Suppressed development of cultured mouse and swine embryos by diethylstilbestrol

Effects of prolonged exposure to the synthetic estrogen diethylstilbestrol (DES) on in vitro development of early mouse and swine embryos were investigated. Two-cell mouse embryos cultured in Whitten's medium (WM) for 192 h were exposed to 10(-4), 10(-7) or 10(-10) M DES dissolved in 1, 10(-3) or 10(-6)% ethanol, respectively. One-cell to eight-cell swine embryos were cultured in WM for 192 h containing 10(-4) or 10(-7) M DES dissolved in 1 and 10(-3)% ethanol, respectively. Embryos cultured in WM containing 1 (0 DES1), 10(-3) (0 DES2) or 10(-6)% ethanol (0 DES3) served as controls. Hatching was inhibited (P less than .05) in mouse embryos cultured in 10(-4) M DES (3.0 +/- 2.1% vs 0 DES1, 25.1 +/- 3.7%). Similar (P greater than .10) percentages of mouse embryos hatched in 10(-7) M DES (36.4 +/- 5.4% vs 0 DES2, 29.1 +/- 5.7%) and 10(-10) M DES (44.4 +/- 4.4% vs 0 DES3, 38.9 +/- 5.3%). Diethylstilbestrol at a concentration of 10(-4) M failed to affect the development of one- to eight-cell swine embryos into blastocysts. However, compared with 0 DES2, 10(-7) M DES reduced (P less than .05) the number of swine blastocysts developing from one- to two-cell (36 vs 78%) and three- to four-cell embryos (50 vs 84%). No significant effects of 10(-7) M DES were detected on the ability of six- to eight-cell swine embryos to develop into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

山羊卵泡颗粒细胞和输卵管上皮细胞共培养对山羊体外受精胚胎发育的影响

试验共分 3组 :对照组为单独培养液 ;第 2组为单层颗粒细胞 ;第 3组为输卵管上皮细胞 ,将山羊体外受精胚胎随机放入以上 3组处理中。结果显示山羊体外受精胚胎在卵泡颗粒细胞中发育到囊胚的比率为 4 1.0 % ,在输卵管上皮细胞中发育到囊胚的比率为 5 8.1% ,而在M199培养液中为 5 .6 %。山羊体外受精胚胎分别与山羊卵泡颗粒细胞和输卵管上皮细胞共同培养时 ,均可提高山羊体外受精胚胎的发育进程。且输卵管上皮细胞比颗粒细胞能显著提高山羊体外受精胚胎的发育进程 (P <0 .0 5 )。     

In vitro development of mouse pronuclear embryos to blastocysts in sequential media with and without co-culture of autologous cumulus cells

The objective of this study was to use mouse embryos as a model system to investigate the effect of co-culture of cumulus cells in Sydney IVF sequential media (Cook) on embryo development, based on the hypothesis that feeder cells in co-culture with a sequential medium could work synergistically to further improve in vitro culture conditions for mammalian preimplantation embryos. The culture systems described here were evaluated by the ability to consistently produce high blastocyst formation rates and high cell number per blastocyst. The role of embryo-to-cell contact was assessed by using Transwell inserts with transparent microporous membranes. Pronuclear embryos of ICR mice were cultured to blastocysts in Cook sequential media, with and without mouse primary cultures of cumulus cells, and with or without inserts. Blastocyst formation rates and cell numbers of in vitro developing embryos in the different culture systems were compared to each other, and to in vivo derived blastocysts. Blastocyst formation rates for Cook medium only was 27.8% (without inserts) and 32.9% (with inserts), whereas Cook-Cumulus cells in identical culture systems was significantly higher at 45.8% (without inserts, P<0.05) and 55.6% (with inserts, P<0.05). When the embryos are suspended above the bottom of the well, for Cook medium significantly lower blastocyst formation rates were observed at 4.2% compared to Cook-Cumulus cells at 17.5% (P<0.05). Mean cell numbers of blastocysts obtained in all co-culture systems were significantly higher (P<0.05) compared to those developing in culture medium only. Although the putative mechanism is as yet unexplained, the improved blastocyst formation rates and cell numbers in co-culture when there is direct contact between the embryo and the cell monolayer suggest that the close proximity between the feeder cells and embryos is in part responsible for these effects.     

Effect of supplemented sericin on the development,cell number,cryosurvival and number of lipid droplets in cultured bovine embryos

Sericin was investigated as an alternative to fetal bovine serum (FBS) for bovine embryo culture. In vitro matured oocytes were developed using 0.05%, 0.1% or 0.15% sericin. The developmental rate, cryosurvival rate and blastulation time of these embryos were compared with those of embryos developed using 5% FBS. The number of lipid droplets was compared among the blastocysts developed using 5% FBS, using 0.05% sericin and in vivo. The rate of cleavage and blastocyst formation was similar among all groups. Blastulation occurred significantly earlier in the embryos developed using 5% FBS than in those developed using sericin at any concentration (P < 0.05). At 72 h after thawing, the cryosurvival rate of the blastocysts developed using 5% FBS and 0.05% sericin were significantly higher compared with those developed using 0.1% and 0.15% sericin (P < 0.05). The blastocysts developed using 0.05% sericin and in vivo produced a significantly fewer number of medium and large lipid droplets than those developed using 5% FBS. These results suggest that the blastocysts developed using 0.05% sericin show characteristics similar to those of the blastocysts developed in vivo and that the use of sericin as an alternative to FBS is feasible.     

Effect of oestrous synchrony between embryo donors and recipients,embryo quality and state on the pregnancy rate in beef cattle

This study aimed to evaluate the effect of oestrous synchrony between donors and recipients and the embryo quality on the pregnancy rate in beef cow recipients. The experiment was performed over two years at an embryo transfer (ET ) centre in Southern Brazil. Ninety Aberdeen Angus cows were subjected to superovulation (SOV ) protocols, resulting in the recovery of 1,048 transferable embryos. Eleven groups were formed with intervals of 6 hr, from ?30 to +30 hr, with respect to recipient versus donor oestrous detection. Evaluation of embryo quality was according to the IETS guidelines. The overall pregnancy rate was 52%. Effects related to donor and recipient oestrous synchronization on pregnancy rate were observed (p  =  .01), ranging from 36% to 50%. The embryo quality rate affected the pregnancy rate, where Grade I resulted in 57% and Grade III in 43% of pregnancy (p  <  .001). The embryonic state (frozen or fresh) showed no (p  >  .05) effect on pregnancy rate: 53% for fresh embryos and 44% for frozen embryos. The odds ratio for explanatory variables causing pregnancy indicated that Grade III embryos had 31% less chance of conception compared to Grade I. Thus, oestrous synchrony between donor and recipient, considering ±30 hr apart, can affect the pregnancy rate along with embryo quality.     

氟对体外培养绵羊成骨细胞RANKL mRNA基因表达影响

为了探讨氟对体外培养的绵羊成骨细胞中核因子кB受体活化因子配体(RANKL)mRNA基因表达的影响,进行了绵羊成骨细胞体外培养,分别加入含浓度为0、10-6、10-5、10-4、5×10-4mol/L氟化钠(NaF)的培养液,并于第6天提取细胞总mRNA,用半定量RT-PCR方法分析RANKL mRNA表达水平的改变.结果表明,10-6、10-5、10-4、5×10-4 mol/L NaF均能刺激体外培养成骨细胞RANKL mRNA表达,当氟化钠浓度为10-5及5×10-4 mol/L时,可以明显促进RANKL mRNA表达(与对照组相比P＜0.01).这说明氟呈剂量依赖性地刺激成骨细胞RANKLmRNA的表达.     

日粮营养水平对育成滩母羊生长发育的影响

将20只健康、体重相近的7月龄滩母羊随机分为5组,进行单笼饲养,设计5种不同营养水平的日粮分别饲喂5组育成母羊,来探究日粮营养水平对育成期滩母羊生长发育的影响。结果表明:试验Ⅳ组的增重最快,其平均日增重最高达(127.22 g/d);育成羊随着年龄的增长,体重增长速度下降。在本试验条件下,随着日粮营养水平的提高,育成滩母羊的总增重、平均日增重、干物质采食量均显著增加(P0.05),而料重比不断下降。各试验组羊只的体长、体高和管围的变化无显著性差异(P0.05),而胸围的增长幅度有显著性差异(P0.05)。结合日增重、体尺指标、生产性能、经济效益等指标,可知试验Ⅳ组的营养水平(CP:15.94%,ME:10.18 MJ/kg)能显著改善羊只生长发育,提高生产性能,获得最高的经济效益。     

Effect of pregnancy and fetal development on sheep liver superoxide dismutase activity

Liver cytosolic CuZn SOD activity of four-week and 12-week pregnant sheep was twice as high as that of their fetuses and almost equal to that of control, barren ewes. By the 20th week of pregnancy activity had decreased by about 70 per cent in the maternal liver and increased to a value similar to that of the controls in the fetuses. The lysosomal CuZn SOD activity remained almost unchanged during pregnancy both in the maternal and fetal livers. Cytosolic and lysosomal CuZn SOD activities of 20-week pregnant sheep and their fetuses showed a similar electrophoretic pattern and low electrophoretic mobility. Hepatic Mn SOD activity increased sharply during fetal development but remained lower than that in both the control and maternal livers. It is proposed that the changes in CuZn SOD and Mn SOD activities are associated with changes in copper metabolism and oxygen utilisation, respectively. The low electrophoretic mobility of CuZn SOD is assumed to be a species specificity.     

Effect of group culture and embryo-culture conditioned medium on development of bovine embryos

We investigated the effect of group culture on bovine embryo development, and also investigated the effect of embryo-culture conditioned medium on developmental competence of individually cultured bovine embryos. Slaughterhouse-derived bovine oocytes were matured and fertilized in vitro. The presumptive zygotes were cultured individually or cultured in groups of 2 to 5 embryos with a constant culture density (5 mul/embryo). After 7 days of culture, the rates of embryos developed to the blastocyst stage were significantly higher (P < 0.05) in group cultures of more than 3 embryos/drop than for embryo culture of 1 or 2 embryos/drop. These results suggest a beneficial effect of group culture may be exerted by possible growth promoting factors secreted by embryos. In the next experiment, we investigated the effect of timing of fresh medium replacement on the development of embryos cultured in groups. The blastocyst formation rate was lower when culture medium was replaced freshly on days 2-4 after fertilization than on days 5-6. The blastocyst formation rates of single-cultured embryos were significantly (p < 0.05) increased by the addition of conditioned medium derived from multiple-embryo culture. These results indicate that group culture promotes embryo development and that embryo culture-derived conditioned medium is effective for supporting development of single cultured embryos.     

Immunohistochemical diagnosis of toxoplasmosis in fetuses and fetal membranes of sheep

A peroxidase antiperoxidase technique was developed for visualizing intact Toxoplasma gondii and its antigenic residues in sections of formalin-fixed, paraffin-embedded tissues from fetal membranes recovered from ewes with induced toxoplasmosis. The peroxidase antiperoxidase method was specific and sensitive, allowing the detection of T gondii antigen even in severely decomposed tissues. Toxoplasma gondii infection was most commonly demonstrated in the cotyledons, heart, lungs, brain, and skeletal muscles of the fetus.     

Effect of lasalocid on the development of Eimeria tenella in chicken embryos

Sporozoites of Eimeria tenella were treated with lasalocid in vitro and their viability tested by inoculating them into the allantoic cavity of 11-day-old chicken embryos. Concentrations of 0.1, 1.0 and 10.0 micrograms ml-1 reduced sporozoite viability, as judged by oocyst production. Injections into the embryos of 5, 50 or 500 micrograms of lasalocid 92-93 h after infection also reduced oocyst production, indicating activity against the later stages of the life cycle.     

氨基酸、维生素对水牛体外受精胚胎体外发育的影响

通过在培养液中添加不同浓度的氨基酸或维生素,探讨其对水牛体外受精(IVF)胚胎体外发育的影响.结果表明:(1)非必需氨基酸可显著提高水牛卵母细胞IVF后胚胎的分裂率,但对囊胚发育率无显著影响;(2)低浓度的必需氨基酸对水牛IVF胚胎的发育具有一定促进作用,但高浓度则有抑制作用;(3)维生素对水牛IVF胚胎发育则有促进作用.在培养液中添加维生素,水牛IVF胚胎的分裂率和第7天囊胚发育率显著提高.     

培养液及血清浓度对山羊孤雌胚胎体外培养的影响

试验比较了在SOFaa，CRlaa，mCRlaa3种培养液中添加不同浓度的成年山羊血清(NGS)对山羊孤雌胚胎进行体外培养的效果。结果表明：在3种培养液中，添加10％的NGS对山羊孤雌胚胎的体外发育效果较好，囊胚率分别可达62．79％(81／129)、53．52%(38／71)、13．64％(12／88)；mCRlaa组囊胚发育率和囊胚细胞数显著低于SOFaa组和CRlaa组，SOFaa组优于CRlaa组．但SOFaa组和CRlaa组间无显著差异。在现有试验条件下，以在SOFaa培养液中．山羊孤雌胚胎的体外培养的第72小时时加入10％的NGS的发育效果较好，囊胚率可达62．79％。     

Attempts to stimulate mitosis in cultured trophoblast cells of cattle embryos

Trophoblast from Day-14 bovine embryos was cultured in medium containing mitogens to determine if the mitotic index could be altered. Trophoblast from each of 15 embryos was cultured in minimum essential medium (Eagles) with 20 % fetal calf serum (control) or in this medium supplemented with pokeweed mitogen (1 %, v/v), phytohemagglutinin (1 %, v/v), concanavalin A (1 %, v/v) or thymidine (2 mg/ml). No mitogenic effect was observed due to any of the treatments. However, mitotic indexes were significantly lower when pokeweed (P < 0.05) or thymidine (P < 0.01) was added to the medium. A highly significant (P < 0.001) variation in mitotic index between embryos was observed.     

Developmental regulation and modulation of apoptotic genes expression in sheep oocytes and embryos cultured in vitro with L‐carnitine

The objective of this study was to find out the impact of L‐carnitine (10 mM) on developmental regulation of preimplantation sheep embryos cultured in vitro when supplemented in maturation medium and post‐fertilization medium separately. Subsequent objective was to observe the L‐carnitine‐mediated alteration in expression of apoptotic genes (Bcl2, Bax, Casp3 and PCNA) in sheep oocytes and developing embryos produced in vitro. Oocytes matured with L‐carnitine showed significantly (p < .05) higher cleavage (67.23% vs 43.12%), morula (47.65% vs 28.58%) and blastocysts (32.12% vs 13.24%) percentage as compared to presumptive zygotes cultured with L‐carnitine during post‐fertilization period. So it is suggested to use L‐carnitine during maturation than post‐fertilization period. Antiapoptotic and proliferative effects of L‐carnitine were confirmed by inducing culture medium with actinomycin D (apoptotic agent) and TNFα (antiproliferative agent), respectively, with and without L‐carnitine. Oocytes and embryos cultured with actinomycin D and TNFα showed developmental arrest with significant (p < .05) decrease in morula and blastocysts percentage but s upplementation of L‐carnitine to actinomycin D and TNFα induced culture medium showed similar result as that of control . L‐carnitine supplementation during IVM significantly (p < .05) upregulated the expression of Bcl2 and PCNA genes in majority of the developmental stages. Although L‐carnitine upregulated the expression of Bax in initial developmental stages but downregulated at latter part, whereas the expression of Casp3 was upregulated upto 16‐cell stage but after that there was no difference in expression. Expression of GAPDH gene was not affected by L‐carnitine supplementation. In conclusion, L‐carnitine acted as an antiapoptotic and proliferative compound during embryo development and supplementation of L‐carnitine during IVM altered the expression of apoptotic genes in the developmental stages of embryos.