| Sperm-rich ejaculate fractions were obtained from Shanghai White Boar using the gloved-hand method.A series of experiments were conducted to determine the suitable dilution,glycerol balance time,freezing and thawing temperature for the freezing of boar spermatozoa in 5mL straws.The ultrastructure and the in vitro & in vivo fertilization(IVF) capacity of the frozen-thawed semen was also be evaluated.The results are as follows:1.0.1%of the N-AcGA in the dilutionâ… can increase the thawed vitality and acrosome integrity of the sperm,50.00%and 56.60%.4%of the glycerin in the dilutionâ…¡can increase the thawed vitality,plasma membrane integrity and acrosome integrity of the sperm,52.50%,52.08%and 77.67%.The quality of the thawed sperm,which frozen with 10%or 20%of the yolk,were better.Equilibration in the glycerol for 2h can increase the thawed acrosome integrity of the sperm markedly(P＜0.05).The experiment was conducted to selecte the best concentration of the yolk(15%and 20%),glycerine(1%and 1.5%),OEP (0.5%and 1%) and two equilibration time in the glycerol(0 h and 2 h) to carry out mutual compatibility of pig sperm frozen,the results show that the dilution of adding 15%of the yolk,1%of glycerin and 0.5%of the OEP,balance 2 h group could get better vitality(35%) and the NAR(56.38%).There was no significant improvement when 0～0.64 mg/mL glutathione was added to the dilution.2.Six different freezing heights were designed to determine the best frozen rate.The group of 1cm freezing height got the best results not only in the post-thaw motility rate (55.00%),but also in the normal arcosome morphology rate(NAR)(80.08%).Ten group of thaw temperature at 42℃(30 s,40s,50s),52℃(18s,25s,35s,45s) and 60℃(20s,30s,40s) were studyed.There was no significant difference in the post-thaw motility between different thawing temperature and its corresponding thawing time.As a whole,the group of 42℃and 40s,whose mobility was bighter than 40%in 15min,got the hightest NAR (59.50%).3 In the packages of the freeze,5 mL maxi-straw got a little lower mobility(40%), viability rate(49.58%),plasma membrane integrity rate(53.91%) and normal acrosome morphology rate(52.65%) than the 0.25 mL straw,but there was no significantly different between them(P＞0.05).The capacity of in vitro fertilization by frozen boar semen in this experiment was similar to the fresh semen(P＞0.05).From the recipient sows of Shanghai White Pig,Pama Minipig and Fengjing pig,the results getted by frozen-thawed semen from 5 mL straws after artificial insemination were similar to the results of that.4 With ultrastructural observation by scanning electron microscopy techniques,results showed that the membrane after frozen and thawed was bubbly.That ultra-low temperature and thawing could damage the sperm membrane of pig seriously. |
