山羊冻胚分割试验

将冷冻-解冻后的琼脂包被孵出胚泡分两组,分别置于12％蔗糖液(A)和磷酸缓冲液(B)中进行2分切割。在体外培养12小时后,A、B两组的半胚发育率分别为68.9％(31/45)和58.3％(35/60)。A组的冻胚分割效果显著好于B组(P<0.05)。将早期囊胚、扩张囊胚、孵出胚泡和琼脂包被孵出胚泡冷冻-解冻后,在12％蔗糖液中分割为2(A、B、C、D组)。在体外培养12～24小时后,A、B、C、D4组的半胚发育率分别为47％(14/30)、 50％(15/30)、27％(8/30)和70％(21/30)。D组的冻胚分割效果显著好于A、B两组(P<0.05)极显著地好于C组(P<0.01)。将5枚在冷冻前用琼脂包被的孵出胚泡在12％蔗糖液中分割为二后移植于5只受体,结果有4只妊娠,共产半胚羔6只,其中2对为同卵双生。本研究证明,在蔗糖液中分割冻胚可提高半胚体外成活率;分割从琼脂释放出的解冻孵出胚泡可提高山羊冻胚分割的效果。     

生产条件下安哥拉山羊胚胎徒手分割研究

生产条件下,徒手二分割安哥拉山羊桑椹胚和囊胚,并将裸半胚手术移植于受体羊子宫角。１９９３年用０．５％链霉蛋白酶处理胚胎１～６ｍｉｎ软化透明带后,在玻璃培养皿中的含１２．５％蔗糖和５％新生犊牛血清（ＮＣＳ）的ＰＢＳ液内分割,将３３枚半胚移植到２１只受体,有２只受体妊娠。１９９４年用０．２５％链霉蛋白酶处理胚胎０．５～１ｍｉｎ软化透明带后或不经处理而直接在磨砂玻璃皿中的含２０％血清的ＰＢＳ液内分割,移植受体３４只,有１０只受体产羔（２９．４％）,共产羔１１只,其中１对为同卵双生,半胚成羔率为２０．０％（１１／５５）。１９９５年,囊胚不经处理直接在磨砂玻璃皿中的含２０％ＮＣＳ的ＰＢＳ液内分割,半胚成对移植于受体羊黄体侧子宫角,产羔受体率为５０．０％（１３／２６）,共产羔１６只,其中３对为同卵双生,半胚成羔率为３０．８％（１６／５２）,胚胎成羔率为６１．５％（１６／２６）。     

移植奶牛胚胎冷冻后分割成的二分胚生产同卵双生犊牛的实验

对解冻后品质优良的Ａ级囊胚进行分割，并将二分胚移植生产同卵双生犊牛。结果表明：９枚Ａ级囊胚切割成１８枚半胚，胚胎分割成功率１００％。单胚移植受体６头。妊娠２头，妊娠率３３．３％；双半胚移植６头，妊娠２头，妊娠率３３．３％。     

移植奶牛胚胎冷冻解冻后分割成的 二分胚生产同卵双生犊牛的实验

对解冻后品质优良的A级囊胚进行分割,并将二分胚移植生产同卵双生犊牛。结果表明,9枚A级囊胚切割成18枚半胚,胚胎分割成功率100%（9/9）。单胚移植受体6头,妊娠2头,妊娠率33.3%;双半胚移植6头,妊娠2头,妊娠率33.3%。     

小鼠孤雌胚的体内体外发育研究

小鼠孤雌激活卵登攀上培养于ＣＺＢ溶液中可发育至孵出囊胚阶段,而且孤雌胚移植后可在受体子宫内附植发育。单倍体、二倍体孤雌胚发育至桑椹胚的能力相似,但发育于囊胚的能力,后得高于前者。孤雌囊胚体外发育至孵出的比率仅为２０％,但在卵巢切除的受体子宫内期发育至孵出囊胚的比率提高到７３．３％。与受精扩张囊胚相比,孤雌扩张囊肿成纤维细胞饲养层上很难贴壁生长,但延迟孤雌囊胚的内细胞团（ＩＣＭ）呆在细胞饲养层上增殖     

奶牛胚胎分割试验研究

简单分割7～8天奶牛胚胎59枚(117个半胚)。裸半胚成对移给59头奶牛或黄牛,80～90天有27头(45.8％)妊娠。最后24头受体产犊34头,半胚产犊率29.1％(34/117)。有10对双胎,双胎率41.7％(10/24)。比较在不同情况下—7天或8天;晚桑椹或囊胚期;透明带软化处理或不软化处理—分割的半胚,成对移植后受体妊娠率分别是40.0％和57.9％;35.7％和54.8％;48.6％和41.7％。半胚产犊率分别是26.6％和34.2％;23.6％和33.9％;30.0％和27.7％。均无显著差异(P>0.05)。分割优质胚胎得到最好的(35.7％)半胚产犊率。半胚在体外5小时内移植有较高(30.5％)的产犊率。试验探索了奶牛半胚移给远处分散的农户黄牛的可能性。11对奶牛半胚移给11头黄牛有6头(54.5％)妊娠。最后5头受体产下7头奶牛犊,两对同卵双胎。     

不同分割液对水牛胚胎分割效果的影响

探讨了3种不同分割液PBS、PBS+0.2 mol/L蔗糖和PBS+5%聚乙稀吡咯烷酮(PVP)对水牛胚胎分割效果的影响。借助显微操作仪,将体外受精培养的水牛桑椹胚(第5天)和囊胚(第6～7天)分割,体外培养半胚,观察其发育情况。结果显示:在PBS+0.2 mol/L蔗糖与PBS+5%PVP中分割桑椹胚,其分割成功率显著高于PBS(71.67%,67.26%,55.44%)(P<0.05),而半胚的囊胚发育率及囊胚细胞数三者均无差异显著性(P>0.05);在PBS+0.2 mol/L蔗糖与PBS+5%PVP中分割囊胚,其分割成功率显著高于PBS(79.13%,76.73%,65.25%)(P<0.05),而半胚培养的囊胚发育率及囊胚细胞数三者差异均无显著性(P>0.05)。说明在PBS中分别添加0.2 mol/L的蔗糖和5%的PVP有利于提高水牛桑椹胚和囊胚的分割成功率。     

影响和牛双半胚移植妊娠率因素分析

研究和牛胚胎不同胚龄和移植方式对分割双半胚移植妊娠率的影响。结果表明:致密桑椹胚、早期囊胚、囊胚分割后双半胚移植妊娠率分别为60.2%、53.8%和50.0%,随着胚龄的上升妊娠率有下降趋势,各组间无显著差异(P0.05);移植到受体牛同侧子宫角双半胚妊娠率高于子宫角双侧各移植半枚分割胚和整胚(58.6%、41.7%、51.2%)(P0.05),双侧移植双犊率显著高于单侧移植和整胚移植(85.7%、36.9%、0%)(P0.01)。     

不同分割液对奶牛不同阶段胚胎分割效果的影响

本试验探讨了3种不同分割液对奶牛桑葚胚和囊胚分割效果的影响。借助显微操作仪，将发育至第6～8天的体内常规生产的桑葚胚和囊胚进行分割，体外培养半胚，观察其发育情况，选择形态恢复好的半胚与一个囊胚滋养层细胞囊泡（trophoblastic vesicles，TRV）共移植。结果显示，在PBS+0.2 mol/L蔗糖与PBS+5%PVP中分割桑葚胚，其分割成功率显著高于PBS（P<0.05），分别为89.13%、86.73%和69.67%，而半胚的囊胚发育率及移植妊娠率三者均无显著差异（P>0.05）；在PBS+0.2 mol/L蔗糖与PBS+5%PVP中分割囊胚， 其分割成功率显著高于PBS（P<0.05），分别为94.52%、92.52%和70.52%，而半胚培养的囊胚发育率及移植妊娠率三者均无显著差异（P>0.05）；说明在PBS中分别添加0.2 mol/L的蔗糖和5%的PVP有利于提高奶牛桑葚胚和囊胚的分割成功率。     

波尔山羊胚胎移植技术的研究与应用

对 75只波尔山羊超排处理 ,共获 A、B级胚胎 776枚 ,平均 (10 .35± 6 .4 5 )枚 /只 ,鲜胚移植受体 36 7只 ,移植产羔率达 5 5 .6 %。应用 CIDR和 PMSG同期发情处理受体山羊 5 6 0只 ,5 4 9只达到同期发情 ,32 h内同期率为 98.0 4 %。囊胚和扩张囊胚在发情后 7d的移植产羔率分别为 6 2 .2 2 % (84 /135 )和 5 8.2 6 % (6 7/115 ) ,显著高于桑椹胚 (43.18% )和孵化囊胚 (5 1.72 % ) (P<0 .0 1) ,而囊胚和扩张囊胚间、桑椹胚和孵化囊胚间差异不显著 (P>0 .0 5 )。受体山羊的黄体数量直接影响到移植产羔结果 ,黄体数≥ 2的受体山羊移植产羔率显著高于黄体数为 1的受体山羊移植产羔率 (P<0 .0 1) ,分别为 6 4 .83% (94 /14 5 )和 4 9.5 5 % (110 /2 2 2 )。PMSG注射时间 (在取 CIDR前 2 d或当天注射 )和剂量对受体羊的黄体数和移植产羔率均无显著影响 (P>0 .0 5 )     

Increase in calf production by the transfer of bisected bovine embryos

A total of 132 embryos were recovered from 17 superovulated donor cows 7 d after estrus. Seventy-four embryos were selected and assigned to 2 treatment groups. The number of whole embryos that were directly transferred (Group A) and bisected (Group B) were 44 and 30 embryos, respectively. Sixty demi-embryos were produced from 30 morulae to blastocyst-stage embryos that were bisected. One hundred-three embryos, including whole and demi-embryos without zonae pellucidae, were nonsurgically transferred. Only one whole or demi-embryo was transferred to each recipients. The pregnancy rate for whole embryos (A) was 63.6% (28/44), while for demi-embryos (B) it was 74.6% (44/59). There was no significant difference between the pregnancy rates of whole embryos (A) and bisected embryos (B) transferred 7 d after estrus. Forty-three calves including the 14 sets of identical twins were obtained from 30 original embryos (143.3%) using the embryo bisection technique.     

山羊类ES细胞的分离与克隆

采集山羊交配后6～8d的桑椹胚、囊胚和孵化囊胚,将桑椹胚和囊胚分别放在小鼠原代胎儿成纤维细胞(PMEF)饲养层和同源原代胎儿成纤维细胞(PGEF)饲养层上比较其脱带时间及脱带率。脱带后,将各自一半胚胎切割,把含ICM的半胚分别放在相应饲养层上进行培养,另一半整胚在各自饲养层上继续培养,而孵化囊胚直接于PGEF饲养层上培养。当ICM增殖一定程度时进行传代,以比较其类ES细胞分离与克隆的效果。结果表明,在2种不同饲养层上,囊胚的脱带时间均短于桑椹胚,囊胚的脱带率均高于桑椹胚,而饲养层的种类对胚胎的脱带时间以及脱带率影响不大。脱带切割囊胚不论在PMEF还是在PGEF饲养层上,其贴壁时间均短于脱带整胚及孵化囊胚,而贴壁率高于脱带整胚,与孵化囊胚相似。脱带整胚及脱带切割胚在PMEF饲养层上所获类ES细胞只能维持3代,而在PGEF饲养层上,脱带切割半胚和孵化囊胚所获类ES细胞传至5代。由此认为,对脱带后的胚胎进行切割处理,有利于ICM的贴壁和增殖;应用同源原代胎儿成纤维细胞饲养层培养系统,有利于类ES细胞的分离与克隆。     

Bisection of bovine morulae and blastocysts from superovulated Danish dairy cows

Sixtyfour compacted morulae and blastocysts were bisected with a microscalpel. The majority of the demi-embryos (n = 122) were reinserted into separate zona pellucidae (ZP) before non-surgical transfer to 113 synchronized recipients, as singles (n = 98) (DE-S) or in pairs (n = 30) (DE-P). Thirty non-manipulated embryos (E) were transferred during the same period and served as controls. Pregnancies were diagnosed by rectal palpation 4-7 weeks after transfer. The pregnancy rates for DE-S, DE-P and E were 32%, 53% and 40%, respectively (P greater than 0.05). A substantial number of abortions were recorded between 50 and 250 days of pregnancy among the recipients with DE-S. The fetal survival rate for DE-S was reduced to 21% and significantly lower (p less than 0.05) than the survival rates of DE-P (43%) and E (40%). The quality of DE and the presence of ZP did not significantly influence the results. No conclusive reasons for the fetal loss could be found but different possibilities are discussed.     

奶牛新鲜和冷冻胚胎分割移植试验

用简单方法,分割7～8日龄新鲜牛胚胎(1分为2),裸半胚成对移植给66头受体,90天妊检,移植妊娠率为56.1％(37/66)。除6头流产和尚有5头待产外,已有26头受体产犊35头,其中有9对同卵双胎,双胎率为34.6％(9/26),半胚产犊率为29.2％(35/120)。对影响成对半胚移植妊娠率和半胚产犊率的诸多因素如胚胎质量,胚胎在体外停留时间、胚胎发育阶段、受体牛品种、黄体状况等进行了较系统的研究。同时对冷冻胚胎进行了分割试验,移植妊娠率为45.5％(5/11),已产3头犊牛。对快速冷冻和常规冷冻胚胎分割后的移植妊娠率进行了比较,分别为25.0％(1/4)和57.4％(4/7)。     

奶牛冷冻胚胎分割和分割后半胚冷冻技术

用简易分割方法,分割奶牛冷冻胚胎16枚,获得半胚32枚,分割成功率为100％(16/16),可移植半胚29枚,移植于16头受体牛,到第3个情期未返情者经直肠检查有6头妊娠,妊娠率为37.5％(6/16)。其中,在分割前或者分割后经恢复培养0.5～2h再移植的10头受体牛,5头妊娠;而未经恢复培养,分割后直接移植的6头受体牛中,只有1头妊娠。移植后3个月,直肠检查确定2头流产。已有1头受体黄牛生出1头奶牛牛犊(母)和1头受体奶牛产1头奶牛牛犊。其余的2头妊娠受体牛将于9月份产犊。此外,用简易分割法分割奶牛胚胎5枚,得到半胚10枚,裸半胚直接冷冻,解冻后回收可移植半胚5枚,移植于4头受体牛,无一头妊娠。结果表明,冷冻胚胎的分割半胚优于分割后冷冻半胚移植效果;冷冻胚胎分割前或者分割后恢复培养移植优于未经恢复培养而直接移植;简易分割法可应用于冷冻胚胎的分割。     

Relationship between time elapsed after human chorionic gonadotropin administration and developmental stage in porcine embryos collected from prepubertal gilts

We examined the relationship between the time elapsed after human chorionic gonadotropin (hCG) administration and developmental stage of porcine embryos after collection. Prepubertal gilts, 7 to 8 months old, were given 1500 IU equine chorionic gonadotropin (eCG) intramuscularly, followed by 500 IU hCG 72 h later. The treated gilts were inseminated artificially on Day 1 (Day 0=the day of hCG administration) and on Day 2. Embryos were collected surgically on Day 6 (140, 144, and 147 h after hCG administration) or on Day 7 (164, 168, and 171 h), and the developmental stages of the collected embryos were examined. From 75.2% (276/367) of the prepubertal gilts treated with hormones, we collected an average of 20.7 embryos per gilt with normal morphology. At 140 h after hCG administration, morulae (54.4%) could be collected. At 144 h, morulae and early blastocysts (57.7% and 28.9%, respectively) were collected. By 147 h, the proportion of embryos at the blastocyst to expanded blastocyst stages had increased (10.0%). From 164 h to 171 h, expanding or expanded blastocysts of more than 200 microm in diameter and hatched blastocysts could be collected. The proportion of hatched blastocysts increased from 3.2% (164 h) to 41.0% (171 h). These results suggests that although the number of ovulations differed among gilts, porcine embryos at the appropriate stages can be collected efficiently by controlling the time elapsed between hCG administration and embryo collection.     

Transfer of Bovine Blastocysts Derived from Short-term In Vitro Culture of Low Quality Morulae Produced In Vivo

The aim of this study was to evaluate if blastocysts arising from in vitro culture of Grade 3 bovine morulae produced in vivo can promote acceptable pregnancy rates when transferred into recipients. Embryos of different stages and qualities were recovered from superovulated Bos taurus and B. indicus donors. Grade 3 morulae were cultured in either Holding Plus® or TCM‐199 (supplemented with 10% bovine fetal serum) media for 24 h at 38.5°C. After this culture period, the resulting blastocysts were morphologically classified (Grades 1, 2 and 3) and transferred into recipients previously synchronized with the donors. Non‐cultured Grades 1 and 3 morulae were used as control. Pregnancy diagnosis was carried out 60 days after embryo transfer and the data were analysed by logistic regression, considering variables, such as embryo quality (Grade), donor breed, culture medium, donor‐recipient synchrony and seasonality. Embryo quality was the only variable, showing significant effect on the pregnancy rate. Pregnancy rates for non‐cultured Grade 1 and 3 morulae, and blastocysts arising from cultured Grade 3 morulae were 58.1% (n = 31), 17.1% (n = 35) and 51.1% (n = 47), respectively (p < 0.05). There were no statistical differences between non‐cultured Grade 1 morulae and cultured blastocysts. Pregnancy rates for Grades 1 (65.0%) and 2 (60.0%) were higher than Grade 3 (29.4%) cultured blastocysts (p < 0.05). It was concluded that short‐term in vitro culture is a very convenient method of identifying morphologically low quality morulae with higher chances of continuing development after the transfer into recipients.     

Matching of embryo stages and grades with recipient oestrous synchrony in bovine embryo transfer

The efficacy of matching embryos with recipients on the basis of embryo stage and grade and donor-recipient oestrous synchrony was investigated using the records of 13,663 embryos that were collected and transferred at a commercial embryo transfer centre. The selection of early blastocysts for exact oestrous synchrony cows was effective and resulted in the highest pregnancy rates. Selection of early morulae was effective for recipients in oestrus after the donor but not when transferred into exact and negative recipients. The matching of late morulae with recipients in oestrus after the donor was not effective and had no influence on pregnancy rates. The selection of late, hatched and collapsed blastocysts for transfer into recipients in oestrus before the donor was ineffective and pregnancy rates were higher in exact and +12 hour recipients. Pregnancy rates declined 23.6 per cent in quality grades 1 to 4 whereas the range between stages was 13.3 per cent. Higher quality embryos of all stages gave the highest pregnancy rates. Examination of pregnancy rates of grades within stages suggested that the more developed the embryo the more difficult it is to grade. The difference in pregnancy rates between exact and -24 (6.9 per cent) and +24 (4.8 per cent) hour recipients was small and declined a further 4.7 per cent and 9.8 per cent in -36 and +36 hour recipients. Grade 3 and 4 embryos tolerated asynchrony better than grade 1 and 2, and early morulae tolerated asynchrony better than the other stages. It was concluded that the matching of certain embryo stages with the donor-recipient oestrous synchrony is advantageous but not always possible.