Transfer of pig embryos to different uterine sites.

Embryo transfer in pigs normally involves surgery. In connection with the development of nonsurgical or endoscopic transfer techniques, it is important to know whether the uterine site to which embryos are transferred has an effect on the success rate. In the present investigation, prepubertal donor gilts were treated with 1,500 IU of PMSG and, 72 h later, with 500 IU of hCG. Gilts were artificially inseminated 24 and 36 h after hCG injection. Embryos at the expanded blastocyst stage were collected from donor gilts. Recipient gilts were treated synchronous with the donors, using 1,000 IU of PMSG followed, 72 h later, with 500 IU of hCG. After a maximum of 3 h in vitro, embryos (n = 15 to 20, mean = 17.3) were transferred surgically to the middle of the uterine horn, to the caudal quarter of the uterine horn, or to the uterine body. Recipients were slaughtered between 28 and 34 d after transfer. The pregnancy rate of the recipients was low when the embryos were deposited in the uterine body (12%), compared with the middle (88%) or the caudal quarter of the uterine horn (81%) (P < .01). The corresponding average number of viable fetuses per pregnant recipient was 8.2 in the uterine body, 5.6 in the middle, and 4.5 in the caudal quarter. Average survival rate of embryos after transfer to the middle of the uterine horn was 41% vs 29 and 3% after transfer to the caudal quarter or the uterine body, respectively (P < .01). Hence, the uterine body seems to be an unsuitable site for embryo transfer in pigs. These results may explain the unsatisfactory results achieved with nonsurgical embryo transfer in the past.     

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。     

切割取样对胚胎发育的影响

体视显微镜下,采用徒手持金属刀片和自制的玻璃切割针分别对小鼠和牛胚胎进行切割取样,并对取样后的胚胎进行体外培养48h,其发育率分别为76.7%(46/60)和80%(16/20),两者差异不显著(P>0.05);奶牛新鲜胚胎切割取样后,胚胎移植妊娠率47.1%(8/17),比对照组妊娠率59.0%(23/39)差异显著(P<0.05)。冷冻-解冻胚胎切割取样后移植妊娠率42.9%(6/17)虽然也比对照组50%低,但是差异不显著(P>0.05)。     

Ultrasonographic monitoring of 103 recipient mares of different reproductive status during the first 30 days after embryo transfers

Ten pluriparous mares were used as donors to supply embryos which were transferred into 103 recipients, 31 of which were nulliparous, 34 were pluriparous and lactating, and 38 were pluriparous and non-lactating. The embryos were recovered eight days after ovulation and pregnancy was confirmed by ultrasound six days after the transfer; the length of the embryos was measured ultrasonographically on days 12, 14, 16, 18, 20, 25 and 30 after the embryo transfer. One hundred and fifteen of 200 flushes provided embryos, 12 being degenerate and 103 being viable embryos. From the 103 embryo transfers carried out, 51 pregnancies were confirmed by ultrasound within 30 days; 16 of the 31 nulliparous recipients became pregnant, 16 of the 34 pluriparous lactating recipients and 19 of the 38 pluriparous non-lactating recipients. There were no significant differences between the groups of mares in the mean (sd) rate of growth of their embryos between 12 and 30 days of gestation.     

Evaluation of the uterine environment and embryos of prepubertal gilts

A series of three experiments was conducted to test the functional status of the uterus and embryos in prepubertal gilts. In Exp. 1, gilts were induced to ovulate by treating with gonadotropins followed by hCG 72 or 96 h later, and were artificially inseminated 24 h after hCG. Five of the 10 gilts treated at 120 d of age, but none of the gilts treated at 100 of age, maintained pregnancies. We next tested the function of the uterine environment by transferring embryos from postpubertal females into gilts of various ages that had been induced to ovulate but not inseminated (Exp. 2). Pregnancy rate at d 50 of gestation was 44% (4/9) for 100-d-old recipients, 67% (2/3) for 140-d-old recipients, and 60% (3/5) for postpubertal recipients (P > 0.20). Therefore, uteri of 100-d-old gilts are able to maintain pregnancies with conceptuses from postpubertal gilts. In Exp. 3, embryos from 100-d-old and postpubertal gilts were transferred into postpubertal recipients. Uterine horns of recipients were surgically separated before transfer, and embryos from 100-d-old and post-pubertal females were transferred to opposite horns of some recipients (experimental). Other recipients received embryos from postpubertal females in both uterine horns (control). When examined on d 50 to 60 of gestation, three of five control gilts were pregnant and three of seven experimental gilts were pregnant (P > 0.50). In experimental recipients, the survival of embryos from 100-d-old gilts was 38% (8/21) compared to 57% (15/26) for embryos from postpubertal gilts (P > 0.30). Because all uterine horns of pregnant recipients contained fetuses, these results support the hypothesis that embryos from 100-d-old gilts are able to initiate and maintain pregnancies in the uteri of postpubertal gilts. Therefore, the uterine environment of 100-d-old gilts provides an environment that supports development of embryos produced by postpubertal gilts, and the embryos produced by 100-d-old gilts can survive and develop in the uteri of postpubertal gilts. It was only the combination of embryos and uteri of 100-d-old gilts that did not permit pregnancy to be maintained.     

季节对杜泊羊体内胚胎生产与移植效果的影响

为了研究季节对杜泊羊体内胚胎生产效率及胚胎移植妊娠率的影响,于2016年选取内蒙古赛诺草原羊业有限公司种羊场经产纯种杜泊母羊作为供体,采用同期发情、超数排卵及腹腔镜人工输精等方法对供体羊进行处理后,利用手术冲胚的方法获得纯种杜泊羊胚胎,同时对受体羊进行同期发情处理和胚胎移植。经统计分析,2016年全年该公司共有放栓供体4 241只,超排处理4 180只,配种供体4 131只,冲胚供体3 987只,供体可用率为94.01%;冲出胚胎总数为23 516枚,可用胚胎18 002枚,胚胎可利用率为76.55%,平均每只供体能获得可用胚胎4.52枚。供体在10月、11月、12月冲胚所获得的平均冲胚数和平均可用胚数明显高于4月、5月、6月(P<0.05),其中10月的平均冲胚数和平均可用胚数最高(P<0.01),分别为7.04枚和5.62枚。将获得的部分胚胎用于胚胎移植,移植单胚怀孕受体数为4 446只,妊娠率为53.67%;移植双胚怀孕受体数805只,妊娠率65.02%。此外,受体在1月、3月、6月、12月受胎率较其他月份高(P<0.05)。该研究可为杜泊种羊超数排卵、胚胎移植和肉羊的产业化生产提供一定的数据参考。     

The transfer of fresh and frozen embryos in an elite swamp buffalo herd

To investigate the development of fresh and frozen swamp buffalo embryos after transfer to synchronized recipients, 14 fresh embryos and 28 frozen embryos, collected from Thai swamp buffalo cows of an elite herd at the Surin Breeding Center, were transferred nonsurgically to 31 synchronized recipients buffalo cows. One fresh embryo was transferred to each of 14 recipients. Twenty eight frozen embryos were transferred to 17 recipients of which 7 cows received I embryo, 9 cows received 2 embryos and 1 cow received 3 embryos. Pregnancy was diagnosed by real time B-mode ultrasonography one month after transfer and confirmed by rectal palpation one and two months later. The pregnant cows were kept under observation until calving. The results of fresh embryo transfer showed that 5/14 (35.7%) were pregnant after 30 days, 4/14 (28.6%) remained pregnant until the 3rd month and 2/14 (14.3%) calved. With the frozen embryos, only one cow which received three embryos became pregnant and remained so for 3 months although the embryo did not survive to full term. The overall pregnancy rate using frozen embryos was 5.9% (1/17). The study demonstrated the possibility of performing embryo transfer in elite buffalo herds for genetic improvement, however the use of frozen embryos needed further investigation.     

Practical Experience with the Treatment of Recipient Mares with a Non‐Steroidal Anti‐Inflammatory Drug in an Equine Embryo Transfer Programme

As part of a commercial embryo transfer programme, 20 embryos were transferred to spontaneously synchronous or synchronized recipient mares. In 14 cases, embryo recipients were treated with non‐steroidal anti‐inflammatory drugs (NSAID), receiving flunixin meglumine i.v. at the time of transfer and vedaprofen orally twice a day on the 3 days after embryo transfer, while six embryos were transferred to untreated mares that served as controls. Out of the 14 recipient mares treated with NSAID, 11 (79%) were pregnant at 6–8 days after transfer and in 10 mares, the pregnancy was continued. From the six untreated recipients, only one became pregnant but underwent early embryonic death between day 14 and 35 after ovulation. In conclusion, pregnancy rate in NSAID‐treated recipients is higher than that in untreated recipients and above reported average values, indicating that treatment of recipient mares with NSAID helps to increase pregnancy rates after transcervical transfer and can be recommended for equine embryo transfer.     

Non-surgical transfer of bovine embryos

Bovine embryos obtained from donors six to nine days after oestrus were transferred non-surgically at a rate of one per recipient using a sterile insemination instrument, protected from contamination by the vagina with a plastic sheath. The percentage of recipients pregnant increased with the age of embryo transferred and for day 6 and 7 embryos was 33% compared to 58% for day 9 and 8 embryos. This difference approached statistical significance. Bacterial contamination of the instrument on withdrawal after transfer was not related to the success or failure of pregnancy. Maintenance of pregnancy to term and calving appeared to be normal. It is suggested that this method could be used for the routine transfer of eight and nine day embryos.     

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％。     

Effect of serum-free co-culture and synchrony of recipients on development of cultured sheep embryos to fetuses

The percentage of sheep embryos that continued to develop after collection and immediate transfer on d 2 after estrus was similar when phosphate-buffered saline with 10% fetal calf serum (PBSFCS, 45%), physiological saline (50%), or tissue culture medium 199 supplemented with 10% fetal calf serum (M199FCS, 47%) was used to flush embryos from oviducts. Co-culture of sheep embryos for 3 d with oviductal cells tended (P = .1) to reduce the percentage of embryos that developed to fetuses after transfer compared with those embryos transferred immediately. Tissue culture medium 199 supplemented with .3% BSA (M199BSA) was an adequate substitute for M199FCS for culture of sheep oviductal cells if tissue culture wells were pretreated with fibronectin. Estradiol in concentrations from 10 to 1,000 pg/ml and progesterone at concentrations of 1 or 10 ng/ml in M199BSA failed to stimulate embryo development during 3 d of co-culture beyond that seen in co-culture with M199FCS or M199BSA without added steroid. Transfer of sheep embyros co-cultured for 3 d in M199BSA or M199FCS to recipients synchronized with donors resulted in about 19% of the embryos developing to fetuses, whereas transfer to recipients that were in estrus 24 h after donors resulted in 33% of embryos developing to fetuses. The significant (P less than .05) improvement for delayed recipients may reflect the relatively lesser developmental rate of co-cultured embryos compared with that of embryos in vivo. Embryo development into fetuses was similar after co-culture in M199FCS or M199BSA co-cultures; therefore, serum is not required for the co-culture of sheep embryos.     

和牛超数排卵及胚胎移植效果观察

为了研究和牛超数排卵和胚胎移植的效果,试验以和牛作供体,荷斯坦青年母牛作受体,采用PG+FSH方案对7头和牛供体进行超数排卵处理,用非手术法取胚并进行鲜胚移植。结果表明：采用PG+FSH方案头均获得胚胎数为(11.29±4.86)枚,可用胚胎(5.86±3.27)枚(A级和B级);鲜胚移植(A级和B级)妊娠率为51.22%。说明采用该方案对和牛供体进行超数排卵,并进行鲜胚移植的效果较为理想。     

Donor and recipient rat strains affect full-term development of one-cell zygotes cultured to morulae/blastocysts

The present study was conducted to examine the developmental potential to offspring of rat embryos cultured from 1-cell to morula/blastocyst stage. Pronuclear zygotes from Wistar x Wistar or (SD x DA) x Wistar strains were cultured in modified rat 1-cell embryo culture medium (mR1ECM) for 96 h in 5% CO(2) in air at 37 C. The proportion of the 3-way cross hybrid zygotes developing into morula/blastocyst stage (74%) was higher than that of the Wistar zygotes (66%). Day-5 morulae/blastocysts developed in vitro were transferred into Day-3 or -4 pseudopregnant recipients of Wistar or SD x DA strain. The transfer of cultured embryos resulted in the birth of offspring at 13-59%, while that of non-cultured control blastocysts showed birth rates of 35-65%. The best offspring rate of cultured embryos (59%) was obtained when the hybrid 1-cell zygotes were cultured in mR1ECM medium and transferred into the 2-days earlier uteri of SD x DA recipients. These results suggest that genetic background of recipients as well as donors is a possible factor affecting full-term development of rat morulae/blastocysts derived from 1-cell stage zygotes cultured in vitro.     

波尔山羊胚胎移植影响因素的分析

波尔山羊胚胎移植效果受诸多因素的影响，供体状况、药物、季节等因素使供体超排效果产生较大的差异，而受体状况、胚胎质量等对受体的妊娠率产生较大的影响，这些因素阻碍了波尔山羊胚胎移植的产业化应用。本文对以上影响波尔山羊胚胎移植效果的因素进行了分析，以期为提高胚胎移植效率提供依据，从而促进波尔山羊胚胎移植产业化进程。     

Embryo transfer from cattle infected with bluetongue virus

Embryos recovered nonsurgically from donor cattle during the peak of bluetongue viremia were surgically transferred to seronegative recipients 7 to 8 or 10 to 11 days after the onset of donor estrus. Virus was isolated from the uterine flushing medium recovered from 11 of the 20 donors. Bluetongue virus was not isolated from the blood of any of 39 recipients, nor did any recipient seroconvert to the virus following transfer. The number of recipients that became pregnant after transfer of embryos from infected donors (21 of 39) was not significantly different from contemporary controls. Virus antigen was not detected by immunofluorescence in any of 63 embryos and oocytes recovered from viremic donors. These results indicate that under standard embryo transfer conditions, transmission of bluetongue virus from infected donors to uninfected recipients is unlikely to occur.     

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。     

Propagation of elite rescue dogs by somatic cell nuclear transfer

The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6‐dimethylaminopyridine (DMAP) (SOF‐DMAP) or porcine zygote medium (PZM‐5) with 1.9 mmol/L DMAP (PZM‐DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM‐DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF‐DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6‐DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies.     

波尔山羊胚胎数量、质量与受体移植妊娠率的关系

本文就波尔山羊胚胎数量，质量与受体移植妊娠率的关系进行了探讨。结果表明：胚胎质量优劣是直接影响胚胎移植成功率的关键因素。移植胚胎数量因品种不同有差异。移植双胚，奶山羊受体胚胎成羔率53．4％，明显高于成都麻羊胚胎成羔率36．3％，成都麻羊单胚成羔率为66．7％。     

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.     

胚胎移植试验羊群发情规律的研究

为了有效提高绵羊体细胞克隆和转基因等研究工作的效率,缩短胚胎移植试验羊群中空怀个体的利用周期间隔,试验对采用不同移植方法的胚胎移植受体及供体绵羊进行了移植前后的发情规律研究。结果表明:采用自制孕激素海绵栓和进口阴道孕激素释放装置(CIDR)对绵羊进行同期发情处理时发情率均接近90%,且92%以上的个体发情起始时间均集中在撤栓后24～48 h。在胚胎移植试验结束后,未参加胚胎移植个体和子宫角法未移植受体第2情期发情起始时间主要集中在上次发情后的14～20 d,而输卵管法未移植受体和输卵管法冲卵供体的发情时间则比较分散,呈现明显延后状态。未参加胚胎移植个体和子宫角法未移植受体人工授精后的受胎率接近往年羊场的平均水平,输卵管法未移植受体的受胎率略有下降,且输卵管法冲卵供体的受胎率明显降低。