| College of Animal Science and Technology, Gansu Agricultural University, Lanzhou.China, 730079 SUMMARYStudy on optimum cryopreservation procedure ,semem diluent, factors influenced the fertility, mechanism of decreasing the fertility of frozen-thawed ram spermatozoa and establishing the method of semen quality evaluation was conducted through series experiments. The results showed that:l.The ram spermatozoa optimum cryopreservation procedure was established evaluated by the targets such as frozen-thawed semen motility, survival index, intact acrosome rate and GOT release: (1) Dilution rate is 1 : 3 of semen : extender,(2) Sperm diluted with two-step dilution method ,the semen was firstly diluted by I extender without glycerol under 38 then diluted by II extender containing glycerol at 4,(3) Equilibration method is 200ml water under 38 for 2.5h,(4) pellet of spermatozoa cooled at minus 110 to 120, (5) Semen pellet thawed at 38 with 5(M vitamin Bn per two pellets.The equilibration method of 200ml water at 38 is significant better than eight layers gauze,50ml water, 100ml water and 150ml water at 38癈 for frozen-thawed ram semen (PO.05) .Low thawing temperature for semen quality is better than higher thawing temperature (>55 or 65 ) . The optimum glycerol concentration in diluent is 4.5%. GOT, LDH and ALP release in two-step dilute method is lower than that of in one-step dilute method.2.Nine of optimum extenders was established : (DThe ingredient of I extender include 3.0285g Tris, 1.6593g citrate, 2.5673g sucrose, 15% egg yolk,4.0% glycerol ,penicillin and streptomycin lOOmg respectively. (2)The ingredient of II extender is adding 0.75g fructose in I extender. (3) in extender formed by adding 6ml vitamin Bg in II extender. (4) IV extender formed by adding 6ml vitamin E in II extender. (5) V extender formed by112adding 6ml vitamin Bi2 in II extender. (6)VI extender formed by adding 0.3g L-glutamicacid in II extender.WVfl extender by adding 20mg sorbitol in II extender. (8)Vffl extender by adding 6ml Astragalus membranaceus (a kind of Chinese herbage medicine) in II extender.(9)DC extender formed by adding 6ml vitamin Be, 6ml vitamin E and 6ml Astragalus membranaceus on the basis of II extender .Extender containing sucrose can provide more cryoprotective ability to frozen-thawed ram spermatozoa than extender containing lactose, glucose and fructose (PO.05) ,and decreased the GOT release (PO.05 "> .The intact acrosome of sperm can be increased when adding 0.75g fructose in the Tris-sucrose-egg yolk extender (P<0.05) .Frozen-thawed sperm motility can be increased and GOT, LDH and ALP release can be decreased by adding vitamin E, Bn and Be in semen extender. In addition, vitamin E and Be can provide effective protection for frozen-thawed sperm acrosome and increase survival index of frozen sperm. The general survival time of frozen-thawed sperm cab be prolonged up to 35h in the way of adding Astragalus membranaceus in semen extender. The hyperactivated motility (HAM) of frozen-thawed spermatozoa was induced on condition that adding Houttuynia cordata (Chinese herbage medicine) to extender. The results showed that the optimum target such as motility ,survival index and intact acrosome of frozen-thawed spermatozoa from 0.41.3.69 and 41.73% in beginning increased up to 0.52,6.83 and 54.59% respectively, while GOT, LDH and ALP release from 369.01,1577.68 and 403.47UL"1 in beginning decreased to 210.37,932.16 and 403.47 UU1.3.Non-return rate (30d) of I , III, IV, Vffl, DC extender is 30.95%,50.00%,38.75%,48.08% and 60.61% under cervical insemination. Non-return rate is positive correlated to motility, number of spermatozoa inseminated, intact acrosome rate, depth of insemination and cervical mucus, negative correlated to GOT, LDH and ALP release of spermatozoa.4.The mechanism of declined fertility of inseminated frozen-thawed ram spermatozoa was preliminarily expounded on the basis of comparison of sperm ultrastructure of fresh and frozen-thawed spermatozoa. The sperm was defined five types acco...
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