| The objective of this research was to establish a high fertility and operability of rooster semen cryopreservation technique, for the use of frozen semen of long-term preservation of rare poultry genetic resources, to provide technical support of introduced germplasm resources. The rare local black silkies chicken were used as the material of this research,rooster sperm cryopreservation of freezing procedures were optimized on this experiment,specially in the freezing and thawing rate, glycerol removal, different concentrations of glycerol, artificial insemination(AI) methods. The experimental results were as follows:1.Different sterilization methods, dilution strike of chicken semen and bacteriostasis experiment results:? The four chicken semen dilution FEB,BPSE,L,LT were selected in this experiment, it should be treated with autoclave and filtration sterilization,at a 1:2 dilution ratio preserved in 5? and the vitality of sperm form 0 to 168h were observed. The results showed that the storage effect of the same dilution with different sterilization methods had no significant difference. The preservation effect of the descending sequence was: BPSE>LT>L>FEB diluent.? The above four dilutions were performed using 1:1 dilution and 1:10 dilution experiment preservation in 5?, the vitality of sperm form 0 to 168h were observed. The results showed that the storage effect of the same dilution with different dilution ratio methods had significant difference. The preservation effect of the descending sequence was:LT>BPSE>L>FEB diluent.? The antibacterial experiment results showed that the addition of appropriate amount of penicillin and streptomycin to the dilutions stored at -20? for a period of time, E. coli was still highly sensitive to the diluents.The freezing rate optimization experiment was carried out with the laboratory self-made portable freezing device. ? The one-step freezing method, The sperm was put in the liquid nitrogen surface freezing for 3-5min effect was the best, after thawing vitality could reached 0.7. ? The two-steps freezing procedure, pad high method (10cm):The first step freezing fumigation was 3-5min, then on the liquid nitrogen surface freezing time was about 5min could obtained better freezing effect,after thawed, semen vitality could reached 0.8-0.6;? The two-steps freezing procedure (distance liquid nitrogen surface 17cm), the Semen vitality was significantly better than all of other freezing rate. The first step with -15?-6?/min of the rate of fumigation 1?5min?Then with -60?-27?/min of the rate of fumigation to 1?5 min can obtain best frozen effect,after thawed sperm viability can reached over 0.8. With optimized freezing procedure and 6% concentration of glycerol production of straw frozen semen, the maximum fertility and hatchability (FH) reached 77.63% and 73.38%.? Thawing effect of different temperature were as follows:5?>10?> 40?> 50?, the best thawing temperature was 5?,5&10? had no significant difference.3. The glycerol remove, glycerol concentration, artificial insemination methods results:? The glycerol (11%) was removed by stepwise methods,The fertility order of the 4 groups was group 1:2 (61.01%)>group 1:1(52.37%)> group 1:4 (41.48%)> non-washed group (6.29%). The fertility of group 1:2 was significantly higher than that of group 1:1 (P<0.05), and it was extremely higher than that of group 1:4 (P<0.01) and non-washed group. 4) The fertility of non-washed group was extremely lower than that of any other groups (P<0.01).? 2,4,6,8,11% five different concentrations of glycerol was adopted as a cryoprotectant. The results showed that:five concentration group fertility rate was as follows: 6% group (77.63%)>4% group (75.1%)>8% group (70.09%)>11% group (57.43%)>2% group (34.8%),4?8% glycerol concentrations of straw semen fertility rate had no significant difference, but it was significantly higher than that of 2% and 11% groups.? The 6% glycerol of frozen semen for artificial insemination experiment, different transmission precision fertility rate and emergence rate order was:group 0.4ml(70.15%)>group 0.2ml(67.08%)>0.3ml{deep vagina group(65.51%)>superficial vagina group(65.51%)}> group 0.1ml(50.23%). The difference was not significant among group0.2ml?group0.4ml. The fertility of group 0.1ml was significantly lower than the other groups.The fertility of deep vagina group 0.3ml was higher than that of superficial vagina group 0.3 ml, but they had no significant difference.In conclusion, The experimental parameters were used in this research.LT dilution solution was used as the base fluid for cryopreservation of rooster semen.The two-steps freezing procedure was:first step distance liquid nitrogen surface 17cm, freezing rate of -14 ?-9?/min for 4min, the second step first 15s will tube frame to fall slowly to the surface of the liquid nitrogen, and then on the surface of the liquid nitrogen, freezing rate of -60? 25?/min for 2min,after that immersed staws into liquid nitrogen.Then the staws were thawed in 5? water for 3min,The glycerol (6%) was removed by washing the semen with stepwise dilution of 1:2,deep vaginal artificial insemination (0.3ml semen)were adopted, could obtain 77.63% fertilization rate. This experiment is easy to operate, high stability, obtain high fertility,reached the advanced level of the similar studies in the world. |
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