FROZEN SEMEN

SEMEN 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 ejaculate Massage method: 􀂄 Rectal massage of vesicular glands and ampullae

SEMEN 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

SEMEN EVALUATION Semen Evaluation 1) Macroscopic examination 􀂄 Volume 􀂄 Color 􀂄 Consistency 􀂄 pH 􀂄 Odor 2) 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

SEMEN EVALUATION Semen Evaluation

SEMEN EVALUATION Semen Evaluation

SEMEN EVALUATION Semen Evaluation

SEMEN EVALUATION Semen Evaluation

SEMEN EVALUATION Semen Evaluation

SEMEN EVALUATION Semen Evaluation

SEMEN EVALUATION Semen Evaluation

SEMEN EVALUATION Semen Evaluation

SEMEN EVALUATION Semen Evaluation

SEMEN 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

SEMEN DILUTION Properties of semen extender: a. Isotonic 􀂄 -salts b. Buffered (PH 6.5) 􀂄 phosphate 􀂄 citrate 􀂄 Tris c. Cold shock protection 􀂄 Lecithin and lipoproteins find in egg yolk and milk d. Nutrients 􀂄 egg yolk and milk fructose and glucose e. Antibiotics 􀂄 Gentamycin 􀂄 Tylosin 􀂄 Lincospectin 􀂄 Penicillin + Streptomycin f. Protection for freeze and thaw 􀂄 -glycerol g. Preserve fertility 􀂄 -Known and unknown factors found by trial and error

PROCESSING A) semen collection 􀂄 do not let temperature drop below 35c B) Antibiotic treatment 􀂄 3-5 minutes before dilution C) pre-dilution 􀂄 start at 35c 􀂄 No glycerol yet 􀂄 up to half final volume D) cooling 􀂄 Cool to 5 c 􀂄 Slow cool over 2 to 4 hours E) Final dilution 􀂄 cooled extender + glycerol F) Equilibration 􀂄 - allow glycerol to act 􀂄 -minimum of 4 hours

PROCESSING G) Packaging 􀂄 Plastic straws - 0.5ml in US - 0.25ml in Europe (French straws) 􀂄 Glass ampoules 􀂄 Old method 􀂄 Pellets 􀂄 No package 􀂄 Most success in swine H) Freezing 􀂄 use of liquid nitrogen vapors first then drop into the Nitrogen -196C I) Storage 􀂄 Dry ice and methanol (old) 􀂄 Liquid nitrogen is now used 􀂄 Do not let tanks dry.

SEMEN 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 poorly ventilated surfaces 􀂄 5-store only the amount of semen needed for six months

SEMEN TANK MANAGEMENT

HANDLING 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 transferred from tank to tank or when handling semen within the neck while trying to locate a specific unit of semen 􀂄 Thermal injury to sperm is permanent and can not be corrected by returning semen to liquid nitrogen.

SEMEN HANDLING PRACTICES TO MINIMIZE THERMAL DAMAGE 1- Transfers of semen between tanks must be coordinated and rapid 2- Avoid unnecessary searching and exposure of semen to dangerously high temperature within neck region 3- Use of Tweezer to transfer the straw to the bath. Quickly lower the rack semen and canister into the tank body.

THAWING OF FROZEN SEMEN 􀂄 Straws are thawed at 35 C for 12 seconds for 0 .5ml straw or 6 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 proper thawing time has passed to prevent excessive worming of the semen after removal of straw carefully 􀂄 Dry the straws with a clean tissue 􀂄 Inspect the straws and discard any with cracks or defective Seals

THAWING OF FROZEN SEMEN

ASSESSMENT QUALITY OF FROZEN SEMEN Motility 􀂄 40% / 3 (Bovine) of progressively motile spermatozoa are the minimal acceptable motility for frozen semen 􀂄 Number of motile spermatozoa per inseminate depend on breed and fertility level of each bull 􀂄 For most bulls each inseminate should contain at least 10 million motile sperms after thawing regardless of how semen is packaged 􀂄 The influence of the method of thawing on post-thaw motility will also be in the number of motile spermatozoa per inseminate. Acrosomal integrity 􀂄 Spermatozoa with deteriorated or damaged, acrosome unlikely to be capable of fertilization 􀂄 Examination of spermatozoa under differential interference phase contrast microscope 􀂄 The sample is incubated at 37 c and aliqutos are removed for evaluation after 0.2, 4, 8 hours 􀂄 For semen in egg yolk citrate extender, the semen diluted with two volumes of 0.2% glutaraldehyde in phosphate –buffer saline.

INSEMINATION 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 themorning Cite of insemination 􀂄 In anterior half of the cervical canal or just anterior to internal os in the body of the uterus