2SEMEN COLLECTION Methods of semen collection Artificial vagina: Bull, stallion, buck, ram, dog sometimes poor Stimulate natural copulation Temperature at 45C ( worm water) Pressure (air and worm water) Lubrication (harmless to sperm and not excessive amount)Electro-ejaculation: used in bull and rams increase Voltage in pulses (1-10v) low voltage ---accessory gland secretion and high voltage ---ejaculation semen volume higher and sperm concentration lower get urine often poorer quality ejaculateMassage method: Rectal massage of vesicular glands and ampullae
3SEMEN COLLECTION Precautions During semen collection Precautions must be considered during collection of semen by AV to obtain good quality and quantity semen: Adjust temperature (45c) AV must be clean, dry and sterilized Do not use any detergents or harmful chemicals to sperm Inner sleeve of AV must be free from any leakages Use of adequate amount of harmless lubricants Adjust the pressure inside AV to stimulate thrust Wash and clean perineal and perpetual regions Avoid the collection in dirty place Teasing the bull by false mount and foreplay Protect the semen collector from cold shock by hand or wool cotton piece
6SEMEN EVALUATION Semen Evaluation 1) Macroscopic examination Volume Color Consistency pH Odor2) Microscopic Examination Assessment of motility Mass motility Individual motility Assessment of sperm morphology Sperm abnormalities life and dead sperm ripe and unripe sperm -pathological cells Assessment of sperm concentration use of hemocytometer Automatic counter
18SEMEN EVALUATION Semen Evaluation 3) Special laboratory tests Measurement of hygienic quality visual examination catalaze test Measurement of metabolic activity Methylene blue reduction test Electrical activity of semen Fructolysis index Resistant tests resistant to sod chloride sol.1% resistant to cold shock viability test
19SEMEN DILUTION Properties of semen extender: a. Isotonic -salts b. Buffered (PH 6.5) phosphate citrate Trisc. Cold shock protection Lecithin and lipoproteins find in egg yolk and milkd. Nutrients egg yolk and milk fructose and glucosee. Antibiotics Gentamycin Tylosin Lincospectin Penicillin + Streptomycinf. Protection for freeze and thaw -glycerolg. Preserve fertility -Known and unknown factors found by trial and error
21PROCESSING A) semen collection do not let temperature drop below 35c B) Antibiotic treatment 3-5 minutes before dilutionC) pre-dilution start at 35c No glycerol yet up to half final volumeD) cooling Cool to 5 c Slow cool over 2 to 4 hoursE) Final dilution cooled extender + glycerolF) Equilibration - allow glycerol to act -minimum of 4 hours
22PROCESSING G) Packaging Plastic straws - 0.5ml in US - 0.25ml in Europe (French straws) Glass ampoules Old method Pellets No package Most success in swineH) Freezing use of liquid nitrogen vapors first then drop into the Nitrogen -196CI) Storage Dry ice and methanol (old) Liquid nitrogen is now used Do not let tanks dry.
24SEMEN TANK MANAGEMENT Avoid excessive movement or abuse of tank Monitor nitrogen levels routinely and keep a record of nitrogen loss Store the semen in an area with good light but out of direct sunlight.Observe tank daily. Once the tank fails, Nitrogen is lost very rapidly. Keep the tank elevated above the concrete floor or other wet and poorlyventilated surfaces 5-store only the amount of semen needed for six months
26HANDLING SEMEN WITHIN TANK In the typical farm semen tank , dangerous temperatures (-32 to 112 C)in the upper half of the neck of tank Exposure to these temperature can occur when the semen is transferredfrom tank to tank or when handling semen within the neck while trying tolocate a specific unit of semen Thermal injury to sperm is permanent and can not be corrected byreturning semen to liquid nitrogen.
27SEMEN HANDLING PRACTICES TO MINIMIZE THERMAL DAMAGE 1- Transfers of semen between tanks must be coordinated and rapid2- Avoid unnecessary searching and exposure of semen to dangerouslyhigh temperature within neck region3- Use of Tweezer to transfer the straw to the bath. Quickly lower the racksemen and canister into the tank body.
28THAWING OF FROZEN SEMEN Straws are thawed at 35 C for 12 seconds for 0 .5ml straw or6 seconds for 0.25 ml straw Do not linger with the straw in the neck of LN storage unit or in the air Remove the straw from the thawing bath immediately after the properthawing time has passed to prevent excessive worming of the semen afterremoval of straw carefully Dry the straws with a clean tissue Inspect the straws and discard any with cracks or defective Seals
30ASSESSMENT QUALITY OF FROZEN SEMEN Motility 40% / 3 (Bovine) of progressively motile spermatozoa are the minimalacceptable motility for frozen semen Number of motile spermatozoa per inseminate depend on breed and fertilitylevel of each bull For most bulls each inseminate should contain at least 10 million motile spermsafter thawing regardless of how semen is packaged The influence of the method of thawing on post-thaw motility will also be in thenumber of motile spermatozoa per inseminate.Acrosomal integrity Spermatozoa with deteriorated or damaged, acrosome unlikely to be capable offertilization Examination of spermatozoa under differential interference phase contrast microscope The sample is incubated at 37 c and aliqutos are removed for evaluation after0.2, 4, 8 hours For semen in egg yolk citrate extender, the semen diluted with two volumes of0.2% glutaraldehyde in phosphate –buffer saline.
31INSEMINATION Use recto-vaginal technique in cow Time of insemination Cow in estrus in the morning must be inseminated in the evening Cow in estrus in evening must be inseminated in themorningCite of insemination In anterior half of the cervical canal or just anterior to internal os in the body ofthe uterus