体外模拟人体细胞癌变的进展

癌症的发生是一个多基因决定和多阶段演进的过程 ,首先是某些基因发生突变并不断积累 ,引起细胞分化生长失控。然而这些突变必须克服机体设置的细胞增殖障碍、应激产生的染色体基因修补机制以及多种抑癌基因的作用。在染色体受到损伤时 ,这些抑癌因子会激活表达 ,调控基因转录以抑制肿瘤生长 ,所以只有当排除了抑癌因子的多重作用后 ,一个正常细胞才能逐渐突破防线而转化成为一个肿瘤细胞[1] 。经过多年研究 ,科学家虽已搞清了主导肿瘤转化的相关基因及其在转化过程中所起的作用 ,但能否根据这些已经了解的细胞转化机制 ,在体外模拟肿瘤的发…     

纳豆激酶粗提液的体外溶栓抑菌实验

目的探讨纳豆激酶粗提液的体外溶栓、抑菌作用.方法以大豆为培养基,米曲霉为菌种,发酵制得成熟纳豆,用不同饱和度(NH4)2SO4对粗提液进行盐析,所得沉淀溶于生理盐水中,用纤维蛋白平板法测定其活性,确定提取纳豆激酶的分级沉淀范围.用体外溶栓法测纳豆激酶粗提液的体外溶栓作用.用平板打孔法及纸片扩散法测不同浓度纳豆抑菌物质的体外抑大肠杆菌作用.结果纳豆激酶的(NH4)2SO4分级沉淀范围选择在60%.纳豆激酶粗提液对血块的溶解作用较同样活力大小的尿激酶强.纳豆抑菌物质粗提液对大肠杆菌都具有一定的体外抑制作用,当浓度大于50%,抑菌圈直径随着浓度的增高而增大.结论纳豆激酶粗提液具有较好的体外溶栓作用,并对大肠杆菌具有一定的抑制作用.     

家猫的胚胎工程

家猫是惟一一种没有被列为珍稀或濒危的猫科动物。通过家猫的胚胎工程研究,对保护其它濒危猫科物种有重要的借鉴意义。本文描述了家猫的一般生殖特点,着床前的胚胎在体内的发育概况;综述了近年来对家猫的超数排卵,卵母细胞的体外成熟,体外受精,胚胎的体外培养,胚胎移植,冷冻保存和胚胎克隆等方面的研究进展。     

山羊体外受精的研究

通过在山羊卵母细胞体外成熟培养液中添加不同的血清和不同浓度的卵泡液 ,在体外成熟培养液中培养不同的时间 ,以及采用不同的精子获能方法来摸索效率较高的体外受精方法体系。在山羊卵母细胞体外成熟培养液中添加FCS、EGS及不同浓度的卵泡液 ,成熟培养时间分别为 16、2 0、2 4和 2 7h ,山羊新鲜精液用钙离子载体法和肝素法进行获能处理后用于体外受精 ,比较其体外成熟率和体外受精率。添加FCS、EGS和 2 0 %卵泡液组的成熟率无显著差异 ,显著高于添加 10 %和 3 0 %卵泡液组的成熟率 ;但添加FCS和EGS组的受精率显著高于添加10 %、2 0 %、3 0 %卵泡液组的受精率。培养 2 4h组和 2 7h组的成熟率显著高于另外两组 ,而培养 2 7h组的受精率显著高于其余各组。用钙离子载体法处理的山羊精子的顶体反应率和体外受精率显著高于肝素法。EGS可以代替FCS添加于成熟培养液中 ,对COCs的成熟率和受精率没有明显的影响 ,但卵泡液不能完全代替FCS的作用。培养时间为 2 7h ,精子用钙离子载体法处理获能后能得到较高的体外受精率     

体外进化方法的原理与应用

体外进化方法的原理与应用王琛金由辛（中国科学院上海生物化学研究所分子生物学国家重点实验室,上海２０００３１）关键词体外进化方法在核酸研究早期,ＤＮＡ和ＲＮＡ的信息贮存和传递功能备受关注,而对它们的高级结构所发挥的生物功能却鲜有问津。Ｒｉ－ｂｏｚｙｍｅ...     

体外培养的小鼠骨髓肥大细胞的初步研究

肥大细胞是一种广泛存在于哺乳类动物体内的细胞。它分布于机体各个系统而非局限于某个组织,同时胞浆中又含有可合成、贮存和释放多种活性介质的颗粒,因而在功能上具有相当的重要性。肥大细胞在变态反应、寄生虫感染等过程中的作用已得到了较充分的认识。近10年来肥大细胞的研究更取得了显著的     

卵泡内环境对猪卵泡卵体外成熟和发育的影响

研究卵泡内环境对猪卵母细胞体外成熟、受精及受精卵体外发育的影响。主要结果如下：直径≥5mm、4－4.9mm、3－3.9mm和2－2.9mm的卵泡卵母细胞体外成熟率分别为90.5%、89.7%、85.4%和67.4%,体外受精后,卵母细胞的发育能力随卵泡直径的增大而增强,直径≥5mm和4－4.9mm卵泡卵的2－细胞、3－4－细胞发育率显著高于直径2－2.9mm的卵泡卵（P＜0.05或0.01）。体外成熟培养36h、42h和48h,直径2－2.9mm卵泡卵的体外成熟率,体外受精后的卵裂率差异不显著（P＞0.05）。在体外成熟培养液中添加5％或15％的不同直径卵泡的卵泡液,各组间卵母细胞的体外成熟率,受精卵的体外发育率均无显著差异,结果表明：卵泡大小对猪卵母细胞体外成熟、受精及受精卵体外发育有重要影响。     

Vitrification of rat embryos at various developmental stages

The effect of developmental stage on the survival of cryopreserved rat embryos was examined. Wistar rat embryos at various developmental stages were vitrified by a 1-step method with EFS40, an ethylene glycol-based solution, or by a 2-step method with EFS20 and EFS40. After warming, the survival of the embryos was assessed by their morphology, their ability to develop to blastocysts (or expanded blastocysts for blastocysts) in culture, or their ability to develop to term after transfer. Most (91-100%) of the embryos recovered after vitrification were morphologically normal in all developmental stages. However, the developmental ability of 1-cell embryos was quite low; exposing them to EFS40 for just 0.5 min decreased the in vitro survival rate from 76 to 9%. The survival rates of 2-cell embryos and blastocysts, both in vitro and in vivo, were significantly higher with a 2-step vitrification process than with a 1-step vitrification process. Very high in vitro survival rates (94-100%) were obtained in 4- to 8-cell embryos and morulae in the 1-step method. Although survival rates in vivo of 4-cell (40%) and 8-cell (4%) embryos vitrified by the 1-step method were comparatively low, the values were similar to those obtained in non-vitrified fresh embryos. When morulae vitrified by the 1-step method were transferred to recipients, the in vivo survival rate (61%) was high, and not significantly different from that of fresh embryos (70%). These results show that rat embryos at the 2-cell to blastocyst stages can be vitrified with EFS40, and that the morula stage is the most feasible stage for embryo cryopreservation in this species.     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.     

Vitrification of in vivo and in vitro produced ovine blastocysts.

Although cryopreservation of bovine embryo has made great progress in recent years, little achievement was obtained in ovine embryo freezing, especially in vitro produced embryos. However, a simple and efficient method for cryopreservation of sheep embryos will be important for application of ovine embryonic techniques such as in vitro fertilization, transgenic, cloning and etc. In this study ovine blastocysts, produced in vivo or in vitro, were cryopreserved by vitrification in EFS40 (40% ethylene glycol (EG), 18% ficoll and 0.5 M sucrose) or GFS40 (40% glycerol (GL), 18% ficoll and 0.5 Mol sucrose). In vitro produced, early blastocysts were directly plunged into liquid nitrogen (LN2) after preparation by one of the following procedures at 25 degrees C: (A) equilibration in EFS40 for 1 min; (B) equilibration in EFS40 for 2 min; (C) equilibration in EFS40 for 30 s following pretreatment in 10% EG for 5 min; (D) equilibration in EFS40 for 30 s following pretreatment in EFS20 for 2 min (E) equilibration in GFS30 for 30 s following pretreatment in 10% GL for 5 min. The survival rates observed after thawing and in vitro culture for 12 h were A 78.0% (39/50), B 50.0% (26/52), C 93.3% (70/75), D 92.0% (46/50) and E 68.0% (34/50). Survival rates were not significantly different for treatments C and D (p>0.05), but those for groups C and D were significantly higher than for A, B and E (p<0.05). After 24 h in vitro culture, hatched blastocyst rates were A 28.0% (14/50), B 21.1% (11/52), C 49.3% (37/75), D 48.0% (24/50), E 32.0% (16/50) and control 54.0% (27/50). The hatching rates for groups A, B and E were significantly lower than the control (p<0.05) in which early IVF blastocysts were cultured in fresh SOFaaBSA medium following treatment in PBS containing 0.3% BSA for 30 min, but for groups C and D it was similar to the control (p>0.05). The freezing procedures A, B and C were used to vitrify in vivo produced, early blastocysts recovered from superovulated ewes. The survival rates of frozen-thawed in vivo embryos were A 94.7% (72/76), B 75.0% (45/60) and C 96.4% (54/56) and for group B was significantly lower than for the other two treatment groups (p<0.05). Hatched blastocyst rates were A 46.0% (35/76), B 26.6% (16/60), C 51.8% (29/56) and the control 56.7% (34/60) in which early blastocysts from superovulation were cultured in fresh SOFaaBSA medium following treatment in PBS containing 0.3% BSA for 30 min. The hatching rate for treatment B was significantly lower than for the control (p<0.05) but did not differ between groups A, C and the control (p>0.05). Frozen-thawed embryos vitrified by procedure C were transferred into synchronous recipient ewes. Pregnancy and lambing rates were similar for embryos transferred fresh or frozen/thawed for both in vivo and in vitro produced embryos. These rates did not differ between in vivo and in vitro embryos transferred fresh (p>0.05). However, for frozen-thawed embryos, both rates were significantly lower for in vitro than for in vivo produced embryos (p<0.05).     

冷冻前后的低渗应激对小鼠孵化囊胚存活率的影响

The objective of this study to evaluate the effect of hypotonic stress on developmental potential of hatched blastocysts perivitrification. Hatched mouse blastocysts were vitrified in liquid nitrogen after equilibration in 10% or 20% GL for 5 min and in GFS40 for 30 sec respectively, the survival rates were 93%-97% after the frozen-thawed embryos were cultured in vitro for 24 h. There were no statistical difference between the frozen and the fresh group (P > 0.05). In order to evaluate effects of hypotonic stress on developmental abilities, fresh hatched mouse blastocysts were respectively exposed to 1.00 x, 0.50 x, 0.30 x, 0.25 x and 0.20 x PBS for 30 min, then cultured in mKRB for 24 h, the survival rates were 98%, 99%, 92%, 92% and 50% respectively. The rate in 0.20 X PBS group was significantly lower than in other groups (P < 0.01). When frozen-thawed embryos were directly treated with different osmotic solutions, the survival rates were 88%, 72%, 58%, 11% and 0 respectively in 1.00 x, 0.50 x, 0.30 x, 0.25 x and 0.20 x PBS group. The rate in 1.00 x PBS group was significantly higher than in other groups (P < 0.05). However, when frozen-thawed embryos were first cultured in vitro for 12 h, then exposed to 1.00 x, 0.50 x, 0.30 x, 0.25 x and 0.2 x PBS, the survival rates were 98%, 94%, 82%, 58% and 26% respectively. There was no statistical difference between 1.00 x and 0.50 x PBS group (P > 0.05). Although the rate in 0.30 x, 0.25 x and 0.20 x PBS group was significantly lower than in 1.00 x group(P < 0.01), it was significantly higher than in the same treatment group without in vitro culture(P < 0.05).     

Vitrification of in vitro cultured porcine two-to-four cell embryos

The objective of this experiment was to evaluate the effect of a 5-day period of in vitro culture of two-to-four cell porcine embryos up to the blastocyst stage on their ability to survive vitrification and warming. In order to increase the cooling rate, superfine open pulled straws and Vit-Master((R)) technology were used for vitrification. Two-to-four cell embryos were collected from weaned sows (n=11) on day 2 (D0=onset of estrus). Some embryos (N=63) were vitrified within 3h after collection, warmed and cultured for 120h (Group V2). Additionally, 81 two-to-four cell embryos were cultured for 96h in order to obtain blastocysts; these were then vitrified, warmed and cultured for 24h (Group VB; N=65). The remaining two-to-four cell embryos were used as controls and thus not vitrified (control embryos; N=70) but were cultured in vitro for 120h. The V2, VB and control embryos were evaluated for their developmental progression and morphology during culture. All embryos (V2, VB and controls) were fixed on the same day of development in order to assess the total number of blastomeres. The survival and blastocyst formation rates obtained from V2 embryos were very poor (9.6+/-0.7% and 3.2+/-0.5%, respectively). The survival and hatching rates of VB embryos (75.0+/-0.69% and 33.6+/-0.13%) were lower (p<0.001) than those obtained with control embryos (89.1+/-0.8% and 47.5+/-0.12%). Hatched VB embryos had a lower (p<0.01) total cell number than hatched control embryos (70.3+/-4.5 versus 90.6+/-3.2, respectively). There was no difference between expanded VB and control blastocysts. In conclusion, blastocysts derived from in vitro culture of two-to-four cell pig embryos could be successfully vitrified using SOPS straws and Vit-Master.     

Vitrification of Mouse Embryos at Various Stages by Open-Pulled Straw (OPS) Method

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure—that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method—that is, embryos were first pretreated in 10%E+10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E+10%D for 0.5 min, exposed to EDFS30 for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.     

Vitrification of mouse embryos at various stages by open-pulled straw (OPS) method

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure-that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method-that is, embryos were first pretreated in 10%E + 10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E + 10%D for 0.5 min, exposed to EDFS30for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.     

Successful vitrification of bovine blastocysts,derived by in vitro maturation and fertilization

Bovine blastocysts were produced through maturation, fertilization, and development in vitro. For vitrification, solutions designated EFS, GFS, and PFS were prepared; these were 40% ethylene glycol, 40% glycerol, and 40% propylene glycol, respectively, diluted in modified phosphate-buffered saline (PBS) containing 30% Ficoll + 0.5 M sucrose. The embryos were exposed to the solutions in one step at room temperature, kept in the solutions for various times, vitrified in liquid nitrogen, and warmed rapidly. When the embryos were vitrified in EFS solution after 1 or 2 min exposure, the postwarming survival rate, assessed by the reexpansion of the blastocoel, was 74–77%. However, when the exposure time was extended to 3 min or longer, this rate dropped to 7–0%. This reduction was attributed to the toxicity of ethylene glycol. Of the embryos vitrified in GFS solution, 53% survived when they were cooled after 1 min exposure; as the duration of the exposure increased, the survival rate increased, reaching a peak (72%) at 4 min. The rate then decreased gradually with exposure time. In PFS solution, embryos surviving after vitrification were recovered only with 1 min exposure (33%), reflecting the high toxicity of propylene glycol. After vitrification in EFS or GFS solution, two embryos were nonsurgically transferred into each of 14 recipient animals. Of the 14 recipients, ten (71%) became pregnant; two resulted in early stillbirths, four recipients delivered twins (four alive and four stillborn), and two delivered single live calves, demonstrating the effectiveness of this simple vitrification method for the cryopreservation of in-vitro-produced bovine blastocysts. © 1993 Wiley-Liss, Inc.     

The effect of equilibration time on survival and development rates of mouse pronuclear-stage embryos vitrified in solid surface (SSV) and convential straws: in vitro and in vivo evaluations

The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage (PN) mouse embryos. A novel vitrification technique (solid surface vitrification, SSV) was compared with a convential one in straws both for cryosurvival and obtaining progeny from cryopreserved PN mouse embryos. In the SSV method, 15-20 PN embryos were exposed to vitrification solutions for approximately 20 sec after equilibration, and then they were dropped in 2 microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. In the straws method, groups of 5-10 PN embryos were loaded in a single straw after equilibration. In experiment I, it was compared the effect of the vitrification solutions alone, without vitrification. No reduction was detected in survival, cleavage and blastocysts rates and the lowest development rate was obtained from hatched blastocyst for 20 min equilibration (24.5%). In experiment II, SSV method resulted in significantly higher survival and cleavage rates than that of in-straw vitrified 15-20 min group (87% vs. 60%, 83% vs. 67%, respectively; P < 0.05). There were no statistical differences among any of the blastocyts groups. However, there was a statistical difference in hatched blastocysts between 15 to 5, 10, and 20 min (P < 0.05). In experiment III, it was found no major effect among equilibration time periods in toxicity groups according to the mean cell number of blastocysts developed from PN embryos. But, there was a significant differences between 15 min SSV and 10 min in straw vitrified according to the mean cell number of blastocysts developed from PN embryos following vitrification (P < 0.05). The good results were obtained from 15 min equilibration group for SSV and 10 min equilibration group for straw vitrification. In the last experiment, embryo transfer after vitrification and toxicity was investigated. There were significant differences between SSV and straw just on the rate of pups born (30% and 20.5% respectively; P < 0.05). In conclusion, vitrification of PN mouse embryos by SSV can result in high rates of in vitro development to expanded and hatched blastocyst stage and in vivo development to live pups.     

Effects of extracellular matrices and growth factors on the development of isolated porcine blastomeres.

The effects of extracellular matrices and growth factors on the development of isolated blastomeres derived from intact 4-, 8-, and 16-cell porcine embryos (termed, respectively, 1/4, 1/8, and 1/16 blastomeres) were investigated in vitro and in vivo. Blastomeres were incubated in extracellular matrix components fibronectin (FIN) or swine skin gelatin (SSG)-precoated culture dishes containing either modified Krebs' Ringer Bicarbonate solution (mKRB) supplemented with 10% heat-inactivated lamb serum, or Hanks' solution supplemented with 10% heat-inactivated newborn calf serum (NBCS) or Waymouth medium supplemented with 10% NBCS or in noncoated dishes in mKRB supplemented with either insulin (10, 100, or 1,000 micrograms/ml), transferrin (10, 100, or 1,000 micrograms/ml), or cAMP (0.2 or 2.0 micrograms/ml). Cultures observed at 24-h intervals and morphological development was recorded. Blastomeres were classified into three categories according to their morphology: (1) regular blastocysts, (2) trophectodermal vesicles, or (3) no development. After 96 h, culture was determined; the overall diameter of the blastocysts was determined and the nuclei were counted. Blastomeres/blastocysts did not adhere to the bottom of the culture dishes coated with extracellular matrices. Blastocyst formation rate was highest when FIN/mKRB was used and reached 44.3%, 41.8%, and 36.5% for 1/4, 1/8, and 1/16 blastomeres, respectively. The respective blastocysts contained an average of 31.2 +/- 5.8, 58.2 +/- 8.4, and 18.5 +/- 3.5 nuclei and had an overall diameter of 250.0 +/- 10.1, 235.0 +/- 12.8, and 172.5 +/- 13.7 microns, 1/8 blastomeres displayed a better (p less than 0.05) growth rate than 1/4 and 1/16 blastomeres, and 1/8 blastomeres in FIN/mKRB grew better (p less than 0.01) when cultured in an open system than in a microdrop under oil (35.5% vs. 5.0% blastocysts). Neither cAMP nor transferrin had a significant stimulating effect on blastocyst development of 1/8 blastomeres when mKRB plus serum was used as the medium. Insulin supplementation (10 or 100 micrograms/ml) to mKRB plus lamb serum significantly (p less than 0.05) stimulated blastocyst formation rate compared with controls (58.6% and 38.9% vs. 17.7%, respectively; 32.6 +/- 2.5, 58.5 +/- 11.8, and 45.1 +/- 4.6 nuclei, respectively). Transfer of 457 blastocysts grown for 24 h in FIN/mKRB to 16 recipients gilts led to three pregnancies, and two litters were born from 1/8 blastomere-derived blastocysts following 116 days of gestation.4+ is