Minimum Standards

for

Production of Frozen Semen

April' 2005

CENTRAL MONITORING UNIT Department of Animal Husbandry & Dairying Ministry of Agriculture, Government of India

CONTENTS

ParticularsPage

1 .Standards for Genetic Merit of Breeding Bulls1

2. Physical Examination2

3. Quarantine2

4. Testing of Bulls3

5. Vaccination Schedule3

6. Culling of Bulls and Semen Doses due to Specific Diseases3

7. Housing4

8. Management of Bulls4

9. Semen Collection5

10.Handling of Semen, Processing & Freezing610 (A).Premises610(B).Equipment810 (C).Personnel Hygiene910(D).Diluents910(E).Evaluation & Processing910(F).Colour Specifications1010(G).Printing of Straws1110(H).Post-thaw Motility1110(1).Quality Checks for Frozen Semen1110(J).Information System1210(K).Semen Storage12

11. Cleaning and Sterilisation of Laboratory Equipment13

12. Summary of Sterilisation15

13. Quality Control of Consumables15

14. Manpower Requirement for Semen Production16

Annex.

I.Tuberculosis Management18

_II.Jhone's Disease (JD) Management19

III.Brucellosis Management20

0IV.Infectious Bovine Rhinotracheitis (IBR) Management21

%V.Bovine Genital Campylobacteriosis Management22

"VI.Bovine Trichomoniasis Management23

VII. Foot and Mouth Disease Management24

VIII. Feeding Growing and Mature Bulls25

IX. List of Laboratory Equipment for Frozen Semen Stations27

X. Qualifications Recommended for Semen Station Personnel29

XI. Job responsibilities of the QCO30Drawing Plan of a Model Semen Station32-33

MINIMUM STANDARDS FOR PRODUCTION OF FROZEN SEMEN

Artificial Insemination with frozen semen has been proved to be the best tool world wide for genetic improvement through dissemination of superior germplasm. This objective can be achieved only if the frozen semen used in Al programme conforms to the quality standards. For production and distribution of quality semen, it is most important that the bulls used in Al programme satisfy pedigree norms, bulls are disease free and semen is harvested and processed in accordance with the standard protocols. The least protocols required for production of quality semen are covered in this manual. Failure to observe these guidelines could lead to production of poor quality semen making it unfit for distribution to AI centres.

The minimum pedigree standards for bulls in the Al programme are given in the following table.

1. Standards for Genetic Merit of Breeding Bulls

Breed

Dam's Lactation yield (K

gs)

Sire's daughters' averages of the bulls

Farm bom

Imported (minimum)

Fat %

Min number of records

Average First Lactn yield (Kgs.)

Reliability (%)

First

Best

Pure HF

4500

5600

9000

3.5

30

4000

85

Pure Jersey

3000

3750

6000

5

30

2500

85

Sahiwal

2400

3000

—

4

20

1500

80

Red Sindhi

2000

2500

—

4.5

20

1500

80

Gir

2400

3000

—

4.5

20

1500

80

Kankrej

2000

2500

_

4.5

20

1400

80

Tharparker

2000

2500

—

4

15

1400

75

Hariyana

1600

2000

— .

4

15

1400

75

Rathi

1600

2000

—

4

20

1500

80

Ongole

1100

1600

—

4

_

—

_

Deoni

300

1000

—

4

_

. —

—

Khillar

380

500

—

4

_

—

—

Dangi

400

530

—

4

—

—

—

Amritmahal

400

500

—

4

_

—

—

HFCross-F2

4000

5000

~

4

30

2500

85

Jersey CB- F2

2800

3500

—

4.5

30

2000

85

Sunandini

2600

3000

3.5

30

3500

85

Murrah

2400

3000

—

7

30

1800

85

Mehsana

2400

3000

—

7

30

1800

85

Nil! Rav!

2400

3000

—

7

30

1800

85

Jaffrabadi

2800

3500

— r

8

30

2000

85

Surti

1600

2000

7

30

1600

85

Dam's milk yield for F1 crosses will be as that of the indigenous dam's i.e. Gir, Sahiwal, Kankrej, Red Sindhi, etc.

Minimum Standards

Average lactation yield (305 days) of HF and Jersey for the year 2002 for the following countries available from ICAR booklet are as below:

Breeds

New Zealand

Australia

Canada

US

France

Israel

HF

3361

6027

8534

9447

6608

10463

Jersey

NA

4381

6048

4619

NA

NA

The above information would be updated every year for information of all concerned so that this data can be used during import of bulls.

2.Physical Examination

Before inducting new bulls in the semen station, a thorough physical examination shall be conducted by an accredited Official / Veterinarian to ensure that the bulls do not display clinical symptom(s) of any infection or any contagious diseases. Prior to introduction of new bulls to the sperm station, breeding soundness examination shall also be carried out. Standards for scrotal circumference and weight gain index for various breeds shall be fixed by initiating age wise (monthly) recording of scrotal circumference and body weight, by the semen stations.

Before introducing to semen station, all bulls shall be karyotyped to rule out any chromosomal defects.

Specific tests may also be conducted for genetically transmitted diseases like Bovine Leukocyte Adhesion Deficiency (BLAD), Cttrullinemia and Deficiency of Uridine Monophosphate Synthase (DUMPS) for HF and HF crosses.

3.Quarantine

A minimum quarantine period of 60 days is compulsory before bringing new bulls in a semen station (incase of bulls are brought from known sources, 30 days quarantine is sufficient). This period shall be divided in two periods of 30 days during which a series of compulsory examinations and tests are carried out. Only after favorable results from the health control point, the bull shall be admitted to the semen station.

a). In the quarantine station new bull/s shall be housed for minimum of 60 days in a place which is effectively separated from (preferably at a distance of 5 km) the facilities occupied by resident bulls 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 bull in quarantine station will be tested against major contagious diseases before its entry to resident herd e.g. TB, JD, Brucellosis, Infectious Bovine Rhinotracheitis (IBR/IPV), Campylobacteriosis and Trichomoniasis. All tests shall be done by an accredited agency or disease diagnostic laboratory.

c) If any bull is found positive for TB, JD and Brucellosis, the positive bull shall be removed immediately without retesting. The remaining bulls shall be further tested twice for that particular disease at an interval of 60 days. If any bull is found positive, all animals in that batch shall be culled. As regards animals positive for IBR, culling shall be made based on isolation of virus for IBR-IPV.

Minimum Standards

d) During quarantine period, the bulls shall be vaccinated against FMD, HS, BQ, Theileriosis and Anthrax. However, vaccinations against bacterial diseases shall be done only if there is an outbreak or prevalence of a particular disease.

Once the quarantine period is over, all bulls shall be introduced to the resident herd. The resident herd would also go through periodical testing and vaccinations as per the schedule listed in the manual.

4. Testing of Bulls

Testing protocols for bulls against Tuberculosis, Johne's disease, Brucellosis, IBR, Campylobacteriosis and Trichomoniasis are given in Annex I to VI. As per OIE guidelines, the breeding bulls should be free from above mentioned diseases. Though Johne's disease is not a sexually transmitted disease but from the herd health point of view, bulls found positive should be removed and therefore it has been included in the MSP.

5. Vaccination Schedule

The bulls shall be vaccinated against FMD, HS, BQ and Anthrax. However, vaccinations against bacterial diseases shall be done only if there is an outbreak or prevalence of a particular disease.

Theileriosis - to be vaccinated once in lifetime in exotic and crossbred bulls.

To reduce lay off time, the bulls shall be vaccinated on the rest day or the day after completing semen collection. Sexual rest may not be required unless otherwise febrile condition is noticed.

The semen station shall arrange for carrying out ring vaccinations for all animals against FMD, HS and BQ within a radius of 10 km around the semen station. Vaccinations against HS and BQ shall be carried out in the areas having incidence of these diseases.

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

Diseases

Bulls

Semen doses

FMD

Retain

Last one month doses to be discarded, refer Annex- VII

Brucellosis

Castrate & remove

To be discarded since the last negative test

TB

Remove

To be discarded since the last negative test

JD

Remove

To be discarded since the last negative test

IBR/IPV

Remove based on specific symptoms

To be discarded since the last negative test

Campylobacteriosis

Treat and retain

To be discarded since the last negative test

Trichomoniasis

Treat and retain

To be discarded since the last negative test

Minimum Standards

The semen station must remove bulls (within 48 hours) which are positive for above

diseases, poor libido, poor semen quality, incurable lameness, etc. Besides, the

semen station shall also cull those bulls which have completed six years of

productive period or 2 lakh semen doses, whichever is achieved earlier.

7. Housing

Bull sheds shall have spacious individual pens with adequate loafing area, manger

and water trough with access to drink water all time. Provision for adequate shade

around the bull shed shall be given priority. The roof shall be made of asbestos or

suitable materials. During summer cooling system with sprinklers and fan is required

particularly for the buffaloes and exotic bulls. Disinfectants like formalin or phenyl

based compounds shall not be used in the bull sheds. Alternatively compounds

containing Gluteraldehyde shall be used. Weekly spraying of Sodium Bicarbonate

(4%) solution shall also be practiced. The floor should be sterilized at least once a

year by a blowlamp or by burning straws. At one comer of the farm there shall be

isolation shed for separating ailing / sick bull for treatment. Bulls once diagnosed

suffering from infected disease, shall be removed immediately from semen station for

safety of other bulls.

8. Management of Bulls

The objective of daily care of bulls is to ensure a satisfactory state of cleanliness. For proper management of bulls the following points shall be considered:

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

b) The coat of the bulls shall be kept clean and generally short. The hooves shallbe 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 role. If cut too short, it may cause irritation of thepreputial mucosa.

d) Bulls shall be brushed and groomed regularly, and where necessary specialattention shall be given to the underside of the abdomen, a day prior tosemen collection. Also, prepuce shall be washed with sterile normal salinesolution.

e) In the event of obvious soiling, careful cleaning of the preputial orifice and theadjoining areas with soap or a detergent is recommended; followed bythorough rinsing and drying.

f)Scientific feeding schedule shall be followed for the bulls. A general guidelineis attached as Annexure - VIII. Semen station shall carryout routine qualityanalysis of feed and fodder for arriving at a balanced ration.

Minimum Standards

9. Semen Collection

a) Ideally, the floor of the collection yard shall be made of concrete layer at adepth of one foot from the ground level. Mixture of sand and limestone shallbe used to fill up to ground level and pressed firmly. If it is not possible torenovate the entire collection arena, at least the mounting area shall havesand and limestone mixture for proper footing of bulls. Alternatively, goodquality rubber mat (with interlocking arrangement) or coir mat shall be put intoconcrete groove of the mounting area for adequate cushioning effect. Aftercollection, the area must be thoroughly cleaned and odorless disinfectantsolution (Colloidal iodine) be sprayed. A dusty floor shall be avoided toprevent dust falling on the AV / semen samples.

b) On the day of collection, before taking semen collection, the bulls shall beproperly washed and cleaned. After the bulls are brought to collection arena,before taking collection, the prepuce shall be cleaned externally with asterilized napkin soaked in normal saline to remove any sand or dustparticles. For each bull a separate napkin shall be used.

c) The person responsible to carry out preputial wash must use disposablegloves and separate sterilized nozzle for each bull to avoid transmission ofIBR infection from one bull to another.

d) The bulls shall be sexually prepared by giving two / three false mountsfollowed by two minutes restraint and the total time limit should be not morethan 12 minutes. The duration of each false mount shall be for 1 to 2 minutes.

e) Sterilized bull aprons shall be used to avoid penis touching hindquarter of thedummy.

f) Preferably veterinarians shall take semen collection. If semen is collectedby staff, a veterinarian shall remain present to supervise the collectionprocess. While taking collection it shall be ensured that AV is not thrusted onpenis of bull, instead penis should be guided to AV.

g) Before every collection, the semen collector shall either wash his hands with0.1% Savlon solution or use disposable gloves or do both. The semencollector shall not touch the penis.

h) Immediately after collection, the AVs shall be thoroughly cleaned by non-spermicidal neutral detergent. Separate AVs shall be used for each mounting. 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 shall be covered with felt / water jacket (plastic bottle filled with warm water at 34° C) to avoid cold shock. The open end of sterilized AVs shall be covered with aluminum foil, which would be removed at the time when bulls are ready for giving semen.

i) Preferably 10° size AVs shall be used for cattle and 8 to 9" size for buffaloes to ensure semen is ejaculated in cone. For buffaloes goat AVs can also be used. The cone shall be of top quality Neoprene rubber.

Minimum Standards

j) Use of lubricant shall be avoided. If it is extremely essential to use lubricant, separate sterilized glass rods shall be used for smearing K-Y Jelly on each AV.

k) The AV shall not to be shaken after ejaculation; otherwise lubricant and debris may mix with the semen samples.

I) The entry of visitors/ staff / labourers shall be strictly prohibited in the collection arena at the time of semen collection and inside the semen laboratory.

m) Protective clothing (barn coat) and gumboots shall be used by the Veterinarians and personnel while taking semen collection. Gumboots and bam coat should be washed daily immediately after completion of semen collection work.

n) Semen must be obtained from a bull having normal libido. While taking two ejaculations the semen station shall keep a gap of 10 to 12 minutes between two ejaculates, depending upon the refractory period of the bull. (To harvest more quantity of semen, interval between two ejaculates shall be kept for 40 to 45 minutes. After taking first ejaculate, the bull shall be taken back to the shed for feeding and watering and again brought back for taking second ejaculate. However, care shall be taken to use a fresh & sterilised apron during second ejaculate)

o) Semen stations must follow the norm of two ejaculates per collection and two collections per bull per week for taking annually at least 90 collections and 180 ejaculates from each adult bull.

10. Handling of semen, processing & freezing

10 (A) Premises

a) Sufficient trees shall be planted and lawns prepared around the semenstation to reduce dust.

b) The ceiling and walls of the laboratory shall be made up of non-porousmaterials. All cracks and crevices shall be sealed to control pests andinsects.

c) Entry of persons to the laboratory other than laboratory personnel shall bestrictly restricted. Airlock system or anti-room shall be provided to avoiddirect entry to the semen-processing lab.

d) Lab windows shall be made of single sheet glass with fixed aluminiumframe. The glass panes shall be plastered with sun control films to avoiddirect sunlight. The doors shall be kept closed, especially during dilutorpreparation and semen processing.

e)Intake areas of the air conditioners shall be -disinfected weekly and ifpossible, split type air conditioners with remote temperature controlmechanism should be installed to maintain the room temperature at

Minimum Standards

20°C. The number of ACs to be fixed to sustain this temperature shall depend on the size of the processing room. Maintaining this temperature is most important to achieve the best results when single step dilution method is followed for freezing semen. Alternatively, HEPA filters shall be installed near intake area of air conditioners. The flow of air from AC must not be towards the front side of the Laminar Air Flow Unit. Adequate number of dial thermometers shall be kept in a few places in the laboratory to check the room temperature.

f) Sink drains shall be decontaminated routinely with a disinfectant.Preferably, the sink shall not be placed in the semen processing room.

g) The floors shall be preferably made up of vitrified tiles. Floors andhorizontal surfaces shall be cleaned and mopped with a disinfectantsolution, as dirt and dust, which settle on these surfaces, are the mainsources of contamination.

h) Unwanted furniture, equipment and materials shall not be kept in the laboratory as they only provide additional area for dust and spores to collect.

i) Appropriate no. of germicidal UV lights (2470 A) in respect of area of laboratory, laminar airflow unit, apron and laboratory footwear cabinet shall be fixed with a common operating switch outside the laboratory. These lights shall be kept 'on' at least for 8 hours prior to commencement of work in the laboratory and shall be switched off before beginning work The date of installation of the UV lights shall be noted to facilitate replacement as the life of UV tube is of 2000 hours. A logbook should be maintained for timely replacement of UV lights.

j) Laboratory shall be fumigated weekly for 2 hours with 12 ml of 37% formaldehyde solution in 100 ml water per cubic meter at weekends, using humidifier. After fumigation, switch on the exhaust fans for clearing formalin fumes. However, this should be supported by monitoring of lab environment by bacterial load test. The bacterial ioad shall be measured every week to monitor pollution of the laboratory atmosphere.

k) (Mixing formalin and water with potassium permanganate crystals*. *WARNING: the correct relative concentration of these two components is essential to avoid a violent reaction. It is therefore recommended that this method is NOT used. (Reference; Bid-safety Unit, Health and Safety Department, University of Edinburgh, August 2003)

I) The work platform, the parts of equipment and other items to be handled during processing of semen, shall be cleaned with 70% alcohol or Glutaril (Qualigen). It is advisable to repeat cleaning schedule after completing processing of semen.

m) Clean laboratory footwear, apron, hand gloves, mask and caps shall be compulsorily put on while working in the laboratory.

n) Eating, drinking, smoking, etc. shall be prohibited in the laboratory and unnecessary conversation should be discouraged. Besides, entry of persons shall be strictly restricted.

Minimum Standards

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 provision for indirect or diffused lighting inside the semen processing room. Care shall also be taken not to switch on tube lights in CH cabinet and laminar airflow unit.

10(B) Equipment

a) The exteriors of all equipment and furniture shall be cleaned weekly. Theequipment shall be kept covered by plastic covers when not in use.

b) The pre-filter of Laminar Airflow unit shall be cleaned weekly. Routineservicing and OOP testing twice a year will ensure efficiency of HEPAfilters. Alternatively culture plate test shall be carried out at frequentinterval to assess bacterial load of the air passing through the filters.

c) Digital photometer / Computer aided Spectrophotometer shall be validatedwith Haemocytometer readings for sperm concentration twice a yearseparately for cattle and buffalo (10 samples each).

d) The automatic semen straw filling and sealing machine shall bethoroughly cleaned, immediately after use.

e) The microscope lens shall be gently cleaned daily with a piece of cottonsoaked in a mixture of ethyl and methyl alcohol (1:1).

f) The bio-freezer shall be defrosted and thoroughly cleaned and dried,immediately after use.

g)Incubators to maintain artificial vagina shall be cleaned anddisinfected daily with 70% alcohol.

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 have precise temperature reading.

j) The Liquid Nitrogen containers returning / received from foreign countries and contagious disease prone areas shall be disinfected thoroughly with 4% soda solution and finally with 1 to 4% formaldehyde. Excess liquid nitrogen available as a result of filling the containers with canisters and goblets, shall never be brought inside the semen processing room.

k) The refrigerator meant for storing eggs, antibiotics and buffer shall not be used for storing vaccines and other materials. All such materials shall be stored at a place away from semen lab. The refrigerator used for storing eggs, etc. shall be sterilized every week using alcohol swab.

Minimum Standards8

10 (C) Personnel Hygiene

Clothing, skin and hair of laboratory personnel are the sources of the contamination. Hence all should wear laboratory aprons and footwear all the time while they are in the laboratory. Hands shall be washed with soap and water and rinsed with 70% alcohol, before commencing work in the laboratory. The bull attendants must undergo test for TB every year. Restricted entry inside the semen processing room and freezing room shall be strictly adhered to.

10 (D) Diluents

a) All disposable and reusable supplies coming in contact with the semen and dilutor must be sterile and devoid of toxins and pyrogens.

b) 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.

c) Glass wares, collection tubes, etc. shall not be handled from their rim / mouth.

d) Pipetting shall be done away with, instead adjustable micropipettes and

disposable tips shall be used.

e) After adding all the components of buffer viz. TRIS, Citric Acid, Glycerol and

Fructose in double, preferably triple distilled water, it should be again

sterilized. If buffer is prepared on the previous day and stored in the

refrigerator then antibiotics are to be added on the next day morning after

warming it at 34°C.

f) Antibiotics in diluents: Combination of antibiotics, in diluents, which can

control Mycoplasma like Tylosin, Lincospectin and Gentamycin may be used.

Alternatively, a combination of Penicillin and Streptomycin shall be used.

g) The eggs used for making dilutor must be fresh. The eggs shall be stored in

refrigerator after wiping with dry cotton. Just before preparation of dilutor,

eggs shall be wiped with 70% alcohol. To avoid Mycoplasma infection, eggs

shall be purchased from known sources.

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

(autoclaved) standard filter papers (Whatman No. 1/ Borosil) and yolk

membrane shall be punctured using sterile glass rod or Pasteur pipette or

sterile straws under the Laminar Flow Unit. Only fresh semen extender/dilutor

shall be used because changes in the pH of stored extender are considered

to be responsible for the deterioration of some nutrient components. Day old

extender shall not be used.

10 (E) Evaluation & Processing

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

Minimum Standards

b) The tube containing the freshly collected semen should be capped withaluminum foil as soon as it is placed in the pass box before transferring to thelaboratory. The collection tube shall be kept capped until processed.

c) After examination of sperm concentration and initial motility, semen samplesshall be primarily diluted with dilutor maintained at 34° C. In the case ofbuffalo semen, examination of mass activity is mandatory because semensamples with zero mass activity may not give better result and usuallydiscarded after freezing. Alternatively, a small drop of fresh semen shall beexamined under a cover slip to check motility and to assess the semensamples.

d) Sperm concentration shall be checked preferably by a digital photometer withauto dilutor, manufactured by a reputed company. The photometer shall becalibrated separately taking 10 readings each for cattle and buffalo semen, atleast once in six months, with haemocytometer readings. Semen samplesshowing less than 500 million / ml sperm concentration shall be discarded.

e) Semen samples selected for freezing should have minimum 70% initialmotility. Final dilution of semen keeping a minimum of 20 million spermatozoaper dose shall be done in appropriate flasks with the dilutor maintained at 34°C.

f) Filling and sealing of semen shall be done under Laminar Flow Unit usingsterile straws, filling nozzles and fresh rubber tubings. Rubber tubings shallbe used once only. Reuse of rubber tubes is not recommended. Consideringthe advantages the French Mini Straws has over French Medium straws, thesemen stations shall use French Mini straws.

g) Unused straws shall be repacked (air-tight) under Laminar Unit beforestorage.

h) Immediately after use, all the glass wares, rubber wares, plastic tips and other reusables shall be immersed in neutral detergent solution (to be kept in a plastic tub near the Laminar Unit).

10(F) Colour Specifications :

All semen stations shall follow the following colour codes :

1.Jersey-Yellow

2.Holstein-Pink

3.Indigenous-Orange

4.HF Crossbred-Pistachio Green (light green)

5.Jersey Crossbred-Salmon

6.Sunandini-Blue

7.Buffalo-Grey

If anyof above colour is not available then transparent colour is used.

Minimum Standards10

10 (G) Printing of Straws

Information regarding bull number, breed, name of the organization, year, batch no. (as per the day of the year), etc. shall be printed on straws preferably after their filling and sealing. After printing the ink gets instantly dried. If filled straws are printed and racked, the actual number of straws can be easily counted. While printing and racking, the room temperature shall be maintained at 20 ° C.

AH semen stations shaii foiiow the following printing abbreviations:

Jersey-JYFarm No. / Name

Holstein-HFBreed

HF Cross-CBHFName of Institute

Jersey Cross-CBJYBatch No. / Date of Prodn.

Sunandini-SUN

Sahiwal-SAH

RedSindhi-RS

Kankrej-KANK

Gir-GIR

Tharparker-THAR

Rathi-RATHI

Haryana^-HAR

Murrah Buffalo-MBF

Surti Buffalo-SBF

Jaffrabadi Buffalo-JBF

Mehsana-MSNB

10 (H) 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. 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, shall not check the post thaw motility. For a minimum concentration of 20 million per dose, minimum acceptable post thaw motility shall be 50%. Semen doses below 50% progressive motility shall be discarded.

10 (I) Quality Checks for frozen semen

This includes (i) Quarterly testing of random samples from each batch for bacterial load using standard plate count (The standards for acceptable colony forming unit in processed semen is 5000 CFU per ml as per OIE norm. If the bacterial load exceeds the OIE limit, the semen doses are to be discarded.) (ii) Hypo osmotic swelling test (HOST) - every day four to six samples (iii) Incubation test - everyday four to six samples (iv) Acrosome integrity test by Giemsa staining - for all bulls at least once in a quarter shall be mandatory. Alternatively wet smear of semen shall be examined using DIC microscope (v) Percent Intact Acrosome - all bulls to be covered once a quarter (vii) Sperm Concentration -randomly two samples per week each for cattle and buffalo

Minimum Standards11

Validation of photometer shall be done once in 6 months, at least 10 samples each for cattle and buffalo. Neat semen shall be examined at an interval of every six months for morphological abnormalities particularly for crossbred bulls. Morphological examination of sperm of young bulls must be carried out (at least six samples at weekly intervals) before introducing them in the herd. Semen should not be used if the sample contains a total abnormality of more than 20% and head and mid-piece abnormality ( alone) of 7%.

10 (J) Information System

a) The semen stations shall use a suitable software to record data pertaining tovarious activities and also should have online facility for the same. The semenstations producing more than one million doses may introduce software thatcan identify and trace the bulls and their ejaculates, production, storage anddispatch of semen (barcode system).

b) Volume of semen, density, motility, sperm concentration, dilution rate, totalextended volume, post-thaw motility (24 hrs), total number of dosesproduced, etc. shall be maintained. Pre-freeze and post-thaw motility shall bechecked for new and problematic bulls.

c) Miscellaneous information regarding actual reason(s) for not donating semen,undesired percentage of gross morphological defects, semen pH, presence ofdirt, dust, blood, pus, etc. in semen samples.

d) Details of semen supplied to various agencies including post-thaw motility atthe time of dispatch.

e) Fertility data of bulls, conception rate, records of the progeny associated withany genetic defect, percent male / female born, etc.

f) Report on microbiological examination of semen samples.

g) Records of all quality tests for neat and frozen semen samples.

10 (K) Semen Storage

To avoid accidental spread of diseases, the semen station shall follow the procedure of preserving semen doses for at least 30 days after production. Semen doses produced before 30 days from the date of dispatch shall only be supplied for field use.

After checking post-thaw motility, if found acceptable, frozen semen doses would be kept in temporary storage for 7 days. After the 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.

Liquid Nitrogen shall be replenished at regular intervals depending on the liquid nitrogen evaporation rate of the container.

Minimum Standards12

11 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 water (5 to 7 changes) to completely remove detergent residues and other impurities. Appropriate procedure for sterilization of different materials, used in the semen station, is given below:

1. Laboratory and other areas

a.Materials

1. Fumigator: Aerosol formation disinfector

2. Formaldehyde Solution 37 % w/v

3. Lab dimensions

4. Lab area in cubic meters

b.Procedure

1 . Review of the microbial count indicates the necessity of fumigation

2. Calculate the area in cubic meter, which is to be fumigated

3. Calculate the formaldehyde solution using dosage 12 ml of 37% w/v of theformaldehyde solution in 100 ml water per M3

4. Decide the no. of fumigators to be used (based on area)

5. Put total quantity of formaldehyde solution in all the fumigators

6. See the doors and windows are sealed properly

7. Switch off the exhausting fans and other fans and switch all the fumigatorsfrom outside switch

8. Evaporate full formaldehyde solution and switch off fumigators aftercompleting fumigation (about 2 hrs.)

9. Ensure that all the dampers of exhaust area, ducts are shut off duringfumigation

10. Switch on the exhaust fans / air handling systems after 2 hours of fumigation

11. Inspect the room for the absences of formalin fumes before the personal entry

12.Expose the settle plates and record the microbial count

1 3. Review the microbial count so as to establish the effectiveness of fumigation

c. Maintain a register to record observations on microbiological load in semen processing lab.

2. Artificial Vagina (AV)

a) Cone from the AV and water from AV jacket shall be removed beforewashing.

b) Cones and AVs shall be cleaned thoroughly with a soft sponge brush underrunning tap water and then soaked in warm neutral cleaner for about 30minutes, followed by proper rinsing in warm and clean water and lastly rinsedthree times with double distilled water.

Minimum Standards13

c) For sterilization, fully assembled AVs shall be autoclaved at 5 p.s.i. pressurefor 20 minutes. During sterilization the AV - valve shall be kept open.Alternatively, use AV sterilizer (using double distilled water in the sterilizer) forproper sterilization of AVs.

d) Finally the AVs shall be stored overnight in an incubator at 45° C.

e) To achieve best cleaning effect, AVs shall be cleaned immediately after use,preferably by non-spermicidal neutral detergent.

3.Glasswares

a) The glasswares shall be washed thoroughly with running tap water andsoaked in warm, non-spermicidal neutral detergent solution for about 30minutes.

b) Using appropriate nylon brush, the glasswares shall be cleaned and rinsedwith running tap water. The collection tubes shall be brushed at least 3 timesand thoroughly cleaned and rinsed with distilled water.

c) Finally the glasswares shall be rinsed three times with double distilled waterand allowed to dry by keeping them inverted on a blotting paper.

d) The open end/s of the dried glasswares shall be covered with aluminium foiland sterilized in hot air oven at 160° C for 1 hour or at 180° C for 30 minutes.

4.Rubber wares

The washing and cleaning procedure of rubber wares is similar to that of glasswares. 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-sensitive rubber wares shall be packed and sealed in specific polythene bags and sterilized in Ethylene Oxide gas sterilizer. 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).

5.Distilled Water

Triple glass distilled water or Milli-Q purified water shall be autoclaved at 15 p.s.i. fop 45 ^minutes and used for preparation of the dilutor. The Triple glass distilled water or Milli-Q purified water stored for more than 5 days shall not be used for buffer preparation.

6.Buffer

Buffer shall be sterilized by autoclaving at 5 p.s.i. pressure for 20 minutes. After autoclaving buffer shall be cooled and stored in refrigerator.

7.Bacteriological Media

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

Minimum Standards14

8. Filter Papers

A bunch of clean filter papers of standard brand like Whatman 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.

12 Summary of Sterilization a) Autoclave

Sr.No.

Item

Pressure (p.s.i.)

Time (Min.)

1.

Artificial Vagina

5

20

2.

Buffer

5

15/20

3.

Plastic Tips

5

20

4.

Filter Papers

5

20

5.

Bull Apron

5

20

6.

Thermo-resistant Rubber wares

3-4

10

7.

Bacteriological Media

15

15

8.

Distilled Water

15

15

9.

Surgical Equipment

10

10

(The rubberwares can withstand above pressure and duration provided the quality is

good)

b) Hot Air Oven

Sr.No.

Item

Temperature

Time (min.)

1.

Glass wares

160°C/180°C

60/30

2.

Filling Nozzles

160° C/ 180° C

60/30

c)AV Steriliser

Wherever Autoclave is not used, AVs and rubber cones shall be sterilised using AV sterilizer. After sterlizer starts boiling, 30 minutes vapour sterilisation shall be done.

d)Ethylene Oxide Gas Steriliser

The exposure time for gas sterilization is inversely proportional to the gas concentration under which the equipment is exposed in the sterilizer. Commonly used concentration of ethylene oxide is 900 mg per litre of cubic space for a period of six hours. All items sterilized with gas must be aired adequately before use. A minimum exposure of 72 hours at room temperature with adequate ventilation is recommended.

13 Quality Control of Consumables Chemicals

The chemicals of only highest purity of either, Analytical Reagent (AR) or Graded Reagent (GR), from reputed manufacturing companies shall be used. Whenever a new chemical is to be introduced in the routine process, it is recommended to

Minimum Standards

15

examine the post-thaw revival rates after conducting few spilt ejaculate trials (maintaining a control) with the new chemical. Assay of chemicals shall be >99%, having less impurities.

Straws

1. Straws manufactured by reputed companies are safer to use for production ofquality semen. While buying straws package volume and microbial load instraws shall be checked randomly from the consignment. In addition, someempty straws should be placed in auto filling & sealing machine and run themachine for sealing the straws. In case any foul smell, it should be presumed thatthe straws are manufactured from poor plastic which could be toxic to thespermatozoa and can even result in reduced motility on long storage.

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

3. The straws should be intact (without cracks / dents etc.) during and after freezing/thawing.

4. The movement of straws along the printing machine should be free and printshould be clear and sharp. Print should not fade as a result of freezing andsubsequent thawing.

5. The use of dark coloured straws should be avoided, as they are not transparentenough. Not only is filling / racking inconvenient, it is also difficult to distinguishbetween filled / 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 on lowest quotation basis. To produce top quality semen, it is better to use AR / GR consumables manufactured by reputed companies whose products are reliable.

14 Manpower Requirement for semen production

Designation

Up to 5 lakh doses

5-10 lakh doses

10-25 lakh doses

Above 25 lakh doses

QIC

1

1

1

1

vo

1

2

2

3

QCO

1

1

1

1

Lab Technician

.—

2

3-4

4-5

Lab Attendant

2

3

3-5

4-6

Office Assistant

—

1

1

1

Bull Attendant

1 person per 7- 8 bulls

Minimum Standards

16

The manpower structure suggested above, is meant only for semen production. For dispatch of semen, facility should be created preferably away from semen station and operated by other person/s not responsible for semen production. The GOI / Department of AH / Livestock Boards / NGO / Private agencies / Union and Federation shall review the requirement of manpower position for each semen station and finalise the staff structure for recruiting additional manpower. After recruitment, all new persons shall be trained at any of the recognized institutes. Once trained, they shall continue to work in the semen station at least for five years.

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 SAG-Bidaj, CFSP&TI-Hessarghatta, KLDB-Matupatty, etc. As semen production activity is an extremely technical work, frequent transfer of personnel could be detrimental in maintaining the quality of semen. Therefore, before considering transfer, in the interest carrying out good work it is essential that proper replacement is identified at least six months in advance and is trained in semen production technology.

Minimum Standards17

Annex-1

Tuberculosis Management

Name

Delayed Hypersensitvity -Single Intradermal test

Reagent

Jovine tuberculin PPD

Reagents from

VRI, Izatnagar

Screening Test details

Testing at

Where animals are housed by RDDL/CDDL/NDDB

Positive result criteria

As per OIE norms Negative : 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 ymph node, 72 hours post-inoculation. Inconclusive : Increase in skin thickness more than 2mm & less than 4mm, absence of above clinical signs, 72 hours post-inoculation. Positive : Increase in skin thickness 4 mm or more, or presence of dinical signs viz. exudation, necrosis, pain, inflammation of the lymphatic duct of that region or the lymph node, 72 hours post-inoculation.

Eligible animals

All animals above 6 months of age, have shown a negative result to at least two tuberculin test carried out at an interval of 6 months

Frequency of testing

Positive herd

Minimum 60 days after culling of last positive animal

Negative herd

Annual test is minimum. Six months (± 1 week) after last whole herd negative testing, desirable.

Action on finding a positive bull

Animal

Immediate isolation and elimination

Semen

Destroy semen doses since last negative test

Tuberculosis free herd (OIE)

Herd found negative on two consecutive tuberculin tests at an interval of 6 months, the first being performed 6 months after the slaughter of last affected animal

Quarantine

Duration of quarantine

Minimum 90 days

Test schedule

Two tuberculin tests, minimum interval of 60 days between tests.

Minimum Standards

18

Annex - II

Johne's Disease (JD) Management

Name

Delayed Hypersensitivity test (Skin test)

Reagent

Johnin PPD

Screening test

Reagents from

IVRI, Izatnagar

Details

Testing at

Where animals are housed by RDDUCDDL/NDDB

Positive result criteria

Increase in skin thickness of over 4mm (discrete circumscribed swelling), 72 hours post-inoculation

Eligible animals

All animals above 6 months

Frequency of testing

Positive herd

Minimum 60 days after culling of last positive animal

Negative herd

Annual test is minimum. Six months (± 1 week) after last whole herd negative testing, desirable.

Action on finding a positive bull

Animal

Immediate isolation and elimination

Semen

Destroy semen doses since last negative test

Quarantine

Duration of quarantine

Minimum 90 days

Test schedule

Two Johnin tests, minimum interval of 60 days between tests.

Minimum Standards

19

Annex- III

Brucellosis Management

Screening Test

Name

ELISA, RBPT+CFT

Details

Sample

Serum

Reagents from

CDDL / RDDL

Testing at

CDDL/ RDDL/NDDB

Eligible animals

• Ail above one year

• In females 14 days after calving or abortion

Frequency of testing

Positive herd

30 to 60 days after culling of last positive animal

Negative herd

Exactly one year (± 1 week) after last whole herd negative testing

Negative herd (optional)

Where the disease has been maintaining a very low profile (less thanl % positive) quarterly or six monthly sample could be collected to minimize losses

Action on finding a positive bull

Animal

Immediate isolation and elimination after castration

Semen

Destroy semen doses since last negative test

Brucellosis free herd (OIE)

Herd found negative on two consecutive annual tests

Quarantine

Duration of quarantine

Minimum 30 days

Test schedule

Two tests, Serum ELISA, interval of 30 days between tests. Only negative animals to be allowed to mix with the rest of the herd.

Additional testing at sexual maturity

Serum ELISA before bulls are used for semen collection and distribution forAI

Minimum Standards

20

Annex- IV

Infectious Bovine Rhinotracheitis (IBR) Management

Screening

Name

ELISA /SNT

test details

Sample

Serum

Testing at

CDDL / RDDUNDDB

Eligible animals

All animals

Frequency of testing

Positive Herd

Whole herd test, 30 to 60 days after culling of positive animals. Six monthly, after the herd become negative.

Negative herd

Exactly one year (± 1 week) after last whole herd testing

Where the disease has been maintaining a low profile (less than 5 % positive) quarterly or six monthly sample could be collected to minimize losses

Action on finding a positive bull

Animal

Immediate isolation and elimination after castration

Semen

Destroy semen doses since last negative test

IBR free herd (OIE)

^

Whole herd tested negative on two consecutive occasions at an interval of 2 to 12 months between tests

Quarantine

Duration of quarantine

Minimum 30 days

Testing

Two tests, Serum ELISA/ SNT, interval of 21 days between tests

Additional testing at sexual maturity

Serum ELISA/SNT before bulls are used for semen distribution in field Al programmes

Minimum Standards

21

Annex- V

Bovine Genital Campylobacteriosis Management

Screening test details

Name

Bacterial isolation & identification

Sample

Preputial washing, semen

Testing at

RDDL/CDDUNDDB

Eligible animals

All male animals

Prevention

Annual sheath lavage

Frequency of testing

Positive herd

30 days after culling of positive animal.

Negative herd

Exactly one year (± 1 week) after last whole herd negative testing

Action on finding a positive bull

Animal

Treat the animals

Semen

Destroy semen doses since last negative

test

Quarantine

Duration of quarantine

Minimum 30 days

Test schedule

One test if age is less than 6 months, else 3 consecutive tests at weekly intervals.

Minimum Standards

22

Annex - VI

Bovine Trichomoniasis Management

Screening test details

Name

Agent isolation & identification

Sample

Preputial washing

Testing at

RDDL/CDDUNDDB

Eligible animals

All male animals

Prevention

Annual sheath lavage

Frequency of testing

Annual

Action on finding a positive animal

Animal

Treat the animals

Semen

Destroy the semen doses since last negative test

Quarantine

Duration of quarantine

Minimum 30 days

Test schedule

One test, if age is less than 6 months, else 3 consecutive tests at weekly intervals.

Additional testing at sexual maturity

Protozoa isolation before bulls are used for semen distribution forAI

Minimum Standards

23

Annex-_V!l

Foot and Mouth Disease Management

Action during FMD outbreak

Animal

solate diseased animals till recovery, do not cull.

Semen

Semen from FMD infected bulls : Destroy semen collected during one month

before onset of outbreak. Do not collect

semen from bulls during the outbreak and

three months after the last case of FMD

recovered in the farm.

Infected animals must be given 90 days rest

Semen from healthy bulls maintained in FMD infected farm:

Destroy semen collected during one month

before onset of outbreak. Do not collect

semen from bulls during the outbreak and

one months after the last case of FMD

recovered in the farm.

Semen could be used other than the above

mentioned periods, if there is no new case of

FMD develops during three months period

after last FMD case recovered in the farm.

Semen for export

Test each batch by virus isolation and PCR

Quarantine

Vaccination - as per manufacturer's recommendations.

Vaccination

Oil vaccine

As per manufacturer's recommendations in farm and 10 km around the farm

Test for seroconversion, by collecting serum on the day of vaccination and 21 days later.

Minimum Standards

24

Annex-VIII

Feeding Growing and Mature Bulls Daily nutrient requirements of growing and mature bulls

Body wt

gain/day

DM/day

c.P.(g)

TON (kg)

Ca(g)

P(g)

VitA

(kg)

(g)

(kg)

(1000 IU)

Growing bulls

100

750

2.8

390

1.9

11

8

4

150

750

4.3

460

2.7

15

11

6

200

750

5.7

530

3.4

18

14

8

250

750

7

610

4

21

16

10

300

750

8.2

680

4.6

23

17

13

350

750

9.3

760

5.2

24

18

15

400

700

10.2

820

5.7

25

19

17

450

600

10.4

875

5.8

26

20

19

500

400

10

885

5.6

26

20

21

550

250

10

845

5.6

25

19

23

600

100

9.8

800

5.5

24

18

26

Maintenance of mature breeding bulls

500

-

8.3

640

4.6

20

15

21

600

-

9.6

735

5.4

22

17

26

700

-

10.9

830

6.1

25

19

30

Daily ration for Bulls

Body wt.

Calf starter

C.F.

B.P.F.

Hay

Green Fodder

(kg)

(kg)

(kg)

(kg)

(kg)

(kg)

Growing bulls

100

2

-

-

0.5

6-8

150

-

-

2

0

8-10

200

-

-

2

0.5

15

300

-

.

2

1

adljb_.

400

a)

-

2

3

ad }ib.

b)

• -

2.5

-

3

ad lib.

500

a)

-

-

2.5

2-4

ad lib.

b)

-

3

-

2-4

ad lib.

600

a)

-

-

2.5

2-4

id Kb,

b)

-

3

-

2-4

ad lib.

Mature breeding bulls

500

a)

-

2.5

-

2-4

ad lib.

b)

-

-

2

2-4

ad Kb,

600

. .. .. ho _ ._

?nn

.. rin - ,- - ...

Minimum Standards

25

Note : 1) Mineral mixture should be supplemented as follows :

2)

50 g mineral mixture for bulls up to 200 kg body weight

70 g mineral mixture for bulls between 200 to 350 kg body

weight.

100 g mineral mixture for bulls above 350 kg body weight

Fresh water should be made available 24 hrs.

Green fodder requirement of 10 mature bulls would be approx. 125 MT per year, which can be grown in 1 hectare of land by intensive farming.

* Source: Ranjhan, S.K (1980). Animal nutrition & feeding practices in India,'2nd Ed., p196-212

Nutrients available in feed & fodder

Calf starter

C.F.

B.P.F.

Green fodder

Hay

DM%

90

90

90

20-25

90

CP%

22-23

18-19

22-23

5-6

5-6

TDN%

70

62-64

65-68

55-60

55

Minimum Standards

26

Annex - IX

List of laboratory equipment for Frozen semen stations (A). Processing Lab

Sr. No.

Items

< 5 lakh

5 to 10 lakh

10 to 20 lakh

> 25 lakh

1

Phase contrast microscope with Biotherm (Nikon/ Olympus)

1

1

2

2

2

DIC Microscope with bio-therm

Nil

Nil

1

1

3

lide warmer

1

1

1

1

4

^to / Eppendorf Pipette

3

3

5

6

5

Waterbath (IMV)

1

2

2

2

6

aminar Air Flow Unit

1

2

2

3

7

CCTV

Nil

1

1

1

8

MV make Photometer

1

2

2

2

9

PH Meter

1

1

1

2

10

Auto filling-sealing machine (Mini)

1

2

2

2

11

Straw Printing Machine

1

1

Nil

Nil

12

et Printer

Nil

1

2

2

13

Cold Handling Cabinet- Split

1

2

2

4

14

Biological freezer

1

1

2

2

15

Freezing Racks (Mini)

30

60

80

100

16

Distribution Ramp (Mini)

1

1

1

1

17

ncubator

2

2

3

3

18

Autoclave

2

2

2

2

19

Hot Air Oven

2

2

2

4

20

Double glass distillation plant

2

2

2

2

21

Mlipore water purifying equipment

Nil

1

1

1

22

ETO Steriliser

1

1

1

1

23

Magnetic Stirrer

1

1

1

2

24

Electronic Weighing balance

1

1

1

1

25

WM bulk FS storage Container with freezing Grill

2

2

2

2

26

Bulk FS Storage Container

1

2

3

5

27

Geyser

2

2

2

2

28

Vacuum Cleaner

1

1

1

1

29

Voltage Stabiliser (Central)

As per req.

As per req.

As per req.

As per req.

30

FS storage Cont (12000 Mini st)

5

10

12

15

31

LN Storage Cont. (50 Lt. Cap)

10

15

20

20

32

PC (to connect with Microscope), Printer & UPS

1

1

1

2

33

Fumigator/Humidifier

1

1

2

2

34

Thawing Unit (IMV)

2

2

2

2

35

LN Pump/Transfer device (IMV)

2

2

2

2

36

SS Forceps -18"

6

6

6

6

37

Refrigerator

1

2

2

2

Minimum Standards

27

(B). Miscellaneous Equipment

Sr. No.

Items

< 5 lakh

5 to 10 lakh

10 to 20 lakh

>25 lakh

1

AV Stand

5

5

5

5

2

Test Tube Stand

5

5

5

5

3

Auto Pipette Stand

2

3

5

5

4

Plastic Tips Holder

2

3

5

5

5

Spirit Lamp

2

2

2

2

6

Timer/ stop watch

2

2

2

2

7

Scissors Straight- 6°

2

4

4

4

8

Hot Air Blower/ Hair Dryer

1

2

2

2

9

Trolley for Movement of LNC

5

5

10

10

10

Anaesthetic Trolley

1

1

1

2

11

PC with Printer + Software (SSMS)

1

1

2

2

12

Glass wares

In sufficient quantity

13

Air-conditioner with Hepa filter

1 ton for 100cft area with remote

(C). Semen despatch section

1

Phase Contrast Microscope with biotherm

1

1

1

1

2

IMV Waterbath

1

1

1

1

3

Thermocol box

2

2

2

3

4

Long Forceps

8

8

8

8

(D). Quality Control Laboratory

1

Phase Contrast Microscope

1

2

IMV Water bath

1

3

Laminar Airflow Unit

1

4

Autoclave

1

5

Hot Air Oven

1

6

Incubator with digital Temp

1

7

Haemocytometer

2

8

Refrigerator

1

9

Gas connection

1

10

Water bath

1

11

Glass ware/Media/ chemicals

Adequate

12

Table.Chair, Almirah

11

13

Misc. Items

11

Minimum Standards

28

Annex - X Qualifications recommended for Semen Station personnel

1.Officer-in-charge

MVSc. in Animal Reproduction / Livestock Production & Management, trained in semen production.

2.Senior Veterinary Officer

MVSc. in Animal Reproduction / Livestock Production & Management, trained in semen production.

3.Veterinary Officer

MVSc. in Animal Reproduction / Livestock Production, trained in semen production/ Livestock management.

4.Quality control Officer

MVSc. in Animal Reproduction / Vety. Microbiology, trained in carrying out all tests to determine quality of semen samples as per MSP.

5.Lab Technician

Bachelor degree in Science / Microbiology / Bio-chemistry, in semen production and quality control. Persons having post-graduation degree are preferable.

6.Semen Collector

Diploma in livestock management or stockman training course with two years experience in livestock farm / semen laboratory.

7.Lab Attendant/ Bull Handler

High school / 8th standard, should be able to read and write and with two years working experience in semen station/ bull station/livestock farm.

For semen & LN Distribution

1.Stockman (Semen & LN Distributor)

Stockman with two years experience, trained in transfer of straws & distribution

2.Stockman (Semen Despatch}

Stockman with two years experience, trained in examination of semen under microscope.

Minimum Standards29

Annex - XI

Job responsibility of the

The QCO shall be responsible for the quality control work pertaining to semen production and any other related work as may be required from time to time. He shall be responsible to carryout the following:

For adult bulls as and when there is report about poor semen quality, particularly of crossbred bulls

- Young bulls four to six times before putting for regular collection. Thereafter, based on the report of poor quality of semen

Depending on the report of poor quality of semen or certain abnormalities, such bulls shall be kept under observation and semen samples examined twice a week for 4 to 6 weeks. Semen from such bulls shall not be frozen until cleared by the QCO.

a). Bacteriological tests:

Neat semen Frozen semen

Lab environment

AV washing-

b). Morphological abnormalities :

Morphological abnormalities -

Randomly two samples daily

Randomly eight samples daily, all bulls to be

covered once in a quarter

Plates shall be placed daily at different

location in the processing room, AV room,

washing room, pass box, Incubator, Laminar

airflow unit ,etc. to check bacterial load

Randomly two AVs once a week.

c). Acrosome integrity test:

Frozen semen samples of all adult & young bulls in each quarter

d). Validation of photometer

Sperm concentration of neat semen shall be measured by Haemocytometer separately for cattle & buffalo bulls for validation of IMV photometer (as per MSP) - twice a year

e). Sperm concentration of frozen semen sample :

- Randomly two samples per week each of cattle and buffalo.

Minimum Standards

30

f). pH measurement:

g). HOST

h). Incubation Test

j). Percent intact acrosome

- washing of sterilized glassware / rubberware shall be checked for pH once a week

· everyday four samples after 24 hrs. PTM

· every day 4 to 6 samples

- all bulls to be covered once a quarter

Semen samples frozen routinely shall not be checked by those who are involved for semen processing. Semen samples shall be checked by the QCO for PTM after 24 hours of freezing. The QCO will be the final authority to accept or reject semen samples. In addition, he will be testing the quality of straws or any other consumable materials used in semen station.

NB. Depending on the work load, the QCO should be assisted by a Lab technician and Lab attendant.

arb:C/alldoc/Min Stn/Min. Sid. for Govt.doc (April1 2005)

Minimum Standards

31

N.D.D.B.

ANAND 388001

SEMEN BANK TOP PLAN

DRAWING NO. LAB 01.A4

SEMEN BANK-LAB.01A4

28-11-02

N.D.D.B.

ANAND 388001

BULL SHED GROUND FLOOR PLAN - SECTION

DRAWING NO. AR-02-05.A4

BULL SHED-05.A4

- S3-

28-11-02

'i^jii*^^'.'^'.^^-»>,-,