Development and quality of bovine embryos produced in vitro using growth factor supplemented serum-free system

The influence of a growth factor supplemented serum-free system on the development, gene expression, and cryotolerance of in vitro pro- duced bovine embryos was investigated. To assess the embryo development and gene ex- pression in blastocysts, abattoir-derived oo- cytes (obtained from 3 - 10 or <3 mm follicles) were matured and fertilized in serum-free media and cultured in synthetic oviductal fluid sup- plemented with fetal bovine serum (FBS, 4%), epidermal growth factor (EGF, 10 ng/mL), insulin like growth factor-1 (IGF-1, 100 ng/mL), stem cell factor (SCF, 50 ng/mL) or combinations of the growth factors. Expressions of selected gene transcripts were relatively quantified in the d 8 blastocysts. To assess the cryotolerance, d 4 morulae (derived from 3 - 10 mm follicles and cultured with the supplementation of FBS or combinations of the growth factors) were vitri- fied, thawed and cultured (with respective sup- plementations). Total cell number and DNA frag- mentation in blastocysts derived from the vitri- fied morulae were assessed through TUNEL assay. The rate (%) of cleavage, blastocyst and expanded/hatched blastocyst did not differ among the culture medium supplementations within the follicle size of 3 - 10 mm (range 65.1 ± 4.3 - 75.4 ± 3.9;22.4 ± 3.9 - 36.4 ± 3.6;and 11.2 ± 2.9 - 23.3 ± 3.2, respectively) or <3 mm (range 59.3 ± 4.2 - 74.5 ± 3.7;15.0 ± 3.5 - 28.7 ± 4.5;and 9.3 ± 2.8 - 17.3 ± 2.7, respectively). Nevertheless, significantly lower (P < 0.05) cleavage and blastocyst rates with FBS and lower blastocyst rate with SCF supplementations were observed for the oocytes derived from <3 compared to 3 - 10 mm follicles. The expression patterns of BCL-2, BAX, HSP1A1, GJA1 and BIRC5 tran- scripts varied significantly (P < 0.05) in all cases, except for BIRC5 in the blastocysts derived from 3 - 10 mm follicles. Following thaw and culture, the development (%) of vitrified morulae into expanded/hatched blastocysts was lower (P < 0.01) with the supplementation of growth fac- tors compared to FBS. In contrast, total number of cells and DNA fragmentation index in blas- tocysts were not different among the treatments. In conclusion, the growth factor supplemented serum-free system was satisfactory for in vitro bovine embryo production. Nevertheless, the system was not efficient when embryos were derived from <3 mm follicles and cultured with SCF. Additionally, gene expression patterns and cryotolerance of the embryos were affected with the treatments of growth factors compared to serum.

Evaluation of an internal control in a multiplex-PCR assay for sex determination of <i>in vitro</i>-produced bovine embryos

The use of an internal control in a multiplex-PCR assay for sex determination of In Vitro-produced bovine embryos was evaluated in biopsies of groups of 54 fresh and 44 frozen embryos. The internal controls used were the primers BOV 1 and BOV 2, which amplify a product with 626 base pairs (bp) of bovine mitochondrial DNA ND5 gene. The primers BRY.4aF and BRY.4aR were used for bovine Y chromosome sequence amplification. The specificity of multiplex- PCR reactions realized in biopsies corresponding to about 20% of each fresh embryo (10 male and 10 female) by means of confirming the sexing in the remaining embryo content (~80%) presented 100% specificity. Amplicons of the internal control and Y chromosome were both amplified until dilution corresponding to 6.25% of total extracted DNA from a male embryo. Sex determination was possible in 53 (98.1%) fresh embryos and 40 (90.9%) frozen embryos. The products related to the Y chromosome and mitochondrial DNA were simultaneously amplified in 34 (63%) fresh embryos and 27 (61.4%) frozen embryos, showing a male embryo. The female sex, distinguished by internal control amplification only, was detected in 19 (35.2%) and 13 (29.5%) biopsies, respectively, of fresh and frozen embryos. In one (1.8%) and four (9.1%) biopsies of fresh and frozen embryos, respectively, neither product was amplified, most likely due to the absence of embryonic cells or the presence of embryonic cells going through apoptosis. The multiplex-PCR assay developed in this work showed avoided the limitation of a lack of an internal standard, and was also sensitive, specific, and efficient in reaction failure identification. This technique shows great potential for use on a commercial scale in routine sex determination of In Vitro-produced embryos.

Effect of heat stress on the maturation,fertilization and development rates of in vitro produced bovine embryos

Heat stress is one of the main reasons for reproductive performance decrease in cattle, resulting in severe economic losses. The aim of this study was to evaluate the effect of heat stress during maturation, fertilization and development of in vitro produced bovine embryos. Cumulus oocyte complexes (COCs) were obtained by follicular puncture from slaughterhouse ovaries and after identification, were divided into four groups: control (CG), exposed 1 (EG1), exposed 2 (EG2), and exposed 3 (EG3). The oocytes of the group CG and CG3 were cultured at 38°C and the oocytes of group EG1 and EG2 were cultured at 40°C during the maturation period (24 hours at 5% CO2 in air). After the maturation period, oocytes of group CG, EG1, EG2, and EG3 were fecundated with frozen thawed semen. The oocytes of CG, EG2 and EG3 groups were cultured at 38°C, and the group EG1 was cultured at 40°C (18 hours at 5% CO2 in air). After that, the CG and EG2 groups were cultured in SOF at 38°C and the groups EG1 and EG3 at 40°C during embryonic development. The embryos were evaluated for cleavage, morula and blastocyst rates by optical microscopy. In control (CG) and EG3 groups, the oocytes showed uniform expansion of cumulus cells, classified as moderate to high, with brown color and uniform appearance of the ooplasm. In the oocytes exposed to 40°C (EG1 and EG2) we observed a decrease in the expansion of cumulus cells, and the same showed rounded appearance and retraction of the ooplasm with dark coloration. The control group (CG) had 68.23% ± 2% of cleavage, 50.16% ± 2% morulas, and 43.28% ± 1% blastocysts. Whereas the EG2 had 31.46% ± 2% cleavage, 35.64% ± 2% morula, and no blastocysts development. The EG3 had 3.7% ± 2% cleavage, and no embryo production. These data suggest that in all stages of exposure to heat stress, the embryos and the gametes are susceptible, leading to a decrease in embryonic development.

High-quality Cleavage Embryo versus Low-quality Blastocyst in Frozen-thawed Cycles:Comparison of Clinical Outcomes

This study compared the clinical outcomes of the frozen-thawed cycles of high-quality cleavage embryos with low-quality blastocysts to provide a reference for the choice of frozen-thawed embryo transfer schemes and to improve clinical pregnancy rates.A retrospective analysis was performed on the clinical data of patients undergoing frozen-thawed embryo transfer at the Reproductive Medicine Center of Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology from 2016 to 2017.In total,845 cases were divided into a high-quality cleavage embryo group(group A)and a low-quality blastocyst group(group B).Each group was further divided into subgroups based on the number of transplants.Group A was categorized into two subgroups comprising of 94 cases in subgroup Al(1 high-quality 8-cell group)and 201 cases in subgroup A2(2 high-quality 8-cell group).Group B was divided into four subgroups consisting of 73 cases in subgroup B I(D53BC group),65 cases in subgroup B2(D54BC group),110 cases in subgroup B3(D63BC group),and 282 cases in subgroup B4(D64BC group).The pregnancy outcomes and neonatal outcomes between the groups were compared.The clinical pregnancy rates(56.72%and 60.00%)and live birth rates(47.76%and 46.15%)in subgroups A2 and B2 showed no significant differences,but these rates were significantly higher in subgroups A2 and B2 than in the rest subgroups(P<0.05).The multiple birth rate(26.32%)in the subgroup A2 was significantly higher than that in the rest subgroups(P<0.05).There were no statistically significant differences in the abortion rates among all groups(P>0.05).In terms of neonatal outcomes,there were no statistically significant differences in the proportion of premature births,sex ratios,and birth defects among the low-weight and gigantic infants(P>0.05).Transplanting two high-quality cleavage embryos during the frozen-thawed embryo transfer cycles could significantly increase clinical pregnancy rates and live birth rates,but at the same time,it also increased the risks of multiple births and complications to mothers and infants.The D54BC subgroup had the most significant advantages among all groups(P<0.05).The rest low-quality blastocysts had clinical outcomes similar to the single high-quality cleavage embryo group.

Tumor necrosis factor alpha inhibits in vitro bovine embryo development through a prostaglandin mediated mechanism

Mastitis or other infectious diseases have been related to reduced fertility in cattle. Inflammatory cytokines such as tumor necrosis factor α （TNFα are released in response to infection and may have negative effects on embryo development, in the current study the effect of exposure to TNFα on the development of in vitro fertilized bovine embryos was examined. Indomethacin, a prostaglandin synthesis inhibitor, was used to determine if blockade of prostaglandin synthesis would alter the effects of TNFα Ovaries were obtained from a local abattoir and immature COC were isolated from 2-10 mm follicles, in vitro matured and fertilized. After fertilization, groups of presumptive zygotes were randomly placed into either control development medium, medium containing 25 ng/mL TNFα or medium containing 25 ng/mL TNFα plus 1 μg/mL indomethacin. The proportion of blastocysts formed was assessed at day 7 of culture. Fewer embryos exposed to TNFα alone reached the blastocyst stage （17.5 ± 2.4%, P 〈 0.01） compared with controls （30.5 ± 2.4%） or embryos developed in TNFα plus indomethacin （25.8 ± 2.8%）. There was no difference between control embryos and embryos developed in TNFα plus indomethacin. These results indicate that TNFα is inhibitory to the in vitro development of bovine embryos and that this inhibition may be mediated by prostaglandins because it can be blocked by indomethacin.

Insulin-like Growth Factor-I (IGF-I) in Reproduction System of Female Bovine

Insulin-like growth factor-I （IGF-I） plays a key role in female reproduction, because it has the effect of anti-apoptosis improving cell proliferation, transformation and differentiation. This paper reviewed the effects of IGF-I on ovary, follicle growth, acquisition of oocyte competence and preimplantation embryo viability, and then summarized different points about IGF-1 for reproduction system

Effects of Feeding OmniGen-AF^®during Superovulation on <i>in Vitro</i>Development of Embryos Recovered from Donor Beef Cows

Embryo quality is crucial when selecting embryos for transfer. Variation in quality may be attributed to poor oocytes, semen, stress, inflammation, and potential immune system dysregulation. OmniGen-AF® (OG) feeding supports immune system function and animal health. Our laboratory recently reported lower percent degenerate embryos recovered and increased plasma progesterone in beef cattle donors fed OG during superovulation. In vitro development of embryos recovered from donor cows fed OG prior to collection is presented here. Embryos were recovered from 24 beef cows assigned to four treatment groups: 0 g OG/hd/d and 200 mg Folltropin®-V (FSH) (0/200);0 g OG/hd/d and 400 mg FSH (0/400), 56 g OG/hd/d, 200 mg FSH (56/200) and 56 g OG/hd/d and 400 mg FSH (56/400). Good to excellent quality early blastocysts were cultured for 8 d. and development through hatching, embryonic volume and plasminogen activator (PA) production were quantified. The complete protocol was repeated 90 - 120 d later as Replicate 2. Optimal development was observed by embryos recovered from 0/200 cows where percent blastocysts hatching was greater (P < 0.05) compared to 56/200 and 0/400 cows and embryonic volume was greatest (P < 0.05) in Replicate 1. However, percent blastocysts hatching from 0/200 cows was similar (P > 0.10) to 56/400 cows and embryos recovered from 56/400 cows in Replicate 1 produced more (P < 0.05) PA compared to all other groups. For cows superovulated with the standard 400-mg FSH dose, feeding OG supported in vitro embryo development similar to that observed for 0/200 cows.

Genomic DNA methylation patterns in bovine preim-plantation embryos derived from in vitro fertilization

By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of bovine zygotes and preimplanta-tion embryos derived from oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and embryo in vitro culture (IVC). The results showed that: a) paternal-specific demethylation occurred in 61.5% of the examined zygotes, while 34.6% of them showed no demethylation; b) decreased methylation level was observed after the 8-cell stage and persisted through the morula stage, however methylation levels were different between blastomeres within the same embryos; c) at the blastocyst stage, the methyla-tion level was very low in inner cell mass, but high in trophectoderm cells. The present study suggests, at least partly, that IVM/IVF/IVC may have effects on DNA methylation reprogramming of bovine zygotes and early embryos.

Sexing Bovine Embryos Using PCR Amplification of Bovine SRY Sequence

This study analyses the bovine SRY DNA sequence by direct sequencing procedure, followed by the designation of the PCR primers specific for bovine SRY. Using PCR amplification of bovine SRY gene, the embryo sex was determined. The results of the embryo sex identification were confirmed after the embryo transfer and pregnancies.

牛体外胚胎生产过程中培养液添加物研究进展

牛体外胚胎生产是一种广泛应用于畜牧生产和生物研究的现代繁殖技术,在促进肉牛和奶牛品种改良以及高效繁育中发挥着关键作用。随着分子生物学、生物化学和细胞生物学技术的不断发展,牛体外胚胎生产技术已取得了长足进步。然而,当前牛体外胚胎生产技术仍存在胚胎生产效率低和胚胎发育潜能差等瓶颈问题,在很大程度上限制了该技术的规模化推广应用。本文系统综述了牛体外胚胎生产中经典添加物和最新添加物的研究进展,旨在为优化构建不同添加物组合的技术体系提供参考,以期更加高效地获得牛体外优质胚胎。

LIF调控奶牛子宫内膜上皮细胞容受性基因表达研究

接受态的子宫内膜对于奶牛胚胎的成功植入至关重要。为探讨白血病抑制因子(LIF)对奶牛子宫内膜容受性的调控机制,试验将奶牛子宫内膜上皮细胞分为对照组、LIF处理组、LIF+STAT3共处理组,研究了LIF对子宫内膜容受性的作用和STAT3信号通路对奶牛子宫内膜容受性的调控。采用实时荧光定量RT-PCR和Western blot检测容受性相关基因HOXa10、VEGF和炎症相关基因TLR4、NF-κB的mRNA和蛋白表达变化。结果表明:与对照组相比,LIF处理组奶牛子宫内膜上皮细胞的容受性相关基因HOXa10、VEGF的表达量显著增加(P<0.05),炎症相关基因TLR4、NF-κB的表达量显著增加(P<0.05)。LIF+STAT3共处理组的HOXa10、VEGF的表达量与LIF处理组比较显著降低(P<0.05),与对照组无显著差异(P>0.05);TLR4、NF-κB的表达量与LIF处理组比较无显著差异(P>0.05),显著高于对照组(P<0.05)。说明LIF通过激活STAT3信号通路调控子宫内膜上皮细胞HOXa10、VEGF的表达,能够增强子宫内膜的容受性。

Factors affecting early embryonic development in cattle:relevance for bovine cloning

Female infertility represents a major challenge for improving the production ef?ciency in the dairy industry. Historically, fertility has declined whereas milk yield has increased tremendously due to intensive genetic selection. In vivo evidence reveals about 60% pregnancy loss takes place during the ?rst month following fertilization. Meanwhile, early embryo development is signi?cant for somatic cell nuclear transfer in cattle as a large proportion of cloned embryos fail to develop beyond periimplantation stage. Oocyte quality is of utmost importance for the early embryo to develop to term for both fertilized and cloned embryos. Epigenetic reprogramming is a key process occurring after fertilization and critical roles of epigenetic modi?ers during preimplantation development are now clear. Incomplete epigenetic reprogramming is believed to be a major limitation to cloning ef?ciency.Treatment of cloned embryos with epigenetic modifying drugs(e.g., Trichostatin A) could greatly improve cloning ef?ciency in both mice and cattle. Recently, the rapid progress in high-throughput sequencing technologies has enabled detailed deciphering of the molecular mechanisms underlying these events. The robust ef?ciency of genomic editing tools also presents an alternative approach to the functional annotation of genes critical to early development.

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。

鉴别牛早期胚胎性别PCR方法引物的设计与筛选

根据牛Y 染色体特异重复序列、睾丸特异蛋白基因以及性别决定基因序列设计合成5对公牛Y 染色体特异引物, 依据牛骨胳肌α肌动蛋白前体基因和微卫星 DNA 序列设计合成 4 对牛 DNA特异引物(内标引物)。单重PCR扩增牛基因组DNA,筛选出4对牛Y 染色体特异引物和1对牛DNA特异内标引物。将不同的 Y 染色体特异引物与内标引物组合,多重PCR扩增牛基因组DNA、已知性别的牛成纤维细胞和克隆胚胎,筛选出 2 个可用于牛早期胚胎性别鉴别的PCR引物组合: B34/A12 和B78/A12。

牛早期胚胎性别鉴定PCR反应体系的优化研究

根据牛SRY基因序列设计合成2对巢式PCR引物作为性别鉴定引物,根据牛酪蛋白基因序列设计了一对引物作为内标引物建立了牛胚胎性别鉴定的PCR反应体系。同时对常规PCR和巢式PCR在牛早期胚胎性别鉴定中的实用性进行比较。15头公牛、13头母牛的DNA样品检测结果表明:使用巢式PCR公牛可以扩增出205bp的SRY基因片段和403bp的酪蛋白基因片段,母牛只能扩增出403bp的酪蛋白基因片段;而使用常规PCR时公牛扩增出255bp的SRY基因片段和403bp的酪蛋白基因片段,母牛只能扩增出403bp的酪蛋白基因片段,其性别鉴定结果和实际完全一致。由于巢式PCR只需10个细胞就可以在紫外透射分析仪下看到扩增结果,而常规PCR则需要20～30个细胞,所以胚胎性别鉴定时使用巢式PCR效果更好。试验采用巢式PCR鉴定了10个奶牛胚胎的性别,同时还对血清是否会对试验结果产生影响进行了研究。

牛体外受精胚冷冻保存的研究

对牛卵泡卵母细胞体外受精（ＩＶＦ）１６８ｈ的致密桑椹胚、囊胚用常规快速冷冻法、预冷和不预冷的超快速冷冻法进行了冷冻保存试验。结果表明：ＩＶＦ囊胚采用含１０％甘油的常规快速冷冻法、含２．１ｍｏｌ／Ｌ甘油和０．２５ｍｏｌ／Ｌ蔗糖预冷５ｍｉｎ的一步冷冻法及含２５％甘油和２５％乙二醇预冷５ｍｉｎ的玻璃化冷冻法等３种方法进行冷冻保存，解冻后的继续发育率（６８．０％，５９．０％，６５．７％）均无显著差异（Ｐ＞０．０５），可用快速、简便、预冷的一步冷冻法或玻璃化冷冻法替代常规快速冷冻法；ＩＶＦ致密桑椹胚可用一步冷冻法（２．１ｍｏｌ／Ｌ甘油＋０．２５ｍｏｌ／Ｌ蔗糖）和玻璃化冷冻法（２５％甘油＋２５％乙二醇或２５％甘油＋２５％１，２－丙二醇）进行预冷的超快速冷冻保存；冻前预冷（５ｍｉｎ）能显著提高ＩＶＦ囊胚的冻后形态正常率和继续发育率（Ｐ＜０．０５）；ＩＶＦ囊胚冷冻—解冻后的继续发育率高于ＩＶＦ致密桑椹胚。

Percoll法处理牛精子对体外受精胚胎发育的影响

牛冷冻精液解冻后，在Ｐｅｒｃｏｌ梯度液中４００ｇ离心２４ｍｉｎ，有效地获得了５７．８７％±８．０％的精子回收率；对照组上游法精子回收率仅为１５．８７％±１．９％（Ｐ＜０．０５）。Ｐｅｒｃｏｌ和上游２种方法分离的精子在受精液中８、１８ｈ后的活率，分别为６０％、１０％和７０％、２０％。２种浓度Ｐｅｒｃｏｌ法分离的精子对不同成熟处理的卵母细胞体外受精（ＩＶＦ）卵裂率的影响：精子最终浓度为２×１０６／ｍＬ时，成熟组Ⅰ（ＬＨ＋ＦＳＨ）和成熟组Ⅱ（ＰＭＳＧ＋ｈＣＧ）的卵裂率分别为５６．１９％和５８．１６％；精子最终浓度为５×１０６／ｍＬ时，２组的卵裂率分别为５９．３０％和６２．７７％，卵裂率在精子浓度不同组间及添加不同激素组间的差异不显著。本试验还研究了２种方法处理的精子对不同成熟处理组的卵裂率和囊胚率，用Ｐｅｒｃｏｌ法处理的精子（２×１０６／ｍＬ）在成熟组Ⅰ获得了５７．１６％的卵裂率和２５．５８％的囊胚率，而上游法分别为６１．９５％和１９．０２％，２种方法相差不显著（Ｐ＞０．０５）；在成熟组Ⅱ获得了６２．１８％的卵裂率和２４．７７％的囊胚率，而上游法分别为７１．１８％和２０．９６％，２种方法的卵裂率相差显著（Ｐ＜０．?

不同血清对常温和冷冻保存牛体外受精胚胎的影响

本研究探讨了不同血清对常温和冷冻保存牛体外受精胚胎的影响。常温 (2 0℃ )保存胚胎的试验结果表明 :用添加 1 5 %胎犊血清 (FCS)和 1 5 %阉公牛血清 (SS)保存的胚胎所获得的孵化率无显著差异(80 .5 %和 82 .6% ,P>0 .0 5 ) ,添加这两种血清与添加 0 .4%牛血清白蛋白 (BSA)和不添加血清的比 ,胚胎的孵化率有极显著的差异 (80 .5 % ,82 .6% ,63 .5 %和 61 .7% ,P<0 .0 1 )。冷冻胚胎的试验结果显示 :冷冻液中加有 1 5 % FCS、 1 5 % SS和 0 .4% BSA与不含血清的单纯磷酸缓冲液 (PBS)比 ,解冻后胚胎的孵化率有极显著的差异 (81 .5 % ,76% ,73 %和 60 .5 % ,P<0 .0 1 )。而这 3种血清之间 ,胚胎的孵化率无显著差异 (P>0 .0 5 )。由上述两个试验的结果说明 :廉价而又易采集的阉公牛血清 (SS)

HA对牛卵母细胞体外成熟和早期胚胎体外发育影响的研究

在TCM-199中添加一些确定的辅助成分,探讨在无血清培养基中添加不同浓度HA对牛卵母细胞体外成熟及早期胚胎体外发育的影响。结果表明,在卵母细胞成熟培养液(TCM-199-m)中添加4.0mg/mL的HA时,卵母细胞的成熟率和卵裂率与对照组(BSA)无显著差异(P>0.05),分别为85.14%,83.57%和89.47%,85.24%,但显著高于其他各浓度组(P<0.05),表明HA在卵母细胞无血清成熟培养中可代替BSA。在受精卵体外培养液(TCM-199-c)中添加HA时,囊胚发育率与对照组(BSA)差异不显著(P>0.05),分别为27.64%和26.51%,但显著高于TCM-199-c组(P<0.05),表明HA对牛胚胎体外发育具有明显的促进作用,在无血清培养中可以代替BSA。当在TCM-199-c+HA中添加少量的BSA时,囊胚发育率(31.19%)显著高于其他各组(P<0.05),表明HA欲完全代替BSA还有不完善之处,有待进一步研究。