some website wrote:Semen Collection and Processing
Semen can be collected by electroejaculation. Prior to beginning the stimulation procedure, the penis is externalized manually or by gentle traction with a cable loop. The penis can be maintained in a position external to the penile sheath by a hand-held loop of cotton gauze placed around it. A 5 cm diamater, three electrode electroejaculation probe is inserted in the animal’s rectum. A series of mild electrical stimulations will be applied through this probe to cause ejaculation into a warm, water-jacketed artificial vagina or collection vial placed over the tip of the penis. The stimulations consist of three series of 18 stimulations each. The first stimulation in each series will be at two volts and each subsequent stimulation will increase in 0.5 volt increments to 10 volts which ends the series. Each stimulation lasts 2-3 seconds. Some movement of the rear legs will occur during stimulation.
Semen will be collected in a warmed, water-jacketed artificial vagina or a 50 ml plastic collection vial. Following completion of the collection, a 0.5 ml sample of raw, unextended semen can be stored frozen in liquid nitrogen for disease testing if appropriate. The remaining semen should be evaluated, processed and frozen in 0.5 ml artificial insemination straws for storage in liquid nitrogen.
Semen Evaluation and Processing
Immediately following semen collection, total ejaculate volume will be determined and a 0.5 ml sample of raw semen will be set aside for disease testing. Antibiotics are then added: 2% final concentration from stock solution of: polymyxin B (61 mg/l), dihydrostreptomycin (1 g/l), penicillin-G (500,000 units/l), in accordance with NAAB (National Association of Animal Breeders, Columbia, MO) recommendations for venereal pathogen control.
Initial evaluations include analyses of % motility, sperm concentration, morphology and progressive status. Morphology is determined from examination of > 100 cells prepared with Therio Stain and classification of sperm as normal or abnormal, including abnormal head (macrocephalic, microcephalic, degenerate), abnormal midpiece (bent residual, cytoplasmic droplet), abnormal flagellum (bent, coiled). Sperm concentrations are estimated by dilution (1:100) into a blood-diluting pipette (BD-WBC-Unopette, Becton-Dickinson Co., Rutherford, NJ) and counting with a Neubauer hemacytometer. Progressive status is based on a rating scale of 0-5 (0 = no movement, 1 = little movement, 2 = movement and poor forward progression, 3 = slow forward progression, 4 = steady forward progression, and 5 = rapid forward progression).
Following these initial evaluations, semen is diluted 1:0.5 with non-glycerated cryodiluent/extender and cooled to 5 ° C. over a 1.5 hr incubation period prior to addition of the remaining non-glycerated extender and an additional incubation at 5 ° C. for 0.5 hr. Extension volumes are determined by adjustment for the % motility and % normal morphology to obtain a concentration of 120 x 106 viable sperm/ml following the non-glycerated extension (first extension). The equation utilized to determine the volume following the first extension and the volume of glycerated extender to be added is as follows:
Total Volume Following First Extension and Volume of Glycerated Extender =
(Semen Volume: ml) (conc.; x 106) (Motility) (Morphology) divided by 120 x 106
The final glycerated extension (7% glycerol final concentration) is added in three steps at 20-minute intervals (25% of the volume during the first step, 25% during the second step and 50% during the third step) to obtain a final concentration of 60 x 106 viable sperm/ml. Extended semen will then be subjected to a final equilibration for one hour at 5 ° C. prior to loading into 0.5 ml straws and freezing.
Only semen samples of > 40% motility, > 2.0 progressive status and > 150 x 106 sperm/ml will be utilized for cryopreservation procedures. A standard field cryopreservation procedure is employed using a liquid nitrogen (LN2) vapor freezing procedure. Liquid nitrogen vapor freezing involves an equilibration of straws in liquid nitrogen vapors (-159 ° C.) for 10 minutes prior to placing in liquid nitrogen storage.
Semen Post-Thaw Evaluations
Semen can be evaluated at 5-7 days after freezing and being placed into liquid nitrogen storage. Semen is thawed (n = 2-8 samples per bull) in a 37 ° C. water bath for 30 seconds and evaluated immediately (0 hr) and following incubation for two hours at room temperature. Post-thaw evaluations includes analyses of % motility, morphology and progressive status.
