山羊早期胚胎发育的初步研究

本实验以黑龙江地方山羊为材料,经FSH超数排卵后,在不同时间屠宰母山羊,并冲洗输卵管及子宫,获取新鲜卵及各发育时间的胚胎。实验中发现山羊的排卵时间为发情开始后约30小时。受精卵的第一次卵裂的发生在排卵24小时以后。2细胞、4细胞、8细胞、16细胞、桑椹及胚泡期胚胎所处的时间分别为排卵后约32～42、48～52、62、72、96小时以及7～8天。16细胞期以前的胚胎移行于输卵管中,桑椹胚及胚泡则移行于子宫中。在每一时间从每只羊中所收集的胚胎基本上处于几个相邻的发育时期。在桑椹胚的动物极有明显的突起,这可能是内细胞群已开始形成的标志。到胚泡期时,胚胎体积增大,透明带变薄。正在孵化或已经孵化的胚泡中,内细胞群外端无滋养层细胞包围。     

Relationship between plasminogen activator production and bovine embryo development in vitro

The relationship of plasminogen activator (PA) production to cell stage, cell number and changes in overall diameter and zona pellucida thickness for bovine embryos developing in vitro was determined. Late morulae to blastocysts (n = 80) were collected nonsurgically from naturally mated, estrous-synchronized, superovulated crossbred beef cows. Embryos were cultured, one embryo per 25-microliters microdrop, for 6 d. At 24-h intervals, embryos were evaluated for stage of development and transferred to fresh microdrops; media were recovered for PA analysis. In addition, embryo diameter and zona pellucida thickness were measured with an ocular micrometer. Plasminogen activator production was determined using a caseinolytic assay with urokinase as the standard. Changes in diameter, zona pellucida thickness and PA production per 24-h interval for each embryo were plotted, and the graphs were cut out and weighed. Sixty-one embryos (76%) completed the hatching process. Total PA production was correlated positively (P less than .005) to embryonic size (r = .40), developmental stage (r = .35) and cell number (r = .35) and negatively, but weakly, correlated to zona pellucida thickness (r = -.13; P = .267). Hatched embryos produced more total PA than embryos that did not hatch (.140 +/- .011 vs .070 +/- .019 g; P less than .01). These results suggest that as embryonic size and cell number increase and development progresses, bovine embryos liberate more PA.     

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.     

利用IVM/IVF技术生产试管犊牛的研究

本研究利用卵母细胞体外成熟(IVM)、体外受精(IVF)技术,体外生产牛胚胎,以获得试管牛.结果表明:卵母细胞体外培养22～24h后,其成熟率为85％;体外受精率为83％.体外受精卵分别在颗粒细胞单层(GCM)和输卵管细胞单层(OCM)上培养,其胚胎最后产量(以授精时卵母细胞的数目为基数)分别为19％和29％,差异极显著(P<0.01).若体外受精卵先在GCM中培养72h后,再将已发生卵裂的(>4细胞)胚胎移到OCM中培养,其胚胎最后产量为35％.由此表明,早期胚胎在其发育过程中至少存在着3个发育相期,即1～8细胞、8～16细胞和16细胞～囊胚3个阶段,各阶段需要不同的培养环境.IVM/IVF胚胎移植到受体黄牛体内后,其足月时的妊娠率为15％.第一头试管犊牛已于1993年4月18日凌晨产出.     

Interaction of bluetongue virus with preimplantation embryos from mice and cattle

Preimplantation embryos from mice and cattle were exposed to bluetongue virus in vitro to determine whether the virus would replicate in these early embryos and, if so, what pathologic consequences would ensue. A high proportion of zona pellucida-free, 2-cell embryos and morulae from mice, and morulae from cattle became infected. The infection was rapidly cytopathic in embryos from both species. Indirect immunofluorescence was used to demonstrate accumulation of virus antigen in the blastomeres of these embryos. The zona pellucida of both murine and bovine embryos provided effective protection from virus present in culture fluid.     

腺病毒介导的高效生产水牛转基因胚胎条件的摸索

试验旨在探究腺病毒感染水牛颗粒细胞和胚胎的最佳条件,以提高转基因水牛胚胎的生产效率。以水牛颗粒细胞和体外发育的胚胎为研究对象,分别用0、1×10~0、1×10^-1、1×10^-2、1×10^-3、1×10^-4、1×10^-5 GFU/mL腺病毒感染水牛颗粒细胞,获得最佳感染浓度后,在最佳浓度条件下分别感染24、48、72、96 h,摸索最佳感染时间,用倒置荧光显微镜观察试验结果。利用腺病毒感染水牛颗粒细胞的最佳浓度和时间分别感染2细胞和4细胞期胚胎,对胚胎的最佳感染条件进行摸索,分析胚胎分裂率、囊胚率和转染囊胚率。结果显示,1×10^-2 GFU/mL浓度和48 h感染时间可获得水牛颗粒细胞的最佳腺病毒感染效率。腺病毒感染2细胞和4细胞期无透明带和非完整透明带胚胎后胚胎发绿光,而感染完整透明带胚胎后不发光,2细胞期胚胎感染后停止发育,4细胞期开始感染的非完整透明带胚胎可继续发育至囊胚。于4细胞期分别感染完整透明带组、非完整透明带组和无透明带组水牛胚胎,结果显示,非完整透明带转染组在囊胚率和囊胚转染效率上均优于其他组。综上,非完整透明带,1×10^-2 GFU/mL和48 h为感染浓度和时间,4细胞期为感染起始期的腺病毒介导的水牛转基因胚胎生产方法能够实现目标基因在水牛胚胎中的高效表达,从而达到提高转基因水牛胚胎生产效率的目的。     

Susceptibility of bovine naked 2- and 4-cell embryos and hatched blastocysts produced in vitro to infection with noncytopathogenic bovine viral diarrhea virus.

To investigate the susceptibility of early bovine embryos to noncytopathogenic bovine viral diarrhea virus (NCP BVDV), 2- and 4-cell embryos produced in vitro from which zona pellucida had been removed by pronase treatment, and hatched blastocysts were exposed to 10(6) TCID50/m/ of NCP BVDV No. 12 strain. The virus was detected in all embryo samples immediately prior to cultivation but not in the medium. After 24-hr culture, the virus was isolated from four media and two embryo samples in four experiments in the blastocyst group, and the viral antigen was demonstrated in the cytoplasm of the embryo cells by the immunofluorescent technique. By contrast, no virus was recovered from, or viral antigen detected in samples from the 2- and 4-cell embryo group in any of the experiments, even though they were exposed to the virus after removal of the zona pellucida. These findings suggest that 2- and 4-cell embryos are unlikely to be susceptible to NCP BVDV, but that blastocysts are capable of being infected with the virus. hatched blastocyst, noncytopathogenic bovine viral diarrhea virus.     

家猫超数排卵的研究

用促卵泡激素(FSH),孕马血清促性腺激素(PMSG)和人绒毛膜促性腺激素(HCG)建立家猫有效的超数排卵方法。FSH与PMSG的超排效果相比,前者明显优于后者。FSH 50单位/只的超排效果最好,最多者可达23个胚胎。胚胎回收率为68.18％。受精后前4天在输卵管可收集到1—、2—和4—细胞胚胎,第5天以后可在于宫收集到8—、16—细胞胚胎,第6天开始收集到桑椹胚。     

波德代羊超排效果的研究

选择60只波德代羊进行超排效果研究。结果表明:两次超排所获得的胚胎总数分别为7枚和8枚,有效胚占回收总胚分别为61.90%、87.50%;对不同年龄的波德代羊超排后,所获得的总胚胎数差异不显著;开始配种后的第6~7天冲胚,能获得较多的桑椹胚,第8天冲胚时将获得较多孵出囊胚;从绵羊两侧卵巢所观察到的黄体数和所回收的胚胎数差异不显著;人工授精能显著提高超排供体母羊授精率和胚胎质量;用FSH恒量注射和PMSG两次注射法较其它方法能明显提高所获得总胚胎数和有效胚胎数。超排技术对波德代羊超排效果理想,为进一步的胚胎移植提供了大量优质胚胎。     

Transfer of Bisected Cattle Embryos within the Same Zona Pellucida*

Cattle embryos at the compact morula and blastocyst stages were bisected by two different methods. In some embryos the embryo bisection was performed outside of the zona pellucida and both halves were replaced into it. Other embryos were bisected within the zona pellucida and no pipetting of the halves was required. They were transferred to synchronized recipient cows and the pregnancy rates and twinning rates were evaluated.     

The blastocyst production rate and incidence of chromosomal abnormalities by developmental stage in in vitro produced porcine embryos

The present study was conducted to determine the relationship between embryonic development speed at different stages (the cleaved stage at 52 h and the blastocyst stage at 6 days post insemination) and incidences of chromosome abnormalities in in vitro produced porcine embryos. Porcine oocytes were collected from 3-6-mm ovarian follicles obtained at a slaughterhouse and matured in modified NCSU-37 medium for 44-46 h. Following in vitro fertilization with a final concentration of 1 x 10(5) sperm/ml for 3 h, all oocytes were cultured in vitro for 52 h. Day-2 (52 h after insemination) embryos were classified according to their cleaved stages into 2-cell, 3- to 4-cell, 5- to 8-cell, and >8-cell stages; these were cultured separately for additional 4 days (Day 6). The resultant Day-6 blastocysts were classified according to the morphological diameter into 3 grades: Grade A, expanded blastocysts; Grade B, expanding blastocysts; and Grade C, early blastocysts. They were then analyzed chromosomally. The 3- to 4-cell and 5- to 8-cell embryos had significantly high blastocyst development rates (46.1 and 36.9%, respectively), and these blastocysts contained significantly more cells (40.2 and 42.4 cells, respectively) than those derived from 2-cell embryos and >8-cell embryos (28.6 and 26.5 cells, respectively). The incidence of chromosomal abnormalities was significantly higher in the blastocysts derived from 2-cell and >8-cell stage embryos than in the blastocysts derived from the other stage embryos. Furthermore, the grade A blastocysts had the lowest incidence of chromosomal abnormalities (35.3%) and contained the most cells (48.7 cells). Porcine in vitro production (IVP) yielded a high blastocyst rate and an excellent embryo quality when 3- to 4-cell and 5- to 8-cell stage embryos were selected on Day 2 after insemination. The same criteria yielded a higher quality of expanded blastocysts based on the stage of embryo development and morphology.     

Morphological aspects of in vivo cleavage in Myocastor coypus (coypu)

The objective of the present work was to characterize the in vivo cleavage stage of Myocastor coypus embryos. For this purpose a colpocytological follow-up and controlled mating of 18 females were performed. Specimens from the beginning of the first cleavage to the acquisition of a morula appearance were considered to be in cleavage stage. Embryos in cleavage were collected between days 3 and 6 post-coitus. Of the collected embryos, 80% presented an even number of blastomeres and the remaining 20% an odd number. Embryos from 3 to 7 cells were blastomere associations in a spherical disposition within the zona pellucida. Blastomeres were spherical or ovoid, presenting slight flattening in areas contacting with other blastomeres. Embryos of 8 and 9 cells were as a group of blastomeres slightly elongated, surrounded by a spherical zona pellucida. The percentage of peri-vitelline space occupied by the embryonic mass ranged from 74.1 to 95.8% for all the substages. The cleavage pattern, developed in the oviduct, was of a rotational holoblastic type and asynchronic.     

Transfer of canine embryos at various developmental stages recovered by hysterectomy or surgical uterine flushing

In dogs, embryo transfer (ET) techniques such as induciton of excessive ovulation and synchronization of estrus have not progressed well. Therefore, using embryos at various developmental stages, ET was investigated in dogs from a beagle colony in which the ovulation days were close, as estimated by the progesterone level. Embryos were, recovered 8-11 days after ovulation (4-9 days after mating) by excising the oviducts and uteri (excision method) in 16 animals and by surgical flushing of the uteri at laparotomy (surgical method) in 3 animals. In 24 dogs with -4 to +2 days of difference in the timing of ovulation between donor and recipient dogs, 1-10 embryos at the 8-cell to blastocyst stages were transferred per animal. The mean embryo recovery rate by the excision method (97.1%) was significantly higher than that by the surgical method (42.5%) (p<0.01). Twelve (57.1%) of 21 animals with -1 to +2 days difference in ovulation day became pregnant after the transfer of 8-cell to blastocyst stage embryos. Although 3 dogs with -4 to -2 days of difference of ovulation day underwent ET of morula or compacted morula, none of these dogs became pregnant. The mean ratio of the number of newborns to the number of transferred embryos was only 51.9%. The mean duration of the period between ovulation and delivery in the pregnant recipients was 65.8 days, which tended to be longer than that in natural mating. These results demonstrate that pregnancy can be induced by ET at the 8-cell to blastocyst stage in dogs with -1 to +2 days difference in ovulation day.     

Glycosaminoglycans improves early development of zona-free 8-cell rat embryos to blastocysts in a chemically defined medium, but not the pregnancy rate following transfer of the blastocysts

The objective of the present study was to clarify the possible role of the zona pellucida (ZP) in early development of rat embryos and to determine the effect of glycosaminoglycans on the development of ZP-free 8-cell embryos before or after embryo transfer at the blastocyst stage. Eight-cell embryos were divided into three groups comprised of, 1) intact controls, 2) embryos with the ZP was removed with acidic solution and 3) pairs of ZP-free 8-cell embryos aggregated in a small hollow. These embryos were cultured in a chemically defined mR1ECM for 24 h. Developmental ability to the blastocyst stage and mean cell number in the blastocyst was lower in ZP-free embryos than in intact controls. When these blastocysts were transferred, the farrowing rate and efficiency of embryos developed to term were also lower in ZP-free embryos, but not in the aggregated ones. Supplementation with hyaluronan (HA; 63-250 μg/ml) or heparan sulfate proteoglycan (HS; 15 μg/ml) significantly improved blastocyst formation of ZP-free embryos and the cell number in the blastocyst by reducing the incidence of apoptosis. However, there were no beneficial effects of HA or HS on farrowing and newborn rates after transfer of the blastocysts. In conclusion, the ZP plays roles in maintaining successful development of early rat embryos at least from the 8-cell stage not only to the blastocyst stage but also to posttransfer stages. Glycosaminoglycans, such as HA or HS, appear to contribute to successful cleavage during early development to the blastocyst stage but may be insufficient to maintain the posttransfer survival of ZP-free embryos.     

Effects of BSA and fetal bovine serum in culture medium on development of rat embryos

Rat 1-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (mR1ECM) or in mR1ECM supplemented with BSA (4 mg/ml; mR1ECM-BSA) or fetal bovine serum (FBS; 10%, v:v; mR1ECM-FBS) instead of polyvinylalcohol. There was no difference in percentages of embryos that developed to the 2-cell to blastocyst stages between mR1ECM and mR1ECM-BSA, but in mR1ECM-FBS, no development beyond the 2-cell stage was observed. When embryos were transferred to mR1ECM-FBS from mR1ECM after 24 to 64 h of culture, development of embryos to and beyond the 4-cell stage was inhibited. However, when transferred after 80 h of culture, more embryos developed to blastocysts and hatching or hatched blastocysts than in embryos cultured in mR1ECM. When 8-cell embryos and early morulae obtained after 72 and 80 h of culture in mR1ECM, respectively, were cultured in mR1ECM-FBS, a higher proportion of early morulae developed to the blastocyst stage than did 8-cell embryos. When morulae obtained after culture in mR1ECM or mR1ECM-BSA were transferred to recipient females, there was no difference in proportions of fetuses obtained. However, a higher proportion of blastocysts cultured in mR1ECM-FBS developed to fetuses compared with those obtained in mR1ECM. These results indicate that BSA has neither deleterious nor beneficial effects on development of rat 1-cell embryos. In contrast, FBS has deleterious effects on early cleavage of embryos but it promotes more rapid development of morulae to blastocysts, resulting in better quality blastocysts.     

Effects of heat stress on the redox status in the oviduct and early embryonic development in mice

This study examined the association between redox status in the oviduct and early embryonic death in heat-stressed mice. In Experiment 1, non-pregnant mice were heat-stressed at 35 C with 60% relative humidity for 12, 24, or 36 h, and the maternal redox status was verified by measuring the levels of reactive oxygen species (ROS) and free radical scavenging activity (FRSA) in the oviduct, and thiobarbituric acid reactive substances (TBARS) and glutathione peroxidase (GSH-Px) activity in the liver. In Experiment 2, zygotes were collected from mice heat-stressed for 12 h on the day of pregnancy, and their developmental abilities were assessed in vitro, along with the intensity of DNA damage at the 2-cell stage. The TBARS value and GSH-Px activity in the liver, and ROS level in the oviduct were significantly higher in heat-stressed mice, and this increase appeared to depend on the duration of the heat stress. Maternal heat stress significantly reduced the percentage of zygotes that developed to the morula and blastocyst and the total cell number in the blastocyst. In addition, DNA damage at the 2-cell stage was significantly higher in maternally heat-stressed embryos. These results suggest that heat stress induces systemic changes in redox status in the maternal body, and the resultant increase in oxidative stress in the oviduct is possibly involved in heat stress-induced early embryonic death .     

Improved survival of vitrified in vivo-derived porcine embryos

An efficient cryopreservation protocol for porcine morulae was investigated with three types of vitrification having different cooling rates (Exp. 1). Survival of embryos vitrified after removal of cytoplasmic lipid droplets was also examined by means of the minimum volume cooling (MVC) method (Exp. 2). In Exp. 1, the morula stage embryos were vitrified with a 0.25 ml plastic straw (ST-method), gel loading tip (GLT-method) and the MVC-method, respectively, and stored in liquid nitrogen after which they were warmed in sucrose solutions with cryoprotectants being subsequently removed in a stepwise manner. In Exp. 2, morulae were centrifuged with 7.5 microg/ml cytocharasin B at 12000 x g for 20 min to polarize the cytoplasmic lipid droplets that were then removed from the embryos by micromanipulation (delipation). Both those delipated at the morula stage and the intact embryos at the morula to blastocyst stages were vitrified by the MVC-method. In vitro survival of the vitrified embryos was assessed in both experiments by culturing in NCSU-23 + 10% FCS for 48 h. In vitro developments of vitrified embryos after warming to blastocysts were 20% (6/30) for the ST-method, 39% (18/46) for the GLT-method, and 60% (26/43) for the MVC-method. Embryo survival was further improved by vitrification after delipation (95%, 35/37) compared to intact vitrified morulae (24/42, 57%, P<0.001) and blastocysts (23/31, 74%, P<0.05). Moreover, the number of cells in blastocysts (92 +/- 25) derived from the delipated-vitrified morulae was comparable to those derived from intact control non-vitrified embryos (103 +/- 31). Our results demonstrate that vitrified porcine morulae have the highest survival when using the MVC-method in conjunction with delipation.     

Oviduct epithelial cell co-culture of early porcine embryos.

One- to 16-cell porcine embryos were cultured in either Whittens medium supplemented with bovine serum albumin and fetal calf serum (WM) or in the same medium with porcine oviduct epithelial cell co-culture (WM-Poec). All stages of embryos cultured in WM-POEC had higher cell counts after 144-168 h of development than did embryos in WM. There was however, no significant difference in blastocyst formation rate of embryos cultured in WM-POEC over those cultured in WM. A high proportion of the embryos entering culture at the 1-2-cell were able to pass the 4-cell block stage in both WM and WM-POEC, 81% and 77%, respectively. In both media, most of the 1-2-cell embryos arrested their development at the compacted morula stage and failed to blastulate while embryos initiating culture at the 4- and 8-16-cell embryos formed blastocysts in culture at a rate of 80-90%.     

Apoptosis-independent Poor Morphology of Bovine Embryos Produced by Multiple Ovulation

In multiple ovulation and embryo transfer (MOET) programmes in cattle, a considerable number of morphologically poor-quality embryos continue to be produced; this is one of the limiting factors of the technique. Apoptosis has often been implicated in developmental arrest and fragmentation; these are regarded as poor traits of embryonic quality in mammalian pre-implantation embryos. In the present study, apoptosis was assessed in morphologically poor-quality embryos in comparison with good-quality embryos that were recovered from a MOET programme. Retarded embryos (two to 16 cell stage), morulae with severe fragmentation and morphologically good-quality morulae recovered from superstimulated cows at day 7 post-insemination were subjected to TdT-mediated dUTP nick-end labelling (TUNEL) and Hoechst staining. Cell nuclei that showed both TUNEL staining and apoptotic morphology were considered to be apoptotic. Apoptotic index (AI) was calculated as the percentage of apoptotic cells per embryo. Fifteen of 17 retarded embryos and 10 of 15 morphologically poor-quality morulae did not show signs of apoptosis. The mean AIs in the morphologically poor-quality embryos (two to 16 cell stage, 2.2%; poor morulae, 1.3%) were as low as that in the good-quality embryos (2.9%). These results suggest that another mode of developmental arrest and/or fragmentation that is independent of apoptosis occurs in morphologically poor-quality embryos recovered from MOET programmes.     

Bovine oocytes grown in serum-free medium acquire fertilization competence

We previously found that bovine oocytes 90-99 microm in diameter in early antral follicles grew to nearly their final size in serum-free medium, with some of the oocytes acquiring the nuclear competence to reach the second metaphase. In the present study, we examined the competence of the fertilization and pre-implantational development of the oocytes grown in serum-free medium. Bovine early antral follicles, 0.4-0.7 mm in diameter, were collected mechanically using fine forceps, embedded in collagen gels, and cultured in serum-free medium for 16 days. Grown oocytes which were enclosed by granulosa cells and did not show disintegrated ooplasm were recovered as normal oocytes, were transferred to the maturation medium, and then inseminated with spermatozoa. Ten to 12 h after insemination, 28% (41/145) of the oocytes were penetrated by spermatozoa. Of the penetrated oocytes, 18 (12%) formed a female and a male pronuclei, and 10 (7%) had a female pronucleus and an enlarged sperm head. Among the abnormally penetrated oocytes (13/41), 10 were penetrated by multiple spermatozoa and 3 were penetrated by a spermatozoon at the first metaphase stage. Of the 106 inseminated oocytes grown under serum-free conditions, 8 oocytes had cleaved and developed to the 2-cell stage 48 h after insemination, and 3-4-cell embryos and 5-8-cell embryos were observed after 72-96 h. However, no embryo developed to the blastocyst stage within 8 days. These results indicate that bovine oocytes grown in serum-free medium can be fertilized, but acquire insufficient embryonic development competence under the employed culture conditions.