No 3-252/201 B_AHT(RGM)

Government of lndia
Ministry of Agriculture and Farmers Welfare
Department of Animal Husbandry & Dairying

Krishi Bhawan, New Delhi

Dated 20.03.2019
Subject: Proceedings of the Workshop on Frozen ]5
Semen production held on
'tG January 2019 and the
Revised Minimum Standards for Bovine
Frozen Semen.

The undersign with the approval of competent

authority is directed to circulate
the approved Proceedings of the workshop
on Frozen semen production herd on .r6
January 2019 and the revised Minimum standards
for Bovine Frozen semen.

a$*s\:'fa\1
(Bhushan Tyagi)
Assistant Commissioner (AH)
Distribution to:
(D Principal Secretary/Secretary , Department of Animal Husbandry, All state
Governments
(ii) Executive Director, NDDB, Anand
(iii) Managing Directors, AII State Milk Federations
(iv) Director , Department of Animal Husbandry,
All state Governments
(v) CEO/MD, State Livestock Development Board
(vi) ln-Charge of all Semen Stations
(vli) Chairman, CMU
(viii) Member Secretary, CMU

Copy for kind information to:

PPS to Sec (ADFyppS to AHC/pS to

JS(C&DDYDC(RGBYDC(BSYAC(BRYAC(HRK)/AC(SNYLO(CB)
 !.Tri.rqi

GOVERNMENT OF INDIA
MINISTRY OF AGRICUTTURE AND FARMERS WELFARE
DEPARTMENT OF ANIMAL HUSBANDRY & DAIRYING

Proceeding of the Workshop

on Bovine Frozen Semen
Production
Held on l-6 fanuary 201,9
NASC Complex Pusa, New Delhi
 MINUTES OF THE WORKSHOP ON BOVINE
FROZEN SEMEN PRODUCTION
UNDER THE CHAIRMANSHIP OF ANIMAL
HUSBANDRY COMMISSIONER (AHC)
HELD ON 16TH JANUARY 2019 AT NASC COMPLEX,
PUSA, NEW DELHI
A workshop under the chairmanship of Animar Husbandry
commissioner (AHC) was
held on 16th January 2o1g atNASC comprex, pusa,
New Derhi to discuss the guiderine
on semen production under pcrc Act, 200g, covering Ar
service providers & technicians
under PC|C Act, 200g and guiderines for evaruation
of semen stations and Ar rraining
institute.

The meeting was attended by the Joint Secretary (CDD),

DADF; ADG (AH), |CAR,
DARE; chairman cMU of semen station; Directors
of state Animar Husbandry
Department; cEos, Livestock Deveropment Board/Agency;
semen station officers and
DADF officers. Detailed list of participants is placed
at Annexure_l.

Joint secretary (cDD), DADF wercomed a, the participants

and briefed the house
regarding the present scenario of bufis avairabre
in the country. He focused on
maintaining High Genetic Merit disease free bulls
in the semen station.

The chairman addressed the house and briefed

regarding the agenda of the meeting.
He congraturated punjab state for coming up
with a Bovine Breeding Act and suggested
that other states may also come up with such regulation-
He informed the house that
Department is in the process of issuing executive
order/guideline for Semen production
under the ambit of prevention and contror of rnfectious and contagious Diseases
in
Animals Act (PCrcDA), 2009 to provide a regar
tooth for imprementation of Bovine
Breeding Policies in the state.

The ADG (AH), lcAR, DARE agreed with the Department's

step towards issuing
executive order/guiderine for semen production
under the ambit of prevention and
contror of rnfectious and contagious Diseases in
Animars Act (pcrcDA),2009 and
production of High Genetic Merit disease
free buils. He added that the semen stations
managed by lcAR are also to be evaluated
by the cMU for semen station.

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osp ileLls Jovo 'fulunoc eql ut suouels uewes rc! locoloJd prepuels
wnwtutw pue locoloJd uoque^eld asees/o onss/ //eq{s lovo eql,,

s/v\olloJ se paserqdai aq Aeur ered aqt leql palse66ns seM ll

,, JO|CU|O

Jo \uet aql ilopq Jou lueuutaAog aJeJS pauJacuoc p aa4eluasetdet

auo Jseq p pue syadxe Jo srs/suoc 'steqlo 6uowe ileqs qclq/A suorie;s
uewas lo duuoltuow pue uoqenp^a )o! yun buuoyuoyy p4uaC elnlnsuoo
oEe ileqs JOVO 'fuNnoc eql ut suoueJs uauas rcJ pcolud prepuels
wnwtutw pue locolord uoque^aJd aseas/o anss, i/eqs tovo aql,,
(rn) ou e.red 6urrnol1o1 aq1 ut pe1sa66ns e:e sa6ueqS .
s00Nnoocnoou
a'r sarJoleroqel paurluapr eql lle ul ujJollun aq lleqs aseaslp ds[/\l
Burlsal .ro1 pasn lry 6uusa1 eq1 sllnseJ lsal ut r$tultollun aql utelutEut oI .
sraqurau nylc pauracuoc
oql qlyr^ real rtena peJeqs aq lleqs sllnq lo podal 6ut1sa1 eseastp autlnog .
:eur;aprnB aq1 ur pelerodrocut aq

og pa1sa66ns arern 6uuvro;;o; pue aut;aptn6paplo Uetp aq1 o1 paat6e asnoq aq1
-locolord prepuels unuJull/l
? locoloJd uouua^ald oseaslo aLll ,v\ollol
pue n!!3aql ,{q saseasrp lle roJ aalJ aseaslp pauluac ale {aq1 lll} l3e aql Jepun
'ureql lsure6e uorlce alel lleqs retruJo 1ue1adu.toc7.to1corlo aql pue '6002 ]cv
voclcd Jo 0z uortcas rapun ,seaJe palceJul, se peleall aq ol aJe suouels uaulas
paJolsr6arun llv 'sasop uaues ales / Ilddns ol aq lleqs suollels uaulos
pa/\Aolle

parelsrOa: lpuo 1eq1 paprcep serv\ y '600Z'(VOCtCa) lcv sleuruv ur saseasl6

snor6eluo3 pue snorlcalul Jo lolluoC pue uotlua^ald rapun 6e eted ut apeu.t
suorsrnord rad se pansst eq llt/vl autlaptnoTtaplo antlncaxe leql paurolul se/v\ ll
:600Z lev VOCIOd rapun uollcnpo;d ueues uo au;;ap;nb 6u1nss1 't

'6urlaau aql

lo epuabE Eurrvrollol eql uo uorleluasatd lauq e aneb ggyq 'JauotssluituoC luelslssv

'asnoq eql ur uorssn3srp astr* epuabe fiq paino1lo1 sert 6utlaau eq1
 constitute centrar Monitoing unit for evaruation and
monitoring of semen
sfaf/ons which shall among others, consisls of expefts
and at least one
representative of concerned State Government not
below the rank of
D i re cto r/J o i nt D i rect o r/E q u i v a I e n t. "

ii. covering Ar service providers & technicians under pcrc

Act, 2009:
The house agreed the covering of Ar service providers
& technicians under pcrc
Act, 2009 and the following suggestions were made:
o All Al Technicians to be registered in INApH with
unique lD
o CMU accreditation of Al Training lnstitute will be valid
for 3 years.

iii. Guidelines for evaluation of private semen stations

The house agreed that there is no need for separate guiderine
for private semen
stations. Private semen stations to be evaruated as per
the MSp of Bovine
Frozen Semen production:

' Any semen station producing more than 1 rakh doses

annualy wi come
under the purview of CMU (SS).
o A fee for evaruation of private semen station may be
decided by cMU to
meet evaluation expense of the CMU team.

tv. Delay in disease testing at RDDLs/CDDLs:

o lt was decided that ail identified disease testing rabs
RDDUCDDUNDDB
to perform disease testing of MSp diseases for the semen
station bulls on
priority and deriver timery report. Further, the possibirity
of incrusion of
more number of raboratories for testing of MSp diseases
to be exprored by
CMU in coordination with DADF.

i 3
)
Chairman, CMU for evaluation of Semen Station, explained the existing Minimum
Standards Protocol (MSP) and the proposed revision in the MSP for Semen Production.
After detailed deliberation the modification in the MSP was accepted by the house
which is placed at Annexur*ll. The major revisions incorporated are as follows:

. Rework on the table made for assessing the Bulls genetic worth in revised MSP
and send for consent to all stakeholder.
o Karyotyping should continue as it is once in life time test.
. Minimum period between tlvo ejaculate should be at least 15 min.
. lt is recommended to preferably collect two ejaculate per day collection and two
days of collection per week from a healthy bull.
o Pattern of Printing Semen Straw from left to right should be clearly stated in the
MSP document.
. Quality Control Test to be conducted daily.
. lt is suggested to modify the manpower requirement as "lt is recommended that
Semen station in-charge should have at-least 5 years experiences in bovine
semen production and should not be transferred frequently. lncumbent should be
positioned for 5 years."
. All Semen Stations not to conduct MSP disease testing of their Bull Herd from
their own state lab or their own agency. The MSP disease testing should be done
by third party. CMU to only consider results of third party disease testing of bulls.
. Request has come to include IBR testing of Semen doses through RT-PCR by
semen station itself. DADF will take final call on it.
o ICAR-|VR| not regularly supplying antigen for testing TB and JD. DADF to take
up the issue with ICAR.

Director, CFSP& Tl, Hessarghata, Karnataka presented the report of CMU evaluation
and the evaluation status of the Al Training lnstitutes.

l4 l-
 Assistant commissioner, Nationar Livestock Mission, DADF
exprained the Minimum
standards Protocor for semen production in smal Ruminants
and standard operating
Procedure for Ar in Goats. Folowing recommendations
were made by participants of
the workshop :

while fixing the standards/criteria for serection of High Genetic

Merit (HGM)
Breeding Rams/Bucks, records like, individual bucUram data, pedigree
information, progeny/ offspring performance, etc. can also
be taken into
consideration.
semen samples selected for freezing should have minimum 70%
initial
progressive motility.
Finar dirution of semen, with a minimum of 40 mirion progressivery motire
spermatozoa per frozen thawed dose of 0.25 mr for raparoscopic
method of Ar
and a minimum of 'r 00 milion progressivery motire spermatozoa per
dose of 0.25
ml for cervicar or intra-cervicar method, shal be done in
appropriate frasks with
the dilutor maintained at 34 0 C.
a ln case of sheep, both cervicar / trans-cervicar and Laparoscopic
intra-uterine
methods and in
case of Goat , only Cervical / trans_cervical method are
recommended.

The house ended with vote of thanks to the Chair

'l
5
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 ANNE xOR E. I
ATTEND ANCE SHEET

WORKSHOP ON SEMEN PRODUCTION

16TH JANUARY 2019
NASC COMPLEX. PUSA. NEW DELHI
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WORKSHOP ON SEMEN PRODUCT]ON
16111 JANUARY 201g
NASC COMPLEX, PUSA, NEW DELHI

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NASC COMPLEX PUSA NEW DELHI

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 ATTENDANCE SHEET
WORKSHOP ON SEMEN PRODUCTION
16" JANUARY 20t9
NASC COMPLEX PUSA, NEW DELHI
'besignati6n
sl. Name & Address Mobile No. Email Signature
No. I 1

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Fr{Sa .i-q=

Minimum Standards

For
Production of Bovine Frozen Semen

2019

DEPARTMENT Or. ANIMAL HUSBANDRY, DAIRYING & FISHERIES

MINISTRY OF AGRICULTURE & FARIUERS WELI'AR.E
G,OVERNMENT OF. INDIA
KRISHI BHAWAN, NEW DELHI
Edited & (2"d) Revised in 2018-19

Page I of 75
 h'/ a IIMU FOR PRODUCTIO N OF BOVINE FROZEN SEMEN
Artificial Insemination with frozen semen has been proved to be the
best toor
worldwide for mass genetic improvement through dissemination
of superior
germplasm. This objective can be achieved only if the frozen
semen used in
A'I programme conforms to certain prescribed quality standards.
For
production of quality semen, it is most important that
the bulls used in A.I
programme meet prescribed norms, and are disease-
free and that the
semen is harvested and processed in accordance with the
standard
protocols' The minimum essentiar protocols required
for production of
qua-lity semen are described in t].is manual. Fa ure
to observe these
guidelines may result in production of poor quality semen
making it unsafe
as well as unfit for use in breeding through artificial insemination
programme.

I Staadards for Genetic Merit of Breeding Bulls

High Genetic Merit (HGM) Buls meeting the breed characteristics

and
production standards shourd preferably be procured
from the Govt approved
Progeny Testing programmes being run professionally
following the
Minimum Standard protocols and Standard Operating procedures AS
approved by DADF, Ministry of Agriculture and Farmers
weifare, Govt. of
India. In case, such bulls are not available or the pT programme
for a
particular breed is not being rul, the buns for semen production may be
procured based on the dam's standard ractation yield.
The reliability of the
data available with respect to performance recording must
be kept in view.
The minimum dam's ractation yield for different breeds
is given in Tabre
2'The Lactation yierd may be arrived at by recording the
animal at monthly
interval continuously for l 1 times or until the animar
becomes dry but not
less than 8 recordings. standard Lactation yietd of
the animal subjected to
performance recording should be calculated using
the Test Interval Method
(A4) described at Section 2. 1.s. I of the International
Agreement of Recording
Practices published by International committee for Animar Recording
(ICAR)'whenever possible parentage of a bull should
be got verified prior to
its procurement using DNA linger printing

Page 2 of75
As a very few bulls coming from genetic improvement programmes are being
inducted in the semen stations at present, it would not be possible to have
breeding values for all bulls of different breeds maintained by the semen
stations. However to ensure that high quality genetics get into the semen
stations, a simplilied procedure giving weightage to different ways the bulls
are obtained could be adopted to assess the genetic worth of a semen
station. At present bulls are obtained in the following different ways:

1. A young bull produced through nominated mating of dam, selected

from top recorded dams, using semen of a top progeny tested sire
(Bulls obtained from Progeny Testing Projects (PT)) provided by
Breeding Value Estimation Committee of GOI.
2. A young bull produced through nominated mating of dam, selected
from top recorded dams, using semen of top quality bull (not proven,
HGM bull) (Bulls obtained from Pedigree Selection (PS) Projects)
3. Young bull produced from a dam having complete lactation record and
known sire
4. Young bull produced from a dam having complete lactation record and
unknown sire
5. Young bulls produced from a dam with a single day record and known
slre
6. Young bulls produced from a dam with a single day record and
unknown sire

Weightages proposed for the above mentioned six different ways of obtaining
young bulls and a procedure for calculating a genetic worth (out of 100
points) of a semen station having bulls obtained from the six sources is
shown in the Table below:

Page 3 of 75
Table: 1 Weightage for different categories of bu[ for calculating
score
for genetics in CMU evaluation
No. of
bulls at 7o of bulls
s the of I
semen dillerent \lleighted
N Source of bulls station categories trIeightage I

A young bull produced

through nominated mating of
dam, selected from top
recorded dams, using semen I

of a top progeny tested sire 100 3s.3 1.0 .)J.JJ

(Bulls obtained from progeny I

Testing Projects (p?)) provided I

1 by Breeding Value Estimatron

Committee of GOI. " I
I I

A young bull produced I

through nominated mating of

dam, selected from top
recorded dams, using semen I

of top quality bull (not proven, 50 76.7 0.8 | 1r.6e I

HGM bull) (Bulls obtained
from Pedigree Selection (pS) I

2 and Progeny Testing (pT)

Projects)**
I
Bulls sire known and their
mother are selected based on 50 16.7 0.8 r1.6s I

complete lactation records $

B ull's sire not known and
4 mother has complete
lactation 40 13.3 0.0 0.0 I

record gg
5 Mother single
record and sire 50 16.7 0.0
known 0.0
I
I
6 Mother single record and sire
not known
10
I
3.3 0.o I o.o I
I
Total bulls 300 I
I
loo.o I
56.71
Genomic Breeding Value to be includ ed a-fter complete
development
and va-lidation of Genomic Serection chip by a committee
constituted
by DADF.
The score card prepared for cMU Evaruation shalr have
at least 5o%
weightage of total score for the Genetic worth of the
Semen station.

Page 4 ol75
Note: " PT projects follow the GoI'PT SOP; top 10%o of active proven bulls
and top 57o of recorded females based on breeding values (provided by
Breeding Value Estimation Committee constituted by Govt. of India.) are
used for selection of young bulls

"Bull's sire is an HGM bull and mother is from top 1O% of recorded females
(Minimum 50O females under recording either from institutional farms or
fie1d)"

** PS projects follow GoI's PS SOP; top 5% recorded females and HGM bulls
are used for producing young bull calves or bulls obtained from bull mother
farms.

$ BuU's sire is an HGM bull and mother is from top 10% of recorded females
(Minimum 50O females under recording)

$$ lvtother is from top 10% recorded females (Minimum 5OO females under
recording)

*** In case of draft breeds. oroductivi W will not be a consideration. Scoring

will be as oer phenotlpic or breed characters.

@ Bull's sire is an HGM bul1

- If Exotic bulls, meeting guidelines for Import of exotic germplasm

circulated by DADF, are used for semen production, such bulls should be
put in category l.

- If Exotic bulls, meeting guidelines for Import of exotic germplasm

circulated by DADF, are used as sire of the bull, the sire should be
considered among top l0olo proven bulls and the bull thus produced should
be put to category according to dam's records available. (i.e., if dam is a
selected, then bull should be considered in category 1, if dam have only 305
day milk yield based on 10 monthly per day records, the bull should be
considered in category 2 and if dam is having only one record, the bull
should be put in category 5, etc.)

Page 5 of 75
 - If a bull is produced through ET, the parameters applicable
for sire and
dam should be used for putting the buls in different categories i.e.
if the
sire is among top proven bulls and donor is selected based on top
recorded
dams, the bull should be put in category 1 and so on.

(This arrangement ui come in force u).e.f. lst Apit 2020, after compretion
of
remaining 2017-18 eualuation uhich u,till be based on unreuised
MSp (2012)).

Table: 2 Standards for Dam,s lactation

Dam' s Lactation yield (Kgs)

Breed First Best Fat o/"

Holstein Friesian 7000 10000 3.5

I

Jersey 5000 6000 5.0

Sahiwal 2400 3000 4.O

Red Sindhi 2000 2500 4.5

Gir 2400 3000 4.5
Kartkrej 2000 2s00 4.5
I

Tharparkar 2000 2500 4.O

Hariana 1600 2000 4.O

Rathi 1600 2000 4.O

I

Ongole 1100 1600 4.O

Deoni 800 1000 4.O

Khillar 380 500 4.O

Dangi 400 530 4.O

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Amritmahal 400 500 4.O

HF Cross- P2 5000 6000 4.O

Jersey Cross- F2 3500 4500 4.5

Sunandini 2500 3000 3.5

Murrah 2400 3000 7.O

Mehsana 2400 3000 7.O

Nili Ravi 2400 3000 7.O

Jaffrabadi 2800 3500 8.0

Surti 1600 2000 7.O

Banni 2400 3000 7.O

Bhadawari 1300 1600 8.O

Pandharpuri 1300 1600 7.O

The sta-ndard for Dam's lactation yield for Fl cross bulls will be the same as
that of respective indigenous bull dam i.e. Gir, Sahiwal, Kankrej, Red
Sindhi, etc.

For Breeds not mentioned in above table, concerned state government may
notify the min. Dam's lactation details and Breed code.

For imported bulls and embryos, tle standards for import of qerrnplasm as
prescribed in the "Guidelines for export / imoort of bouine dermDlasm" issued
and amended from time -to -time bu DADF, Ministru of Aqianlfure and
Fanners Welfare, GOI shall be applicable.

2 (af . Bull Identilication

a). Unique identification number having 12 digits with bar code shall be
practiced for identihcation of bulls across all Semen Stations. It should be

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 compatible for registration under INAPH, presently being managed by
NDDB, Anand.

b). The bar coded ear tag with unique 12 digit raser printed number
w l be
physica-lly applied to the bun and will remain on it for rife.
If the burl is sold
to other SS, tJle tag no. i.e the bull identification will remain
unchanged.
c)' In case the tag falrs, is 10st or destroyed, it will be changed
with a new 12
digit Bull ID following standard procedure of "Ear Tag change, under
INAPH.

d). In addition, each burl will be assigned an arphanumeric ID by the

semen Station incorporating three character semen Station (SS)
code (table
o4l;2 to 4 character breed code (tabre 0s) and rast six digits of the
unique
I.D. or name given to bull. For Example: BAS-HFCB_32019g; ALM-HF_
ARJUN. etc. This will be a mandatory and w 1 have to be printed
on the
straw' In case, a bulr is sord to other SS, the new ss w,l assign
a fresh
alphanumeric ID.

e). Management of data for semen production and artirrciar

insemination wilr
become easier by capturing rea-l time bull detail, using bar
coded semen
straws and ear tags.

2. (b). Physical Examination

Before procuring new bull calves/bulls for the semen station,

a thorough
physical and andrologicar examination sharl be conducted
by an experienced
Veterinarian with respect to breed characteristics, general
health and
suitability as a breeding bull.

Standards for scrotal circumference and weight gain index

for various
breeds need to be evolved and in particular for indigenous
breeds by
recording the scrotal circumference once in three months
and body weight
once a month, by the semen stations. For every new
bull calf procured, the

Page 8 of 75
measurement of scrotal circumference and body weight needs to be initiated
immediately.

Prior to introduction of any bull for semen collection, breeding soundness

examination should be conducted and documented by a competent
professional.

3. Karyotying and testing for Genetic disorders/ diseases

It is necessary that all bulls be karyotyped to rule out any chromosomal

defects. In addition, breed specific Genetic disorders such as Factor XI
deficiency syndrome, Bovine Leukocyte Adhesion Deliciency (BLAD),
Citrullinemia, and Deficiency of Uridine Monophosphate Synthase (DUMPS)
will be tested in HF ald HF crossbred bulis and as a precaution in Jersey
and their crosses (to rule out possibility of HF blood/crossing at any stage)

4. Quarantine
A minimum quarantine period of 60 days is compulsory before bringing new
bulls into a semen station. Only after favourable results from the health
control point, the bulls shall be admitted to the semen station. Relevant
definitions are given in Annexure- I

a) In the quarantine station, new animals shall be housed for a

minimum of 6O days in a place which is effectively separated and
away from (preferably at a distance of 5 km) the facilities occupied by
resident bulls. Manpower deployed and all equipment used in
handling, feeding, watering and cleaning the new bulls shall not be
shared with the resident herd(s).

b) Each new animal in quarantine station will be tested against major

contagious diseases before its entry to resident herd namely TB, JD,
Brucellosis, Campylobacteriosis, Trichomoniasis, Infectious Bovine
Rhinotracheitis and Bovine Viral Diarrhoea. All tests shall be done by
Page 9 of 75
 an accredited agency or disease diagnostic laboratory as indicated
in
Annexure- 2.

c) During the quarantine period, the bulls shall be vaccinated against

FMD, HS, BQ, The eriosis and Anthrax. However, vaccinations
against bacteriar diseases shall be done onry if there is an outbreak
or
prevalence ofa particular disease in the area.
once the quarantine period is over, all bulls shall be introduced
to the
young bull rearing station or to the Semen Station depending
upon the age
of bulls.

*The rocedure and duration r arantine in di rent sittt ations is nln

Annerure- 34, 38, 3C & sD.

5. Testing of Bulls
The testing protocols/ procedures for bulls against T\rberculosis,
Johne,s
disease, Brucellosis, campylobacteriosis, Trichomoniasis,
Infectious Bovine
Rhinotracheitis and Bovine Viral Diarrhoea are given in
Annexure- 4 to ro.
The breeding burls shourd be free from above mentioned
diseases. Though
Johne's disease is not a sexually transmitted disease
but being a chronic,
infectious and incurable disease, it has been included
and the breeding
bulls found positive for Johne's need to be removed. The
bul1s in the
quaraatine/ rearing station and the resident herd
should go through
periodical testing and vaccinations as per the schedule
listed in the manual.
6. Vaccination Schedule

The bulls shail be vaccinated against FMD, HS, Be,

Theileriosis and
Anthrax' However, vaccinations against bacterial diseases
sharl be done onry
if there is an outbreak or prevalence ofa particular disease
in the area.
Theileriosis - Exotic and crossbred bulls shafl be vaccinated once
in their
lifetime.

Page 10 of 75
Guidelines issued bv the De T) artment of Animai Husband T_\/ and D a 1n:t n g.
Ministrv of Aericulture and F armer Welfare for progres sive IBR BVD control
and as amended from time-to-time shall be follow edi n letter and spirit bv all
semen stations.

Besides, the semen station are advised to cull those bulls which have
completed eight years of productive period or 3 lakh semen doses, whichever
is earlier unless the bull is of exceptional genetic merit. In addition, the
bulls with poor libido, poor semen quality, incurable lameness, etc. may also
be culled on regular basis.

8. Housing

Bull sheds shall have spacious individual pens with adequate loafing area,
manger and water trough with access to drinking water all time. Adequate
shade around the bull shed shall be provided. The roof shall be made of
asbestos or suitable materials. During summer, cooling system with
sprinklers and fans is required particularly for the buffaloes and exotic
bulls. Disinfectants like formalin or phenyl based compounds shall trot be
used in the bull sheds. Alternatively, compounds containing Gluteraldehyde
may be used. Weekly spraying of Sodium Carbonate (4% solution) shall also
be practiced. The floor should be sterilized at least once a year by a blow
lamp or by burning straws. At one corner of the farm, there shall be an
isolation shed for separating ailing / sick bull(s) for treatment. Bull(s) once
diagnosed suffering from any of the infectious diseases shall be removed
immediately from semen station to contain its spread to other bulls.

There should be separate staff and separate bio-security arrangements for

semen station and female herd, if any.

9. Maoagement of Bulls

Proper management of breeding bulls at all times is essential to keep them

in good health and to ensure a satisfactory state of cleanliness. The
following guidelines should be considered:

a) The bulls shall be kept under hygienic conditions at all times.

Page 13 of75
 b) The coat of the bulls shall be kept clean and generally short. The
hooves shall be regularly trimmed.

c) The length of the tuft of hairs at the preputial orifice, which is

invariably soiled, shall be cut to about 2 cm. The hair would not be
removed altogether, because of its protective ro1e. If cut too short,
it
may cause irritation of the preputial mucosa.

d) Bulls shatl be brushed and groomed regurarly, and where necessary,

special attention shall be given to the under- belly, a day prior
to
semen collection.

e) cleaning of the prepuce with sterile normar sarine solution prior

to the
day of collection can be practiced if the microbial load in frozen
semen
is beyond the prescribed rimit otherwise occasional cleaning is
advised
as per need. The person carrying out preputial wash must use
disposable gloves and separate sterilized tozzle for each bull
to avoid
transmission of infection from one bull to another.
f) In the event of obvious soiling, careful clea,ing of the preputiar
orifice
and the adjoining areas with soap or a detergent is recommended
followed by thorough washing and drying.

g) scientific feeding schedule shal be foflowed for the bu[s.

A general
guideline is attached as Annexure- 12. Semen station
is advised to
carryout routine quality analysis of feed and fodder to ensure
balanced ration.

1O. Semen Collection

a) Ideally, the floor of the collection yard shalr be made of concrete

rayer
at a depth of one foot from the ground level. Mixture of sand
and
limestone shail be used to fill up to ground lever ard pressed
firmly. If
it is not possible to renovate the entire collection arena, at least
the
Page 14 of 75
To reduce lay off time, the bulls shall be vaccinated on the rest day or on the
day after completing semen collection. Sexual rest may not be required
unless febrile condition is noticed.

The semen station shall arrange for carr5ring out ring vaccinations of all
cloven footed animals including swine against FMD within a radius of 10 km
around the semen station. Vaccinations against HS and BQ shall be carried
out in the areas having incidence ofthese diseases.

7. Culling of Bulls and Semen Doses due to Specific Diseases

Diseases Bulls Semen doses

I

FMD Retain Last one month's doses to be

I
discarded, refer Annexure- 11

Brucellosis Castrate & remove FS doses in stock to be discarded

as per prescribed
I

since the last negative test

scientihc method

TB Castrate & remove FS doses in stock to be discarded

I as per prescribed since the last negative test
scientihc method

JD Remove as per FS doses in stock to be discarded

I

prescribed since the last negative test

I
scientihc method
I

Infectious i). Castrate and i).Test each batch / ejaculate since

I

Bovine Remove for IBR - last negative test by RT-PCR. Semen

Rhinotrache free S.S. and found positive shall be destroyed by
itis (IBR) retest all incineration /autoclaving.. Use only
remaining bul1s Semen that has tested negative by
until all tested
Page ll of 75
 negative. RT-PCR.
I
I
I
ii) Isolate the bull i

and process and

ii) Test each batch / ejaculate by
store semen
RT-PCR. Semen found positive shall
separately for IBR
positive be destroyed by incineration/
semen
autoclaving.. Use only Semen that
stations. All IBR
has tested negative by RT-pCR.
positive SS should
aim at becoming
IBR negative as
soon as possible.
Only IBR sero-
negative bulls will
be introduced at
SS I

Bovine Viral Isolate and remove Destroy by in cineration

Frozen
Diarrhoea Persistently Semen doses of the pI positive bulls.
(BVD) Infected bulis.
Only PI negative
Animals will enter
the SS

Campylobac Isolate and remove FS doses in stock to be discarded

teriosis since the last negative test

Trichomono Isolate and remove FSd oses in stock to be discarded

S1S
since the last negative test
I I

The semen station must remove bulls (within 4g

hours) which are positive
for Brucellosis, TB, JD, IBR ( IBR negative SS) and pI
BVD.

Page 12 of75
 mounting area shall have sand and limestone mixture for proper
footing of bulls. Alternatively, good quality rubber mat (with
interlocking arrangement) or coir mat shall be put into concrete
groove of the mounting area for adequate cushioning effect. After
collection, the area must be thoroughly cleaned and odorless
disinfectant solution (Colloidal iodine) be sprayed. A dusty floor shall
be avoided to prevent dust falling on the A.V / semen samples.

b) On the day of collection, before collecting semen, the bulls shall be

properly washed and cleaned. After that, the prepuce shall be cleaned
externally with normal saline and a steril2ed paper napkin or
sterilized cloth napkin soaked in normal saline to remove any sand or
dust particles. For each bull a separate napkin shall be used.

c) The person carrying out preputial wash must use disposable gloves
and separate sterilized oozzle for each bull to avoid transmission of
infection from one bu11 to another. Preputial washing should be
carried out if the bull is soiled otherwise it is better to avoid.

d) Semen collection should be individualized based on the bull behavior.

e) Sexual preparation (number of false mounts and restraint) of the bulls

may not be generalized but decided based on the behavior of the
individual bulls. For this purpose, the sexual behavior of the
individual bulls sha,ll be studied and documented

f) As a general rule, bulls shall be sexually prepared by giving two /

three false mounts followed by restraint. The gap between two
ejaculates shall be preferably approximately fifteen minutes to half an
hour depending on the bull. Second ejaculate shall be taken following
proper stimulation and preparation of bulls.

Page l5 of 75
 g) Sterilized bull aprons shall be used to avoid penis touching
hindquarter of the dummy.

h) Before every collection, the semen collector shall wash his hands with
suitable antiseptic solution or use disposable gloves or do both. The
semen collector shall not touch the penis.
i) Semen should be collected either by the Veterinarian himself or by a
suitably trained technician / staff under his close supervision. while
taking collection, it shall be ensured that AV is not thrust on penis of
bull, instead the penis is gently guided into the AV.

i) Immediately after collection, the AVs shail be thoroughly creaned by

non-spermicidal neutral detergent. separate AVs shall be used for
each ejaculation. The AV shall be changed even if the bull has
inserted its penis without successful ejaculation. The same AV shall
not be used twice. The AVs shall always be kept inverted and the
collection tube shal be covered with felt f water jacket (plastic bottle
frlled with wa-rm water at 34o c) to avoid cold shock. The open end of
sterilized AVs shall be covered with aluminum fo , which wourd be
removed at the time when bull is ready for giving semen.

k) Appropriate size AVs, ranging from g-14",shal be used for cattle and
buffaloes ensuring that the semen is ejaculated in the cone. The cone
shall be of good quality Neoprene rubber/Silicone rubber.

l) use of lubricant shall be avoided. If it is extremely essential to use

lubricant, separate sterilized grass rods shalr be used for smearing K-y
Jelly on individual AVs.

Page 16 of 75
 m) The AV shall not to be shaken after ejaculation and carried to the pass
box with open end slightly inclined downward to avoid flowing down
and mixing of iubricant and debris with the semen samples.

n) As soon as the {irst ejaculate is taken, the bull apron should be

removed and dipped in the plastic tub filled with warm detergent
solution. For second ejaculate, a fresh, sterilized bull apron should be
used.

o) The entry of visitors and staff /labourers (otJler than those involved in
semen collection) shall be strictly prohibited in the collection arena at
the time of semen collection.

p) Protective clothing (barn coat) and gumboots shall be used by the

veterinarians and staff during semen collection. Gumboots and barn
coat should be washed immediately after completion of semen
collection work.

q) Semen stations must follow the norm of a minimum of two ejaculates

per collection and two collections per bull per week, resulting into at
least 90 collections and 180 ejaculates annually from each adult bull.
However, a maximum number of collections per bull would depend on
the individual capacity of the bull.

11. Handling, processing & freezing of semen

11 (A) Premises

a) Sufficient trees shall be planted and lawns prepared around the

semen station to minimize dust in the premises.

Page 17 of 75
 b) The ceiling and walls of the laboratory shall be made up of non_
porous materials. A11 cracks and crevices shall be sealed to control
pests ald insects.
c) Entry of persons to the laboratory, other than laboratory
personnel, shall be strictly restricted. Airlock system or anti_room
shall be provided to avoid direct entry to the semen_processing
laboratory.

d) Laboratory windows shall preferably be made of fixed double glass

sheet with aluminum frame. The glass panes shall be plastered
with sun control films to avoid direct suniight. The doors shall be
kept closed, especially during dilutor preparation arrd semen
processing.

e) Preferably cassette tlrpe or, split type air conditioners fitted with air
puriffing system with remote temperature control mechanism
should be installed to maintain the room temperature at 2O"C _

22"C. T}:e number of ACs to be fixed to sustain this temperature

shall depend on the size of the processing room. Maintaining this
temperature is al essential requirement to achieve the optimum
results when single step dilution method is followed for freezing
semen. The flow of air from AC must not be towards the front side
of the Laminar Air Flow Unit. Adequate number of thermometers
shall be kept at suitable locations in the laboratory to monitor
room temperature.

Alternatively, central cooling with 10 to 15 air changes should

be
fixed, especially for tJre semen processing laboratory. This helps
to
control the bacterial load in the semen_processing laboratory and
in removing obnoxious odour. The processing laboratory should
ideally maintain around 55yo relative humidity.

Page 18 of75
0 Sink drains shall be decontaminated routinely with a disinfectant.
Sink shall not be placed in the semen processing room.

The floors shall be preferably made up of vitrified tiles. Floors and

horizontal surfaces shall be cleaned and mopped with a
disinfectant solution, as dirt and dust, which settle on these
surfaces, are the main sources of contamination.

h) Unwanted furniture, equipment and materials shall not be kept in

the laboratory as these only provide additional area for dust and
spores to accumulate.

i) Appropriate number of germicidal W lights (247 O Al may be

provided in the laboratory_(9p!&neU, laminar airflow unit, apron
and laboratory footwear cabinet etc with a common operating
switch outside the laboratory. These lights shall be switched bn'at
least 8 hours prior to commencement of work in the laboratory and
shall be switched 'ofl' before beginning the work. The date of
installation of the UV lights shall be recorded to facilitate timely
replacement as the life of W tube is of 2000 hours. A logbook
should be maintained for UV lights.

i) The laboratory shall be fumigated twice a week with Cold

Fumigant, using humidifier.

k) The efficacy of Fumigation should be regularly monitored by

undertaking bacterial load test (pre and post funigationl of the
laboratory environment. The bacterial load shall be measured every
week to monitor contamination of the laboratory atmosphere.

Page 19 of 75
 l) The working platforms, the exposed parts of equipments and other
items to be handled during processing of semen, shall be cleaned
with alcohol (Iso Propvll or Glutaril. It is advisable to repeat
7 Oo/o

the cleaning exercise after completing processing of semen.

m) Clean laboratory footwear, apron f coats, hand gloves, mask and

caps shall be compulsorily put on while working in the laboratorv.

n) Eating, drinking, smoking, etc. shall be strictly prohibited in the

laboratory. Unnecessary conversation should also be discouraged
in the laboratory. Besides, entry of unauthorized persons shall be
strictly restricted.

o) Long exposure of semen to ultraviolet rays, visible light in direct

sunlight and white florescent light causes chromosomal damage
and hence, direct exposure to such sources of light shall be
avoided. Hence, there shall be a provision for indirect or diffused
lighting inside the semen processing room. care shan also be taken
not to switch on tube lights in cold handling cabinet and laminar
air flow unit (LAFU). However, at the time of filting and sealing of
straws in LAFU, diffused light could be used.

11 (Bl Equipment

a) The exteriors of all equipment and furniture shal be creaned weekry.

The equipment shall be kept covered by plastic covers when not
in
use.

Page 20 of 75
b) The pre-filter of Laminar Airflow unit shall be cleaned Ouarterlv.
Routine servicing and DOP testing twice a year will ensure efliciency
of HEPA filters. Alternatively, culture plate test to monitor bacterial
Ioad of the air passed through the filters shall be carried out at weekly
interva-ls to assess its efficacy/ functioning.
c) Digital photometer / Computer aided Spectrophotometer shall be
validated with Haemoc5.tometer readings for sperm concentration
twice a year separately for cattle and buffalo (20 samples each).
d) The automatic semen straw hlling and sealing machine sha-ll be
thoroughly cleaned, immediately after use.

e) The microscope lens shall be gently cleaned daily with a piece of

cotton soaked in a mixture of ethyl and methyl alcohol (1: 1) or a
mixture of SOVo ethyl alcohol and 207o ether.

f) The bio-freezer shall be defrosted and thoroughly cleaned and dried,

immediately after use.

Incubators to maintain artificial vagina shall be cleaned and

disinfected regularly wittr 7 Ooh alcohol (lso Propyl).

h) Single distilled water shall be used in autoclave and thermo-controlled

water bath. The water bath shall be cleaned and filled with single
distilled water on a regular basis.

i) The thermometer kept immersed in water bath shall be cleaned daily

to ensure recording of correct temperarure. Alternately, water bath
fitted with digital display temperature indicator should be used.

Page 2l of 75
 i) The Liquid Nitrogen containers returned / received from outside shall
be disinfected thoroughly with 4%o sodium carbonate solution and
frnally with I to 4o/o formaldehyde.

k) The refrigerator meant for storing eggs, a.tibiotics and buffer shall
only for storing t-hese materials. while vaccines, medicines and other
materials shall be stored at a place away from the semen laboratory.
The refrigerator used for storing eggs, etc. shall be cleaned and
sterilized every week using alcohol swab.

l) The following equipment should be validated/ calibrated by

Manufacturer/ supplier or by NABL certifred laboratories once in a
year::
i. Thermometers
ii. Water Bath
iii. Weighing Balance
iv. Incubator
v. Autoclave
vi. Hot Air Oven
vii. Slide Warmer
viii. Micropipettes
ix. pH Meter
x . Cold Handling Cabinet
xi. Laminar Air Flow Units
xii Biological Freeznr
xiii. Microjet Ink printer
xiv. Filling & Sealing Machine
xv. Photometer
xvi. Triple distillation unit

m) All equipment used in semen processing should be covered under

Annual Maintenance Contracts

Page 22 of 75
11 (Cf Personnel Hygiere
Clothing, skin and hair of laboratory personnel are the recognized
sources of contamination. Hence, everyone should wear laboratory
aprons, footwear and take other necessary precautions all the time while
working in the laboratory. Hands shall be washed with soap and water
and rinsed witn' 7 Oo/" alcohol before commencing work in the laboratory.

The bull attendants and officials co mlng in rezular contact wi th bulls

must und test for Tuberculosis (TB) and Brucello sls every Year.

The other staff working in farm should be tested for TB and Brucellosis
once in two years.

11 (Df Diluents

a) Buffer and diluents should be prepared in a separate classified room/

zone.

b) All disposable and reusable supplies coming in contact with the

semen and dilutor must be sterile and devoid of toxins and pyrogens.

c) Prolonged storage of purified water is not recommended because water

purity deteriorates progressively over a period of time as heavy metals
leach from some glass and plastic storage vials / containers.

d) Glassware, collection tubes, etc. shall not be held from their rim /
mouth.

e) Manual pipetting shall be replaced with automatic adjustable

micropipettes with sterile disposable tips.

Page 23 of75
 f) After adding all the components of buffer viz. TRIS, Citric Acid,
Glycerol and Fructose in ultra-pure water (Autoclaved) or triple glass/
double distilled water it should be sterilized by microhltration
(Optional). If the buffer is prepared on the previous day, it should be
stored in the refrigerator and antibiotics are to be added next dav in
the morning after warming it to 34.C.

g).Antibiotics in diluents: A combination of peniciliin and

Streptomycin may be used in diluents. However, it is better to use a
combination of Gentamycin, 'Illosin, Lincomycin & Spectinomycin
(GTLS), having the additional benefrt of controlling Mycoplasma. The
altibiotics should preferably be of rissue culture grade or laboratory
grade and not those used for therapeutic purpose as injections.

h) Only fresh eggs shall be used for making dilutor. The eggs shall be
stored in refrigerator after wiping with dry cotton. Just before preparation
of dilutor, eggs shall be wiped with ZO%o alcohol (Iso propyl). To ensure
the quality, eggs shall be purchased from known sources.

i) The required quantity of yolk shall be separated from albumin on

sterile (autoclaved) standard firter papers (No. r) and yolk membrane sharl
be punctured using ster e grass rod, pasteur pipette or sterile
straws/
tip
under the Laminar Air Frow unit. only fresh semen extender/dilutor
shall be used since some constituents may get deteriorated during
storage. Even a day old extender should not be used.

11 lEf Evaluatioa & processing

a) The tube containing the freshly collected semen should be capped

with aluminum foil as soon as it is placed in the dynamic pass
box
before trarsferring to the raboratory. The colection tube
shourd
preferably remain capped until processed.

Page 24 of 75
b) As soon as the neat semen is received, it shall be kept in a thermo-
controlled water bath at 34o C under Laminar Air Flow Unit, after
recording the volume of semen.

c) After examination of sperm concentration and initial motility as soon

as possible, the semen samples shall be primarily diluted (l :1) with
dilutor maintained at 34"C.
After dilution of semen in the ratio of 1:1, it shall be extended further
(l-rnal) in appropriately sized flasks after with dilutor / flasks
maintained at the same temperature (34"C), under the Laminar air
flow unit. The neat semen sarnples should not be allowed to get
accumulated for long time in water bath to minimize adverse effects
on viability.

After the linal dilution the flask should be kept at room temperature
for lO-15 minutes so that temperature reaches 2O-22"C.

d) Sperm concentration shall be checked by a digitat photometer with

auto dilutor, manufactured by a reputed company. Semen samples
having concentration of less than 5O0 million I rnl sperm
concentration shall be discarded. The dilution unit of photometer
should be placed under laminar air flow unit.

The volume of straws should be checked as it may va4r between

manufacturer I type ol straw. While determining the dilution rate as
per the photometric reading, the correct volume of the French mini
straw should be fed to the photometer. The volume of randomly drawn
straws from a day's production shou-ld be checked as part of quality
assurance and be documented. Each straw (dose) should possess a
minimum of 2O million spermatozoa.

Page 25 of75
 e) Semen samples selected for processing should have a minimum of
7 Oo/o initial progressive motility.

f) Filling and sealing of semen shall be done under Laminar Air Flow
Unit using sterile straws, frlling nozzles and disposable rubber
tubing's. Rubber tubing's sha-ll not be reused in any case.
g) unused straws shall be repacked (air-tight) under Laminar Air Flow
Unit before storage. Immediately after use, all the glass ware and
other re-usable articles shall be immersed in lukewarm neu trai
detergent solution (in a plastic tub near the Laminar Air Flow Unit).

h) The freezing should be carried out as per the recommended protocols

for freezing cattle and buffalo semen. After freezing gets over, the
straws should be collected from the racks using scoop tongs. The
operator should wear appropriate protective gloves to avoid frost
injury

1l (Ff Colour Specifrcations of straws:

All semen stations shall folrow the foilowing colour codes for filiing of
semen in straws:
Table: 3 Colour Specifications

Breed Colour

Holstein Pink/Rose
I

HF Crossbred Pistachio Green (light green)

Jersey Yellow
I
I

Jersey Crossbred Salmon

I

Indigenous cattle Orange

I I

Page 26 of 75
 Sunandini Blue

Buffalo Grey

If any of above mentioned colour is not available, then transparent straws

shall be used.

I I (Gf Printing of Straws

Each Semen Station shall print certain key information on the straw in the
following Sequence, starting from the factory end.

1. SSBull ID - Alphanumeric
2. Breed of Bull
3. Semen Station name
4. Batch No with Ejaculation No in Parentheses
5. Dam's Lactation Yield and source ( e.g. 3380- PT)
6. Brand image
7. Brand Code

Items serially numbered from 1 to 5 shall be mandatory for all stations.

Items 6 and 7 may be optional.
NOTE - canister holder-label may preferablv have information of Bull No..
Breed, Source-PT/PS/ ET/lM/O, Dam lactation yield/breeding Value and
Name of Semen Station

Table: 4 Three Character Semen Station Codes

Sr. Semen Station State Type ss

No. Code

1 ABC, Salon I Uttar Pradesh Trust ABC

I

2 AMUL I
Gujarat Coop. AMU
I

Page27 of75
 3 BAIF Maharashtra Trust BAF

4 Bassi Rajasthan Coop. BAS

I

5 Bhopal Madhya Pradesh I Govt. BHO

i

6 Banavasi Andhra Pradesh Govt BNV

7 CFSP&TI, Karnataka GOI CSF

Hessarghatta

8 Dhoni Kerala Govt. DHO

9 Banas new Gujarat Coop DSU

I

10 Jagudan Gujarat Coop. DUR

I I

11 Haringhata West Bengal Govt. HGT

I
1a Hisar Haryana Govt HIS
I

13 Nandini Karnataka Coop. KMF

I

l4 Karimnagar Andhra Pradesh Govt KNG

I

15 Mattupatty Kerala Govt. MUT

I

I6 Nabha Punjab Govt. NBH

17 NJF, Ooty Tamil Nadu Coop. NJF I

I

18 FSPS, Ooty Tamil Nadu Govt OTY

I

19 Patan Gujarat Govt. PAT

20 SAG Bidaj Gujarat Trust BDJ

2l Salboni West Bengal Govt. SAL

)a Rishikesh Uttaranchal Govt. ULD
I

23 Anjora Chhattisgadh Govt. ANJ

I l
I

Page 28 of75
24 Aurangabad I Maharashtra Govt AUR

25 Babugarh Uttar Pradesh Govt. BAB

I

26 Beldanga West Bengal Govt. BEL

I

27 Bhatian Punjab Coop. BHA

28 Chitale Maharashtra Pvt CHI

I

29 Jind, BAIF Harvana NGO JND

I

30 Cuttack
I
I oaisna To.*. CTK
I

a1
Deep Frozen Semen Uttar Pradesh Govt. RKH
Station,Rehmankhera,
Lucknow

Dharwad Karnataka Govt DHA

I

JJ Echenkottai Tamil Nadu Govt EKT

34 Gurgaon Haryana I
Govt GUR
I
I
I

35 Hakkal, Jammu Jammu & Govt. HKL

Kashmir

36 Hosur Tamil Nadu Govt. HOS

I

J/ Jagadhari I
Haryana Govt. JAG

38 Barapeta (Khanapara) Assam Govt. BPI

I

39 Kulathupuzha Kerala Govt. KUL

40 Nagpur Maharashtra Govt. NAG

I

4l Nandyal Andhra Pradesh Govt. NDL

I I

42 Palampur Himachal Govt. PLM

Pradesh
I
I
I

Page29 of75
 +J Pune Maharashtra Govt. PNE
I
I

44 Rohtak Haryana Coop. ROH

I
I

45 Ropar Punjab Govt. ROP

I

46 SLBTC, Hessarghatta Karnataka Govt. SLB

I

47 Srinagar Jammu & Govt. SRI

I

Kashmir
48 SSCC, Hessarghatta Karnataka Govt. SSC
I
I

49 Vizag Andhra Pradesh Govt. VZG

50 Germplasm Station, Rajasthan Coop. NWA

Narwa, Jodhpur I I

51 Central Semen Bank, Meghalaya Govt SLG

Upper Shillong. I
I I I

52 Alamadhi Semen Tamil Nadu Trust ALM

I I
Station, Chennai
I

53 Rahuri Semen Station, Maharashtra Trust RHR I

Ahmednagar.
I

54 Hisar Bovine Research Haryana Private HBR

I
I

Breed Code formation

Sr.No. ss Bull ID Breed Code formation

1 Every Breed has its own two character code as Breed Code. For Exp._
Jersey:.Iy or Red Sindhi= RS etc. For Non-Descript breed use ND as
breed code. Refer trailing table for breed code list.

Page 30 of 75
Sr.No. SS Bull ID Breed Code formation

2 If there is no cross with the breed then that breed has lo0o/o blood
Ievel of its own. For e.g Breed code for 1007o pure Jersey animal will
be 'JY' . Therefore, SS Bull ID of a 1007o pure jersey bull at ABC
Semen Station will be ABC-JY-34'.

1
For crossbred, having two or more than two type of breed involve,
Breed code and SS BuI1 ID will be formed in the following order.

(a) In case exotic breed is available then breed code

ofthe exotic breed
having maximum Toage will be preferred and concatenated with 'CB'.
Eg. (1) a crossbred bull having HF(75%) and Sahiwal(2S%) breed will
have breed code 'HFCB'and SS Bull ID as ABC-HFCB-97'. E;gl2l a
crossbred bull having HF(25%) and Sahiwal(75%) breed will have
breed code 'HFCB'and SS Bull ID as ABC-HFCB-90'. Eg (3) a
crossbred bull having HF(SO%) and Sahiwal(SO%) breed will have
breed code 'HPCB'and SS Bull ID as ABC-HFCB-971'. So the breed
code in all the three cases will remain same as 'HFCB'

(b) Incase, exotic breed is not available then breed code of the
indigenous breed having maximum Toage will be considered and
concatenated with 'CB'. For e.g. a crossbred bull having
Sahiwal(SO%), Red Sindhi(2S%) and Gir(2\o/ol breed will have breed
code 'SHCB'and SS Bull ID as ABC-SHCB-97'.

Table: 5 Breed codes

s.N. SpeciesName BreedName CODE

Buffalo
1 Bhadawari BW
2 Buffalo Jaffarabadi JF
\) Buffalo Marathwadi MD
4 Buffaio Mehsana MH

Page 3l of 75
s.N. SpeeiesName BreedName CODE
5 Buffalo Murrah MR
6 Buffalo Nagpuri NP
7 Buffalo Nili-Ravi NR
8 Buffalo Pandharpuri PD
9 Buffalo Surti SU
10 Buffalo Toda TD
11 Buffalo Banni BN
12 Buffalo Chilika CK
13 Buffalo Kalahandi KD
).4 Buffalo Bargur BG
15 Buffalo Luit(Swam p) LU
16 Cattle HF HF
17 Cattle Jersey JY
18 Cattle Amritmahal AM
19 Cattle Bachaur BC
20 Cattle Bargur BR
2t Cattle Dangi DN
22 Cattle Deoni DO
23 Cattle Gaolao GL
24 Cattle Gir GR
25 Cattle Hallikar HK
26 Cattie Hariana HR
27 Cattle Kangayam KY
28 Cattle Kankrej KN
29 Cattle Kenkantha KK
30 Cattle Kherigarh KG
31 Cattle Khillar KH
32 Cattle Krishna Valley KV
JJ Cattle Malvi MV
34 Cattle Mewati MW
35 Cattle Nagori NG

36 Cattle Nimari NM
Cattle On le OG
38 Cattle Ponwar PW
39 Cattle Punganur PG
40 Cattle Rathi RT

Page 32 of 75
 41 Cattle Red Kandhari RK
42 Cattle Red Sindhi RS
43 Cattle Sahiwal SH
44 Cattle Siri SR
45 Cattle Tharparkar TH
46 Cattle Umblachery UB
A7 Cattle Vechur VC
48 Cattle Motu MO
49 Cattle Ghumusari GH
50 Cattle Binjharpuri BH
51 Cattle Khariar KR
52 Cattle Pulikulam PU
53 Cattle Kosali KS
54 Cattle Malnad Gidda MG
55 Cattle Belahi BL
56 Cattle Gangatiri GT
57 Cattle Badri BD
58 Cattle Ladakhi LD
59 Cattle Konkan Kapila KP
60 Cattle Lakhimi LK

11 (Hf Post thaw motility

After freezing, the semen straws shall be stored in a separate container.

Post-thaw motility of semen should be examined at 24 hours (after
freezing). Differences in observations shall be updated and recorded for
the purpose of accepting a particular batch of semen doses. Whenever
there is any doubt, post-thaw motility shall be examined by two
experienced persons. Preferably, the person involved in evaluation of neat
semen, shali not check the post thaw motility. For a minimum
concentration of 20 million per dose, minimum acceptable post thaw
motility shall be 507o. Semen doses below 50% motility shall be

Page 33 of 75
 discarded. semen samples with circular a,d Jerky movements and with
any other abnormal motitity shall be discarded.

11 (Il Quality Checks for frozen semerl

The quality control measures are sub divided into mandatory and
optional tests and include continuous monitoring of semen production at
various steps/sub processes essential for quality semen production. It shall
be mandatory to document quarterly suamary of each test including the
number of samples tested and the number of sampres not meeting
standard with the follow up action taken, The quarterly summary will
be authenticated by the Officer iu-charge ofthe Semen Station.

Part-A Mandatory Testing

1.O Quality Control ofSub processes

2.O Quality Control ofSub products
3. O Quality Control ofEnd product
4.O Feedback & Fertility Testing
Part- B Optional testing

Mandatory Testing

1.O Oualitv Control of Sub processes:

1.I Microbial load Testins

Sr. I Sub Process Name of Frequency Action to be taken

I

no. test to be
I carried out I

Page 34 of 75
 1.1(af Sterilization Microbial Daily, Failed test -Re-
[,oad random wash and steril2e
I
Testing of sampling, a the whole lot of
Glassware minimum of Glassware again on
two glass the date of result
wares assuming that the
same error in
sterilization process
I I is continuing.
I

1.1(bl Dilution & Microbial Daily two Failed Test -

Filling/sealing load media plates Appearance of (even
I
Testing to be exposed a single) CFU
in each following incubation
I
laminar flow for 48 hrs
I
unit
I Action taken -
calibration &
maintenance check
of LAFU
I

r.1(c) AV Microbial Daily, Failed sample -Re-

Load raldom wash and sterilize
I

Testing sampling of a I
the whole lot of AVs
I
I of AV Wash minimum of again on the date of
I I two ready to result assuming that
I
I
use AVs the same error in
I
sterilization process
is continuing.

1.2 Calibration of Equipment:

Sr Activity Frequency Method

no
i

Page 35 of 75
 1.2(af Calibration of Annual Outsourced
I

Equipment from NABL

accredited lab
I

1.2(bl Calibration of Six monthly By the

Photometer manufacturer
I I

for filters and

equation

1.3 Validation Process :

Sr no. Activity lFrequency lfretnoa Action to be taken

1.3(af Random checking Six monthly As per If variation, I
I

of sperm (cover all SOP unacceptable

concentration ia
straws bulls) > Dilution
107o;
I

activity, straws &

Calibration to be
taken up with AMC
Provider
1.3(bl Validation of Six monthly As per If variation,
Photometer (cover all SOP unacceptable
I

bulls) >2O7o; Calibration

to be taken up with
I

I I
AMC Provider

2.O Ouality Contro I of Sub Prod ucts

Sr Sub Test to Frequency Action to be taken I

no. Product be carried I

out
2.rlal Diluter pH- Each lot of Failed sampie to be
I testing Buffer & diluter discarded recorded and

Page 36 of 75
 prepared details to be given in
quarterly report.
I

QtH ofbuffer should not

I
be belout 6.7 and not to be
aboue 7. 1)

3, O Oualitv Control ofEnd product

3.1Frozen Semen S traws:

Sr. no. Name of test to Frequency Action' to be taken

be carried out

3.1(af Incubation Test Daily, randomly As per interpretation

selected ejaculates table given below
(10 samples) to be
tested before passing
to storage

(Where large samples

are to be tested for
trIM 05 samples for
incubation)

Interpretation of Incubation test

Incubation- Observation and Action taken

I

Time

O min (PTMI Pass sample = >l= 5O%o

I
I

Failed sample to be discarded, recorded and details to be

given in quarterly report

I 12O min I Pass samPle =

rl= 2O"/o

Page 37 of 75
 Failed sample to be discarded, recorded and details to be
given in quarterly report

Note :
Bulls with more than 3O7o ejaculates being discarded in a month
may be subjected to additional tests at the discretion of the Oflicer In-
charge.

3.2 Microbial load of FS Straws

Sr, End Name of Frequency Method Action' to be

I I

no. Product I test to be taken I

carriea out
I I

3.2lal Frozen Bacterial Daily, As per Failed test -

Semen load Test random SOP I
If all three
Straws sample of
samples have
FS straws
higher No of CFU
of three
than the MSP ;
I Bulls
i the entire batch
I
(All bulls to be Re- tested .

to be Failed samples
I
completed to be discarded,
in quarter) recorded and
I

details to be
I

given in
quarterly report

3.2(bl Monitoring Incubation 2-4 As per Faiied sample to

I
I

Page 38 of 75
 of Semen cuE Post- (random) SOP be discarded,
Quality thaw samples in recorded artd
under motility storage for details to be
Long more than given in
I

Storage six months Quarterly report

I

(and
I
I
thereafter

tt
I

every six
I

tt months)
subjected
to PTM &
I
Incubation
i
I
test as
I
above

3.21 Monitoring PIA To be As per Failed sample/

cf of Quality decided by SOP batch to be
of frozen Sperm discarded,
semen I
station/
QCO (each
I
bull at
least twice
annually)
I

3.2(d) Monitoring HOS Test To be As per Failed sample /

of Quality decided by SOP batch to be
of frozen Sperm discarded,
semen I station/
I
QCO (each
bull at I

I
I
least twice

I
I
annually)

Page 39 of75
3.3 Use of Equipments for Advance qualitv Check: (Vlherever
laboratory is equipped)

Sr no. Activity Frequency Method Action to be

I
I

taken I

3.3(af CASA Evaluation Monthly As per number of I

of neat/ frozen SOP samples to be

semen tested should be

decided by Sperm
I
station/ QCO

I I

3.3(bl Evaluation by Monthly As per I

Do I

Flow c5rtometer SOP I

I

Page 40 of75
 4.O Feedback & Fertility Testins:

Activity F.requency Action to be taken

I

Compilation of Quarterly; preferably Bulls having less than

I
authenticated changing bulls every 307o Conception rate
I

conception data based I

quarter- should be subjected to

on at least 5OO A.I/bull detailed investigation
for a minimum, of 2Oo/o before culling
of the bulls in semen
production
represeuting all breeds I

at the sperm station

(Note-Data can be obta ined

using INAPH application )

Part- B Optional testing:

Name of Frequency iMethod Action to Remarks

Test be taken I

Sperm To be decided As per SOP As per the Conditional

Morphology by Sperm advice of
I testing station/ QCO QCO

Live - Dead To be decided As per SOP I

A S per th e Conditional
count by Sperm i advice of
station/ QCO
lo""

11 (Jf Information Systen

In order to facilitate the information system, all the bulls maintained by

the semen station must be identified using unique 12 digit, bar coded
system compatible with INAPH and each bull be given an alphanumeric

Page 4l of 75
identification by the respective SS as described in earlier section to
enable on-line reporting and real time data capture of semen production
and artilicial insemination.

The semen stations shall use suitable software to record data pertaining
to various activities and also should have online facility for the same. The
software should be able to identify and trace the bulls and their
ejaculates, production, storage and dispatch of semen etc.

a) Record for volume of semen, motility, sperm concentration, dilution

rate, total extended volume, post-thaw motility (24 hrs after freezing),
and total number of doses produced, etc. shall be maintained.

b) Miscellaneous information regarding actual reason(s) for not donating

semen, undesired percentage of gross morphological defects, semen
pH, presence of dirt, dust, blood, pus, etc. in semen samples shall be
noted and recorded.

c) Details of semen supplied to various agencies, including post-thaw

motility at the time of dispatch, shall be recorded.

d) Fertility data of bulls, conception rate, records of the progeny

associated with any genetic defect, percent male I female born, etc.
shall be noted and recorded.

e) Data/ record of microbiological examination of semen samples shall

be maintained.

f) Record of all quality tests for neat and frozen semen samples shall be
maintained.

Page 42 of 75
11 (Kl Semen Storage

To avoid accidental spread of diseases, tJ.e semen station shall follow the
procedure of preserving semen doses for at least 30 days (quarantine)
after production. Frozen semen doses produced at least 3O days prior to
the date of dispatch should only be supplied for AI.

After checking post-thaw motility and found acceptable, frozen semen

straws shall be stored in temporary storage container for 3O days. After
temporary storage, the semen goblets shall be transferred to the bulk
storage containers with proper recording of position in the canisters.
After each dispatch, records redefining the position of remaining doses
shall be updated.

Ttvo reference samples of the doses dispatched should be drawn and

retained for six months or a screen shot of randomly selected sarnple
should be stored and a soft copy of which should be given to the
customer

The goblets containing the semen should be well identified and

precaution should be taken to see that each goblet has sufficient space
for liquid nitrogen. Mini straws need special care and should not be
exposed above liquid nitrogen even for a short time (10 seconds) as they
get warm faster and any exposure causes irreversible damage to sperm
viability.

Liquid Nitrogen shall be replenished at regular intervals depending on

the liquid nitrogen evaporation rate ofthe container.

12 Bio-security

Page 43 of 75
 The risk of disease spread has grown manifold with increasing number
of bulls maintained at the semen production center. With the expected
higher risk, implementation of strict bio-security measures at the semen
stations assumes greater significance. Every semen station should have
a well defrned Bio-security protocol put in place across all its activities.
There shall be a designated Bio-security Officer (Veterinarian) at each
Sperm Station. The premises should be demarcated into high, medium
and low risk zones.

The detailed. protocol lor Bio-securitg circulated. to all SS mcy be

refefted for strict compliance.
13 Cleaning and Sterilisation

All the items to be washed shall be initially cleaned with running tap
water and soaked in warm neutral detergent for at least 30 minutes.
These items will then be thoroughly cleaned under running tap water
using a brush. Filling nozzles shall be cleaned with pressure using 20 ml
syringe. These materials shall be rinsed thoroughly with de-ionized/
distilled water (3 changes) to completely remove detergent residues and
other impurities. Appropriate procedure for sterilization of different
materials, recommended for use in the semen station follows.

13.1 Laboratory and other areas

cold fumigation solution is ideal for fumigation of laboratory and other

areas. It should be done as per SOP/ manufactures guidetines.

13.2 Artifrcial Vagina lAVl

Page 44 of 75
 a) cone from the AV and water from AV jacket shall be removed before
washing.

b) Cones and AVs shall be cleaned thoroughly with a soft sponge brush
under running tap water and then soaked in warm neutral detergent
for about 3O minutes, followed by proper rinsing in warm and clean
water and then three times rinsing with double distilled water.

c) For sterilization, fully assembled AVs shall be autoclaved at 5 p.s.i.

pressure for 20 minutes. During steriiization, the valve of AV shall be
kept open. Alternatively, use AV sterilizer (using double distilled water
in the sterilizer) for proper sterilization of AVs.

d) Finally, AVs hlled with water and covered with aluminium foil, at both
ends shall be stored overnight in an incubator at 45o C.
e) To achieve best cleaning effect, AVs sha11 be cleaned immediately after
use, preferably by non-spermicidal neutral detergent.

13.3 Glassware

a) The glassware shall be washed thoroughly with running tap water and
soaked in warm, non-spermicidal neutral detergent solution for about
30 minutes.
b) Using appropriate nylon brush, the glassware shall be cleaned and
rinsed with running tap water. The collection tubes shall be brushed
at least 3 times and thoroughly cleaned and rinsed with distilled
water.

c) Finally the glassware shall be rinsed three times with double distilled
water and allowed to dry by keeping them inverted on a blotting paper
or a drying stand made of SS/ plastic.

Page 45 of 75
 d) The open end/s of the dried glassware shall be covered with
aluminium foil and sterilized in hot air oven at 160'C (holding) for one
hour or at 180'C for 30 minutes. One item should be wrapped with
newspaper and its mild charring will indicate proper sterilization.

13.4 Rubber wares

The washing and cleaning procedure of rubber wares is similar to that of

glass ware. Care shall be taken to clean the rubber wares with sponge
brush instead of nylon brush. Plastic tips shall be cleaned by water jet
with force using a syringe. Sterilization technique, however, differs owing
to the thermo-sensitivity of the rubber items. Thermo-resistant rubber
wares shall be sterilized by autoclaving at 3 - 4 p.s.i. for 10 minutes.
(The rubber tubing for semen filling shall not be reused).

13.5 Distilled Ulater

Fresh triple glass distilled water or Ultra pure water shall be autoclaved
at 15 p.s.i. for 15 minutes and used for preparation of the dilutor.

13.6 Bulfer

Buffer shall be sterilized by microfiltration on 0,2 pm membrane

fi1ter. (optionaJ)

13.7 Bacteriological Media

It is to be autoclaved at lS p.s.i. pressure for 15 minutes.

13.8 Filter Papers

Page 46 ol75
 A bunch of clean filter papers of standard brand - No. 1 (thrashed to
remove dirt, if any) shall be wrapped in thick cotton cloth for sterilization
in an autoclave at 5 p.s.i. pressure for 20 minutes. Alternately. these can
be sterilized drv in suitablv sized dishes in hot air oven at 180
desree Centiqrade for 30 minutes.

14 Summary of Sterilization
Table:6 Autoclave
Sr.No. Item Pressure Time
I
(p.s.i.l (Min.f

1 Artificial Vagina 5 20

2. Piastic Tips 5 20

3 Filter Papers 5 ci

4 Bull Apron 5 20

5. I Thermo-resistant Rubber J-+ 10

wares
6. Bacteriological Media 15 15

7. Distilled Water 15 15

8. Surgical Equipment 10 10
I I

(The rubber wares can withstand above pressure and duration

provided the quality is good)

Table: 7 Hot Air Oven

Sr.No. Item Temperature Time

(min.)

Page 47 of 75
 1 Glass wares 1600C/ 1800C 60l30
2. Filling Nozzles 1600C/ 1B0oC 60l30

cf AV Steriliser

wherever Autoclave is not used, AVs and rubber cones shan be sterilised
using AV sterilizer. After sterilizer starts boiling, 30 minutes vapour
sterilisation shall be do

d) Validation of Sterilization

To ensure that potentiany infectious agents are destroyed;

the efficacy of
sterilization regimes may be subjected to validation through _physical,
Chemical or Biological tests, if required.

15 Quality Control of Consumables

Chem icals
The chemicals of only highest purity of either, Analltical
Reagent (AR) or
Guaralteed Reagent (GRf, from reputed manufacturing companies
shall be
used' whenever a new chemical is to be introduced in the routine
process, it
is recommended to examine the post-thaw revival rates after
conducting a
few spilt ejaculate trials (maintaining a controt) with the
new chemical.
Assay of chemicals shall be >99%o, having iess impurities.

Straws

1. Straws manufactured by highly reputed companies

are safer to use for
production of qua,lity semen. While buying straws,
package voiume and
microbial load in straws shall be checked randomly
from the
consignment. In addition, some empty straws should
be praced in filling

Page 48 of 75
 and sealing machine and the machine should be run to see the sealing
quality of the straws. In case of any foul smell, it should be presumed
that the straws are manufactured from poor plastic which could be toxic
to the spermatozoa and can even result in reduced motitity on long
storage.

2. The factory plug should not be loose. The factory seal should be
impenetrable and the seal formed should be homogeneous and compact.

3. The straws should be intact (without cracks / dents, etc.) and should not
get damaged during filling / sealing and after fteezing thawing.
/
4. The movement of straws along the printing machine should be free and
print should be clear and sharp. print should not fade as a result of
freezing and subsequent thawing.

5. The use of dark coloured straws should be avoided, as they are not
transparent enough. Due to this, it is diffrcult to distinguish between
lilled / semi-filled straws.

6. Movement of the factory plug should be free.

7. Straws should be routinely checked for microbial load.

Note: The semen stations should avoid purchase of consumables/ straws

simply on lowest quotation basis. The quality should never be
compromised in view of the stakes involved. Only time-tested
products may be used keeping in view the storage for a very long
period. For example: To produce top quality semen, it is better to use
AR / GR reagents manufactured by reputed companies (for eg-
Sigma, Merck, Himedia etc ) whose products are reliable and time-
tested. This is true with other consumables used for semen
production as well.

Page 49 of 75
16 Manpower Requiremeat for semen production
Tabie: 8

Designation Up to >10-25 >25-50 >5O lakh Mega

10 lakh lakh doses Semen Stn,
lakh Doses Doses l0m doses
doses

Page 50 of 75
 Officer In-charge 1 1 1 1

QCO/QAO 1 1 1 1 1

Vet. Officer 1 2 3 J-+ 5-6

Agriculture 1 1 1 1 1

Officer
Data Mgmt. 1 1 1 1

Ofhcer
Accts. & Adm 1 1 t-2 1-2
Oflicer I
I

Office Assistant 1 2 o
5 6-7

Livestock I 2 3 4 5
Assistant I

Agri. Assistant
I

Lab Technician I
1 2 3-4 I s-6 8-10

Vehicle/Tractor 1 2 3 4 5
Driver I I

Lab Artendant a
I
2 3-5 7-8 10-t2

Bull Attendant 1 person per 7- 8 bulls

Agri. Labourers l5-2O I 10O acres depending on

mechanization level

The manpower structure suggested above is meant only for semen/fodder

production. For other activities, manpower may be positioned as per the
need. For dispatch of semen, facility should be created preferably away from
semen station and operated by person/s not involved in semen production.
The GOI / Department of AH / Livestock Boards / NGO / Private agencies /
Cooperatives owning the semen station shall review the requirement of
manpower position for each semen station and finalize the staff structure for

Page 51 of 75
recruiting additional manpower. After recruitment, all new persons shali be
trained at any of the recognized institutes. Once trained, they shall continue
to work in the semen station at least for five years.

It 15 reco nded that Semen station ln-chars e should have at least 05

vears experience in bovine semen production

Refresher training / visit to other semen lab: technical exposure of semen

station personnel working in the semen lab must be arranged compulsorily
once in two to three years at reputed institutions like CFSp&TI -
Hessarghatta, KLDB - Mattupatty, and other stations using state of the art
technologr etc. As semen production activit5z is a highly professional
/technical work, job rotation of personnel could be detrimental in
maintaining the quality of semen. Therefore, personnel working in a semen
station should not be transferred at least for five years. If it is inevitable, a
proper replacement should be identilied at least six months in advalce and
shall be trained in semen production technologr.

Page 52 of 75
 Annexure- 1

DEFINITIONS FOR USE IN THE HEALTH PROTOCOL

Bull Adult maie cattle or buffalo used for collection of

semen. Teasers and other animals resident in the
semen stations are also subjected to similar
disease testing, vaccination and medications for
I

I
maintaining their health status.
Bull Calf A male cattle or buffalo which has not yet
reached sexual maturity.
Known Animals originating from a semen station or
health status rearing station that is strictly complying with the
guidelines mentioned in the MSP.
MSP diseases MSP diseases are the set of diseases - the
I
causative organism of which should not be
present in the semen - or preferably in the bu1l.
I

These diseases include Bovine Bruceilosis,

Tuberculosis (TB), Paratuberculosis (JD), Bovine
Genital Campylobacteriosis, Trichomoniasis and
Foot and Mouth Disease (FMD).
Quarantine A farm where bulls or bu1l ca,lves are isolated and
station examined to assess the health status before
shifting to the semen station or rearing station. A
series of clinical and laboratory examinations,
vaccinations and medications etc. are
undertaken during quarantine.
Rearing A farm where bull-calves or young bulls, coming
station from quarantine station are reared till they attain
sexual maturity and subsequently get shifted to
semen station. A series of clinical and laboratory
examinations, vaccinations and medications etc.
are undertaken during the stay of bull calves in

Page 53 of 75
 the rearing station to maintain their health
status.
Semen A farm along with semen processing facilities
station where adult bulls are housed for semen
collection and processing. A series of ciinical and
laboratory examinations, vaccinations and
medications etc. are undertaken during the stay
of bulls in the semen station to maintain their
hea,lth status.
Unkaown Animals originating fro m village or farm where all
health status the animals of the farm or the viliage have not
been tested against the MSp diseases

Page 54 of 75
 Annexure- 2

Details ofthe tests to be conducted I

Disease Test Sample Tested by

I

oflicers of
Brucellosis ELISA Serum CDDL/RDDL/
I
NDDB/ State
Veterinary
I
Universities
TB- DTH- Intra-dermal on Semen station/
Tuberculin PPD the bull CDDL/RDDL/
NDDB/ State
Veterinary
I
Universities
JD- DTH- Johnin Intra-dermal on Semen station/
PPD the bull CDDL/RDDL/
NDDB / State
I
Veterinary I

I Universities I

Trichomoniasis Agent Preputial CDDL/RDDL/

identification washings / NDDB/ State
semen Veterinary
I

Universities
Bovine Genital Agent Preputial CDDL/RDDL/
Campylobacterio identification washings NDDB/ State
sis Veterinary
I

Universities
FMD ELISA Serum PD-FMD,
I

Mukteshwar
I
and its
I I
I
laboratories/

Page 55 of75
 NDDB /Stare
Veterinary
I

I
I

I
Universities
IBR ELISA Serum for CDDL/RDDL/
Real time- PCR ELrSA (9 NDDB/ State
months age) Veterinary
Semen for RT- Universities
PCR
BVD ELISA 2 times Serum CDDL/RDDL/
at 30 days NDDB/ Stare
I

interval I
Veterinary
(RT-PCR up to 6 I
Universities
I

I
months age)

* TB and JD testing at Quarantine station as well as Rearing station

shall
be performed by the oflicers of the semen station. However, the testing at
the semen station shall be done by the officers of the GDDL/RDDL/NDDB/
NABL accredited State Veterinary Universities and approved by CMU

Page 56 of 75
 Annexure- 3A

Quarantine Guidelines

A. Quarantine of adult bulls of unknown health status

Quarantine Minimum 60 days or long enough to allow at least
period two tests for MSP diseases to be performed during
quarantine with a minimum interval of 30 days
between the two tests. In case ofTB and JD the
interval between the two tests should not be less
I
than 62 days.
Shifting of Within 3O days from the date when the last test was
bulls from performed and all bulls were found negative.
the
quarantine
Action on Brucellosis, TB, JD, Cull / remove the positive bull
finding a Bovine Genitai ald put all the remaining
positive Campylobacteriosis, bulls under extended
result Trichomoniasis, IBR, quarantine.
BVD (Pr)
Extended For a period of minimum 60 days or long enough to
quarantine allow at least two tests for the diseases mentioned
above to be performed, from the day last positive bull
was culled/ removed. Perform one test within the last
30 days of the extended quarantine.
Action on During Quarantine, if the bulls are housed and
finding a managed
positive . Individually - Remove only the positive bull.
during . In groups (not more than 3 animals in each
extended group) - Remove all bulls in t1le group in which
quarantine positive was detected.
o Free and not in groups- Remove all the bulls.

Page 57 of 75
 B. Quarantine of adult bulls of known health status
Quarantine Minimum 30 days or long enough to a11ow at least
period one test for all MSP diseases
Shifting of Within 30 days of the last negative test
bulls from
the
quarantine
I

Action on Same as in Annex- 3A

finding a
positive
result
Extended For a period ol minimum 3O days from the day iast
quarantine positive bull was cufled/ removed. perform one test
wittrin the last 30 days of the extended quarantine.
Action on Same as in Annex- 3,{
finding a
positive
during I

exterded
quarantine

Page 58 of75
 Annexure- 3C

C. Quarantine of adult bulls to be shifted between the farms

managed by the same administration
f For shifting between semen stations for semen production
f From a rearing station that implernents Quarantine
(Annexure-3D) before allowing entr5r of ca,lves for rearing
Quarantine Minimum 30 days or sufhcient to a-llow at least one test for
I
period MSP diseases
Shifting of Within 30 days of the last negative test
bulls from
the
quarantine
Action on Same as in Annexure- 3A
finding a
positive
result
Extended For a period of 30 days from the day last positive bull was
quarantine culled/ removed. Perform one test within the last 30 days
of the extended quarantine.
Action on Same as in Annexure- 3.4
finding a
positive
during
extended
quarantine

Page 59 of 75
 Annexure- 3D

D, Quarantine of calves above 2 months of age

Quarantine Minimum 60 days or sufficient to al1ow at least two
period tests for each of the MSP diseases to be performed
with a minimum interval of 3O days between the I

tests. In case ofTB and JD the intervai between the

two tests should not be less than 62 days.
Shifting of Within 30 days of negative results.
calves from
quarantine
Action taken TB, JD Remove the positive calf and
on finding put all the remaining calves
positive calf under extended quarantine.
Bovine Genital Tests conducted only on calves
Campylobacteriosis older than 6 months,
and Trichomoniasis Remove the positive calf and
put all the remaining calves
under extended quarantine.
Brucellosis, Remove the positive calf
IBR, irrespective of age and put all
BVD (Pr) the remaining calves under
extended quarantine.
OR
For Brucellosis and IBR: If the
positive calf is less than 9
months old, isolate the calf till
it is 9 month old and retest.
Calf positive at retesting
should be removed.
Extended For a period of mini mum 60 days from the day last
quariantine positive calf was removed. perform one test within the

Page 60 of 75
 last 30 days ofthe extended quarantine.
Action on Same as in Annexure- 3A
finding a
positive
during
extended
quarantine

Page 6l of 75
 Annexure- 4

Disease testing and manageme[t of Bovine Tuberculosis in Semen

Station

Name of test Delayed Hypersensitivity - Single Intra Dermal (SiD)

Test

Reagent used Bovine tuberculin PPD

Manufacturer IVRI, Izatnagar

Testing done On site, where animals are housed

Result Positiae: Increase in skin thickness of 4 mm or

criteria more, or presence of clinical signs viz. exudation,
necrosis, pain, ald inflammation of the lymphatic
duct of that region or the lyrnph node, 72 hours
post-inoculation.

Negatiae'. Increase in skin thickness less thal 2

mm & without clinical signs viz. exudation,
necrosis, pain, inflammation of the lymphatic duct
of that region or the lymph node, T2 hours post-
inoculation.

fnconclusioe: Increase in skin thickness more t_han

2mm & less than 4mm, absence of above clinical
signs, 72 hours post-inoculation_ Bull with
inconclusive result should be immediately isolated.
Only if tlle animal is negative during the testing in
isolation, it should be brought back to the semen
station.

Eligible Animals above 2 months of age

I
animals

Page 62 of 75
 Action to be Immediate isolation and removal from herd (within
taken on 2 days)
Positive
animal

Frozen sernen Destroy frozen semen doses of the positive animal

doses of the since the last negative test.
positive
animal

Positive herd Testing not before 42 days after culling of last

testing positive anima-l.

Negative herd Six monthly (t 1 week) testing after last whole herd
I

testing negative testing.

TB free herd Herd found negative on two consecutive tubercuhn

tests carried out at an interval of 6 months, the frrst
being performed 6 months after the culling of last
I

affected animal.

If frequency of testing is more than two in a year,

I
the testing should establish that all animals in the
herd have been negative for the last 6 months
beginning from 6 months after culling the last
affected animal.

Page 63 of 75
 Annexure- 5

Disease testing and management of para tuberculosis (JD) in Semen

Station

Name of test Delaye d Hypersensitivity - Single Intra Dermal (SID)

Test

Reagent used Johnin PPD

Manufacturer IVRI, Izatnagar

Testing done On site, where animals are housed

I

Result criteria Positiue.' Increase in skin thickness of 4 mm or

more, or presence of clinical signs viz. exudation,
necrosis, pain, ald inflammation of the ll,rnphatic
duct of that region or the lymph node, 72 hours
post-inoculation.

Negatioe: Increase in skin thickness less than 2

mm & without clinical signs viz. exudation,
necrosis, pain, inflammation of the lymphatic duct
of that region or the lymph node, T2 hours post_
inoculation.

Inconclusiae: Increase in skin thickness more than

2mm & less than 4mm, absence of above clinical
signs, 72 hours post-inoculation. Bull with
inconclusive result should be immediately isolated.
Only if the animal is negative during the testing in
isolation, it should be brought back to the semen
station.

Eligible Animals above 2 months of age

animals

Action to be Immediate isola tion and removal from herd (within

Page 64 of 75
 taken on 2 days)
Positive
anilnal

Frozen semel Destroy frozen semen doses of the positive animal

doses of the since the last negative test.
positive
animal

Positive herd Testing not before 42 days after culling of last

I
testing positive animal.

Negative herd Six monthly (t 1 week) testing after last whole herd
testing negative testing.

JD negative Herd found negative on two consecutive Johnin

I
herd tests carried out at an interval of 6 months, the first
being performed 6 months after culling of the last
affected alimal.

If frequency of testing is more than 2 in a year, the

testing should establish that all animals in the herd
have been negative for the last 6 months beginning
from 6 months after culiing the last affected animal.

Page 65 of 75
 Annexure- 6

Disease testing and management of Bovine Brucellosis in Semen

Station

Name of test Er.zyrr.e Linked Immunosorbent Assay (ELISA)

Sample Serum
required
I

Eligible A11 alimals. However, animals up to 9 months of

animals age may have maternal antibodies. I

Action to be Immediate isolation ald removal from herd after

taken on the castration (within 2 days)
positive
animal

Frozen semen Destroy frozen semen doses of the positive anima1

doses of the since the last negative test.
positive
animal

Positive herd Testing 30 to 60 days after culling of last positive

testing animal.

Negative herd Six monthly (t 1 week) testing after last whole herd I

testing negative testing.

Brucellosis Herd found negative on two consecutive annual

free herd tests. I

If the frequency of testing is more than one in a

I
year, the testing should demonstrate that the herd
has been negative for the last one year

Page 66 of 75
 Annexure- 7

Disease testing and management of Bovine Genital Campylobacteriosis

(BGCf in Semen Station

Name of test Agent -Identification

Sample required Preputial washing/ semen

I

Eligible animals Animals above 619 months of age of Exotic and

their crosses and 24-30 months in bul1s of
indigenous breeds including Buffalo ( when
I
there is free penile movement)

Positive animal Immediate isolation and removal from herd

(within 2 days)

Frozen semen Destroy frozen semen doses of the positive

doses of the animal since the last negative test.
positive animal

Positive herd Minimum of 30 days after treatment/ culling of

testing last positive animal.

Negative herd Annual (t I week) testing after last whole herd

testing negative testing.

I
Bovine Genital A11 alimals are negative on two consecutive
Campylobacterio annual testing.
sis free herd

Page 61 of 15
 Annexure- 8

Disease testing and management of Bovine Trichomonosis in Semen

Station
Name of test Agent -Identification

Sample Preputial washing

required

Eligible Animal s above 6/9 months of age in Exotic and

animals their crosses and 24-30 months in bu1ls of
indigenous breeds including Buffalo ( when there is
free penile movement)

Action to be Immediate isola tion and removal from herd (within

taken on 2 days)
Positive
animal

Frozen semen Destroy fro zen semen doses of the positive animal
doses of the since last negative test. I

positive
animal

Positive herd Minimum of 3O days after treatment/cuiling of last

testing positive animal

Negative herd Annual (1 1 week) testing after last whole herd

testing negattve testing

Bovine A11 animal s are negative on two consecutive annual

Trichomonosi testing.
s free herd

Page 68 of 75
 Annexure- 9

Testing and management of Infectious Bovine Rhinotracheitis (IBR) at

semen stations
Name of the test E;nzyrl:e Linked Immuno absorbent Assay (ELISA),
Real-time PCR
Sample (s) Serum for ELISA, semen for real-time PCR
required
Induction of new Only negative animals will be inducted.
animals into A11the animals to be inducted irrespective of their
herd/semen age should be put on hold and inducted only if
stations found test negative after the age of 9 months.
Sero positive Action in order of priority:-
bulls at IBR (i) Immediately cuII sero-positive animals and
positive semen castrate them.
station (ii) If culling is not possibie, immediately isolate the
animal and process and store their semen
separately. Test each ejaculate by real-time PCR
(rt-PCR). Semen positive by real-time PCR shall
be destroyed by incineration. Use only semen
that has tested negative by rt-PCR.
(iii) Test all the animals at three months interval.
Action to be (i) A11 positive bulls culled immediately.
taken on bulls at (ii) Retest remaining bulls at 30 - 60 days after
I
the IBR free culling last positive animals. Repeat (i) & (ii) until
Semen Stations** the remaining herd is tested negative. Therea,fter
test at 6 monthly interval.
(iii) The negative herd should be tested at 6 monthly
interval.
Documentation Records of all the ejaculates collected from sero-
positive bulls, the results of real-time PCR, details of
real time PCR positive ejaculates destroyed and
details of agencies where semen has been
distributed shall be maintained.
**Please refer to the Guidelines for progressive IBR/BVD control issued by
DADF , Govt of India and as amended from time-to-time.

Page 69 of 75
 Annexure- 10

Testing and management of Bovine Viral Diarrhoea (BVDI at semen

stations

Name of the test Enzyrne Linked Immuno absorbent Assay (ELISA) for
detection of antigen (Ag-ELISA)/real time pCR (rt
PCR)
Sample Serum
Induction of new Test th e animai for Persistent Infection (pI) by testing
animals into two times at al interval of at least 30 days by Ag-
herd/semen ELISA. Test by rt-PCR instead ofAg-ELISA for
statious animals up to 6 months of age.
If the animal is positive on both the tests, the arlimal
is considered positive for pl. Only pI negative animals
shall be inducted.
Action to be taken Immediately isolate and cu11
for PI positive
animals
Semen dosses of PI Destroy by incineration of frozen semen doses of the
positive animals PI positive bulls.

Bulls at the semen (i) Test all the b ulls for PI (if not already tested for pl
stations status) by testing two times at an interval of at least
30 days. If the bull is found positive on both the
tests, then the bu1l is considered positive for pI. All pI
positive bulls shall be culled immediately.
(ii) Test all new bulls entering the semen station for
PL Only PI negative bulls should enter the semen
station.

Please refer to the Guidelines for progressive IBR/BVD

contror issued by
DADF , Govt of India and as amended from time_to_time.

Page 70 of 75
 Annexure- 11

Management of Foot & Mouth Disease (FMDf in Semen Station

f.MD outbreak in semen station

Immediate Immediate disinfection of premises and fomites.

action to be
Destruction of contaminated feed & fodder by I

taken
burning.

Frozen semen Destroy frozen semen collected from infected animal

doses of FMD up to one month prior to onset of outbreak.
infected
animal

Action to be
Isolate the affected bull immediately
taken on FMD
Affected bull is treated and rested for 90 days
infected
after recovery from clinical symptoms.
animal
a No semen collection from any infected animal
during the infection and up to 3 months after
last case has recovered in the farm.

Animals in No semen collection from healthy bulls during the

the farm not outbreak and no semen collection up to one month
affected by after the last case has recovered.
FMD

Semen Sale lf frozen semen sale is from the same campus of the
SS where FMD is recorded, suspend semen sale till
30 days after the last case has recovered.

FMD outbreak in areas surrounding the SS

Ring Arrange immediate ring vaccination within a radius

vaccination of 10 Km around the focus of infection starting from

Page 7l of 75
 the perimeter towards the focus.

Disinfection Disinfection of the roadsides adjacent to the farm on

a daily basis.

Movement of Stop all fodder movement through areas of

fodder infection.

Animal Stop animal movement of semen station through

movement areas of infection.

Page 72 of 75
 Annexure - 12
Feeding crowing aud Mature Bulls
Daily nutrient requirements of growing and mature bulls *

gainlda DM/da c.P. TDN

Body wt v v (el lkcl ca (gl P (cl vit. A

(ke) (g) (kel I

(looo rul
I I

Growing bulls

100 7so 2.4 390 1.9 11 8 4

150 750 4.3 460 2.7 15 11 6

l4
200 750 5.7 530 3.4
tt
18
tl 10 8

250 750 7 610 4 21,

l,ul I

300 750 8.2 680 4.6 t7 1a

350 7so I
9.3 760 5.2 24 18 15

400 700 to.2 820 5.7 25 t9 17

I

450 600 10.4 475 5.8 26 20 19

I

500 400 10 88s 5.6 26 20 21

I 550 250 10 84s 5.6 25 19 23

I
I I

600 100 9.8 800 5.5 24 18 26

I

Maintenance of mature breeding

bulls

500 8.3 640 4.6 20 15 2t

I

600 9.6 735 5.4 22 t7 26

Page 73 of 75
 700 10.9 I
830 6.t o< 19 I
30
I
I

Daily ration for Bulls

Body wt. Calf starter c.F. B.P.F. Hay Green Fodder

I

(kgl (ksl (kel (kgl (ke) (ke)

bulls

100 2 I 0.5 6-8

I

150 2 o 8- 10

200 2 0.5 lsl

300 2 I
1 ad lib.
I

400 a) 2 I 3 ad lib.

b) 2.5 a
ad lib.

500 a) 2.5 2-4 ad lib.

b) 3 2-4 ad lib.

600 a) 2.5 2-4 ad lib.

I

b) 3 2-4 ad lib.
I

lli ture breeding bulls

500 a) 2.5 2-4 ad lib.

I
I

b) 2 2-4 ad lib.

600 -do-

Page 74 of 75
 700 do-----------------

Note : f ) Mineral mixture should be supplemented as follows :

5O g mineral mixture for bulls up to 2OO kg body weight

70 g mineral mixture for bulls between 200 to 35O kg
body weight.
10O g mineral mixture for bulls above 35O kg body
weight

2l F resh water should be made available 24 hrs.

Green fodder requirement of ro mature buls wourd be approx. 125 MT per

year, which can be grown in t hectare of land by intensive farming.

Source Ranj han S K (1 9 80 ) An imal nu tri ti o n & fee ding prac ti C e S 1n

India,

2"d Ed., p196-2t2

Nutrients available in feed & fodder

Calf Green
starter c.F. B.P.F. fodder Hay

DM o/o oo 90 90 20-25 90
I

CP o/o ))_oa 18-19 22-23 5-6 5-6

I

TDN % 70 62-64 65-68 55-60 55

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