recent advances in frozen semen technologies in livestock in India and abroad.

1. Advances in Frozen Semen
Technologies and Role of
Hygienic Approach During AI
Towards Fertility Management
in Livestock
Dr. Sharadindu Shil
B.V.Sc. & A.H. (Gold Medallist), MVSc. (Gold
Medallist)
Veterinary Officer
ABAHC, Ratanpur

2. Introduction to Indian Scenario
 India has one of the largest networks of AI centres in the world with about 95000
centres carrying on an average about 650 AI in a year.
 Semen production in the country has increased from 22 million straws (1999-2000) to
83 million straws (2013- 2014) and the number of inseminations from 20 million to 65
million (22 million animals under AI coverage).
 The overall conception rate has also increased from 20% to 35%.
 The field AI delivery systems in our country are still being developed.
 A combination of factors viz. cold chain management of semen doses in the field, skill
of the AI technicians in handling of semen doses etc. could affect quality of the
product once it leaves the production station.

3. Evolution of Frozen Semen Technology
The first successful insemination was performed by the Italian physiologist and priest Abbe Lazzaro
Spallanzani (1784) in a dog
Practical procedure was initiated in Russia in 1899 by Ivanov.
Milanov, another Russian scientist and successor of Ivanov, started large scale breeding programs for
cattle and sheep, and designed and made artificial vaginas.
1937- Danish veterinarians developed the first rectovaginal / cervical fixation method of AI.
1948- Sorenson first time used large sized straws (12mm) made up of polyvinyl chloride.
1949- Polge, Smith and Parkes discovered cryoprotective effect of glycerol in frozen semen technology.
1960-Adler developed the first technique for freezing of semen in straws using liquid nitrogen vapour.
1964- Cassou improved the straws by reducing their size and named it as medium French straws. The
size of the straw was 135 mm long and 2.8 mm diameter with 0.5 ml semen capacity.
1968-Cassou further reduced the size of the straws to the diameter of 2 mm with a capacity of 0.25 ml
and named it as mini French straws.
1972-A plastic straw called mini tube or German straws or ‘Lanshut system’ was developed in
Germany.

4. In India
1939-In India, first
time, AI was done by
Sampat Kumaran at
‘Palace Dairy Farm
Mysore”. He
inseminated large
number of Halliker
cows with semen of
Holstein Friesian and
got 33 cows
pregnant.
1943-The first
buffalo calf through
AI was born at the
Allahabad
Agricultural
Institute.
1951-56-In the first
five-year plan (1951-
56) the Government
of India introduced
150 key village
centres to improve
cattle and buffaloes
in this country.
1956-61-The second
five-year plan (1956-
61) gave a boost to
AI work by
implanting it in 400
key village centres.

6. •Semen Sexing or Sex sorted Semen.
•Sperm Encapsulation
•Sperm Transcriptomics.
•Ovum Pick Up (OPU)
•Seminal Biomarkers
•In Vitro Maturation, Fertilization and Culture
(IVMFC)
•Intracytoplasmic Sperm Injection (ICSI)
•Embryo Transfer Technology (ETT)
•Embryo Cryopreservation
•Embryo Sexing
•Embryogenomics
•Somatic Cell Nuclear Transfer
•Stem Cell Technology
•Transgenics
•Nanotechnology
Recent
Advances in
Assisted
Reproductive
Technologies

7. Semen Sexing or Sex Sorted Semen
 The aim of sexed semen is to produce a
calf of specific sex.
 It is crucial to produce pre sexed
livestock by sperm or embryo sexing
carrying high genetic value animals.
 Huge gap between demand and supply of
semen straws in the country can be met
through production of superior males by
using sex sorted spermatozoa from
superior dams.
 Flow cytometry sorting method is having
about 85-90% efficiency.
 Conception rate of AI using sexed
sperms, with one tenth the sperm number
of non-sexed sperms, is around 70-80%
of that achieved by non-sexed sperms in
heifers.

8. Status of Semen Sexing In India
 In India, PBGSBS, a Government of West Bengal organization, initiated sorting of
semen on 15/08/2009 under RKVY at Frozen Semen Bull Station, Haringhata.
 They reported first male calf named Shreyas, born on 1/01/2011 using sexed semen.
 Later, female calves were also successfully born using sexed semen.
 They are currently in a position to produce 40 straws per day.
 The conception rates observed were 20.7% in cows (37) and 35.3% in heifers (58)
using sexed semen.
 In Punjab, the farmers are charged Rs. 600 per straw with the state subsidizing the
remaining 50% of the total cost of Rs 1200 per straw.
 The ABS India is providing sexed semen of Holstein and Jersey.
 Haryana Livestock Development Board in collaboration with Navasota (USA) is
planning to introduce sexed semen technology in Murrah buffaloes.

9. Application of Semen Sexing Technology In India
 The projected demand of milk by 2020 is estimated to be 191.3 MT.
 To meet the increasing demand increase in the number of elite females can be
achieved by shifting the sex ratio towards females.
 The projected additional frozen semen doses required per year are 48, 9.6 and 52.8
million for indigenous cattle, crossbred cattle and buffaloes respectively.
 By introducing sexed semen, superior bulls could be produced from the limited
number of elite cows available.
 The sexed female sperms could be used in progeny testing.
 There are a large number of unproductive young bulls due to ban on cow slaughter,
competing on limited feed and fodder resources.
 The use of sexed semen can solve the problem of production of unwanted male
progenies.

10. Constraints
 High cost.
 Scanty commercial availability of the sorting technology.
 Lower sorting speed and efficiency.
 30% sperms are rejected during the sexing process.
 Low conception rate: 10-20% lower.
 Low number of elite bulls in India.
 Need of standardization in Indian conditions.
 Lack of skilled manpower.
 Lack of awareness among farmers.
 Requires a different AI gun .

11. Ovum Pick Up (OPU)
 Non-invasive and repeatable technique used for recovering large numbers of compe-
tent oocytes from antral follicles of live animals.
 Repeated OPU can be performed without side effects both in cattle and buffaloes with
a mini-mal stress to the animal.
 In India, the first buffalo calf (Saubhagya) was produced through this technique.
 Less invasiveness and the use of superior animals as oocyte donors in embryo transfer.
 Oocyte retrieval from females at virtually any age or reproductive status, including pre
pubertal heifers and pregnant cows.
 Potential to substantially increase the lifetime productivity of high genetic merit
females, and effectively reduces the generation interval.
 One of the limitations of this technique is the low oocyte yield per ovary and necessity
for sophisticated instrument.

12. Embryo Transfer Technology (ETT)
 Embryo transfer is a technique by which embryos are collected from a donor female and
are transferred to recipient females, which serve as surrogate mothers for the remainder
of pregnancy.
a) To get maximum number of high pedigreed calves from high yielding cows/buffaloes.
These can be selected through Herd Registration scheme.
b) To accelerate genetic gain / year by selecting the calves on the basis of collaterals'
performance.
c) To reduce birth of low potential calves through conventional breeding practices adapted
by farmers
d) To conserve indigenous Germplasm in the form of embryos.
 Allow top quality female livestock to have a greater influence on the genetic
advancement of a herd or flock instead of superior sires.
 A high yielding cow/buffalo produces normally 8-10 high yielding calves during life-
With ET technology we can get 80-200 calves during its lifetime.
 Greatly reducing the risk for transmission of infectious diseases.

14. FROZEN SEMEN STRAW

15. Q
U
A
L
I
T
Y

16. REPRODUCTION
SEMEN
QUALITY
GENETIC
SANITARY
ENVIRONMENT

17. GENETIC QUALITY
• Selection of bulls
•Production
•Hereditary problems
SANITARY
QUALITY
•Diaseases Diagnosis
•Control
•Prevention
•Monitoring
ENVIRONMENTAL
QUALITY
•Animal Welfare
•Nutrition
•Animal management
REPRODUCTIVE
QUALITY
•Bulls Selection
•Andrological evaluation
•Ejaculates selection

18. Testing at Various Level of Calf Selection
Testingat
semen
station
Testingof
dams for
initial
selection
Testing of
dams andcalf
before
procurement
Testing at
pre-
quarantine
station
Testing at
quarantine
station
Testingat
rearing
station
1
2
3
4
5
6

19. Disease Testing at Semen Station
TB & JD
Bulls in
semen
station
Brucellosis
IBR
BGC
Trichomoniasis
BVD
FMD

20. Disease Testing of Bulls at Semen Station
TB & JD : 6 monthly , +Ve cull
repeat after 42 days in +ve herd
Until all negative
Thereafter 6 monthly
Brucellosis : 6 monthly , +ve cull
Repeat after ~60 days in +ve herd
Thereafter 6 monthly
BGC & Tricho : at entry , +ve treat
Repeat after ~30 days after treatment
Thereafter Annually
Bulls in
semen
station
IBR: -Ve at 9 months age
+Ve cull, retest herd 60 days
Until all -Ve
Thereafter 6 monthly
+Ve Bull semen testing for BHV-1
BVD : -Ve for Ag & Ab
ELISA
+Ve cull retest herd 60 days
Until all -Ve
Thereafter 6 monthly

22. Test Protocol Prescribed in OIE and MSP
OIE MSP India
Prescribed Test MSP Tests
Bovine tuberculosis
(TB)
Tuberculintest DTH (TuberculinPPD)
Paratuberculosis (JD) _ DTH (JohninPPD)
Bovine brucellosis BBAT,CF,
ELISA,FPA
ELISA
Trichomoniasis Agentid. Agentid
Bovine genital
campylobacteriosis
Agentid. Agentid
Infectious bovine
rhinotracheitis (IBR)
VN,ELISA,
Agent id.
(semenonly),
PCR
ELISA,
Semen -PCR
BVD-MD Agent id,ELISA ELISA
Foot and mouth
disease
ELISA,VN ELISA

23. Challenges
 There has been no developments with regard to the minimum prescribed tests &
frequency of tests for the product quality post formulation of the MSP.
 The three critical tests prescribed in the MSP for semen quality are-
1) The sperm concentration in the semen dose.
2) Post-thaw motility.
3) The microbial quality.
 Of these except for sperm concentration the other two tests have issues of repeatability
& reproducibility.
 It is probably ‘time’ that the stations raise the bar and move over the minimum
standards prescribed for the semen production and focus on a limited number of tests
that predict fertility with greater accuracy.
 Lack of correlation between the findings of the laboratory tests with the ultimate field
data on fertility
 Less optimization of the different types & the frequency of QC tests to be carried out
in frozen semen.

24. Suggestions
 Should have efficient data management systems to monitor the field programmes.
 The sperm load in the semen dose needs to be comparatively lesser than now in the
Indian scenario.
 Sexed semen should be adopted on a large scale in India.
 Should allow lower loads for high genetic merit bulls with proven high fertility and
higher loads for comparatively lower fertility bulls.
 Breed wise variations in calibration/ standardization of the equipment to be fully
addressed,
 Synergy between the semen stations and the research organizations in the country.
 Policy interventions which facilitate easy adoption of recent advances in the semen
processing techniques from the developed nations.

25. STORAGE OF FROZEN SEMEN
 The frozen semen straws should always be kept submerged in liquid nitrogen in
liquid nitrogen container.
 The semen containers should be periodically topped with liquid nitrogen.
 The semen straws should be kept in a plastic goblet which in turn should be kept in
canister.
 The goblet should not be tightly packed with semen straws.
 There should be space for liquid nitrogen to go inside to maintain the temperature at
lower level.
 Always keep identification slip or colored plastic sticks in the goblet.

26.  The slip or stick should contain the information about the semen straws for easy
and quick identification.
 Don’t keep different breeds’ semen straws in a goblet. The semen straws of same
breed but from different bulls should be stored with proper partition.
 The goblets used must be slightly shorter than straws to enable quick removal of
straws. The commonly used goblets are 12 cm in height.
 A 35 mm diameter goblet holds 85 medium straws and 65 mm goblet holds 300
medium straws.
 The frozen semen should never be touched with hands. The straws should always
be removed with pre-cooled stainless steel forceps.
 Frozen semen is exposed to elevated temperature when the semen is transferred or
taken from a storage container.

27. Contd.
 The increase of temperature is determined by length of time exposed, ambient
temperature, air circulation, level of liquid nitrogen in container and height to
which the canister is raised above the neck.
 The technician must be efficient enough to pick desired straw within 10 seconds.
 This will minimize the fluctuation in temperature of straws while handling.
 If semen is to be transferred from one canister to other, keep both the canisters
submerged in liquid nitrogen kept in a thermo cool box and carry out the transfer
quickly.
 During storage and handling of frozen semen, any rise in temperature above -130 ⁰
C should be avoided.
 The key factor enabling the successful long term storage of frozen semen is low
temperature.

28. Handling During and After Thawing of Frozen Semen
Thaw the semen straw at 37 ⁰ C water bath either horizontally or vertically
 The semen straw should be taken with pre-cooled forceps.
 The straw should be given a jerk to remove all the liquid nitrogen attached
over the surface.
 Don’t thaw more than 2-3 straws at a time.
 After thawing the semen should be used immediately.
 The straw should be wiped thoroughly to remove all the water.
 The semen straw should be cut at laboratory seal end where the air space is
available.
 The AI gun should be loaded correctly after pulling the plunger down.
 The sheath should be applied over the AI gun and the button should be placed
on the sheath.
 The semen should be deposited by pushing the plunger smoothly.

30. Self-assessment Time!
 Wash hands.
 Keep insemination supplies dry and
clean at all times.
 Keep breeding tool box clean and
organized.
 Water bath thermos at 94–98°F for
thawing semen.
 Empty and clean water bath thermos
routinely.
 Correct use of breeding sheaths/
protective rods.
 Keep AI rod clean.
 Wash AI rod.
 Implement sanitary conditions when
preparing the semen straw.
 Protect loaded AI rod from cold shock
and contamination.
 Wear rubber gloves.
 Wipe the vulva with a paper towel.
 Use a paper/towel folded in half and
inserted into the vulva to help prevent
contamination.
 Keep AI tank clean and filled with the
correct level of liquid nitrogen.

31. Infertility
 Sometimes considered as synonymous with sterility or it implies a failure or delay
in producing the annual live calf. The term subfertility is a more appropriate term.
 Semen has no therapeutic effect to “cure” fertility problems in the cow.
 Today’s fertility is a reflection of the cow’s environment and management during
the previous two or three months.
 “It’s easy to list 60 to 80 different factors that can affect the success of a given
insemination, many of which occurred months prior to the insemination date.
Successful managers recognize each day must be used to prepare cows for
tomorrow’s fertility.”

32. Hereditary or Congenital Anatomical
Defects of the Reproductive Tract that
Affect Fertility
 Both congenital and acquired abnormalities of the genital system can influence
fertility.
 Anatomical abnormalities usually affect individual cows or heifers and are
therefore unlikely to have a major influence on fertility in a herd.
 In some cows, because of the severity of the abnormalities, sterility is manifested at
the time of first service period while in some, where the defect is less severe, it may
not be detected until late in life.

33.  Ovarian hypoplasia.
 Segmental aplasia of mullerian duct and imperforate hymen.
 Congenital lack of endometrial glands.
 Double external os of the cervix.
 Uterus didelphys.
 Intersexuality and freemartinism.

34. Acquired Defects of The Reproductive Tract
 Tumours of the ovary, ovaritis,
para-ovarian cysts.
 Ovobursal adhesions and
hydrosalpinx and pyosalpinx.
 Endometritis and pyometra.
 Mucometra or hydrometra.
 Perimetritis & parametritis.
 Abscess of uterine wall.
 Cervicitis and vaginitis.
 Tumours of the cervix and vagina.
 Fibrosis of vagina and cervix.

35. Hormonal Causes of Infertility
 Hormonal or functional forms of infertility mostly affect individual animals
within a herd.
 However, when a larger group of animals in a herd are affected- may be due to
 inherited factors
 nutritional deficiencies or excesses
 social influences which may arise from modern husbandry methods.
 Hormonal diseases may include cystic ovarian degeneration, failure of estrum
or anestrum and repeat breeders.
 Cystic ovarian degeneration
 Anestrum

36. Key to Success
 Don’t practice-“Stick a dose of semen in her just in case.” (Re-insemination aborts)
 The timing of A.I. must ensure that the fertile life of sperm and egg will overlap.
 Distinguish the difference between “first standing mount” and “first observed
standing mount”. 4-14 hr. from “first standing mount” .
 Once daily A.I. program is appreciable.
 Clean hands, clean paper towels, clean equipment and perhaps double sheath
breeding

37. Conclusion
 Artificial Insemination has been long used as a tool for the rapid genetic
improvement of the bovine population in the country.
 Increased adoption of AI is of utmost importance to improve the productivity
of the dairy animals in the country.
 It is necessary that semen production stations equip themselves with the
technology to evaluate and process semen so that there is minimum
deterioration in its inherent quality to fertilize.
 Time has come to design more efficient and effective structures that focus on
adding value to the quality product without increasing costs.
 Each semen production centre must strive to develop and continually
revalidate semen collection and processing protocols using acceptable
viability criteria that are consistent with recommendations.
 In house quality audit and effective implementation of quality management
systems for consistent production of a quality product is required.

38. “We are what we repeatedly
do,therefore, excellence is not a
fact, but a habit”
Aristotles
dr.sharadindu@gmail.com. +91-9007930227

39. • Artificial insemination (AI) is widely accepted as a technology that
can bring about rapid genetic improvement in cattle and buffaloes.
• However, optimum conception rates will only be achieved if the
quality of semen used is good, the insemination is done at the most
appropriate time in relation to the oestrous period, and the
technicians have adequate training and skills in the procedure.
• Although AI is widely used in many Asian countries, the above
factors, together with other socio-economic considerations specific
to smallholder production systems and inadequate infrastructure
for the efficient delivery of AI services, have often led to poor
success rates.
• If these constraints can be overcome, not only would the farmers
and service providers benefit, but the technology would also
become more widely adopted. Wider adoption of AI could then
contribute to better food security and alleviation of rural poverty.