in fertility may result from
decreased uterine pH during the luteal phase of the estrous cycle in cattle fed high
levels of degradable protein. Energy costs for detoxifying ammonia into urea (waste
product resulting from excess protein) may aggravate an existing energy shortage
resulting in reduced ovarian activity. This means that excess protein requires extra
energy to remove it from the body. If cows are in a negative energy balance and are
losing weight, they will lose even more weight because of the excessively high
protein. Cows that lose excessive weight have a harder time re-breeding and probably
won’t until their weight begins to come back. Cows fed excess protein (more than
10-15% above requirements) required more services per conception and had longer
calving intervals. The maintenance requirement of CP was estimated to be 6.27 g/kg
W0.75 whereas CP requirements for milk production and weight gain were 82.3 g/kg
FCM and 0.44 g/g body weight gain, respectively. For examples a cow weighing 350
kg, producing 10 liter of FCM need 1290 g CP and 6.6 kg TDN daily. Approximately
dry matter intake of cow is 10 kg; diet of cow should have 10-13% protein and 65%
TDN. This has to be met by feeding good quality fodders and concentrate to avoid
production problems.

Fat: Cows receiving high level of energy during the postpartum period had increased
LH pulse frequency and decreased the period from parturition to ovulation. Fatty
acids and cholesterol are substrates for reproductive hormone synthesis
(progesterone, prostaglandins). Therefore, the effects of fat may be independent to
those of increased energy availability. In subcontinent animal diets usually contain 2
to 3% fat. Supplementing fat to improve reproduction was initially attempted to
increase the energy density in the diet. High fat diets for cattle contain 5 to 8 % fat.
Exceeding these dietary fat levels >8% impairs rumen function. For examples a 400
kg body weight cow consuming 10 kg dry matter have about 300 g crude fat in their
diet thus there is scope of adding upto 500 g fat in daily diet for high yielders without
any detrimental effect on rumen fermentation. Feeding high fat diets to cycling
heifers and postpartum cows increased progesterone production and the lifespan of
the corpus luteum (CL). Higher progesterone levels during the luteal phase generally
result in improved fertility. Increasing dietary fat also results in increased follicular
growth. Fat content of the diet should be increased above the typical 3% of diet DM
for cows in the early postpartum period in order to meet the energy requirement.
Feeding calcium salts of long chain fatty acids also improved pregnancy or
conception rates have also been reported.

29 
 
 Urea nitrogen and fertility: A high protein intake leads to increases in milk
production in dairy cows. However, at the same time, dairy cows fed high dietary
protein increase blood urea nitrogen (BUN) concentration, which is associated with
reduced reproductive performance possibly due to alteration of intrauterine
environment and low plasma progesterone concentrations. BUN concentrations >20
mg/dl was associated with lowered conception rates in dairy cows. Milk urea
nitrogen (MUN), instead of BUN, could be used as an indicator of urea status in
dairy cows and is more practical in the field. Thus, MUN is a practical and reliable
indicator for protein metabolism in dairy cattle. Urea nitrogen concentrations in bulk
tank milk have been used to predict protein supply and fertility differences between
herds. Heifers fed high rumen degradable protein increase BUN concentrations and
decrease uterine pH and pregnancy rate. In addition, cows fed excess amount of
protein increase BUN concentration as well as decrease uterine pH. Excess rumen
degradable protein has a deleterious effect on embryonic development in lactating
cows, but not in non lactating cows. It is the level of protein <18% in total dry matter
intake in dairy cows is important as Indian cross bred cows have performed well on
50:50 RDP: UDP when fed lower levels.

Minerals: Minerals play an important role in nutrient utilization and several

biochemical functions concerning production and reproduction. The availability of
minerals to cattle depends upon the production system, feeding practices, and
environment. Macro-minerals include: calcium, phosphorus, magnesium, potassium,
sulfur and salt are needed in relatively large amounts in the body. The trace minerals
include: cobalt, copper, iodine, iron, manganese, selenium and zinc and are needed in
very small or “trace” amounts in the body. Animals obtained minerals through the
consumption of natural feeds, fodders and supplementation of inorganic salts in the
ration. Mineral deficiencies and imbalances have long been held responsible for low
production among cattle and buffaloes fed on crop residues in tropical agro-climatic
condition. Mineral deficiencies and imbalances are often cited as causes of poor
reproduction. It is clear that adequate amounts of minerals must be provided, but
little is known about the effects of marginal deficiencies and imbalances. The same is
true of excessive intakes of minerals which may indeed be harmful.
An important concept surrounding macro mineral balance is dietary cation-anion
difference (DCAD). DCAD measures the level of four macro minerals: sodium and
potassium, which are cations and carry a positive charge, and chloride and sulfur,
which are anions and carry a negative charge. The DCAD of diet can be calculated
by equation using the quantities of the salts administered and the measured mineral
concentrations and dry matter intake of all feeds.
DCAD (mEq/100 g DM) = (Na + K) (Cl + S).
Research shows that a negative DCAD prior to calving help in improving Ca
homeostasis of periparturient cow, decreasing the incidence of metabolic disorders
postpartum and increasing early lactation production. A positive DCAD (20 mEq/100
g DM) have been recommonded for Indian cow and buffaloes. A diet having 33
mEq/100 g DM DCAD has promoted feed consumption, water intake and resulted in
greater milk yield and milk fat in early lactating buffaloes.

Calcium (Ca): Ca plays a very importance role in structural and physiological

functions. Ratio of Ca: P between 1.5:1 and 2.5:1 for lactating cows caused no
problems. Lactating cows must be provided with adequate amounts of Ca to
maximize production and minimize health problems. Other function of calcium is to

30 
 
 allow the muscle contraction. A reduction in muscle contractility affects rumen
function; lower dry matter intake thus negative energy balance. This results in
increase in fat mobilization leading to fatty liver syndrome and ketosis. It has been
shown that plasma calcium concentration of 5mg/ml reduces abomasal motility by
70% and the strength of the contraction by 50%. Low calcium concentrations also
prevent insulin production, further exacerbating this situation. Muscle tone in the
uterus will also be adversely affected with cows experiencing prolonged calving and
retained placenta. Uterine involution may also be impaired giving rise to fertility
problems. A major concern in the mineral feeding of dry cows relates to providing
optimum levels of calcium and phosphorus in order to decrease the occurrence of
milk fever. Indian lactating cows need about 0.40 to 0.60% to low yielders and ration
containing 0.75 to 0.80 percent Ca on DM basis should be provided to high
producing cows.

Phosphorus (P): P is the second most abundant mineral element in the body with 80
to 85% of P in the body being found in the teeth and bones. Essentially, phosphorus
is involved in metabolic reaction and energy transfer within the body. It is required
for normal milk production, growth, and efficient use of feed and by the rumen
microorganisms in the digestion of cellulose and synthesis of microbial protein. A
decreased fertility rate, feed intake, milk production, decreased ovarian activity,
irregular estrous cycles, increased occurrence of cystic ovaries, delayed sexual
maturity and low conception rates have been reported when phosphorus intakes are
low. In a field study when heifers received only 70-80% of their phosphorus
requirements and serum phosphorus levels were low, fertility was impaired (3.7
services per conception). Services per conception were reduced to 1.3 after adequate
phosphorus was supplemented. Increasing phosphorus supplementation from 0.4% to
0.6% of the ration had no effect on days to first estrus or services per conception.
However, in some instances, responses have been reported in the field when
phosphorus supplementation was increased to 0.5% or 0.6%. Increasing the dietary
concentration of phosphorus above requirement does not improve reproductive
performance. Scientists have found that reproductive performance was not different
on the lower but adequate concentration of phosphorus when compared to cows fed a
higher concentration (above requirements) of dietary phosphorus. Thus, increasing
the concentration of dietary phosphorus above requirement (more than 0.38-0.40%)
does not improve reproductive performance.

Selenium (Se): Se is involved in normal spermatogenesis and is an essential

component of a range of selenoproteins, including glutathione peroxidase,
thioredoxin reductase and iodothyronine deiodinase. Selenium with GPx also appears
to be involved as a structural protein to provide normal sperm motility. Both
deficiency and excessive selenium have been demonstrated to be detrimental to
normal spermatogenesis. There are two major sources of Se for animals: (1) Se
naturally originating from plants, in the form of seleno-amino acids, including
selenomethionine and selenocysteine; (2) inorganic Se in the form of selenate or
selenite. Even if the physiological requirement for Se is low in an animal, if this is
not met, the anti-oxidant system is compromised, with subsequent detrimental
consequences in terms of animal health (Figure 1). A marginal selenium deficiency
in pregnant animals will lead to abortion, or calves will be weak and unable to stand
or suckle. Selenium supplementation reduces the incidence of retained placentas,
cystic ovaries, mastitis and metritis. Compromised selenium status has also been

31 
 
 associated with poor uterine involution, and weak or silent heats. In males, selenium
supplementation has been shown to increase semen quality. Symptoms of chronic
selenium toxicity include lameness, sore feet, deformed claws and loss of hairs from
tail. In pregnant animals, selenium toxicity will produce abortions, stillbirth and
weak and lethargic calves as selenium accumulate in the fetus at the expense of the
cow. The dietary requirement for selenium by most species is about 0.1 ppm.
Revised requirement of selenium for better immune response in dairy animals is 0.3
ppm.

Figure1. The relationship between mineral status and onset of subclinical and
clinical disease symptoms
Zinc (Zn): Zn is an essential component of over 200 enzyme systems of which the
metabolic action include carbohydrate and protein metabolism, protein synthesis,
nucleic acid metabolism, epithelial tissue integrity, cell repair and division, and
vitamin A and E transport and utilization. In addition, zinc plays a major role in the
immune system and certain reproductive hormones. Zinc is known to be essential
for proper sexual maturity, reproductive capacity, and more specifically, onset of
estrus. Zinc has a critical role in the repair and maintenance of the uterine lining
following parturition, speeding return to normal reproductive function and estrus. In
bulls, a zinc deficiency results in poor semen quality and reduced testicular size and
libido. Zinc has also been shown to increase plasma beta carotene level which is
correlated to improvement in conception rates and embryonic development.
Improved zinc status also improves fertility by reducing lameness, resulting in cows
more willing to show heat and improved mobility and performance of bulls. Severe
zinc deficiency in cattle results in reduced growth, reduced feed intake, loss of hair,
skin lesions that are most severe on the legs, neck, head and around the nostrils,
excessive salivation, swollen feed with open, scaly lesions, and impaired
reproduction. A deficiency of zinc in males reduces testicular development and
sperm production. In females, cycling and conception rate are decreased by zinc
deficiency. Severe zinc deficiency is rare, but has been observed in ruminants
grazing forages. Based on zinc supplementation studies, subclinical zinc deficiency
can result in impaired reproduction and decreased weight gains. The recommended
dietary content of zinc for dairy cattle is typically between 18 and 73ppm depending
upon the stage of lifecycle and dry matter intake. Copper, cadmium, calcium and
iron reduce zinc absorption and interfere with zinc metabolism. Requirement of Zn
in diet of dairy cows is 40 ppm (NRC, 2001).
Copper (Cu): Cu is a necessary component of number of enzymes including
superoxide dismutase, lysyl oxidase and thioloxidase. These enzymes function to

32 
 
 eliminate free radicals that increase tissue susceptibility to bacterial infections,
increase structural strength and elasticity of connective tissues and blood vessels and
increase strength of horn such as in the claw (Hoof), minimizing lameness. Cu has
an important role in the immune system where its compounds are involved in
reducing infections and diseases. Copper deficiency in cattle is generally due to the
presence of dietary antagonists, such as sulfur, molybdenum and iron (Fe) that
reduce Cu bioavailability. Dietary requirements for Cu are greatly increased by high
concentrations of molybdenum and sulfur. Deficiencies of copper have also been
associated with retained placenta, embryonic death and decreased conception rates.
Inadequate Cu status may be related to an increased incidence of infections at
calving, increased severity of infections and a higher SCC than that seen in Cu-
adequate cattle. Reproductive problems that relate to copper deficiency manifest
themselves in inhibited conception rate even though estrus may be normal. Weak
and silent heats have been reported. Dairy cows with higher serum copper levels had
significantly less days to first service, fewer services per conception and fewer days
to open. Proper copper supplementation of the sire is needed for production of
quality semen. Feeding a total of 10 to 15 ppm copper in the ration dry matter or
supplementing with 10 ppm copper should meet dairy cattle needs. If rations contain
antagonists such as elevated iron, sulfur, or molybdenum, replacing 35 percent of
supplemental copper with organic copper sources could improve copper availability.
The following mineral ratios may be helpful in maintaining Cu levels in blood: Zn:
Cu 4:1, Cu: Mo 6:1 and Fe: Cu 40:1.

Manganese (Mn): Mn is a required nutrient for dairy cows; however, clinical

deficiencies are extremely rare because most feedstuffs contain at least marginally
adequate concentrations of Mn. Mn is an activator of enzyme systems in the
metabolism of carbohydrate, fats, protein and nucleic acids. Mn appears to have a
vital role in reproduction. It is necessary for cholesterol synthesis, which in turn is
required for synthesis of the steroids, estrogen, progesterone and testosterone.
Insufficient steroid production results in decreased circulating concentrations of
these reproductive hormones resulting in abnormal sperm in males and irregular
estrus cycles in females. The corpus luteum has high manganese content and may be
affected by level of manganese supplementation. Also, vaginal manganese
concentration is higher in cycling than in anoestrous ruminants. A deficiency in
manganese may be associated with suppression of estrus, cyclic ovaries and reduced
conception rate. The maintenance requirement for absorbed Mn was set at 0.002
mg/kg of body weight (1.2 mg/day for an average Holstein cow), the growth
requirement was set at 0.7 mg/kg of growth, pregnancy requirement was set at 0.3
mg/d, and the lactation requirement was set at 0.03 mg/kg of milk (NRC, 2001).

Iodine (I): Iodine is important in the development of fetus and maintenance of

general basal metabolic rate as it is involved in synthesis of thyroid hormone,
thyroxin, which regulates the rate of metabolism. Iodine deficiency leads to delay in
puberty, suppressed or irregular estrus, failure of fertilization, early embryonic
death, still birth with weak calves, abortion, and increased frequency of retained
placenta in females and decrease in libido and deterioration of semen quality in
males. Inadequate thyroid function reduces conception rate and ovarian activity.
Thus, iodine deficiency impairs reproduction and iodine supplementation has been
recommended when necessary to insure that cows consume 15-20 mg of iodine each
day. Recently, Excessive iodine intakes have been associated with various health

33 
 
 problems including abortion and decreased resistance to infection and disease. Signs
of subclinical iodine deficiency in breeding females include suppressed estrus,
abortions, still births, increased frequency of retained placentas and extended
gestation periods. Calves born to cows that are marginally deficient in iodine are
weak and may be hairless.

Cobalt (Co): Co is needed for proper vitamin B12 synthesis. Maintaining adequate
vitamin B12 status benefits both the dam and offspring. When adequate, sufficient
amounts of vitamin B12 cross the placenta and are present in colostrums. Milk and
colostrums in particular, contain high levels of vitamin B12 which is required for
the conversion of propionate to glucose and for folic acid metabolism. Depletion of
cobalt and vitamin B12 at parturition causes depressed milk production and
colostrums yield and quality. Cobalt deficiency is associated with an increased
incidence of silent heats, a delayed onset of puberty, nonfunctional ovaries, and
abortion. Inadequate cobalt levels in the diet have been correlated with increased
early calf mortality. A cobalt deficiency ultimately results in a vitamin B12
deficiency. Manganese, zinc, iodine and monensin may reduce cobalt deficiency.
The required dietary content of cobalt for dairy cattle is 0.11 ppm.

Potassium (K): K is the third most abundant mineral element in the animal body,
surpassed only by calcium and phosphorus. Potassium concentrations in cells
exceed the concentration of sodium Na by 20 to 30 times. Outside the cell the
reverse is true. Potassium comprises about 5 percent of the total mineral content of
the body. Potassium (K) is an essential cation included in ration formulations for
dairy cows. Feeding high levels of potassium may delay the onset of puberty, delay
ovulation, impair corpus luteum development and increase the incidence of
anestrous in heifers. A lower fertility in cows fed with high levels of potassium or
diets in which the potassium-sodium ratio was too wide. The K requirement for
gestating and lactating sows is 0.20 percent. Potassium requirement increases in
diets with higher Na and chloride (Cl) levels. Ruminants have a higher K
requirement than nonruminants. Potassium is essential for rumen microorganisms.
The single most consistent effect of suboptimal K in the ration of ruminants is
decreased feed intake. Lactating dairy cattle, particularly high producing cows,
require the highest levels of dietary K. Under heat stress, their optimal level of
dietary K can be as high as 1.9 percent, but the normal National Research Council
(NRC) recommendation is 1.0 percent of dietary dry matter.
Chromium (Cr): Cr potentiates insulin action, resulting in increased uptake of
glucose and amino acids by cells in the body. Dietary energy intake in early
lactation does not meet energy demands for maintaining body reserves and milk
yield. Low serum insulin, high serum glucagon and growth hormone, and high
plasma NEFA concentrations in early lactation dairy cows indicate high catabolic
activities and negative energy balance. This leads to increased gluconeogenesis and
glycogenolysis in the liver, and increased mobilization of protein reserves from
muscle tissue. This metabolic pattern is initiated before parturition. Several studies
reported insulin resistance begins before parturition and continues during early
lactation. Thus, during the periparturient period, insulin resistance may be an
important factor in the initiation of catabolic activities. Chromium supplementation
(0.5 ppm) may enhance the action of insulin and, consequently, decrease plasma
NEFA and liver triglyceride concentrations and improve glucose tolerance, which

34 
 
 may result in improvement of performance and production during the periparturient
period.

Salt (NaCl): The daily salt requirements for dairy cattle are met easily by adding 1
percent salt to a grain mix and offering additional salt free choice. Lactating cows
need 2 g salt/kg milk production. Dry cows need 40g salt daily or 0.3% Na per kg
DM. Salt deficiencies can affect the efficiency of digestion and indirectly the
reproduction performance of cows. Sodium and chloride normally do not appear in
feedstuffs in adequate amounts to meet animal requirements and should be provided
free choice at all times.

Vitamins: The vitamin requirements of dairy cows are met by a combination of

rumen and tissue synthesis, natural feeds and dietary supplementation. Factory made
concentrates containing supplemental vitamins greatly reduces infertility due to a
vitamin deficiency. If commercial concentrates are not fortified with vitamin
supplements than extra vitamin must be provided in dairy cow rations when feed
intake is restricted and (or) low quality forage is fed to control or reduce body
condition. To ensure adequate vitamins intake animals must be fed with
concentrates or mixed in a complete dairy cow ration.
Vitamin A: it is one of the fat soluble vitamins and is known to regulate the
development, cellular growth and differentiation, and tissue function. Its metabolites
affect ovarian follicular growth, uterine environments and oocyte maturation.
Vitamin A is required for maintaining healthy tissue in the reproductive tract. In
deficient cattle, delayed sexual maturity, abortion, the birth of dead or weak calves,
retained placenta and metritis have been reported. A deficiency of vitamin A has a
direct effect on the structure and function of pituitary gland, gonads and uterus. The
recommended daily supplementation for dairy cows is 35,000-45,000 units. One mg
of carotene provides 400 IU vitamin A in dairy ration. Carotene is known to be in
high concentrations in fresh green roughages while grains contain relatively low
amounts. Silages, especially alfalfa, contain moderate levels while corn silage is a
poor source. Dry hays, especially alfalfa, are excellent sources of carotene.

Vitamin D: Vitamin D contributes to more than calcium and bone formation in

cattle. The active vitamin D hormone also contributes to immune, reproductive, and
mammary physiology. Multiple tissues and factors also contribute to vitamin D
activity. A concentrates containing supplemental vitamin D in amounts sufficient
should be provided to meet the cow’s requirement of 10,000 IU per day.

Vitamin E: Vitamin E functions as an intra-cellular antioxidant scavenging for free

reactive oxygen and lipid hydroperoxidases, and converting them to non-reactive
forms, thus maintaining the integrity of membrane phospholipids against oxidative
damage and peroxidation. In vitamin E and selenium deficiency condition, these
free radicals accumulate and not only damage cell membranes, but also disrupt
several processes linked to the synthesis of steroids, prostaglandins, sperm motility
and the development of the embryo. The current NRC (2001) requirement for dairy
cows is 15 IU/kg of DMI (approximately 150 and 300 IU/d for dry and lactating
cows, respectively). The vitamin E ( -tocopherol) status of dairy cows is one
important component of a well-functioning immune system because of its
antioxidant effects on cows. Feeding vitamin E during dry periods until 30 days
after calving resulted in 80% decrease in clinical mastitis and 60% reduction in

35 
 
 intramammary infections. Supplementation of vitamin E at 1,500 IU daily from 60
day prepartum to 30 day post partum to buffaloes exhibited beneficial effect on
plasma -tocopherol level and total antioxidant activity around parturition and
continuation of its supplementation at 1,000 IU d-1 from 30 to 60 days of lactation
improved post partum reproductive performance of buffaloes.

Conclusion
All nutrients have direct or indirect role in reproduction of dairy animals.
Supplementation of deficient nutrients in ration of dairy cow not only improves
productivity but also reproduction. Deficiency is an area specific problem thus
adjustment in diet must be made according feeds and fodders available, type of
animals, breed or species. A sound knowledge of nutrient requirement of species in
question will help in reducing reproductive problems.

* * * * * * * 
   

36 
 
 Handling of frozen semen at artificial insemination center
Sarvajeet Yadav and Dilip Kumar Swain

Frozen semen technology in modern day AI practices uses straws to preserve the
suitable and superior germplasm for a long period of time in liquid N2.The straw system
not only allows complete delivery of semen during insemination but also permits more
uniform control of the freezing and thawing process as a consequence to an improved
sperm cell recovery. In spite of these advantages, the straw system exhibits limitations in
term of its mishandling or poor handling at AI centers and a consequent reduction in
conception rates have been achieved. A sperm to accomplish the process of fertilization
must be intact, viable and should not have incurred any structural and functional damage
during freezing and thawing process.
The major objectives of semen freezing are-
9 To preserve the sperms at an ultralow temperature of -1960C.
9 To metabolically cease the activities of the sperms.
9 To make the sperms metabolically quiescent along with no transcription and
translation.
9 Long term storage of the sperms with a very less structural and functional
damage.
9 To carry out AI of proven animal sperm.
9 To disseminate the superior germplasm at a faster pace.
9 To conserve the genetic resources of endangered animals.
9 Better utilization of semen and prevention of semen loss.
9 Genetic upgradation of local breeds.
9
The major objectives of thawing are-
9 To bring the sperms to incubation temperature.
9 To make the sperms metabolically active and efficient.
9 To achieve dynamic activities of the sperms in terms of its motility and fertility.
9 To achieve the best possible conception rates after AI.
9 To make the sperms compatible for the process of AI.

STRAWS AND FREEZING OF SEMEN

Straws are the most convenient structures used for the process of semen
cryopreservation. Two types of straws are usually used for storage of semen namely
French midi (0.5 ml) and French mini straws (0.25ml). The 0.5 ml French straws were
the most popular in past, but now a days, the 0.25 ml straws are more popular and
cherished everywhere. Each Package system has a different surface to-volume ratio
which requires unique handling practices so as to prevent damage to the sperms.
Recommendations for handling semen vary, thus whole set of circumstances has resulted
in confusion for veterinarians, inseminators and, in some cases, fostered the notion that
almost any method of handling semen is adequate. Whether this attitude comes from
indifference, ignorance, or confusion, but the end result may be lowered conception
rates. That is why a proper understanding of straws and the mode of freezing practices
should be well understood to achieve the maximum possible conception after AI.

37 
 
WHY HANDLING OF SEMEN NEED TO BE UNDERTOOD
9 To achieve the best quality sperms after freezing and thawing.
9 To optimize the freezing of semen.
9 To prevent the irreversible damages those occur to the sperms during freezing.
9 To enhance the life qualities of the sperms.
9 To enhance the sperm storage as well as to avoid the damages incurred to the
sperms due to ignorance of the persons dealing with semen at AI centers.

Semen storage Tank Management:

The semen storage tank is actually a large, metal, vacuum sealed bottle encased
within an extremely efficient insulation system. The vacuum bottle construction allows
for maintenance of -3200F (liquid nitrogen temperature) when at least 2 inches of liquid
nitrogen are present. Technical advances in design and construction have produced
storage tanks with liquid nitrogen holding times of six to nine months. The maintenance
of very low liquid nitrogen temperature in the inner chamber is due to high quality solid
insulation material and vacuum in the outer chamber. Although the newer tanks are
better insulated, they are still susceptible to damage from mishandling. The inner
chamber containing liquid nitrogen is actually suspended from the outer shell by the neck
tube. Abnormal stress on the neck tube caused by sudden jarring or excessive swinging
motion could crack the tube and result in vacuum loss. Since vacuum is the major
insulation component of the tank, a loss of vacuum causes an increase in temperature
within the inner chamber and a rapid evaporation of nitrogen. Accumulation of frost at
the top of the tank indicates a rapid evaporation of liquid nitrogen. Finally, frost indicates
that the vacuum insulation has been lost and liquid nitrogen has been or is evaporating
rapidly. If this should happen, use a wooden yardstick to measure the amount of liquid
in the tank. If the tank still contains liquid nitrogen, the semen is probably still good but
should be transferred to a good tank immediately. Should the tank be empty of liquid
nitrogen, it is doubtful that the sperms are viable.
Although semen storage tanks are well constructed, they are still susceptible to
damage from mishandling. To avoid follow these simple management practices:
9 Avoid excessive movement or abuse of the tank.
9 Monitor nitrogen levels routinely and keep a record of nitrogen loss. Remember,
even new tanks can have defects and fail.
9 Store the semen in an area with good light but out of direct sunlight.
9 Observe the tank daily.
9 Once a tank fails, nitrogen is lost very rapidly. Have an alternative semen tank
available in case of a failure.
9 Keep the tank elevated above the concrete floor or other wet and poorly
ventilated surfaces.
9 Corrosion of the outer shell will shorten the functional life of the tank and
possibly cause failure.
9 Store only the amount of semen needed for six months.
9 Always be watchful for a lid that is left off and for frost or sweat on the tank.
9 Give particular attention to the neck and vacuum fitting.

38 
 
CR
ROSS SECT
TION OF SEMEN
S TA
ANK
A. Durable, taamper prooff lid design
B. Easy mainttenance lid design
d
C. Superior sttrength, lighhtweight aluuminum con
nstruction
D. High strenngth neck tubbe reduces liquid nitrogen loss
E. Locking
L tabb F. Color-ccoded canistter/lid numb
bering
sysstem
G. Advanced Chemical
C V
Vacuum Rettention Systtem--
dessigned for suuperior vacuum perforrmance overr the life of the productt
H. Spider desiign-- for eassy retrieval and insertio
on of producct canisters
J. Insulation--
I MVE's advvanced insullation system provides maximum thermal
perrformance
Haandling sem men with in tank:
In the typical farmm semen tannk, dangero
ously high temperatures exist in th
he upper
hallf of the necck tube (Figgure 1).
Conversioon table:
+54 °F = 12
1 °C
+36°F = 2°C
+5°F = -155°C
-8°F = -222°C
-40°F = -440 °C
-51°F = -446 °C
-103°F = -75
- °C
-116°F = -82
- °C
-148°F = -100
- °C
-184°F = -120
- °C
-220°F = -140
- °C
-256°F = -160
- °C
-292°F = -180
- °C Fig 1
1: Cross sectiion of semen
n tank and vaarious 
-313°F = -191
- °C temperature
t e gradients across the tan
nk 
-320°F = -196
- °C

Table 1
1: Various teemperature ggradients acrross the tankk 

39 
 
 Frozen semen can be stored indefinitely if it is maintained constantly at very low
temperature. The critical temperature is -80oC. Frozen semen that is exposed to
temperatures warmer than -80oC (even for a short period of time) and then returned to
the tank may be damaged. The extent of damage depends upon how long the semen is
exposed to elevated temperatures. Although it is easy to maintain frozen semen at a safe
temperature, simultaneously, it is also easy to destroy its quality in a few moments of
carelessness.
When extended semen cools during the freezing process, microenvironments are
created within the semen package. Each chemical component of extended semen freezes
or solidifies at a different temperature. Water freezes as temperatures drop below 4ºC
forming ice crystals which remain somewhat unstable at temperatures above -80ºC. This
instability may be due to recrystallization of the ice. Also, as water is converted to ice,
the sperms are exposed to the remaining concentrated solution of salts and other
components of extender which freeze at temperatures considerably below the freezing
point of water. Instability of ice and concentrated solutions are harmful to sperm, and to
prevent this glycerol as a cryoprotective agent has been used and has shown
improvement in sperm storage and minimal loss to the sperms. However, semen must be
kept at temperatures well below critical temperatures where the recrystallization of ice
begins to occur.
In the typical farm semen tank, dangerous temperatures exist in the upper half of
the neck tube. Exposure to these temperatures can occur when semen is transferred from
tank to tank or when handling semen within the neck while trying to locate and thaw a
specific unit of semen. Remember, the larger surface-to-volume ratio of the straw makes
it very susceptible to thermal fluctuation. Transfer of semen between tanks must be
coordinated and rapid. Two people should be involved. Raise canisters only to a level
necessary to locate the appropriate rack of semen. Thermal injury to sperm is
permanent and cannot be corrected by returning semen to liquid nitrogen.
Tips to follow when transferring semen between semen tanks:
9 Have the tanks side by side and as close as possible. If possible,
fill the tanks with nitrogen before the transfer.
9 Have the appropriate canister in each tank in the center position.
9 Transfer the racks quickly (within 3 to 5 seconds). Never touch
the units of semen with bare fingers.

40 
 
Straw retrieval and thawing:
When the 0.5 ml French straw was first introduced in this country, there was
confusion about the optimal thawing method. Recommendations varied among AI
organizations, each of which has a specific method for diluting, cooling, packaging, and
freezing semen in straws. The total processing system determines the optimal rate of
thaw. As a result of considerable research, it is generally concluded that warm water
thaw (37ºC) results in improved sperm cell recovery compared with other methods of
thawing. Success of warm water thaw is due to the fact that sperm are exposed to
critically dangerous temperatures for only a brief amount of time. The rise in temperature
is rapid enough to minimize sperm damage.
A major criticism and concern for the warm water thaw is the danger of heat
shock caused by mishandling the straw following thawing. Heat shock is the permanent
injury to sperm caused by a sudden increase in semen temperature after thawing. It can
occur during preparation of the inseminating device or transport to the cow. If
precautions are taken to prevent heat shock, the advantage of warm water thaw will be
realized. It is important that the temperature of the thaw water be checked immediately
before removing the straw from the tank. Use an accurate, easy-to-read thermometer.
The length of thaw should be at least 35 to 40 seconds.
Some organizations recommend the pocket thaw for straws. This method is
successful for semen processed and packaged by their system. However, the pocket thaw
should not be used for semen packaged in straws from other organizations. The standard
1 ml ampules should be thawed in ice water (5º C) for 10 minutes. The smaller 0.5 ml
ampules can be thawed in warm water for 90 seconds or ice water for three to five
minutes. Semen should be thawed according to the recommendations of the organization
supplying that specific unit of semen.)
Semen may be exposed to these temperatures(as above in fig.) when you try to
locate and thaw a specific unit of semen or when you transfer semen from tank to tank.
The following semen handling practices are recommended to minimize thermal damage:

9 Identify which canister contains the desired semen. A semen inventory

that keeps track of the location of each bull prevents unnecessary
searching that increases semen exposure.
9 Remove the canister from its storage position and raise it until the cane
tops are two to three inches below the top opening of the tank. Locate the
cane you want.
9 On new canes, bend the identification tab up to a 45 degree angle. This
will keep the straw from bending or breaking.
9 Use the forceps to remove one straw at a time from the top goblet. Shake
the straw gently to remove any liquid nitrogen remaining in the straw tip.
The straw should be removed within 10 seconds from the time the
canister is raised into position.
9 Immediately after the unit of semen is immersed in water, return the rack
to the canister
9 Place the straw in 35- 37o C water to thaw for at least 40 seconds.
9 A rule of thumb is to thaw only as many straws as you can use in 10 to 15
minutes. In an emergency situation, a 15 to 20 minute interval between
thawing and insemination will not lower semen quality a great deal.
However, you must maintain the temperature near 35-37o for that time.

41 
 
 9 Avoid thawing more than three straws at once to maintain the correct
thaw water temperature.
9 After all straws in top goblet have been used, remove the goblet from the
cane and discard it.
9 To retrieve straws from the bottom goblet, grasp the cane and raise it until
the straws are even with other cane tops. Hold the cane and canister with
one hand. With your forceps in the other hand, remove the desired straw
and place it in the thaw water. When all the straws are used from the
bottom goblet, remove and discard the cane and goblet. Return the
canister to its storage position immediately after straw removal.

Tips for optimal Thawing:

9 Always keep insemination equipment clean, dry, and warm.
9 Use a thermometer and don't guess at the temperature. Check the
thermometer for accuracy at least every six months with a reference
thermometer.
9 Use an insulated water bath designed for thawing semen, or wide mouth
thermos that is deep enough to immerse the entire straw. Recently,
electronic thawing devices have been developed; these maintain water
temperature accurately between 95o and 98oF. These are convenient to
use when breeding many cows at one time.
9 Never thaw more than one unit of semen at a time. You breed cows
individually; you should thaw units of semen individually.
9 Gently shake the straw as it is taken from the tank to remove any liquid
nitrogen that may be retained in the cotton plug end of the straw.
9 Time the thaw with a watch to avoid guessing.

Handling Semen after Thawing and During Insemination:

Semen is most frequently damaged during handling after it has been thawed and
is being transported to the cow. After thawing, semen temperature must be maintained
as close to 37oC as possible. The handling of thawed semen and preparation of the
insemination rod should be in a sheltered area that can be heated in cold weather. The
following practices will minimize semen damage that commonly occurs with improper
semen handling procedures.
9 While the semen is thawing, warm the insemination rod by rubbing it
briskly with a paper towel. In cold weather, place the warm rod within
clothing so it will be close to your body and maintain its warmth.
9 After the semen is thawed for the required time, dry the straw thoroughly
with a paper towel and protect it from rapid cooling.
9 Adjust the air space in the straw to assure that no semen is lost when the
end of the straw is cut off. This can be done by slightly flicking the wrist
while holding the straw at the crimp sealed end.
9 Transfer the straw to the rod, and cut the tip of the crimp sealed end of
the straw squarely and through the air space. Only sharp scissors or a
specially designed straw cutter should be used to cut the straw. Make
sure to cut the straw "square" to achieve a good seal with the sheath.
9 Wrap the assembled insemination rod in a clean, dry paper towel and
tuck it within your clothing for transport to the cow. Do not place the
rod in your mouth or carry it uncovered in your hand.

42 
 
 9 Inseminate the cow within minutes after the semen has been thawed.
The period of time between removing the semen from the tank and
depositing the semen in the cow should not exceed 15 minutes.

Temperature control after thawing:

Since the straws are thawed in 370C water, you must be able to provide thermal
protection, especially on cold days. Never allow semen to refreeze. Try to keep the
semen temperature near 37 degrees, while you are loading the gun and preparing to
inseminate the cow. Our research has shown that the temperature of semen will change
to the level of environmental temperature in about 30 seconds if held in an unprotected
gun. During hot weather, before loading the straw make sure the insemination gun is not
so hot that it will cook the semen. For maximum thermal protection during cold weather,
the use of individually packaged sheaths is recommended in the following procedure.
9 Remove a sheath from the breeding kit with it still in its individual
package. Insert it down in your shirt for a minute until it's warm. Push
the large open end of the sheath out through the side of the package.
9 Prewarm the gun by stroking it vigorously with your hand five or six
times.
9 Hold the gun tip in your hand to keep it warm.
9 Insert the straw in the gun and cut the straw tip.
9 Remove the warmed sheath from your shirt and quickly assemble the gun,
straw and sheath while the sheath remains in its package.
9 Reinsert the loaded gun back down inside your shirt.
9 When you have the animal secured, your arm inserted in her rectum and
her vulva cleaned and open, remove the gun from the package in your
shirt quickly and insert it into the vagina.
9 This procedure not only thermally protects the semen, but also
hygienically protects the sheath until insemination. An alternative to using
individually packaged sheaths is to wrap the loaded gun with a clean
paper towel.
9 Done correctly, this procedure too, provides thermal and hygienic
protection of semen and equipment.

Insemination Gun Loading:

9 Choose a sheltered area for loading the guns and inseminating cattle to
help curb undesirable semen temperature fluctuations.
9 Before loading the insemination run, warm the barrel if necessary and pull
the plunger back about six inches.
9 Partially remove a plastic breeding sheath from the container so it is easy
to grasp when needed. Make certain no crack exists at the breeding end.
9 Remove the straw from the thaw water and wipe it completely dry with a
paper towel.
9 Check to see that it contains semen from the desired bull and that a small
bubble is at the crimped end.
9 If the bubble is not at the crimped end, gently tap the straw until the
bubble moves to its correct position.
9 If you cannot read the printing on the straw, do not use it. Later, ask your
semen supplier for a replacement.

43 
 
 9 Place the cotton plug end of the straw in the gun. It will stop at exactly
the right depth.
9 Wipe the scissors with a paper towel and cut off the strap 1/4" below the
lab seal. Make a right angle cut to the straw.
9 Slide the plastic sheath over the straw and gun. Be sure the white plastic
ring is half way on the gun. If it is not, it may become tight before the
sheath is all the way on the gun.
9 Firmly tighten the white ring with a twisting motion.

Insemination Equipment:
A liquid nitrogen tank with one or more canister is needed to store straws. In
addition, the following equipment is recommended to insure proper straw handling and
insemination procedures. This equipment should be stored in a clean, dry stainless steel
breeding kit.
9 A set of forceps are recommended for removing straws from the tank.
There are several good types of forceps available.
9 A one pint, insulated, wide mouthed thermos with a dial thermometer is
regularly used to hold thaw water.
9 A good pair of sharp stainless steel scissors are recommended to cut
straws.
9 A French Straw Gun for insemination is hollow with a plunger to expel
the semen.
9 A plastic disposable sheath covers the loaded gun. Sheaths are available
bulk or individually packaged.
9 A small plastic "O" ring on the gun locks the gun and sheath together.
9 A package of paper towels will have many uses during insemination.

Equipment maintenance:
9 Check the thermometer you use in the thaw water occasionally with a
standard mercury or alcohol thermometer. This confirms that its
calibration is correct.
9 Never use an "O" ring gun without the "O" ring.
9 Sheaths left in an extremely hot place such as the dash of an automobile
will warp and expand. Extremely cold sheaths may crack and shatter
when loading the gun. Sheaths with these conditions should not be used.
9 Store your nitrogen semen tank on a wooden pallet in a dry environment.
The nitrogen use rate of your semen storage tank should be measured
periodically. If a tank is losing efficiency, it should be replaced.

Double sheath insemination for disease control:

If vaginitis or urea plasma is a diagnosed problem in your herd, individually
packaged sheaths can be used in a double sheath insemination procedure. This
procedure reduces the number of bacteria carried from the vagina into the uterus during
insemination and may increase fertility. To double sheath breed cows, use the following
procedures:
9 Keep the plastic sheath package on the loaded gun as you withdraw it
from your shirt.
9 Insert the double sheathed gun through the vagina to the point where it
joins the cervix.

44 
 
 9 Pull the package back on the gun through the cervix and deposit semen as
you normally do.

Clean up:
After insemination, there are several clean-up steps you will want to follow.
9 Inspect the gun tip for signs of infection. Make a note for your subject
matter specialist if you see any.
9 Use your thumb and forefinger to snap the white plastic "O" ring loose.
9 Bend the sheath and straw tip at a 90 degree angle, and remove these from
the gun.
9 Tighten the "O" ring back on the gun so it will not get lost.
9 Double check the bull's identity on the straws.
9 Reverse strips your glove so the straw, sheath and manure are trapped
inside. Squeeze out any excess air, tied a knot in the open end and
dispose of the glove.
9 Wipe the gun clean and dry it before returning it to the insemination kit.
9 If the gun becomes dirty after use, take it completely apart, wash with
soapy water and rinse with clean water. Shake the gun dry and
reassemble it.

Inseminating a group of synchronized cattle:

Breeding a group of cattle during a short period of time following estrous
synchronization may present problems for the herd manager and Al technician. Although
artificial insemination of synchronized cows is the final step in a synchronization
breeding program, it is critical to the success of the entire program. It is important that
special consideration be given to proper semen handling and insemination technique to
ensure optimum conception rate.
1. Personnel: Assign each person a job to move the breeding phase of this program
along efficiently. One individual should be responsible for thawing semen and
preparing the inseminating gun. This relieves the AI technician of additional tasks,
allowing concentration on proper AI technique. Additional people could move cattle
to and from the breeding chute. Everyone should be thoroughly instructed in their
specific jobs prior to the breeding.
2. Physical facilities: Ensure an adequate holding area is available where heifers can be
assembled for treatment, heat detection, and breeding. This area should have a
breeding chute or similar arrangement where animals can be treated and artificially
bred in a safe and efficient manner, If possible, the synchronized animals should be
observed for heat following hormone treatment. This would require that the holding
area be equipped with sufficient feed and watering facilities for approximately five
days until all synchronized cows are bred. The breeding chute area should be covered
to protect semen, supplies, records, and personnel from adverse weather.
3. Procedures:
A. Prepare a list of selected matings if numerous AI sires are to be used for
breeding the synchronized cattle. This list could be used in selecting which unit of semen
is to be thawed and inseminated for each particular cow. An inventory system describing
the location of semen from each bull within the semen tank is also desirable.
B. Follow thaw procedures according to the recommendations of the AI
organization supplying the semen. The thaw water must be maintained at the proper
temperature for each dose of semen thawed. NOTE: recent research from Washington

45 
 
State has shown that 10 to 20 straws can be thawed simultaneously as long as the thaw
bath water remains a constant 95ºF and the environmental temperature is not severely
cold. Straws thawed in bulk should be agitated slightly to keep them from sticking
together. Bulk thawing of semen should only be considered when a large group of
synchronized cattle are to be inseminated.
C. Do not prepare the insemination gun too far in advance of insemination. Breed
the cow as soon as possible after the semen is properly thawed and the inseminating
equipment is assembled.
D. Prepare insemination devices in a warm, clean environment near the breeding
chute, but far enough away to avoid excessive dust and debris near the cattle. This will
minimize the chance of contaminating the equipment and semen.
4. Other considerations:
A. Handle animals gently to avoid unnecessary excitement before, during, and
after breeding. Undue excitement may adversely affect sperm transport within
the female reproductive system causing a lower conception rate.
B. Breed animals based on standing heat, remembering to breed the animals 10 to
12 hours after the beginning of standing behavior.
C. Use proper insemination techniques. Sufficiently qualified inseminators
should be on hand if many cows are to be bred over a short period of time.
Consult your Al representative for advice and help in this regard.
The success of a synchronization-breeding program depends on prior planning,
teamwork, and attention to detail by everyone involved.

Where is the weakest link in your pregnancy equation?

When you A.I. bred three cows last evening, what was the probability that they
settled? Will they deliver a calf in about 282 days? What is your probability of success?
There are many variables affecting the outcome of each insemination. If any one
component is limiting, then your A.I. breeding vehicle is not hitting on all cylinders.
Let's identify some of those factors and determine how you cna raise the odds for
pregnancy each time you breed an animal. There are at least four major pieces to the
puzzle: percentage of cows detected in heat, herd fertility, semen fertility and
inseminator proficiency.

Four factors influencing the pregnancy equation

% % % % Number
detected herd semen Inseminator of calves
Case
in heat fertility fertility proficiency born
(A) x (B) x (C) x (D) = (E)

Nearly ideal situation 80 90 95 100 68

Poor heat detection 50 90 95 100 43

Poor inseminator proficiency 80 90 95 75 43

46 
 
Poor inseminator proficiency and
50 90 95 75 32
heat detection

Poor herd fertility 80 50 95 100 38

Poor herd fertility and heat detection 50 50 95 100 24

Pregnancy outcome (A x B x C x D = E); E = per 100 cows

Concluding Remarks
Semen freezing is a gift for the modern day breeding of animals and in
specific for bovines. Lots have been achieved through the frozen semen
technology and many more has to be achieved in future. Lots have been
developed in terms of semen freezing, thawing, extenders, semen tanks and
freezing protocols but still we have not achieved the best possible response out of
freezing. Poor semen handing at AI centers may be one of the most common
factor behind this poor conception rates. The paper discussed some of the quality
control check points for the handling of semen at AI centers and urged their
implementation at a strict basis. Much has to be learned in future regarding the
handling but time demands a strict follow in the instructions to carry out
successful insemination of animals with best quality sperms. Proper training and
education to the veterinarians as well as persons dealing with semen handling
will improve the quality of semen as well as the conception rates in cows. This
will also increase the economy of AI in cows in achieving optimal fertility in
cows.

* * * * * * * 
   

47 
 
 Nutritional management of peri-parturient animals
Shanker K. Singh and Dilip Kumar Swain

INTRODUCTION
Large improvements have occurred in the production and efficiency of the dairy
cow during the last 3 decades worldwide. However, based on veterinary records, these
improvements have unfortunately not been followed by simultaneous improvements in
the disease incidence. Therefore, it is highly relevant to focus on how we may monitor
individual cows to improve animal health.
The transition period is the most critical period in the productive cycle of dairy
cows. This period for parturient dairy cows, which lasts from approximately 3 weeks
before calving to approximately 3 weeks after, is a time during which the cows
experience severe negative energy balance (NEB) due to decreased dry matter intake and
increased energy demand for lactation. The term transition is to underscore the important
physiological, metabolic, and nutritional changes occurring in this time frame. It
constitutes a turning point in the productive cycle of the cow from one lactation to the
next. A number of profound physiologic changes occur in the transition cow that
modifies her metabolism drastically. During the transition from late gestation through
early lactation, most dairy cows undergo major changes in e.g. endocrine regulation that
cause extensive mobilization of body tissue, primarily adipose tissue, in order to meet the
nutrient demands for maintenance and milk production are characterized by increased
circulating concentrations of e.g. non-esterified fatty acids (NEFA) released primarily
from adipose tissue, increased circulating concentrations of beta-hydroxybutyrate (BHB)
reflecting incomplete fatty acid oxidation in the liver, and decreased concentrations of
glucose in the bloodstream. The rapidly increasing demands of the fetus and the
development of the mammary glands, including the initiation of synthesis of milk
components, are causing these changes. The manner in which these changes occur and
how they are managed are of great importance as they are closely linked to lactation
performance, clinical and subclinical postpartum diseases, and reproductive performance
that can significantly affect profitability.
A smooth transition from the late dry period through early lactation has profound
impacts on the success of both the lactation and conception. Most problems that herds
experience during transition manifest as metabolic disease issues, or stem from them.
High incidence rates of retained placenta, metritis, ketosis and displaced abomasums can
be obvious reasons for poor herd performance. Therefore, there is a need to carefully
monitor various factors such as management, adequate feed computation, proper balance
between energy and protein, stress free environment to make the transition period from
dry to the lactation stage as smooth as possible. The present manuscript discusses the
various problems and their effective means required during this period.
PERIPARTURIENT DISORDERS
The period from calving to the first few weeks of lactation is associated with a
high incidence of metabolic and infectious diseases that include milk fever, abomasal
displacement, ketosis, fatty liver syndrome, dystocia, retained fetal membranes, metritis
and mastitis. These diseases are interrelated and one is frequently a risk factor for

48 
 
another. The disorders are further classified in to following subcategories depending on
the causative factor associated with that. The disorders are summarized as follows.
1. METABOLIC DYSFUNCTIONS DURING TRANSITION
Most problems that herds experience during transition manifest as metabolic
disease issues. High incidence rates of retained placenta, metritis, ketosis and displaced
abomasums can be obvious reasons for poor herd performance. Metabolic problems on
most herds usually have multiple component causes. These causes may include many
factors such as pen density or feed access issues, stressful grouping strategies, frequent
group changes, feed issues or body condition problems. Effective monitoring programs
can not only help identify problems but can be used to track the success of management
changes. Major metabolic transitions occur in a cow’s body as she converts from a non-
lactating to a lactating state and undergoes the stress of parturition. Management
transitions such as pen moves and ration changes are additionally occurring. Cows often
fail to adapt to these metabolic and management changes, resulting in 75% of dairy cow
disease incidence during the first month after calving and substantial economic losses to
the dairy industry. Among all of the factors, the role of negative energy balance is
predominant that leads to the occurrence of metabolic disorders.
2. NEGATIVE ENERGY BALANCE AND PRODUCTION DISEASES
The periparturient dairy cow transitioning from pregnancy into lactation,
experiences a dramatic increase in nutrient requirements that cannot be met by feed
intake alone. Thus, the animal experiences a period of negative energy balance and must
mobilize body tissue lipid and protein in order to sustain productive function. Estimates
of demand for glucose, amino acids, fatty acids, and net energy by the gravid uterus at
250 d of gestation in comparison with the lactating mammary gland at 4 d postpartum
indicate approximately a tripling of demand for glucose, a doubling of demand for amino
acids, and approximately a 5-fold increase in demand for fatty acids during this time
frame. Furthermore, the requirement for Ca increases approximately four-fold on the day
of parturition. Delicate coordination of hepatic gluconeogenesis, fatty acid, and calcium
metabolism are of paramount importance if the animal is to avoid metabolic dysfunction.
During the period of periparturient negative energy balance, body fat is
mobilized into the bloodstream in the form of non esterified fatty acids (NEFA). This
circulating NEFA released from adipose tissue enter the liver and have three fates: 1)
they can be completely oxidized for energy via the Kreb’s cycle, 2) converted to BHB, or
3) they can be re-synthesized to triglycerides (TG) where they can either be exported via
very low density lipoproteins (VLDL), or they can be stored in the liver. The liver of
cattle has a very limited capacity for export of TG via VLDL, and therefore, especially
during early lactation, the rapid and extensive influx of circulating NEFA into the liver
can result in a buildup of TG as well as an increased export of BHB into circulation.
Nearly 50% of all cows experience an accumulation of liver TG at this time resulting
into the impairment of liver function.
3. HYPOCALCEMIA DURING TRANSITION
With the onset of copious milk secretion and its attendant drain on plasma
calcium concentrations, calcium homeostasis is maintained by the actions of parathyroid
hormone and 1,25-dihydroxy vitamin D3 via increased bone resorption and intestinal
absorption. During early lactation, these processes cannot fully meet the calcium needs

49 
 
secreted in milk and most cows experience negative calcium balance resulting in at least
some degree of hypocalcemia. Maintenance of calcium homeostasis is critical for many
functions including neuro-muscular excitability, blood clotting, and hormone secretion.
When plasma calcium becomes too low, nerve and muscle function is impaired, resulting
in parturient paresis, or milk fever. Cows with milk fever could suffer decreased milk
yield, lower feed intake and depressed chewing behavior, and increasedrisk of associated
diseases such as dystocia, retained placenta, ketosis, metritis, displaced abomasum, and
mastitis than health animals. Animals that do not develop milk fever efficiently mobilize
bone tissue to contribute to the circulating pools of calcium and phosphorus.
The large increase in calcium requirements also strains the regulatory
mechanisms of the transition cow. Calcium requirements can increase by more than 3-
fold on the very first day of lactation, and this drain continues as milk yield increases
much more rapidly than DMI. As a result, cows selected for high milk yield will nearly
always experience some decrease in available (ionized) blood calcium during the first
week of lactation. Although the adoption of anionic prepartum diets has been quite
successful at reducing the incidence of milk fever, subclinical hypocalcemia can occur
even with careful management of dietary cation-anion difference (DCAD).
Calcium is an important signal transducer in many other cell types, including
immune cells. It was demonstrated that monocytes from cows experiencing
hypocalcemia had low intracellular calcium stores and did not mobilize calcium to the
same extent in response to stimulation. An inability of monocytes to mobilize
intracellular calcium after being stimulated would be expected to dampen functional
responses such as cytokine release and cell proliferation. Such findings may provide a
physiological basis for the long-observed links between hypocalcemia and mastitis in
transition cows.
4. TRANSITION IMMUNOSUPRESSION
Natural immunosuppression occurs in most cows during very late pregnancy and
early lactation. Immune cells involved in both the innate and adaptive immune response
are altered during the transition period. The function of neutrophils, that are part of the
first line of defense against invading microorganisms, is reduced at this time including
chemotaxis, i.e. migration to the site of infection, phagocytosis, i.e. engulfment of the
invading microorganism, and oxidative burst, i.e. killing of the invading microorganism
during phagocytosis. Further, the number and proliferation of lymphocytes is reduced
during the transition period. The changing hormonal environment, especially increases in
circulating glucocorticoids at parturition, has been associated with the naturally
occurring immunosuppression observed at this time.
Host defense mechanisms are also compromised directly because of numerous
physiological and environmental factors during the transition period. Physiological
stresses associated with rapid differentiation of secretory parenchyma, intense mammary
gland growth, and the onset of copious milk synthesis and secretion are accompanied by
a high energy demand and an increased oxygen requirement. This increased oxygen
demand augments the production of oxygen-derived reactants, collectively termed
reactive oxygen species (ROS). Excessive production of free radicals and concomitant
damage at cellular and tissue levels are controlled by cellular antioxidants defense
systems. The oxidative damage to the leucocytes leads to immune dysfunctions and a
consequent immune suppression during transition.

50 
 
 Periparturient negative energy balance has also been implicated in contributing to
immunosuppression. However, negative energy balance alone had little effect on the
expression of adhesion molecules on the surface of bovine leukocytes. Research has
revealed that individual metabolic components associated with negative energy balance,
exacerbate periparturient immunosuppression. Hyperketonemia appears to have multiple
negative effects on aspects of immune function. Ketosis may increase the risk of mastitis
in periparturient immune suppressed cattle because many immune cell types are
negatively affected by metabolite levels typical of a ketotic environment (i.e., low
concentrations of glucose and high concentrations of ketone bodies and NEFA). A
ketotic environment suppressed bovine lymphocyte blastogenesis, decreased the
respiratory burst activity of PMN, lowered the chemotactic capacity of leukocytes,
decreased interferon- and tumor necrosis factor- titers from bovine aorta endothelial
cells, decreased the bactericidal activity of ovine neutrophils and inhibited human T-cell
proliferation in vitro. Furthermore, experimental mastitis in ketonemic cows was more
severe than mastitis in non-ketonemic cows regardless of pre infection chemotactic
response. Impairment of the udder defense mechanism in cows experiencing negative
energy balance seems to be related to hyperketonemia.
Reports of the negative effects of ketosis on immune function may be related to
or compounded by the impact of fatty liver on immune function. Triglyceride
accumulation in the liver is reported to have metabolic effects whereby hepatic ureagenic
and perhaps gluconeogenic capacity is reduced, but fatty liver also affects immune
function. Cows without fatty liver cleared bacterial endotoxin from circulation within 30
min of intravenous endotoxin administration, whereas cows with fatty liver were unable
to clear the administered endotoxin even after 6 h. PMN harvested from the blood or
uterus of periparturient cows with liver triglyceride greater than 40 mg/g had decreased
expression of functional surface molecules and had decreased in vitro functional capacity
(cytotoxicity and reactive oxygen species generation). Cows with fatty liver displayed
decreased neutrophil extravasation in vitro and are having a significantly longer time to
resolve intramammary infection than did cows without hepatic lipidosis.
Periparturient metabolism that has the potential to impact immune competence is
calcium metabolism. Significant quantities of calcium are required for milk synthesis and
an inadequate adaptation to this calcium sink at the onset of lactation results in
hypocalcemia (milk fever). Although it is important for milk synthesis, calcium is also
important for intracellular metabolism and signaling in most cell types, including the
leukocytes of the immune system. Realizing the importance of calcium in leukocyte
activation, low blood calcium around the time of calving could contribute to
periparturient immunosuppression. Recently it is reported that calcium stores in
mononuclear leukocytes are depleted prior to the development of hypocalcemia in the
blood, and that this depletion of intracellular calcium does potentially contribute to
immunosuppression. Interestingly, it appears that intracellular calcium stores are a more
sensitive measure of calcium stress than is blood calcium concentration.
Glucocorticoids (e.g. cortisol), known endocrine immunosuppressants, are
elevated around the time of calving, and have been postulated to be at least partly
responsible for periparturient immunosuppression. Furthermore, changes in estradiol and
progesterone just prior to calving may directly or indirectly affect immunocompetence.
However, changes in any of these steroid hormones do not overlap with the entire period
of immunosuppression, suggesting that other causes are at least partially responsible for
immune dysfunction.

51 
 
 5. GLUCOSE DURING TRANSITION
Glucose is required in large amounts during the transition period as a fuel for the
gravid uterus, mammary gland, peripheral tissues, central nervous system, red blood
cells, gastrointestinal tract, and also is absolutely required for synthesis of lactose, which
largely controls milk volume (Figure 1). It is supplied to the cow through digestion of
starch in the small intestine to glucose, which is then absorbed. At lower starch intakes,
most glucose derived from starch is catabolized by the gastrointestinal tract; therefore,
the cow is mostly dependent on gluconeogenesis in the liver and kidney to meet her
glucose needs. This is especially true during the transition period. Propionate derived
from ruminal fermentation is the principal substrate used to make glucose by liver
followed under normal conditions by amino acids, lactate, and glycerol.
Increased liver glucose production is another adaptation to lactation. Meeting the
increased demand for glucose during the transition period is especially challenging,
because little glucose is absorbed from the ruminant gastrointestinal tract. Over the
course of the first 2 months of lactation, liver glucose production increases by at least 2-
fold and most of this change likely occurs within a week after calving. Several studies
have found decreased capacity for gluconeogenesis in liver slices from cows with fatty
liver and others have shown that induced fatty liver results in decreased activities of
several rate-determining gluconeogenic enzymes. The ability of a cow to successfully
up-regulate gluconeogenesis in early lactation is critical to both avoid metabolic
problems (e.g. ketosis) and to maximize peak milk production, and the negative effect of
fatty liver on gluconeogenesis is one reason that this condition is of concern.
Amino acids make a variable, yet important contribution to gluconeogenesis. All
amino acids except for leucine and lysine can make a net contribution to
gluconeogenesis, and the nonessential amino acids alanine and glutamine have been
reported to be the most gluconeogenic of all amino acids. Similar to propionate,
utilization of amino acids for gluconeogenesis may be somewhat supply-dependent. This
makes sense because the liver is the main site of catabolism of amino acids that provided
in excess of those needed to make milk and body proteins.

Fig-1: Role of Glucose in Transition body metabolism

52 
 
 6. NEFA DURING TRANSITION
The drive to produce milk is given priority over nearly all other physiological
processes during this time, and a number of changes occur to partition nutrients to the
mammary gland. Negative energy balance and homeorhetic adaptations during the
transition to lactation decrease plasma insulin concentration substantially and also
decrease the responsiveness of adipose tissue to insulin. These adaptations lead to
dramatic increases in plasma non-esterified fatty acid (NEFA) concentration, and this
also leads to greater uptake of fatty acids by the liver. This increase in fatty acid supply
to the liver often exceeds the capacity for oxidation, resulting in both ketone body
production and storage of triglycerides. This process can occur quite rapidly, and cows
can develop moderate fatty liver and ketosis in the course of just a few days.
One such interaction is the effect of energy balance on immune function. Nearly
all transition cows experience at least 3 weeks of negative energy balance, a situation in
which they require more energy for maintenance and milk production than is consumed
from the diet. One response to this nutrient imbalance is rapid mobilization of adipose
tissue triglyceride, resulting in elevation of plasma NEFA concentrations by as much as
10-fold. Greatly elevated concentrations of NEFA often result in significant conversion
of NEFA to ketones (e.g. BHBA) in the liver. Recent work has demonstrated that
elevated NEFA concentrations may directly impair neutrophil viability and high BHBA
concentrations can decrease neutrophil function. These relationships may help to explain
at least some of the decrease in immune function during this time of negative energy
balance. Fatty acids are utilized as fuels for skeletal muscle, liver, and perhaps other
organs of the cow. Furthermore, approximately 50% of fatty acids found in milk fat
come from either the diet or from lipoprotein triglycerides in blood. Fatty acids used by
the mammary gland during synthesis of milk fat also can be provided by NEFA in the
bloods that are released during mobilization of adipose tissue (Figure 2).

Fig-2: Role of NEFA in Transition Metabolism

40% of fatty acids in milk fat during the first week of lactation may come from
blood NEFA. Inadequate energy intake to meet energy demands of the cow results in
mobilization of adipose tissue and release of NEFA into the blood; therefore, DMI and
plasma NEFA concentrations usually are inversely related. The major challenge from a
nutritional and managerial standpoint is to minimize both the height and the duration of
the spike in plasma NEFA at and following calving. The liver takes up NEFA in
proportion to their concentration in plasma, but typically does not have sufficient

53 
 
capacity to completely dispose of NEFA through export into the blood or catabolism for
energy. When nutrient intake is insufficient and large amounts of NEFA are released into
the blood, the liver begins to accumulate and store NEFA as triglycerides, thus leading to
fatty liver in varying degrees. A second problem occurs when supply of gluconeogenic
precursors (to supply intermediates for intracellular metabolism) is insufficient to fully
catabolize the breakdown product (acetyl CoA) of -oxidation of fatty acids. Buildup of
acetyl CoA in the cell leads to formation of ketone bodies, which are released into the
blood. Typically, concentrations of ketone bodies in blood lag slightly behind those of
NEFA during the periparturient period, but the shapes of the curves are very similar to
one another.
7. AMINO ACIDS
Substantial attention has been paid over the last several years to protein and
amino acid nutrition of the transition cow. Amino acids absorbed from the
gastrointestinal tract (both microbial protein and ruminally undegradable protein (RUP)
plus amino acids released during turnover of muscle protein form the amino acid pool
and are utilized by the gravid uterus and remodeling mammary gland during late
gestation and in smaller amounts by liver for gluconeogenesis (Figure 3). Amino acids
from the blood pool also are utilized constantly during turnover of all body proteins. At
parturition, amino acid utilization by the lactating mammary gland increases
substantially. At the same time, rate of degradation of skeletal muscle appears to increase
and it has been hypothesized that synthesis of skeletal muscle is essentially shut down.
At the same time, the protein synthetic rate of liver, and perhaps gastrointestinal tract, is
increasing as the cow builds her support organs for lactation. As mentioned above, amino
acids appear to increase their contribution to glucose synthesis in liver during early
lactation (Figure 3). Amino acids are not “stored” in the body as free amino acids—they
are either incorporated into protein, catabolized in some body tissues for energy, or are
taken up by the liver, catabolized, and the nitrogen converted into urea. Perhaps one
advantage of increasing protein supply to cows during the transition period is to provide
intracellular metabolite in liver to facilitate complete oxidation of fatty acids and
decrease synthesis of ketone bodies.

Fig-3: Transition metabolism of Amino acids

54 
 
 Metabolism of individual amino acids also has received some attention over the
past several years. Availability of protected amino acids, primarily methionine and
lysine, has spurred interest in determining their roles in all stages of the lactation cycle.
In theory, both methionine and lysine have significant roles in metabolism of fatty acids
by liver. Lysine is involved in the synthesis of carnitine, which is required for fatty acid
transport during oxidation of fatty acids by liver and other tissues.
Methionine is also involved in synthesis of carnitine, and additionally in
synthesis of choline, which may indirectly limit export of fatty acids from liver in very
low density lipoproteins (VLDL). Results of supplementing methionine and lysine
during the transition period have been mixed. Effects of these individual amino acids on
liver metabolism and metabolic health have not been evaluated. As we learn more about
amino acid metabolism during the transition period, we should be able to more reliably
predict responses to supplementation of protected amino acids to diets of transition cows.
8. NUTRITION
The primary goal in transition cow nutrition has been crystallized in the past
decade: control body condition. No other factor that we can measure is a better predictor
of a disastrous transition period than a body condition score (BCS) of 4 or greater. In
fact, most academics who focus on metabolic disorders during this period would now
advocate a target BCS of 3 or even less at calving, because the consequences of high
BCS have proven to be far more serious than the consequences of low BCS. Cows
suffering from fat cow syndrome, despite having more stored energy to help offset
negative energy balance, experience greater decreases in DMI than healthy cows, have
greater increases in plasma NEFA, and are far more likely to have clinical cases of
ketosis and even infectious disorders. In our opinion, this goal is best met by feeding
relatively low-energy diets throughout the dry period, although a wide variety of
formulations can potentially be used to accomplish this. The devil, of course, is in the
details: preventing excessive sorting, promoting sufficient DMI to meet energy
requirements, and balancing for DCAD.
As is the case for social stress, nutritional needs of close-up heifers can be best
met by housing them separately. Because these heifers are still growing and because they
are less susceptible to fat cow syndrome, it is probably logical to offer them a slightly
higher-energy diet than multiparous cows. Likewise, anionic diets that benefit
multiparous transition cows have been observed to dramatically decrease DMI of heifers.
Heifers rarely experience severe hypocalcemia anyway, so it is best if they are fed diets
without added sources of anions.
9. DISEASE PREVENTION
The immune suppression that cows experience during the transition period
suggests several management strategies that may help to limit disease pressure and
associated stress during this time. Clearly, dairies are interested in reducing pathogen
loads for all cows. However, if there is an opportunity to improve the cleanliness of
certain pens, it would be wise to invest that effort in the fresh pens, since this is where
the majority of mastitis and metritis cases occur. Additionally, vaccination protocols
should be designed to avoid vaccinating cows during the final 3 weeks of gestation, as
the decreased function of the adaptive immune system during this period would limit the
effectiveness of vaccines and produce potentially harmful inflammation during a critical
time.

55 
 
CONCLUSIONS
To reduce the occurrence of production diseases of periparturient cows and
improve their reproductive and lactation performance, following measures could be
adopted.
1. Increase the dry matter intake of the animal to keep the animal in balanced
energy state.
2. Reduce and minimize the negative energy balance so as avoid metabolic
dysfunctions like ketosis.
3. Maintain optimal protein feeding so as to boost the cellular functions.
4. Stimulation of rumen fermentation so as to keep optimal activity of the rumen
functions.
5. Maintain the DCAD balance so as to avoid hypocalcemia and avoid milk fever.
6. Feeding of micro minerals antioxidants to reduce oxidative stress and boosting up
of immune functions of the leucocytes as well as enhancement immune status of
the animal body.
All the above factors can be controlled through proper animal management and
nutrition leading to a more healthy and productive performances by the dairy animals.
Ultimate aim is to ensure that cows calve within a 12-12.5 month interval and produce a
healthy calf and high milk production.

* * * * * * * 
   

56 
 
 Evaluation of frozen semen to optimize the conception rate in bovine
Dilip Kumar Swain 

Brief overview of frozen semen in animal reproduction

Boomed meat and milk industry have demanded the dissemination of superior
germplasm. The mammoth increase in the need of animal product with a limited land and
ration has arguably demanded the alternative techniques to natural breeding and in this
scenario; frozen semen has been emerged as the ray of hope for successful as well as
faster propagation of germplasm. Frozen semen technology has been the pillar behind
artificial insemination and one of the major strengths of assisted reproduction. The
discovery of liquid N2 and different extenders has made it relatively easy for the
preservation of liquid semen. Some of the major advantages of the frozen semen are-
Long term of storage of superior quality of sperms.
Faster propagation of germplasm.
Prevents the loss of semen in excess.
Increase possibility of inseminations by using semen from a bull.
Ease in transport and avoid country or border as a limitation for best semen.
Animal cloning and stem cell technology are dependent on frozen semen.
Up gradation of local breeds with superior sperms.
The mammalian sperm is a deceptively simple and terminally differentiated cell.
Although it seems to have a limited array of functional features, in essence to deliver an
intact haploid genome to an oocyte at fertilization, these functions touch many important
points in physiology and cellular and molecular biology, with implications for animal
production, (in) fertility and toxicology, to name but a few aspects.
Despite the extensive knowledge on spermatology, a definition of what
constitutes a “good” spermatozoon (i.e. a male gamete with high fertilization potential)
remains elusive. Besides the already mentioned species-specific differences, the fact that
ejaculates from the same male may vary according to several factors, and that they are
always very heterogeneous, containing both functional sperm and defective cells, is the
main obstacle for proper analysis. However, there are several aspects of sperm biology
that can be monitored, and which can give clear indications on the potential fertility of a
given sample. Furthermore, the analysis of sperm structure and function may be adjusted
and complemented according to specific needs, be they clinical, teaching or research;
and several methods can be chosen according to both the purpose of the assay, and the
available resources.
Since the advent of AI, researchers have sought laboratory assays that would
accurately predict the fertilizing potential of a semen sample. This goal, however, has
proven difficult to achieve. This difficulty arises from the complex nature of the
problem, dealing with the complexities of the spermatozoon itself, with our ability to
evaluate fertility, and with our ability to manage the females to be inseminated. Part of
the complexity dealing with the spermatozoa, comes from the fact that each
spermatozoon is a multi compartmental cell that must possess many different attributes
to be able to fertilize an oocyte. Each spermatozoon must possess:

57 
 
 Motility: To carryout transit in the female reproductive tract
Active mitochondria to supply the energy necessary for motility and other
metabolic functions
Intact acrosomal membranes that are capable of undergoing capacitation changes
thereby permitting the acrosome reaction to occur, but only at the correct moment
that is during fertilization.
Receptors that permit the cell to bind to the zona pellucida and to the oolemma;
Plasma membranes that permit fusion with the oolemma
A nucleus that is capable of proper decondensation, nuclear reorganization, and
genetic performance to maintain zygotic and embryonic development.

SPERM COMPARTMENTS AND THEIR FUNCTIONAL SIGNIFICANCE

A functional sperm is composed of three main regions: the head, the mid piece,
and the tail. The head contains the nucleus, in which sperm-specific DNA binding
proteins, called protamines, have replaced histones. The presence of protamines allows
for tighter chromatin packaging, and, together with the loss of most of the typical cell
organelles and cytoplasm that takes place during spermiogenesis, this is thought to have
a role in reducing cell volume and increasing the sperm’s aerodynamic properties, thus
potentially facilitating fertilization. It should be noted that these characteristics are also
widely believed to render the sperm transcriptionally inactive, or at least translationally
impaired. Overlaying the nucleus is a large secretory vesicle, the acrosome. This vesicle
contains hydrolitic enzymes thought to aid in sperm penetration through oocyte-
protecting layers, namely the translucent glycoprotein-based zona pellucida.
Release of acrosomal contents, an exocytotic process dubbed the acrosome
reaction, must only take place in the vicinity of an oocyte, and the lack of an acrosome in
any other circumstance signals that the sperm will likely not be fully functional.
However, before the sperm can undergo the acrosome reaction it must be primed by a
series of poorly understood maturation steps involving tightly regulated intracellular
signaling and protein phosphorylation (characteristically on tyrosine residues), which
comprise what is called sperm capacitation. The sperm tail (or flagellum) is paramount
for sperm motility, and is comprised of an axoneme containing a typical 9+2
microtubular arrangement, consisting of nine doublet pairs of peripheral microtubules,
arranged in circle around a central pair.
Other protein structures (the outer dense fibers and fibrous sheath) are arranged
around the axoneme, adding strength and resistance to the tail. The sperm midpiece, as
its name indicates, apparently connects the head and the tail, although it in fact consists
of a variable number of mitochondria wrapped helically around the anterior portion of
the flagellum. It has been assumed that the role of these mitochondria might be to
provide ATP for sperm movement through oxidative phosphorylation. However, it is
also possible that the necessary ATP is produced mostly by glycolitic pathways
throughout the flagellum, with mitochondrial ATP required only in specific
circumstances. It is also very important to note that there are many species-specific
differences, and what holds true for one species may not translate well (or at all) to
another.

58 
 
ACROSOME AND ITS FUNCTIONAL SIGNIFICANCE IN FROZEN SEMEN
In order to fertilize an oocyte, a spermatozoon must first become capacitated and
bind to the zona pellucida of the oocyte. Capacitation is a process in which many
changes occur within the spermatozoon including but not limited to; cholesterol efflux
from the plasma membrane increases in intracellular calcium, bicarbonate, potassium,
protein phosphorylation, and a decrease in intracellular pH. In the best case scenario, a
spermatozoon reaches the site of fertilization and completes capacitation at the time the
oocyte is present. However, cryopreservation results in a loss of lipids from the sperm
membranes and a rearrangement of lipids and proteins within the membrane, which
results in a ‘‘precapacitated’’ spermatozoa, resulting in a reduced fertilizing lifespan for
the spermatozoon. Therefore, assays to evaluate sperm capacitation can be used to
evaluate the normalcy of spermatozoa after freezing and thawing, as well as to monitor
techniques designed to induce sperm capacitation for assisted reproductive technologies.
Since there are many aspects of sperm capacitation many assays have been
developed which monitor one or more parts of the capacitation process. Changes in
membrane fluidity can be measured, using the probe merocyanin 540, as capacitation
occurs. Similarly, changes in intracellular calcium levels using chlortetracycline, Indo-1
AM or fluo-3 AM, changes in protein phosphorylation or the ability of sperm to undergo
an acrosome reaction when challenged with various acrosome reaction-inducing
compounds can be assessed as sperm undergo capacitation.
SPERM MITOCHONDRIA AND ITS FUNCTIONAL SIGNIFICANCE
Several fluorescent probes have been used to evaluate sperm mitochondrial
function. Mitochondrial probes are actively transported into actively respiring
mitochondria, therefore, the more active the mitochondrial respiration, the more probe is
accumulated. Rhodamine 123 was initially used to evaluate sperm mitochondrial
function, but can only differentiate between respiring and non-respiring mitochondria.
More recently, JC-1 has been used to assess spermatozoal mitochondria function. At low
concentrations, JC-1 remains in the monomeric state and fluoresces green. However, at
high concentrations, JC-1 forms aggregates that fluoresce orange. Therefore, JC-1 has
not only the ability to distinguish functional from non-functional mitochondrial, but
permits different levels of mitochondrial function to be determined by intensity of
mitochondrial ‘orangeness’. In support of sperm motility being a measure of
mitochondrial function, the percentages of sperm with functioning mitochondria is
highly correlated to sperm motility, regardless of whether rhodamine 123 or JC-1 is used
to evaluate mitochondrial function.
SPERM DNA EVALUATION AND ITS FUNCTIONAL SIGNIFICANCE
The genetic integrity of the spermatozoan is essential for normal embryo
development. A high level of DNA fragmentation in sperm cells may represent a cause
of male infertility that conventional examinations - sperm concentration, motility
analysis, morphology assessment - cannot detect. Results reported in the scientific
literature show regardless of the assisted reproductive technology used, elevated levels of
DNA fragmentation above the critical threshold will significantly compromise the
possibility of a successful pregnancy.
High sperm DNA fragmentation does not appear to affect fertilisation or the first
or second embryo cleavage stages

59 
 
 High sperm DNA fragmentation can affect embryo cleavage once the paternal
genome is switched on, and subsequent blastocyst development
DNA fragmentation levels are closely correlated with IUI, IVF and ICSI
miscarriage and pregnancy rates
DNA fragmentation is significantly higher in frozen semen
Animals with poor semen parameters are more likely to have high DNA
fragmentation
High sperm DNA fragmentation is also found in animals with normal semen
parameters
Freeze thaw process induces a high amount of oxidative stress to the sperms causing
induction of DNA fragmentation. This instead makes the sperms prone to apoptois and
thereby a reduction in the sperm quality. That is why sperm DNA evaluation is a key
process in frozen semen analysis. This can be achieved by-
Comet assay of the sperms.
Sperm chromatin assay
TUNEL assay of the sperms.
Detection of apoptotic like changes in the sperms through the evaluation of
caspases and various death ligands.

MAJOR SETBACKS OF CRYOPRESERVED SPERMS

Cold stress to the sperms
Formation of inter and intra cellular ice crystals
Free radical generation and oxidative stress to the sperms.
Capacitation like changes in the sperms
Acrosome damage and loss of functional intact acrosome
Loss of membrane integrity and there by osmotic stress to the sperms
Cholesterol loss from the membrane causing reduction in the fluidity of the
sperm membrane
Chromatin cross linking, protein denaturation and lipid peroxidation of sperm
plasma membrane.
Induction of cell death in sperms leading to apoptosis.
Loss of functional proteins from the sperms and a consequent loss of motility and
fertility in the sperms.
Abnormal sperm motility and altered ion channels of the sperms.
Reduction in seminal attributes after freezing.
Enzyme leakage causing loss of sperm function.
Reduced metabolic activity of the sperms.
Protein tyrosine phosphorylation causing precapacitation in the sperms.
Induction of acrosome reaction in the sperms.
Altered ionic equilibrium in the sperms.
Deprotamination in the sperms and hence there is a loss in the chromatin
compaction.
Reduction in the viability of the sperms.
Damage to the sperms at cellular and molecular level causing overall loss of
sperm competence for the process of fertilization.

60 
 
MAJOR SITES OF SPERM DYSFUNCTION DURING FREEZING AND
THAWING
Freezing and thawing of the semen target primarily four structures of the sperms
leading to reduction in the sperm quality. These sites are
Sperm Plasma membrane and its molecular alteration in terms of ion channels
and cholesterol.
Sperm acrosome inducing precapaciation like changes along with acrosome
reaction.
Sperm nuclear DNA causing or inducing the sperm DNA fragmentation,
deprotamination.
Sperm mitochondria and alterations in the mitochondrial function along with
compromised sperm motility.

Logic behind the evaluation of frozen semen:

Freezing process induces a large number of irreversible changes in the sperms
which make the sperms incapable of fertlisation.
Generation of free radicals and oxidative stress to the sperms substantially reduce
the sperm quality.
Multiple alterations occur in the sperms both at cellular and molecular level
leading to a dramatic loss in post thaw sperm quality.
Multiple sites of sperm damage are possible during freeze thaw process as a
consequence there will be a reduction in the sperm fertilizing ability.
Cold stress, stress associated with freezing and alterations in sperms are still not
understood at molecular level and hence a number unknown causes are defined
for the reduction in the sperm quality.

EVALUATION OF FROZEN SEMEN AND ITS SIGNIFICANCE

Correlations between laboratory results and fertility are inconsistent between
studies. Part of the reason for this is that sperm must possess many attributes to fertilize
an oocyte, not merely one or two or even three attributes. Secondly, for most attributes,
there is not a continuum from a minimum to a maximum that affects fertility, but instead
spermatozoa must possess a sufficient amount of each attribute to fertilize an oocyte.
Therefore, when evaluating a specific sperm attribute, we can postulate which samples
are likely to have poor fertilizing capacity, but are unable to determine if a particular
sample will be fertile.
In addition, as mentioned previously, there are many variables (management of
females to be inseminated, age of females to be inseminated, competence of inseminator,
etc.) that have a marked effect on fertility outcome, but have nothing to do with semen
itself. Finally, our ability to determine ‘fertility’ accurately is dependent upon having
sufficient numbers of inseminations from a particular semen sample. Taken together, it is
unlikely that any laboratory will consistently have laboratory assay results that correlate
well with fertility.
Over the past 75 year, researchers developed laboratory assays for many of the
attributes spermatozoa require to fertilize an oocyte, but there are likely many sperm

61 
 
attributes necessary to fertilize an oocyte that are unknown. There may also be problems
inherent in a specific assay that may limit the usefulness of the data obtained using that
assay. In addition, because each spermatozoon requires many attributes to fertilize an
oocyte, an assay measuring only a single attribute will fail to detect spermatozoa
defective in a different attribute, and therefore, overestimate the number of fertile
spermatozoa in a semen sample.
Unfortunately, because these assays evaluate a population mean for each attribute
measured, combining results from two or three different assays on the same sperm
population does little to determine the ‘true number of fertile sperm’ in a population. To
achieve this, we would need to assess all the attributes necessary for fertilization
simultaneously on each individual spermatozoon.
Problems also exist in defining and measuring fertility. Fertility can be described
as the percentage of females from which embryos are flushed after insemination, the
percentage of females that are pregnant a defined number of days after a single
insemination or multiple inseminations during a single heat cycle or several heat cycles,
or the percentage of females that deliver a live offspring after insemination (again single
or multiple inseminations over one or many heat cycles). Each of these is a very different
response and each reflects a different aspect of maternal input that is not likely to be
detected in a semen assay. In addition, for fertility data to be useful, it must accurately
reflect the true fertilizing potential of the semen used.
All together above mentioned events mediate a time dependent irreversible damage
to the sperms and as a consequence to this there is a reduction in sperm quality. The
sperm looses the functional competence to bring out fertilization. These cellular and
molecular changes are not assayed through the routine semen analysis and hence require
sophisticated assays and imaging to evaluate the sperm functions. Following are some of
the assays which are employed to evaluate the sperm functional competence in terms of
sperm fertilization.
Fluorescent based evaluation of sperm viability by employing PI or EBr.
Evaluation of sperm acrosomal integrity by employing FITC-PSA staining.
Evaluation of sperm mitochondrial activity by JC I staining.
Evaluation of sperm DNA integrity by employing various techniques namely
Comet assay, TUNEL assay, Sperm chromatin structure assay (SCSA).
Evaluation of sperm motility through CASA.

OPTIMIZATION OF CONCEPTION RATES IN BOVINES IN SEPCIFIC

REFERENCE TO FROZEN SEMEN EVALUATION
Optimization of conception rates in bovines can be achieved by both male and
female factors. Following are some of the parameters which can be incorporated to
enhance the conception rates in the bovines and in specific reference to frozen semen.
Proper evaluation of semen samples by using multiple automatic and visual
methods.
Evaluation of sperm basic and sub cellular parameters.
Proper evaluation of sperm motility by using CASA.
Evaluation of sperm DNA integrity/Chromatin structure.
Employment of highly sensitive fluorescent based staining of sperm components.

62 
 
 Proper care of the sperm during freezing and thawing.
Prescreening of sperms for the process of freezing.
Selection of best quality of semen along with best ejaculates for preservation.

Concluding remarks
No doubt that freezed semen is the best choice for artificial insemination but
simultaneously one cannot avoid the abnormalities induced by freeze thaw process. This
ultimately compromises the poor fertility response when used and hence time demands
to opt for multiple tests for quality evaluation of sperm samples. Employment of various
staining (fluorescent and non fluorescent) will effectively solve the issue for semen
evaluation along with incorporation of automation will substantially improve the semen
for artificial insemination and the consequence thereof. Some of the basic laboratory
assays for analyzing spermatozoa have been described as well as the spermatozoal
attributes each assesses. Relatively few of these assays are conducted on every semen
sample, and it is unreasonable that many of these assays should be conducted on every
semen sample, due to time and expense. However, these assays might be performed on
selected semen samples from males that are exhibiting low fertility to determine more
precisely what spermatozoal attributes are compromised. Steps may then be taken to
attempt to correct the deficiency, if possible. Unfortunately, it is very unlikely that
results from any single laboratory assay will effectively estimate the fertilizing potential
of a semen sample. However, laboratory assays are important, as they can help to
eliminate poor samples from being used for artificial insemination and to determine what
spermatozoal defects are present in samples with poor fertility. Several laboratory assays
have been developed that evaluate several spermatozoa attributes simultaneously on the
same spermatozoon. Such assays increase the likelihood of more accurately identifying
sub-populations of fertile sperm in a sample and are more powerful than conducting
single assessments of the same sperm attributes on different sub-samples of the semen
sample, which may only still measure two or three attributes. In conclusion, besides
employing routine semen analysis tests, one should employ specialized sperm assays to
get best quality sperms for fertilization. Along with this, optimization of freezing with
the selection of best extenders will enhance the quality of frozen semen opted for
artificial insemination purposes.

* * * * * * * 
   

63 
 
 Current concept in management of post-partum uterine infection in
cattle and buffalo
Anuj Kumar

Introduction
Fertility is one of the important parameter for life time performance of dairy
animal. Regular breeding depend upon the normal function of the reproductive system.
Many dairy herds do not achieve their targets for reproductive performance and incur
substantial economic loss. Reproductive performance is more related to health during
transition period of the cow and timely achievement of subsequent pregnancy. One of the
most significant causes of infertility in cattle is the complex of diseases that includes
retained foetal membranes, puerperal metritis, endometritis, pyometra and other
nonspecific infections of the uterus. These diseases share common etiological factors,
predispose to one another and, to a large extent, share common treatments.
A degree of bacterial contamination of the uterus almost always occurs during, or
immediately after, parturition. Bacterial contamination of the uterus may also occur
during coitus or insemination. Whether or not a persistent infection of the uterus
becomes established depends upon the level of contamination, the animal’s uterine
defence mechanisms and the presence of substrates (such as devitalised tissue) for the
growth of bacteria. Under normal circumstances, there are several mechanisms which
prevent opportunist pathogens from colonising the genital tract. Firstly, the uterus is
protected by the physical barriers of the vulval sphincter and cervix. It should be noted
that, although the vulva may appear of little consequence as a barrier, it is, in fact,
remarkably efficient at preventing faecal contamination of the tubular genitalia.
Secondly, the uterus is protected by local and systemic defence mechanisms; both are
influenced by the reproductive steroid hormones, oestrogen and progesterone. In general,
it is considered that the genital tract is more resistant to infection when it is under
oestrogen dominance, whilst under progesterone dominance it is more susceptible. The
reproductive endocrine system therefore has a significant influence on the resistance of
the genital tract to infection. It is not surprising that on the two occasions when the
physical barriers are breached (i.e. at coitus, or insemination; and at the time of
parturition, especially immediately postpartum) the genital tract is in its most resistant
state, since it is under the dominance of oestrogens and progesterone concentrations are
low.
Uterine bacterial infections are important because they disrupt not only the
function of the uterus, but also the ovary and the higher control centres for controlling
reproductive function in the hypothalamus and pituitary. The inflammatory and immune
response to uterine bacterial infection compromises animal health as well as causing
infertility. Thus, for veterinarians, the diagnosis and treatment of uterine disease is a key
component of fertility control programs. Understanding the mechanisms underlying the
effect of uterine bacterial contamination and the associated immune response on bovine
reproduction is an important challenge for reproductive biologists.
Essentially all peripartum dairy cattle have bacterial contamination of the uterus
for 2 to 3 wk after calving; a substantial minority have at least one form of pathology of
the reproductive tract, ranging from overt systemic disease to chronic inflammation. This
high risk of disease at this time is due to reduced immune function from approximately 2
wk before to 3 wk after calving. The severity of reduced feed intake, negative energy

64 
 
balance and weight loss contribute to the degree and duration of reduced immune
defence. Innate immunity from neutrophils is a primary means of immune response in
the uterus, and neutrophil migration and phagocytic and oxidative activity are associated
with the risk of retained foetal membranes, metritis and endometritis. Although
metabolic (e.g., ketosis and fatty liver) and uterine diseases are excessively common, the
risk factors for disease between herds or even within a herd, in which cows apparently
have similar nutritional and management experiences, are unclear.

Transition period physiology and immunity

Various aspects of immune function are suppressed in dairy cows from 1 to 2
weeks prepartum until 2–3 weeks postpartum. The precise causes of impaired immune
function in transition cows are unclear, although the peripartum drop in energy, vitamin
and mineral intake, negative energy balance and mobilization of body fat and protein,
dramatic changes in progesterone and estrogen levels in late gestation, and the massive
increase in cortisol level at calving appear to contribute. The hormonal and energy
effects of lactation appear to exert an immunosuppressive effect beyond those associated
with parturition itself. Cows in greater negative energy balance have more pronounced
impairment of at least some immune functions. Cows with retained foetal membrane or
metritis have earlier and more profound impairments of innate immunity, starting several
weeks before disease occurs.
Uterine bacterial contamination in cattle is a dynamic situation, with regular
contamination, clearance of bacteria and spontaneous re-contamination during the first
few weeks after parturition, rather than just contamination at the time of calving. Indeed,
the proportion of uterus contaminated with bacteria changes little during the first 2 weeks
after parturition. The postpartum return of ovarian cyclic activity depends upon the
uterine immune response. In particular, luteal phase concentrations of progesterone
suppress the immune response making the uterus more susceptible to bacterial infection.
Early ovulation after parturition and establishment of a progesterone phase before
elimination of uterine bacterial contamination predisposes to the establishment of
pyometra.
It is important to differentiate between the contamination of the uterine lumen
with a range of bacteria and the persistence of pathogenic bacteria with the establishment
of uterine disease, which might be described as uterine infection. Metritis is a severe
inflammatory reaction involving all layers of the uterus: endometrium, submucosa,
muscularis and serosa. Clinically, metritis is characterised by pyrexia ( 39.5 C) up to
10 days postpartum with a fetid purulent vulval discharge, often associated with delayed
involution of the uterus. Endometritis is a superficial inflammation of the endometrium,
extending no deeper than the stratum spongiosum.

Management of postpartum uterine infections

Administration of systemic antibiotics
Irrespective of the mechanisms underlying the infertility caused by uterine
infection, it is important for veterinarians to diagnose and treat uterine disease promptly
and effectively. Diagnosis of metritis within the first 10 days postpartum is associated
with the presence of pyrexia, fetid pus within the uterine lumen, vagina or discharging
from the vulva, and delayed uterine involution. Solely monitoring the rectal temperature
of dairy cows to diagnose metritis is less reliable than including an examination for
abnormal uterine discharge because pyrexia is not consistently associated with
pathogenic bacteria in the uterine lumen. The most important line of treatment for
metritis is parenteral antibiotic. Although oxytetracycline is widely used, it is not the

65 
 
optimum treatment because there is evidence for bacterial resistance to this antimicrobial
and high concentrations of the antibiotic are required to inhibit bacterial growth
(minimum inhibitory concentration, MIC) when tested in vitro. Indeed, cephalosporins
have the lowest MIC values when tested against recognised pathogenic bacteria collected
from the uterus of cows with clinical disease. Furthermore, dairy cows at risk of metritis
are less likely to develop disease if treated with ceftiofur crystalline free acid.
The character and odor of the vaginal mucus can be scored to produce an
endometritis clinical score. Vaginal mucus is assessed for color, proportion and volume
of pus, and odor resulting in an endometritis clinical score ranging from 0 to 6. The
endometritis clinical score reflects the bacterial growth density of recognised uterine
pathogens, but not other bacteria. Thus, the endometritis clinical score is a useful
indicator for veterinarians of the presence and growth density of pathogenic bacteria
contaminating the postpartum uterine lumen. In addition, endometritis clinical score is
prognostic for the likely outcome of treatment. The mucus containing only flecks of pus
(score 1) has similar numbers of bacteria to clear, normal mucus (score 0) 3 or 4 weeks
postpartum and may reflect the healing stages of uterine infection. Estradiol at doses of
5–10 mg per animal has been used therapeutically for postpartum endometritis and is as
effective as PGF2 . However, the interval from treatment to conception was longer with
estradiol treatment than PGF2 or intrauterine antibiotic.

Intrauterine infusion of antimicrobials

Infusion of antimicrobials into the uterus is aimed at achieving high
concentrations at the site of infection. In contrast to systemic administration, intrauterine
administration achieves higher drug concentration in the endometrium, but little
penetration to deeper layers of the uterus or other genital tissues. Substances reported to
be used for IU infusion include tetracycline, penicillin, cephapirin, chloramphenicol,
diluted Lugol’s iodine, gentamycin, spectinomycin, sulfonamides, nitrofurasone, iodine,
and chlorhexidine. Most are not approved for IU use, and in many cases have no
published information on withdrawal times. However, there is evidence to suggest that
IU infusion of several antibiotics result in drug residues in milk. Lugol’s iodine and
oxytetracyline are irritating and are reported to cause coagulation necrosis of the
endometrium.

Prostaglandin therapy
In cyclic cows, PGF2 causes luteolysis of a responsive corpus luteum (CL)
resulting in decreased progesterone level and subsequent estrus, with increased estrogen
level and myometrial contractions. These events are helpfull for clearance of uterine
infection. The precise mechanism by which PGF2 resolves uterine infection is not
known. There is controversy about the possible effect of PGF2 other than to cause
luteolysis and its consequential actions, although PGF2 receptors are apparently present
in the myometrium. There is some evidence that PGF2 may exert a direct short-term
contractile effect on the uterus.

Immunomodulatory substances
Antibiotics used in treatment of uterine infection increased public health concern
about the medicinal residues in edible animal product for human being. Antibiotics also
inhibit or destroy the phagocytic activity of PMN leucocytes responsible for uterine
defence mechanism. An alternative approach could be use of natural substances as a
means of activation of natural defence mechanism in the uterus. These may be used as

66 
 
intrauterine infusion for treatment of uterine infections. Some immunomodulatory
substances used in bovine uterine infection are as follows
1. Bacterial modulins- Standard E. coli LPS, Indigenous E. coli LPS and Bactezria
free filtrate.
2. Homologous plasma enriched with leucocytes
3. Herbal drugs like Neem oil, Garlic extract, Oyster glycogen and Colostral whey

Conclusions
In cattle, postpartum contamination of the uterine lumen is omnipresent, and
persistence of pathogenic bacteria commonly causes clinical disease. The consequence is
infertility associated with delayed ovulation after parturition, persistence of the corpus
luteum once it forms, and lower conception rates. Thus, effective diagnosis and treatment
of uterine disease is essential in reproductive control programs. Uterine infections not
only damage the uterus, but also suppress hypothalamic GnRH and pituitary LH
secretion, and have localised effects on ovarian function. The mechanisms underlying the
effects of infection on the reproductive tract involve the immune or inflammatory
response. Conversely, the reproductive endocrine environment can modulate the immune
response and understanding the integration of the immune and endocrine systems is an
important challenge for veterinarian

* * * * * * * 
   

67 
 
Impact of herd management on reproductive efficiency of dairy animals
Yajuvendra Singh, R. Sirohi, D.N. Singh, Mamta & Ajay Kumar

India is country full of natural biodiversity. Same stands true for the domesticated
livestock. Out of various livestock species being reared, dairy animals especially cattle
and buffalo play the most decisive role to the economy of Indian farmers as well as the
whole country. Though, the population of cattle and buffalo in India is the highest in
world but most of our indigenous breeds are poor in their productive and reproductive
performance.
An important prerequisite for the sustainability of a dairy production system is
that cows must have efficient reproductive performance. This is essential for the
production of the main commodity of interest; milk, as well as to provide replacement
animals. Over and above this, and particularly in smallholder systems, the animals must
also provide other outputs which will directly or indirectly result in economic benefits to
the farmer. These include, in different combinations depending on the system, meat,
draught power, fuel and fertilizer. Since the continued generation of these outputs all rely
on the production of offspring, no dairy production system is sustainable without an
acceptable level of reproduction. Though, it is quite obvious that production
performance is negatively associated with the reproductive performance of dairy
animals, yet, till now all our breeding policies and / or strategies have been formulated
keeping in view the production efficiency of dairy animals whereas reproductive
efficiency could never receive due attention of the breeders or policy makers.

From the viewpoint of reproduction, the main factors which contribute to

economic losses may be delayed puberty, long calving intervals, short productive life
(due to culling for infertility or sterility) and high calf mortality.

Economically viable reproductive events

During the lifetime of an individual female, higher reproductive efficiency yields

more calves for use as replacements or for sale in the herd, as well as more lactations and
therefore more milk. These considerations become more important as rearing conditions
become more intensive, where expenses for labour and feed have to be compensated for
by higher income.

In order to fulfill its role as an economically useful animal to its owner, dairy females
must perform the following functions:

Grow rapidly from birth until puberty

Attain puberty at an early age
Conceive readily to a fertile mating
Produce a viable calf
Produce adequate milk for the calf and extra for sale
Return to estrus early during the postpartum period and conceive again
Continue producing calves and milk at regular intervals till the end of its
productive life.

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The ability of an animal to meet these requirements will depend on many factors as
outlined below.

Puberty

In the female ruminant, puberty is defined as the first behavioural oestrus

accompanied by ovulation and development of a normal corpus luteum (CL) in the
ovary. This is determined by several factors, which are endogenous, for example,
genotype, growth and body weight, as well as exogenous, for example, year or season of
birth, rainfall, nutrition, thermal environment, photoperiod, and rearing method and
diseases. Generally, cattle and buffalo heifers attain puberty when they reach 55 to 60
per cent of their adult body weight. However, the age at which they attain puberty can be
highly variable, ranging from 12 to 40 months in cattle and 18 to 46 months in buffalo.
Thus growth rate and body weight are more important determinants of puberty than age.
Under optimum conditions, exotic cattle and their crosses attain puberty earlier than zebu
cattle, river buffalo and their crosses earlier than swamp buffalo. However, zebu cattle
and buffalo generally have a longer productive life than exotic cattle and some of the
disadvantages of late puberty are compensated for by their longevity.

The main factors which influence the age of attainment of puberty are the
genotype, nutrition, management, thermal environment, year or season of birth, parasites
and diseases.

Sexual cycles and mating

Adult non-pregnant heifers and cows normally undergo regular oestrous cycles,
which have a mean duration of 21 days. The oestrous cycle has four stages: pro-oestrus,
oestrus, met-oestrus and di-oestrus.

During pro-oestrus, under the influence of the two pituitary hormones, Follicular
Stimulating Hormone (FSH) and Lutenizing Hormone (LH), one follicle (rarely two)
from a group of growing follicles in the ovary continues to grow and mature and secrete
estrogens. Estrogens act on the brain of the cow and cause the behavioral changes
characteristic of oestrus or heat. Simultaneously, effects on the reproductive tract cause
changes such as swelling of the vulva, hyperemia of the vagina, secretion of cervical
mucus and increase in uterine tone. The high estrogen levels trigger a large release of LH
which causes ovulation after the end of the heat period. After ovulation the remnants of
the follicle are transformed into the CL which secretes progesterone and prepares the
reproductive tract for pregnancy. Some discharge of blood, termed met-oestrus bleeding,
can be seen from the vagina in most heifers and about 60 per cent of cows. This is in no
way related to whether conception occurred at the preceding ovulation.

The external signs of heat are usually more pronounced in exotic cattle than in
zebu cattle and show least in buffalo. However, marked variations occur between and
within breeds and the signs can range from very weak to very marked.

The duration of heat is usually shorter in tropical cattle breeds (mean 10 hr) than
in temperate breeds (mean 15 hr). The expression of heat signs is also influenced by
environmental factors such as temperature and humidity, social factors such as the
dominance order of an animal in a herd and the presence of disease or painful conditions

69 
 
in the animal's legs or hooves. Heat detection is an important factor in the fertility of
dairy cattle and buffalo. The cheapest and most easily applicable method is visual
observation. In using this method, however, it is essential that the observer is aware of
what to look for and is motivated.

Various aids for heat detection are available and include the following: KaMaR
heat mount detectors and tail paint; movement detectors (pedometers); measurement of
vaginal resistance (which declines at oestrus); examination of vaginal mucus for stretch-
ability (spinnbarkeit) and crystallisation pattern; monitoring of body (or milk)
temperature; and repeated measurement of progesterone concentration. Most of these,
however, are not practicable under smallholder farming systems in developing countries.
The use of `teaser' animals may be applicable in the larger herds with grazing
management systems. Such animals include males which have been vasectomised, penile
deviated or aproned, and females which have been androgenised. They are usually fitted
with chin-ball markers to identify cows which they mount.

Pregnancy and parturition

Fertilization of the ovum occurs in the oviduct (fallopian tube) and the resulting
embryo enters the uterus after four days. The embryo undergoes rapid cell division and
growth. Implantation or attachment to the lining of the uterus occurs progressively
during the period 25 to 35 days after fertilisation. The embryo is called a fetus from 45
days after fertilisation.

The standard method for diagnosis of pregnancy is palpation of the genital tract
through the rectum by a veterinarian, which is usually done from about 50 days after
mating. The criteria used for determining pregnancy by rectal palpation in buffalo are
similar to those in cattle but, due to the longer gestation length, each palpable feature is
first discernible about two to four weeks later, during pregnancy. Other modern methods
such as measurement of hormone levels in blood or milk and ultrasound scanning can
also be used for pregnancy diagnosis.

At the end of gestation the process of calving (parturition) is initiated. It consists

of three stages: dilatation of the birth canal (2-6 hr); expulsion of the fetus (30-40 min);
and expulsion of the fetal membranes (2-6 hr). Under normal conditions, the process of
calving should be completed in about 8 to 12 hours in cattle and six to eight hours in
buffalo.

The postpartum period

After calving, the reproductive tract of the cow goes through a period of recovery
called involution, during which the uterus returns to its non-pregnant size and state. This
is usually completed in 25 to 35 days. However, this process can be delayed if the uterus
becomes infected after calving. This can happen if the cow calves under dirty unhygienic
conditions, or if it has an abnormal delivery, such as dystocia, retained placenta or
prolapse of the uterus.

The sexual cycles are also suppressed during the early postpartum period. The
organs which govern the hormonal mechanisms controlling the sexual cycle, which
include the hypothalamus in the brain, the pituitary gland under the brain and the ovary

70 
 
in the abdomen, gradually recover their cyclic functional status and the cow should
normally show signs of heat within 30 to 60 days after calving. However, a number of
endogenous (genotype, milk yield, age/parity, body condition etc.) and exogenous
(nutrition, suckling, year/season/rainfall, environmental and social) factors influence the
functions of these organs and, as described in the following section, the commencement
of sexual cycles can be delayed and result in low reproductive efficiency.

Reproductive indices

The efficiency of reproduction can be accessed from several measures or

parameters, which are termed reproductive indices. Each index provides information on a
specific aspect of fertility but has its particular value as well as limitations. Therefore, in
order to obtain an assessment of the overall reproductive performance at a given time or
over a longer period, several of these indices need to be used. Some indices are
applicable only to a herd or a population of cows, while others can also be used in
individual animals.

In the case of heifers, the important indices are the age of attainment of puberty
and the age at first calving. The former depends on the time of onset of ovarian activity,
while the latter is influenced by the time of conception. Under free ranging conditions
with access to bulls, heifers will usually conceive soon after puberty. In confined
systems, however, efficiency of heat detection, timing of service and other related factors
will have important influences on the age of first calving.

The conception rate (CR) is the percentage of animals conceiving (based on

pregnancy diagnosis by rectal palpation) relative to the number of animals which have
been served. It is usual to calculate it either as CR to the first service only, or as overall
CR.

The pregnancy rate (PR) is calculated as the percentage of animals becoming

pregnant (usually over a one year period) relative to all breedable females in the herd.

The calving rate is the percentage of breedable females which calve during a
given year. The number of services per conception (S/C) is the total number of services
given to a group of conceiving cows, divided by the number of conceiving cows. These
indices are all influenced by factors related to the cow, the bull or artificial insemination
(AI) and the farming system.

The non-return rate (NRR) is an index often used by AI services to evaluate the
success of their operations. This is calculated as 30, 60 or 90 day NRR and refers to the
percentage of animals which were serviced but did not return for a repeat service within
the specified period, the assumption being that they conceived. Its usefulness is limited
to situations where AI is the sole method of breeding and the recording systems are
foolproof. Under many tropical smallholder systems, the more likely reasons for `non-
return' to AI are the use of a stud bull or sale of the cow when the farmer found out that it
had not conceived to AI. Thus the use of NRR by AI organizations under such conditions
is meaningless.

71 
 
 The calving interval (CI) is possibly the single index which provides most
information on reproductive efficiency, whether in an individual cow or on a herd basis.
This is made up of three components:

i. Interval from calving to first oestrus (postpartum anoestrus period, a)

ii. Interval from first oestrus to conception (service period, b) (a + b = `open period')
iii. Interval from conception to calving (gestation period)
iv. Interval from calving to cessation of milk let down (lactation length)
v. Interval from cessation of milk let down to calving (dry period)

In order to maintain optimum economic benefits under modern intensive dairy

systems, it is generally accepted that the CI should be around one year. Since the average
gestation length is 280 to 285 days, a cow must become pregnant by 80 to 85 days after
calving in order to achieve this. The cow must commence ovarian activity early during
the postpartum period, show heat, conceive readily, carry the pregnancy successfully and
produce a calf. The farmer must detect heat, mate the cow at the correct time and provide
adequate nutritional and other inputs.

Factors affecting reproductive efficiency of dairy animals

Genotype and Environment

The genotype of the animal determines its reproductive potential, but the end
result is influenced by all the factors mentioned earlier, which we can broadly call
environment. Thus a well adapted tropical zebu cow might have a relatively low inherent
reproductive capacity, but may be able to achieve its full potential under the prevailing
conditions. A Holstein Freisian (HF) cow which has been bred for high milk production
and excellent reproductive performance under temperate conditions will, if moved to the
tropics, perform well below its potential capability.

Nutrition and environment

Nutrition has a major influence on the age of attainment of puberty. For a given
genotype of cow or buffalo, heifers that are well fed will grow faster and attain puberty
at an earlier age than those poorly fed. It results in an earlier age of first calving, with
economic benefits to the farmer through more milk and more calves during the lifetime
of the animal.

In dairy cattle, the period of early lactation is particularly stressful, in that the
cow is unable to meet its nutrient requirements for high milk production through the
daily feed intake. Therefore, it draws on stored nutrient reserves (fat deposits) until it
attains a metabolic balance around the second or third month of lactation. Thus cows
calving in poor body condition lose more weight and take longer to regain the lost weight
than those calving in good condition. As a result, such cows also take longer to
commence cyclic ovarian activity than well-fed cows.

Management Practices

Management practices such as suckling of calves can influence the resumption of

sexual cycles after calving. It has been observed that in a traditional system of

72 
 
management, where calves were raised separately from their mothers from the time they
were one week old and allowed access to the cows only once a day for suckling, the
reproductive performance of the cows was excellent.

Research has also shown that, apart from the suckling stimulus, the mere
presence of the calf near its dam, and the degree of direct contact by touch, smell and
sound, can delay the onset of postpartum ovarian activity in the dam. This is thought to
be mediated by substances such as b-endorphin and insulin-like growth factor (IGF).
Therefore a further method of improving reproductive efficiency is to change the system
of calf feeding so as to restrict the interaction between the cow and its calf.

Breeding management

Management decisions of farmers as well as their knowledge and skills have

important effects on the reproductive performance of cows. The farmer must decide on
the age and or weight at which heifers are mated, and the interval after calving when
cows are mated. In the case of heifers, mating too early will result in a longer postpartum
anoestrus period after the first calving, while mating late will result in loss of production
time. Cows which have normal calvings and uncomplicated uterine involution can
normally be mated if they show heat anytime after 45 days, but full fertility may not be
obtained until about 60 days after calving. Delaying mating beyond 60 to 90 days will
result in economic losses in many production systems.
Some smallholder farmers, in few parts of the country, commonly delay mating
for three to six months after calving. This is due to a belief that mating too early will
result in a shortened lactation and lowered milk production. These farmers require
education regarding the advantages of early mating in terms of greater lifetime
productivity of milk and calves by an individual cow.
Management of reproduction for optimum efficiency

Genotypes, records and general management

The genotypes of animals which are raised in smallholdings must be compatible

with the environment and the available resources, including the knowledge and skills of
the farmers. Thus cows must be served with bulls or semen of the appropriate genotype
and replacement stock must be selected on defined criteria. In-breeding should be
avoided. The necessary information must be provided to smallholders. Since most
reproductive traits have a low heritability, it is generally not feasible to select for high
fertility under smallholder conditions. However, it is essential that selection against
infertility takes place by eliminating genetically abnormal or infertile animals from
breeding, by culling. This is not easy in situations where herd size is very small and the
farmer may have only one replacement heifer. In such situations schemes such as
subsidized heifer exchange programs need to be considered.

Records are an indispensable component of modern dairy farming, but are

usually non-existent on most smallholder farms. These are required to construct a wall
calendar on which calvings and services are recorded, which helps in proper selection
and culling of most suitable individuals.

73 
 
 Other aspects which need addressing are: appropriate feeding of calves, heifers
and cows; minimizing the negative effects of suckling and cow-calf interactions;
provision of adequate water for drinking; alleviation of heat stress, particularly in
buffalo; and control of parasites and diseases

Heat detection and mating

The most suitable method of heat detection will depend on the production system.
If cows are continuously tired, there are no opportunities for them to manifest interactive
sexual behaviour. Thus physical signs alone form the criteria and their detection requires
knowledge as well as motivation. In such systems cows should be checked at least three
times per day. Sometimes the only signs visible may be swelling of the vulva and a
discharge of clear mucus. In buffalo even the latter may be seen only on the floor when
the animal is lying down. Observing the response of the animal to pinching of the clitoral
region or to pressure on the lumbar area can also be used for confirmation.

In systems where there are several cows and they are allowed to interact at
pasture or milking time, observations for homosexual and other estrous behaviors should
be carried out at least three times per day, for at least 15 to 20 minutes each time.
Particular attention should be paid to cows that have come into heat earlier, irrespective
of whether they had been mated or not. Such animals must be observed for possible
return to heat, from the 18th day after the previous heat. If they show heat signs again
this usually means they have not conceived so they must be mated again. The most
important aid to heat detection is a record of when a cow has been in heat previously.

Once heat has been detected the next step is to ensure that the cow is mated at the
optimum time, which is during the latter part of the heat period or around 12 to 18 hours
after the start of heat. If hand-mating is done with a bull, two services with an interval of
six to 12 hours is recommended. When AI is used, cows detected in heat during the
morning should be served in the afternoon of the same day and cows detected in heat
during the afternoon or evening should be served the next morning.

Care of pregnant and parturient animals

Pregnant cows should be managed so as to prevent stress and accidents. They

must be dried off two months before the expected calving date (seven months after
conception in cattle and eight months after conception in buffalo) and fed well (steaming
up ration) during the dry period to allow accumulation of body reserves. Cows due to
calve should be washed and moved to a separate clean pen if possible. Reproductive
problems which occur during pregnancy or around parturition can sometimes be very
serious and therefore should receive prompt veterinary assistance.

The postpartum period

The vulval discharges which occur after calving should cease by about 15 days.
Their persistence indicates delayed involution or uterine infection which will require
treatment. This is more likely to occur in cows that have had abnormal calvings.

Cows under good nutrition and management may commence ovarian activity
within one month of calving. However, if the first heat occurs earlier than 45 days after

74 
 
calving, it is advisable to skip this heat and serve at the next one. If the calving and
uterine involution has proceeded without complications, the cow can be served at the
first heat which occurs after 45 days. In cows that have had calving difficulties or
infections during the postpartum period mating should be delayed until about 90 days
after calving.

* * * * * * * 

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