奶牛半胚裸露冷冻保存试验

本试验研究了不同冷冻方法，半胚冷冻前不同培养时间及不同冷冻保护液对奶牛７日龄半胚裸露冷冻效果的影响，结果以１０％（Ｖ／Ｖ）甘油＋１０％（Ｗ＋Ｖ）葡聚糖（Ｔ－５００）＋０．１Ｍ蔗糖＋２０％ＦＣＳ－ＰＢＳ溶液为冷冻保护液，裸露冷冻前培养１．０小时的半胚，获得了８２．４％（１４／１７）的较高存活率。试验结果表明，半胚冷冻前培养时间及不同保护液对奶牛半胚裸露冷冻有显著影响。     

超排山羊卵巢卵母细胞体外成熟和体外受精的研究

以ＴＣＭ１９９＋１０ｍｍｏｌ／ＬＨＥＰＥＳ＋青霉素（６ｍｇ／Ｌ）＋链霉素（５ｍｇ／Ｌ）为基础培养液（ＢＭ），再分别加入不同成分，配成５种卵母细胞成熟液：（Ａ）ＢＭ＋１０％ＥＧＳ；（Ｂ）ＢＭ＋１０％ＥＧＳ＋ＨＣＧ（２．５ｍｇ／Ｌ）；（Ｃ）ＢＭ＋１０％ＥＧＳ＋ＨＣＧ＋Ｅ２（１ｍｇ／Ｌ）；（Ｄ）ＢＭ＋１０％ＦＣＳ＋ＨＣＧ＋Ｅ２；（Ｅ）ＢＭ＋１０％ＥＧＳ＋ＨＣＧ＋Ｅ２＋颗粒细胞（１．５×１０６～３．０×１０６个／ｍＬ）。在３８．５℃、５％ＣＯ２下培养２６ｈ，排卵后第１天卵巢卵母细胞体外成熟率分别为６７．６７％（２０／３０），６０．４２％（２９／４８），８３．６７％（４１／４９）和８０．８２％（５９／７３），排卵后第５天卵巢卵母细胞，在培液Ｄ中体外成熟率为７１．４３％（４５／６３）。排卵后第１天的９８枚卵巢卵母细胞，体外成熟体外受精卵裂率为２１．４３％，２１枚２细胞胚移植５头受体获２头羔羊。研究表明，超排山羊卵巢卵母细胞经体外成熟可获得大量廉价的成熟卵母细胞，并可通过体外受精获得试管山羊     

奶牛胚胎分割与半胚裸露冷冻保存试验

本试验研究了①Ａ、Ｂ、Ｃ三个质量等级胚胎的分割效果；②五种不同冷冻方法冷冻裸露半胚的存活情况；③冻前培养３．０、１．５～２．０、１．０小时的裸露半胚存活率。结果①Ａ、Ｂ、Ｃ级胚胎的分割成功率分别为９５．８％（９２／９６）、７且．４％（７０／９８）、３０％（６／２０），三者间差异极显著（Ｐ＜０．０１）；②半胚冷冻前以２０％ＦＣＳ－ＰＢＳ液培养３．０小时后，以五种不同冷冻方法冷冻，均未获得存活半胚；③半胚冷冻前培养３．０、１．５～２．０、１．０小时后，以添加１０％而聚糖的１０％甘油＋０．１Ｍ蔗糖／ＰＢＳ液冷冻，分别获得了０％（０／４５）、６８．８％（１１／１６）、７５．０％（９／１２）的存活率。结果表明，胚胎质量是影响胚胎分割成功率的关键因素，半胚冷冻前的培养时间是影响裸露半胚存活的重要因素。     

小鼠桑椹胚简易玻璃化冷冻技术再探讨

本试验继小鼠扩张囊胚玻璃化冷冻保存成功后，在室温（２５℃）下利用不同浓度的ＥＦＳ玻璃化溶液，对小鼠的桑椹胚简易玻璃化冷冻技术进行再探讨。结果是胚胎在１０％ＥＧ溶液中预先处理５分钟，再移入事先配置好含有ＥＦＳ３０的０．２５ｍｌ塑料细管中１分钟平衡后直接投入液氮中冷冻，解冻后获得的发育率最高（９４％）。冻胚移植后妊娠率和产仔率分别为５６％（９／１６）及４２％（４９／１１６）。与对照组相比差异不显著（Ｐ＞０．０５）     

牛体外受精胚胎细胞核移植的实验研究

将采自肉联厂屠宰车间的牛卵巢中的卵母细胞置于ＴＣＭ－１９９＋１０％ＥＣＳ（０ｄ）＋ＦＳＨ（１万ＩＵ／Ｌ）＋ＬＨ（５ｍｇ／Ｌ）中培养成熟，用含０．３％透明质酸酶的消化液将卵母细胞周围的卵丘细胞去掉，并以７．５ｍｇ／Ｌ的ＣＢ液处理后，用微吸管吸去透明带内的第一极体及极体下面的部分卵母细胞质，然后将经体外受精并发育至８～１６细胞期胚胎的卵裂球注入去核卵母细胞的卵周隙中，再将移核胚置于电融合槽内进行融合（电场强度为１２００Ｖ／ｃｍ，持续时间为８０μｓ，１次脉冲刺激）。将融合的胚胎移入生长有单层贴壁颗粒细胞的发育培养液（ＴＣＭ－１９９＋１０％牛血清）中培养，观察移核胚的融合率及发育率，部分去核卵母细胞经染色后观察去核率。结果表明：（１）移核胚的融合率为８６．９％（５３／６１）；（２）发育至２～８细胞期的胚胎占培养的移核胚胎总数的２１．３％（１０／４７）；（３）卵母细胞的去核率为６８．２％（４５／６６）。     

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）。     

绵羊冷冻胚胎移植试验

绵羊冷冻胚胎有枚浆冻前Ａ级胚胎解冻后,胚胎等级有所下降,可用胚率为７７．９％,Ａ、Ｂ级胚移植妊娠率分别为３３．３％（１８／５４）和１６．７％（１／６）,差异不显著（Ｐ〉０．０５）;桑椹胚、早期囊胚、中期囊胚、扩张囊胚移植妊娠率分别为２０．８％（５／２５）、４２．９％（６／１４）、３５．７％（５／１４）５ ３７．５％（３／８）,囊胚期胚胎移植妊娠率较高,但差异不显著（Ｐ〉０．０５）     

兔胚胎核移植操作条件的建立

本文对影响家兔胚胎细胞核移植的几个重要因素进行了研究。以不同的融合电场强度对重组胚进行处理，结果表明脉冲参数为１．６５ｋｖ／ｃｍ，脉宽为８５μｓ，脉冲数为１时，融合率达到７７．１％。显著高于其它场强的融合率。当场强增加至２．５０ｋｖ／ｃｍ时，多数重组胚发生溶解。融合液中的离子成份对重组胚的激活有重要影响。以０．３Ｍ甘露醇为融合液时，电激三次的激活率（５０％）显著高于一次（３６％）和二次（２２．２％Ｐ＜０．０５）。当加入１００μｎＣａ２＋时，电激三次后的激活率达到７１．４％，显著高于电激一次（４８％，Ｐ＜０．０１）和二次（６１．３％，Ｐ＜０．０５）。多次电激还可提高重组胚的发育率，当有Ｃａ２＋存在时，三次电激后的囊胚发育率为３１．４％。此外，随着卵母细胞卵龄的增加，重组胚的囊胚发育率下降，而融合胚的碎裂率则增高。当卵龄为ＨＣＧ后２１—２４小时，碎裂率高达５２％。对８－，１６－，３２－细胞期卵裂球的重组胚体外发育能力的研究表明，８－细胞期卵裂球的重组胚之囊胚发育率显著高于１６－及３２－细胞期卵裂的重组胚（３６．４％比２６％、２７．５％，Ｐ＜０．０５）。将部分３２－细胞期卵裂球的重组胚移入受体后，产出了３只核移植     

牛胚胎性别鉴定与取样胚胎移植应用技术的研究

本研究应用ＰＣＲ技术扩增牛ＳＲＹ序列进行奶牛胚胎性别鉴定。经１０９枚鲜、冻胚的移植，获鲜胚移值妊娠率５８．６％（３４／５８），常规冷冻胚胎移植妊娠率４４．４％（１２／２７），一步细管冷冻解冻胚胎移值妊娠率１６．７％（４／２４）。犊牛性别验证与ＳＲＹ鉴定结果均相符合。实验中对胚胎发育时期的划分，胚胎质量评定和胚龄的确定，胚龄与受体发情时间在移植中的关系，胚胎的切割取样，取样胚胎的冷冻进行了研究。     

道塞特、萨福克、特克塞尔三品种绵羊规模化胚胎移植应用研究报告

2003年5月和2004年6月分两批从澳大利亚引进三个绵羊品种：道塞特（Dorset）、萨福克（Suffork）和特克塞尔（Texel）共389只，选择其中116只作为供体母羊进行胚胎移植，使甩CIDR＋FSH递减法对供体超数排卵处理，其中111只供体发情，配种、采胚（反应率95．6％，111／116），平均采胚10．04枚（1114／111），可用胚平均6．23枚（691／111），可用胚率62.03％（691／1114）。将691枚2～16细胞可用胚移植给618只受体小尾寒羊，受体采用PG两次处理法同期处理。妊娠485只，妊娠率78．48％，共产羔537只，产羔率110．72％。     

受体绵羊人工授精后移植胚胎的试验

试验通过使用人工授精后绵羊作为受体进行胚胎移植,以寻找一种更加有效的方法提高绵羊胚胎移植的经济效益。试验中使用FSH对10只无角道赛特绵羊进行超数排卵处理,同时对60只受体小尾寒羊进行同期发情。供体羊在发情配种后4.5～5.0d从子宫角收集胚胎。同时,将胚胎移植到同期发情并进行人工授精的受体羊子宫内。总共有57枚可用胚移植给44只受体小尾寒羊,32只怀孕到分娩,共产下羔羊51只(无角道赛特羔28只,道赛特与小尾寒羊杂种羔23只)。此外,经人工授精但未进行手术移植的7只小尾寒羊产下15只杂种羔羊。移胚植受体妊娠率72.7%(32/44),移胚受体繁殖率118%(51/44),受体利用率88.3%(53/60)。移胚受体总妊娠率和受体利用率均显著高于常规ET组(P<0.01)。与常规胚胎移植相比,受体羊人工授精后移植胚胎不仅提高了无角道赛特母羊的繁殖率,而且提高了受体羊的利用率。     

猪微注射基因导入系统影响受体受孕率及产仔率的几个因素

在应用显微注射、胚胎移植系统技术，进行猪ＯＭＴ／ＰＧＨ基因导入的研究中，对移入受体的胚数，ＰＭＳＧ处理受体以及不同的移植方法（自体移植与异体移植）等影响受体受孕率及产仔率的因素，进行了试验分析，结果表明：（１）移入受体的注射胚数分别为１０—１９枚、２０—２９枚、３０枚以上时，其受孕率为４５．５％、６４．７％和７１．４％，产仔率为８．０％、１９．６％和１４．５％，以移入２０—２９枚效率最高；（２）用ＰＭＳＧ对受体母猪做同期发情处理，其受孕率和产仔率比选择自然发情的受体分别下降２５％和８．５％；（３）采用自体移植的方式，在移入胚数基本相同的情况下（１５枚左右），比异体移植的受孕率和产仔率分别提高１４．３％和６．４％。     

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.     

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)。     

绵羊体外受精卵冷冻解冻后的移植

以含有10％乙二醇的磷酸缓冲液对体外受精的绵羊桑椹胚及囊胚进行防冻和冷冻处理后保存于液氮中。解冻后将胚胎分别以去除防冻剂后移植和连同防冻剂直接移植两种方法,将30枚桑椹胚和19枚囊胚先后以外科手术法移植到28只受体母羊子宫内。结果共有8只受胎,其中4只足月妊娠后产羔4只(♀1,♂3),另外4只分别在移植后2～3个月妊娠中断,其原因尚不清楚。解冻后连同防冻剂直接移植后的受胎率为50％(4/8),明显高于去除防冻剂后移植的结果20％(4/20),(P<0.05)。受体的发情同步化程度不同(-1～+1日)对受胎率无明显影响。     

Use of insulin-like growth factor-I during embryo culture and treatment of recipients with gonadotropin-releasing hormone to increase pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed,lactating cows

An experiment was conducted to determine whether pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed cows could be improved by 1) culturing embryos in the presence of IGF-I and 2) treating recipients with GnRH. Lactating Holstein cows (n = 260) were synchronized using a timed ovulation protocol. Embryos were produced in vitro and cultured with or without 100 ng/mL of IGF-I. On d 7 after anticipated ovulation (d 0), a single embryo was transferred to all recipients with a palpable corpus luteum (n = 210). A subset of recipients (n = 164) was injected with either GnRH or placebo on d 11. Plasma progesterone concentrations on d 0 and 7 were used to determine the synchrony of recipients. Pregnancy was diagnosed at d 53 and 81 by rectal palpation. Among all recipients, transfer of IGF-I-treated embryos increased pregnancy rate at d 53 (P < 0.05) and tended to increase pregnancy rate at d 81 (P < 0.06). Calving rate also tended to be higher for recipients that received IGF-I-treated embryos (P < 0.07). Among the subset of synchronized recipients (n = 190), pregnancy rate at d 53 and d 81 and calving rate were higher (P < 0.05) for IGF-I-treated embryos. The GnRH tended to increase pregnancy rate at d 53 for all recipients (P < 0.08) and the subset of synchronized recipients (P < 0.10). There were no effects of GnRH (P > 0.10) for pregnancy rate at d 81 and calving rate. The overall proportion of male calves was 64.3%. There was no effect (P > 0.10) of embryo treatment or GnRH on the birth weight or sex ratio of calves. Results of this experiment indicate that treatment of embryos with IGF-I can improve pregnancy and calving rates following transfer of in vitro-produced embryos. Further research is necessary to determine whether the treatment of recipients with GnRH is a practical approach to increase pregnancy rates following in vitro embryo transfer.     

Early Pregnancy Diagnosis by Serum Progesterone and Ultrasound in Sheep Carrying Somatic Cell Nuclear Transfer-Derived Pregnancies

Early pregnancy diagnosis and monitoring play an important role following embryo transfer in sheep. The aims of the current study were to investigate (i) the pattern of serum progesterone profiles in sheep carrying somatic cell nuclear transfer (SCNT)‐derived (clone) pregnancies, and (ii) the frequency of pregnancy loss during development following SCNT embryo transfer. Sheep SCNT embryos were made using standard nuclear transfer techniques. Day 7 embryos were surgically transferred to oestrus‐synchronized recipients (n = 27). As a control, normal fertile ewes (n = 12) were bred by natural breeding. Serum was collected from all the ewes on the day of estrus (day 0 sample), 7 days post‐estrus (day 7 sample) and 19 days post‐estrus (day 19 sample) and every 10 days thereafter until lambing or pregnancy loss occurred. Serum progesterone (P4) was assessed using enzyme immunoassay. Pregnancy was confirmed by ultrasound scanning on day 35 of pregnancy followed by subsequent scanning every 10 days. In control ewes, pregnancy rate on day 35 was 83.3% (10/12), whereas in the ewes that received SCNT embryos, it was 22.2% (6/27; p < 0.05). The day 45 pregnancy rate in the control ewes was 83.3%, whereas in the SCNT embryo recipients it was 11.0% (p < 0.05). Hormone analysis revealed that SCNT embryo recipients exhibited a significantly lower P4 profiles at different time points in pregnancy compared to controls (p < 0.05). This study highlights the use of serum progesterone in combination with ultrasound for the investigation of embryo loss and crucial times during development of normal and SCNT embryos in sheep. Further, the serum P4 levels directly reflect the degree of placental development in these two groups.     

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。     

Secondary Corpora Lutea Induced by hCG Treatment Enhanced Demi‐Embryo Survival in Lactating High‐Yielding Dairy Cows

Using a novel in vivo model considering a low developmental competence embryo (demi‐embryo) and a subnormal fertility recipient (lactating high‐yielding dairy cow), this experiment evaluated the effect of human chorionic gonadotrophin (hCG) treatment at embryo transfer (ET) on embryonic size at implantation, embryonic survival and recipient plasma progesterone (P₄) and bovine pregnancy‐specific protein B (PSPB) concentrations until day 63 of pregnancy. Embryos were bisected and each pair of demi‐embryos was bilaterally transferred to recipients (n = 61) on day 7 of the oestrous cycle. At ET recipients were randomly assigned to treatment with 1500 IU hCG or to untreated controls. Higher (p < 0.01) pregnancy rates on days 25, 42 and 63, and embryo survival rate on day 63 were observed in hCG‐treated cows with secondary CL than in hCG‐treated cows without secondary CL and in untreated cows. Pregnancy rates and embryo survival rate were similar in hCG‐treated cows without secondary CL and untreated cows. Embryonic size on day 42 was not affected by treatment with hCG, presence of secondary CL and type of pregnancy (single vs twin). Presence of secondary CL increased (p < 0.05) plasma P₄ concentrations of pregnant cows on days 14, 19 and 25 but not thereafter and of non‐pregnant cows on days 14–21. Treatment with hCG and presence of secondary CL had no effect on plasma PSPB concentrations, which were higher (p < 0.05) in twin than in single pregnancies. In conclusion, secondary CL induced by hCG treatment at ET significantly increased plasma P₄ concentrations, the survival rate of demi‐embryos and the pregnancy rate of high‐yielding lactating dairy cows. Embryos were rescued beyond maternal recognition of pregnancy, but later embryonic survival, growth until implantation and placental PSPB secretion until day 63 of pregnancy were not affected by treatment or presence of secondary CL.     

Transfer of porcine embryos after 3 days of in vitro culture

Two experiments were conducted to determine the viability of porcine embryos transferred after long-term in vitro culture. In Exp. 1, four-cell embryos were kept in culture for 120 h. Embryos that were exposed to fresh culture medium every 12 h survived better than embryos kept in the same medium throughout the culture period. In Exp. 2, four- and eight-cell embryos were cultured in vitro for 72 h before transfer to estrus-induced recipient gilts. Each gilt received, on average, 19 embryos. If recipients were synchronous with donors 3/32 (9%) recipients remained pregnant with an average of 4.0 +/- .6 viable young. If the sexual cycle of the recipients was 24 h behind that of the donors the pregnancy rate was 18/34 (53%) with 4.4 +/- .5 viable young. Average embryo survival rate for the two groups was 1.8 and 12.5%, respectively. A 24-hourly medium replacement during the in vitro culture period had no significant effect on transfer results. When transferring freshly collected blastocysts, pregnancy rate, number of viable young and survival rate of embryos were 6/10 (60%), 7.8 +/- 1.4, and 23.9% for synchronous recipients and 7/10 (70%), 9.3 +/- 1.8, and 32.9% for asynchronous recipients, respectively. Recipients with very high plasma progesterone levels or numerous follicular cysts at the time of transfer were less likely to remain pregnant than others.