Rabbit genomic DNAs derived from Japanese White Rabbits (JWR), Chinchilla Rabbits (CR) and New Zealand White Rabbits (NZWR) were analyzed with random amplified polymorphic DNA (RAPD) technique and the genetic variation among 3 rabbit strains were studied. The results showed that after 140 random primers in 7 sets were used to amplify the rabbit genomic DNA with PCR methods, 10 have been screened out on the preliminary experiments, including BH9,AD1,BH11,AN12,AN14,O19,AD3,AD19,O4 and O14. Two primers (AD1 and BH9) among the 10 are superior to others and could not only amplify the common DNA bands of 3 rabbit strain lines but also the different characteristic bands of each strain. 78 amplified fragments were detected by electrophoresis on agarose gel. Among them, 25 fragments were polymorphic, accounting for 32.1 %. It was showed that the RAPD polymorphisms among rabbit lines were abundant. The genetic similarity between JW rabbits and NZW rabbits was highest (0.8286) among 3 rabbit strains and the lowest genetic similarity between CR and JW is 0.6190. The amplified band patterns also showed that the genetic similarity inner a strain is bigger than among different strains. The RAPD technique could be used as a method to monitor genetic relationship among different rabbit strains.

Objective To establish an optimal method of frozen-thawing mouse sperms.Methods R 18S3 and FERTIUP TM-CPA were used as cryopreservation protection solutions to freeze mouse sperms of DBA/2,C57BL/6J,KM and B6.129S7-Ldlr tm1Her/J four strains.Three thawing methods were used and the rate of in vitro fertilization(IVF)was used to assess the result of cryopreservation of sperms.Results When R 18S3 was used to freeze sperms,there was a significant difference between the rates of IVF in DBA/2(73.3%,88.4%,55.6%)and KM(64.9%,60.2%,39.6%)among three thawing methods(P 0.05),but not significant difference between C57BL/6J(3.0%,10.3%,10.3%)and B6.129S7-Ldlr tm1Her/J(0%,5.0%,0%)(P 0.05).When FERTIUP TM-CPA was used for cryopreservation of sperms,there was a significant difference of the ratios of IVF in DBA/2(33.6%,14.1%,91.6%),C57BL/6J(8.4%,21.0%,4.9%)and B6.129S7-Ldlr tm1Her/J(8.2%,10.0%,28.9%)(P0.05),but not with that of KM(48.1%,48.0%,48.1%)mice(P0.05).Conclusion For DBA/2 and KM mouse sperms,frozen with either R 18S3 or FERTIUP TM-CPA and thawed with a suitable method,both can get an ideal rate of IVF.However,no matter which cryopreservation protective solution and thawing method are used,the rate of IVF of C57BL/6J and B6.129S7-Ldlrtm1Her/J thawed sperms is always relatively decreased.

为建立兔的胚胎库,探索一种效果稳定可靠、方法简捷的冷冻方法,试验选择冷冻管法一步式玻璃化冷冻保存兔胚胎,比较了不同冲胚时间对兔胚胎冷冻保存效果的影响。配种后65h冲胚保存效果好于60h和55h,复苏胚胎囊胚孵化率依次为:63.1%、62.5%和60.7%,差异不显著。不同预处理对冷冻效果的试验中,胚胎在4℃预处理4min保存效果最好,囊胚孵化率为71.5%。在4℃下2min的保存效果(21.6%)低于同组的其它处理,差异显著(P<0.05)。室温1min的保存效果略高于前者(31.0%),但显著低于同组其它处理(P<0.01)。优化冷冻过程以后,比较两品系的冷冻效果,青紫兰兔(Chinchilla Rabbit,CR)有效供体的平均胚胎数高于新西兰兔(New Zealand White Rabbit,NZWR),42.4±8.8vs38.9±5.5枚/只,但差异不显著(P>0.05)。胚胎移植后,新西兰兔6只受体1只死亡,5只妊娠,产活子38只,青紫兰受体5只有4只妊娠,共产活子28只。胚胎活子效率28.8%和25.9%(NZWR和CR),差异不显著。显示冷冻管一步法玻璃化冷冻保存兔胚的方法可以保存兔胚并获得子兔。

Objective To explore the possibility of use of IVF-20 medium in rat in vitro fertilization(IVF) experiments,and provide a practical reference for embryo conservation.Methods Using IVF-20 as capacitation and fertilization medium,four strains of rats,including SD,Wistar,GK and F344 were chosen for the IVF experiment.The resulting IVF embryos were cultured in vitro with mR1EMC medium.Besides,some of 2-cell embryos were transferred into the oviducts of recipients after induction of pseudopregnancy.Results The cleavage rates of SD,Wistar,GK and F344 rats were 83.2%,72.6%,87.8% and 71.6%,respectively.The blastocyst development rate in SD rat was 16.7%,lower but with no significant difference than that(24.5%) of embryos produced in vivo.Both recipients transferred with embryos from IVF established pregnancy,and delivered 10 pups(12.5%).Conclusion IVF-20 medium used in human in vitro fertilization is also suitable for in vitro capacitation and fertilization in rats,resulting in good cleavage rate,as well as blastocyst development rate,and the 2-cell embryos from IVF can fulfill the complete development after transplantation and give birth to normal pups.

Objective To investigate the feasibility of IVF-20 medium on the in vitro fertilization (IVF) in Rats. Methods Using IVF-20 as capacitation and fertilization medium, the IVF in four strains, including SD, Wistar, GK and F344 were conducted. Besides, the embryos from SD rat fertilized both in vitro and in vivo were cultured using mR1EMC medium to test their viability. Result The fertilization rates in SD, Wistar, GK and F344 were 83.2%、72.6%、87.8% and 71.6% respectively. The rate of embryos developed to blastula was 16.7%, having comparative viability of embryos fertilized in vivo (24.5%). Conclusion The IVF-20 medium, which had been used in vitro fertilization of human, is also a suitable medium for the capacitation and fertilization in rats.

Objective To establish an effective method for superovulatory treatment of rats with pregnant mare serum gonadotrophin(PMSG) and human chorionic gonadotropin(hCG). Methods Sprague-Dawley rats at different age(4-5 weeks,67 weeks and 8-9 weeks) were superovulated with PMSG and hCG at a dosage of 10 IU, 20 IU,30 IU,40 IU or 50 IU,respectively.The results of superovulation of those rats were analyzed.Finally,the 2-cell embryos fertilized in vivo were transferred to recipients to observe the rate of pregnancy.Results Rats aged 4-5 weeks showed the best effect of PMSG/hCG at a dosage of 20 IU on superovulation,with an average number of 54.6 per rat,while rats aged 6-7 weeks and 8-9 weeks were 32.7 and 21.5 per rat,respectively.After embryo transfer,the rate of pregnancy was 63.3%,showing no significant difference with that of control rats(60%).Conclusion Rats at 4-5 weeks of age are more sensitive to superovulation,giving a highest number of oocytes with 20 IU PMSG and hCG.Such oocytes obtained by superovulation are good in quality and can complete the whole term development after fertilized in vivo and embryo transfer.

Objective To investigate the factors affecting cryopreservation of mouse sperm.Methods Mice sperm from C57BL/6J,FVB/NJ,B6D2F1 and ICR strains were cryopreserved using 18% raffinose,3% skim milk as cryoprotectant solution,and estimated the survival and fertility of post-thawed sperm from the five strains.The progressive motility of sperms from each strain was affected differently.Results The fertility rate of C57BL/6J,FVB/NJ,B6D2F1,KM and ICR strains were significantly decreased from 81.4%,87.2%,90.4%,82.7% and 79.4%,respectively,of fresh sperm,to 6.7%, 46.0%,76.8%,59.2% and 31.9%,respectively,when using post-thawed sperms(P<0.01).Conclusion The fertility and susceptibility to cryopreservation of sperm from mice of different genetic background differ significantly.

Sperm cryopreservation is a well-established technique for most mammals.Whereas,a successful cryopreservation protocol of rodent spermremains a scientific challenge.Especially for rat, its long tail of sperm makes its sperm cryopreservation very hard.Accordingly,ICSI of rat is not done better than mouse.There are not any reports on rat sperm cryopreservation and Intracytoplasmic sperm injection(ICSI) in China,and the researches were not done widely abroard.The artical made a simple review on the advances of sperm cryopreservation and Intracytoplasmic sperm injection(ICSI) in rat.

To comparative studies on ova superovulated and in vitro fertilization in various genetic background mouse strains,and to explore avenue of establishing the embryo bank in individual mouse genotypes.35 mouse strains(7 inbred,3 congenic 3 closed colony and 22 mutant) were obtained from Shanghai SLAC Laboratory Animal Co.Ltd.Females were superovulated and operated by IVF.The results were the proportion of females yielding ovulation varied markedly among genotypes(11.8～38.7),abnormal oocytes rate was with a range of 0.2% to 32.2%,The average rate of fertility of individual mouse genotypes was 56.23% with a range of 15.2 % to 97.2%.Concluded the fertility rate by IVF in inbred mouse were higher than outbred,mutant,congenic and induced mutant strains.individual mouse genotypes have significant differences in the rate of fertillity(P0.05).