Comparative IFN-τ Secretion after Hatching by Bovine Blastocysts Derived Ex Vivo and Completely Produced In Vitro

The interferon-tau (IFN-tau) secretion levels after hatching by bovine blastocysts derived from in vitro maturated oocytes (Group A) and from in vivo (Group B) were investigated considering embryo quality. Only very homogeneous blastocysts of excellent or good quality were considered from day 7 of culture (Group A) and day 7 after artificial insemination with frozen-thawed from the same bull used for in vitro fertilization (Group B). All embryos were individually cultured into a 50 microl droplet of synthetic oviduct fluid medium with 10% fetal calf serum. After 24-h culture both Group A (n=44) and B (n=40) secreted <54 pm IFN-tau. After 48-, 72-, 96- and 120-h culture, Group A daily secreted 143 +/- 24 pm IFN-tau (n=19) vs 85 +/- 12 pm IFN-tau (n=21) for Group B (p < 0.01), 491 +/- 128 pm IFN-tau (n=29) vs 216 +/- 37 pm IFN-tau (n=23) (NS), 499 +/- 135 pm IFN-tau (n=26) vs 353 +/- 93 pm IFN-tau (n=21) (NS), 559 +/- 136 pm IFN-tau (n=22) vs 333 +/- 75 pm IFN-tau (n=20) (NS), respectively. Taken all together during 5 days, Group A produced per embryo 1690 +/- 290 pm IFN-tau (n=22) vs 982 +/- 182 pm IFN-tau (n=20) for Group B (p < 0.05). For all culture time there were sizable percentages of embryos that did not produce concentrations of IFN-tau above a certain cut-off level, and as such were not used to compute the means. In respect of the embryo quality whatever the groups after days 7-12 of culture, IFN-tau secretions were 1815 +/- 453 pm (n=10) for the embryos of excellent quality vs 1356 +/- 200 pm (n=28) for those of good quality (NS) and 360 +/- 188 pm (n=4) (p < 0.05) for embryos of fair quality. A positive relationship between IFN-tau production and in vitro development of quality I embryos was observed, whatever the embryos origins and, the embryos completely produced in vitro secreted more IFN-tau than the embryos produced in vivo.     

The Effects of Blastomere Biopsy and Oxygen Tension on Bovine Embryo Development, Rate of Apoptosis and Interferon-τ Secretion

A series of experiments was performed to examine the effects of blastomere biopsies on subsequent development of IVF-derived bovine embryos. The first experiment was designed to assess the optimal time for blastomere removal. One blastomere was removed either 48 or 72 h after IVF. Biopsy at 48 h resulted in 17.2% of embryos proceeding to the blastocyst stage, which was lower than when biopsies were performed at 72 h (37.5%, p < 0.05). In the second experiment, embryos were cultured either under atmospheric or 5% O(2) following blastomere removal. Biopsies had no effect on rate of blastocyst formation with 36% of controls and 33.7% of biopsied embryos proceeding to that stage. However, culture under 5% O(2) significantly increased the number of blastocysts from 29.9% to 40.3% (p < 0.05). This effect was significant in both biopsied and control embryos. In the final experiment, biopsied embryos were again cultured under different oxygen tension. Blastocysts were collected and cultured individually for 48 h in medium droplets in their respective O(2) concentration after which time the medium was assayed for concentration of interferon-tau (IFN-tau). Reduced O(2) concentration again significantly increased blastocyst formation from 24.9% to 41.9% (p < 0.05). IFN-tau secretion was not affected by biopsies, but culture under atmospheric O(2) resulted in significantly increased IFN-tau concentration in medium droplets (12274.0 +/- 2825.9 pM vs 5046.5 +/- 2562.2 pM; p < 0.05).     

Effect of Meloxicam on Pregnancy Rate of Recipient Heifers Following Transfer of In Vitro Produced Embryos

The main objective of this study was to determine if administration of meloxicam, a cyclooxygenase (COX) two inhibitor, to heifers in which embryo transfer (ET) is more difficult and requires a greater manipulation of the tract, would be beneficial. Nulliparous recipient heifers were divided in two groups: CON (n = 102), in which animals received 10 ml of saline IM (the same volume of meloxicam) and MEL (n = 105) animals that were treated with meloxicam. According to the degree in passing the catheter, recipients from both groups were classified as Grade I, easy (< 60 s), and Grade II (more than 80 s), difficult. Immediately after embryo transfer, MEL recipients received an injection of 200 mg of meloxicam (10 ml).There was no difference in the pregnancy rates on Day 35 considering animals which presented Grade I cervix independently whether the treatment was performed or not (p = 0.22). There was a statistical difference in the pregnancy rates (p < 0.01) between both groups (49.0% and 66.7% for CON and MEL, respectively) when cervical grade was not considered, on Day 35. Considering the animals that presented Grade II cervix, the pregnancy rate was higher for MEL (21.15% and 78.84%, respectively) in both examinations (p < 0.01).The authors concluded that meloxicam had a positive influence on general pregnancy rate of treated heifers in comparison to non‐treated heifers. It was also observed that pregnancy rate was not influenced by meloxicam administration in Grade I heifers. Treatment increased the pregnancy rate of Grade II heifers.     

性别鉴定后胚胎移植妊娠率影响因素分析

对影响后的性别鉴定胚胎移植妊娠率的因素进行了分析。结果表明,性别鉴定胚胎的鲜胚和冻胚移植妊娠率均低于非性别鉴定胚胎,但二者差异不显著(P>0.05),这表明性别鉴定胚胎在生产中应用是可行的。随着胚胎细胞取样数的增多,其移植妊娠率逐渐下降,且冷冻带来的损伤明显增大;性别鉴定胚胎在体外放置时间小于5 h的妊娠率比大于5 h的高,且差异极显著(P<0.01);性别鉴定胚胎移植时受体的最佳移植时间在发情后的7.5~8.5 d;繁殖力好的受体牛移植妊娠率比繁殖力差的受体牛移植妊娠率高,且差异极显著(P<0.01)。     

Forced Collapse of the Blastocoel Cavity Improves Developmental Potential in Cryopreserved Bovine Blastocysts by Slow‐Rate Freezing and Vitrification

This study was conducted to evaluate the effectiveness of forced collapse of the blastocoel before slow‐rate freezing and vitrification of bovine blastocysts. Cryopreservation of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production using the embryo transfer technique. However, the low efficiency of frozen–thawed embryos survival and further development is a crucial problem. In this study, bovine in vitro and in vivo blastocysts were slow‐rate frozen and vitrified after forced blastocoele collapse (FBC) of the blastocyst cavity by puncturing the blastocoele with a pulled Pasteur pipet. Differences in the developmental potential of frozen–thawed blastocysts derived from FBC and non‐FBC groups were found in both slow‐rate freezing and vitrification. Furthermore, we found that the total cell number of blastocysts in FBC groups was increased and the index of apoptosis in FBC groups was decreased. Consistent with these results, real‐time RT‐PCR analysis data showed that expression of the anti‐apoptotic Bcl‐XL gene was significantly increased by FBC groups, whereas expression of the pro‐apoptotic Bax gene was significantly decreased by FBC groups. Our results also showed that pregnancy outcomes in both slow‐rate frozen and vitrified bovine in vivo blastocysts could be improved by reducing the fluid content after FBC of the blastocyst cavity. Therefore, we suggest that FBC of the blastocyst cavity with a pulled Pasteur pipet is an effective pre‐treatment technique for both slow‐rate freezing and vitrification of bovine blastocysts.     

微量元素锌对牛卵母细胞体外成熟及其体外受精胚胎发育的影响

本研究旨在探讨锌对牛卵母细胞体外成熟及体外受精胚胎发育的影响。首先使用锌螯合剂TPEN去除锌,并检测缺锌对牛卵母细胞体外成熟的影响;然后在体外成熟液中分别添加0(对照组)、0.4、0.8、1.2、1.6μg/mL硫酸锌,探索不同浓度硫酸锌对体外成熟及后续胚胎发育的影响。结果表明:锌元素在体外成熟液中的含量低于牛卵泡液和颈静脉血清(P<0.05);去除体外成熟液中的锌后牛卵母细胞的体外成熟效率下降(P<0.05),且具有时间依赖性,补充适宜浓度硫酸锌后成熟效率恢复;向体外成熟液中添加硫酸锌并未对卵母细胞体外成熟效率产生显著影响,但添加0.8μg/mL硫酸锌成熟后的卵母细胞中活性氧含量显著降低,后续体外受精胚胎的囊胚发育效率显著提高;RT-qPCR分析结果显示,与对照组相比,添加0.8μg/mL硫酸锌成熟后的卵母细胞中抗氧化基因SOD1、CAT、TXN1、PRD1和卵丘扩展基因PTX3、TSG6的表达水平均提高(P<0.05)。研究表明,添加0.8μg/mL硫酸锌可以通过提高卵母细胞内抗氧化酶基因的表达水平,降低卵母细胞内活性氧含量,促进卵丘扩展,从而提高卵母细胞成熟质量和体外受精胚胎的发育效率。     

大幅度提高山区牧场母牛胚胎移植成功率的技术探讨

采用中国荷斯坦、西门塔尔和安格斯牛冷冻胚胎对48头山区牧场母牛实施胚胎移植。结果表明，移植妊娠率达到70．83％，胚胎移植的后代雄性比例明显多于雌性（P〈0．01），自然发情受体牛的移植成功率明显低于经CIDR处理的受体（P〈0．01）。     

Transfer of Beef Embryos in Dairy Cows: Influence of Recipient and Embryo Quality on Pregnancy Rate and Calving Performance

Contents
On three experimental farms 205 deep frozen Blonde d'Aquitaine embryos were transferred into Holstein dairy cows 7 days after a spontaneous heat. The aim of the study was firstly to examine the influence of recipient and embryo quality on pregnancy rate and secondly to study the calving performance of dairy cows carrying beef embryos. Recipients were divided into three groups based on the quality of the corpus luteum (unsuitable, moderately suitable and suitable) established by rectal palpation. Embryos classified as fair, good and excellent, were randomly distributed over the recipients, including those classified as being unsuitable. The overall pregnancy rate was 36% (standard deviation (SD) = 3.3), dystocia occurred in 48% of births (SD = 5.9), gestation was 297 days (SD = 1.2) and birth weight was 52 kg (SD = 2.0). No statistical differences were found in pregnancy rates and calving performance between the groups of recipients or between the classes of embryos. It is concluded that there is no justification to reject recipients on the basis of the quality of the corpus luteum. Furthermore there is no reason to reject fair embryos for transfer. Beef embryos in dairy cows resulted in much dystocia because of high birth weight, and in long gestation periods.     

体外受精后不同时间收集的牛胚胎的冷冻效果

比较了体外受精后不同时间收集的牛囊胚期胚胎冷冻后的存活率。试验一收集受精后第６，７，８和９天出现的胚胎，分３步加入０．７５ｍｏｌ／Ｌ甘油和０．５ｍｏｌ／Ｌ丙二醇，将胚胎装入０．２５ｍＬ细管后，放入已降温至５℃的冷冻机内，立即以２℃／ｍｉｎ的速率降温至－７℃，停留５ｍｉｎ后植冰。植冰后５ｍｉｎ，以０．３℃／ｍｉｎ的速率降温至－２５℃，取出胚胎，并立即投入液氮中保存。解冻时，先让细管在空气中暴露１０ｓ     

营养水平对妊娠早期母猪胚胎存活和瘦素、孕酮分泌及基因表达影响

本试验选用63头长白-大白杂交初产母猪,研究营养水平对初产母猪妊娠早期胚胎存活和瘦素(leptin)、孕酮分泌及胚胎瘦素、瘦素受体(Ob-R)、信号传导和转录活化因子-3(STAT-3)、孕酮受体(PGR)、DNA甲基化转移酶-1(DNMT-1)基因表达的影响.将配种后的母猪随机分到高、中、低3个营养水平组按2倍、1.2倍和0.6倍维持需要(分别记为2.0M、1.2M和0.6M)供给饲粮,妊娠12、25和35 d屠宰母猪收集血清和胚胎,用ELISA方法测定血清中瘦素、孕酮水平,用RT-PCR方法研究目的基因在胚胎中的表达差异.结果表明:①胚胎存活率,妊娠12、25和35 d时,1.2M组均显著高于2.0M组(P<0.05);1.2M组妊娠12 d时与0.6M组间差异不显著(P>0.05),而妊娠25、35 d高于0.6M组(P<0.05);②血清瘦素水平,妊娠12 d时,2.0M组显著高于1.2M组(P<0.05),极显著高于0.6M组(P<0.01);妊娠25和35 d时,2.0M组极显著高于1.2M和0.6M组(P<0.01);1.2M组极显著高于0.6M组(P<0.01).血清孕酮水平,妊娠12、25和35 d时,2.0M组极显著低于1.2M组和0.6M组(P<0.01),1.2M组和0.6M组之间差异不显著(P>0.05);③营养水平对胚胎存活重要基因表达的影响,leptin和ob-R基因mRNA的相对表达量在妊娠12、25和35 d时,2.0M组显著高于1.2M和0.6M组(P<0.05),而1.2M与0.6M组差异不显著(P>0.05).妊娠12和25 d时,2.0M组胚胎STAT-3基因的相对表达极显著高于0.6M组(P<0.01);妊娠35 d时,2.0M和1.2M组极显著高于0.6M组(P<0.01).妊娠12、25和35 d时,2.0M组胚胎DNMT-1的相对表达显著高于0.6M组(P<0.05).妊娠12 d时,各水平组胚胎PGR表达差异不显著(P>0.05),妊娠25 d时,0.6M组极显著高于2.0M组(P<0.01),显著高于1.2M组(P<0.05),妊娠35 d时,1.2M组极显著高于2.0M和0.6M组(P<0.01),0.6M组显著高于2.0M组(P<0.05).以上结果表明在妊娠35 d以内以中营养水平饲喂初产母猪具有较高的胚胎存活率.营养水平显著影响瘦素、孕酮的分泌及lep-tin等基因的表达,瘦素、孕酮分泌过高、过低均对胚胎发育不利,高水平饲喂及严重限饲引起胚胎中leptin、Ob-R、STAT-3、PGR、DNMT-1基因表达变化,降低胚胎存活率.     

滋养层细胞囊泡与奶牛低品质胚胎共移植对受胎率的影响

将奶牛低品质胚胎与囊胚滋养层细胞囊泡（trophoblastic vesicles,TRV）共移植,TRV来源于体外受精培养 7 d、体内培养7 d囊胚的囊胚滋养层细胞。胚胎移植受体牛超声波妊娠诊断共进行2次,即发情的第26～43天和第38～73天。第1次妊娠诊断时试验组（与TRV共移植组）妊娠率（66.7%,16/24）显著高于对照组（34.5%,10/29）（P＜0.05）。在第1次和第2次妊娠诊断期间试验组有3头牛的胚胎死亡。试验组和对照组的分娩率分别为41.7%（10/24）和27.6%（8/29）,两者间无显著差异。两组间受体牛的妊娠期和后代出生重相似。     

The Effect of Species-Specific FSH Administration During In vitro Maturation of Bovine Oocytes on Embryonic Developmental Capability

Veterinary Research Communications -     

Effects of Serum-Free In Vitro Maturation of Bovine Oocytes on Subsequent Embryo Development and Cell Allocation in Two Developmental Stages of Day 7 Blastocysts

Maturation of oocytes and the subsequent outcome of the in vitro production (IVP) are affected by the composition of in vitro maturation (IVM) medium. To determine the use of serum interfering with effects of single molecules, we aimed at developing simplified IVM medium. The experimental IVM media were: (1) M199-medium supplemented with hormones and serum (control), (2) as 1 but serum was substituted with fatty acid-free serum albumin (FAFBSA) and (3) M199-medium without hormonal and serum supplementation (M199). The quality of embryos was assessed on day 7 by morphology and cryotolerance, as well as by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) and differential staining. Results showed that the nuclear maturation was suppressed in M199 group alone. Embryo cleavage and development rates, and the proportion of quality 1 blastocysts were lower in the FAFBSA and M199 groups compared to the control. Differences in the cell allocation of fresh embryos were observed at the blastocyst stage, but not at the expanded blastocyst stage. The control group blastocysts had larger number of cells allocated to the inner cell mass (ICM), and the FAFBSA group blastocysts larger apoptotic cell proportion compared to the blastocysts derived from other groups. After cryopreservation, the reduction of ICM proportion and increase of apoptotic cell proportion of embryos were equal between the experimental groups. In conclusion, exclusion of serum from the IVM media reduces embryo development and may cause perturbations in blastocyst development. Differences in the cell allocation of blastocysts between IVM media may appear only when the developmental stages are taken into account.     

新鲜、玻璃化冷冻牛卵母细胞体外受精囊胚全基因组甲基化模式初探

旨在初步分析新鲜及玻璃化冷冻牛卵母细胞体外受精囊胚全基因组甲基化模式。本研究采用单细胞全基因组甲基化测序技术(scWGMS)检测新鲜、玻璃化冷冻牛卵母细胞体外受精囊胚全基因组甲基化水平和差异甲基化区域(DMR),探讨两者之间DNA甲基化水平上的差异。结果表明,新鲜卵母细胞体外受精囊胚的整体甲基化水平显著高于玻璃化冷冻卵母细胞体外受精囊胚的整体甲基化水平(P<0.05)。采用基因本体分析(GO)和相关信号通路(KEGG)对143个DMRs分析,发现生物学过程主要显著富集在新陈代谢、生长发育、细胞定位、细胞刺激反应等,通路主要富集在生长发育、核酸结合及组蛋白乙酰化上,并筛选出几个与之相关的候选基因(FARP2、PI4KA、FAM3D、NCOR2、ZNF827等)。本研究初步发现,玻璃化冷冻牛卵母细胞体外受精囊胚的全基因组甲基化水平显著降低,且DMR区域主要集中在ATP结合、生长发育及组蛋白乙酰化,为提高玻璃化冷冻卵母细胞体外受精囊胚质量提供信息参考。     

牛不同生长发育时期卵泡酶活性及定位

应用组织化学方法观察牛卵巢卵泡中葡萄糖-6-磷酸酶(G-6-P酶)和碱性磷酸酶(AP)在不同发育时期的活性变化及其在卵泡内的定位。旨在探讨这两种酶的活性同卵泡生长发育的关系。结果表明：不同发育时期的卵泡数量与AP和G-6-P酶活性阳性反应呈极显著正相关。其中酶活性阳性反应有腔卵泡数量多于腔前卵泡；但G-6-P酶活性阴性反应卵泡显著多于阳性卵泡，说明AP活性强于G-6-P酶。腔前卵泡酶活性位置主要在颗粒细胞和卵母细胞上，而有腔卵泡酶活性位置主要在内膜细胞上。     

核移植前激活受体卵子对牛核移植卵体外发育的影响

本研究探讨了核移植前对受体卵子进行激活、细胞融合开始时间及供体受精卵细胞周期调节对核移植卵体外发育的影响。其结果显示核移植前对受体卵子激活组的细胞融合率与对照组没有差异 ,但重组胚胎的卵裂率、8～ 16细胞期胚胎及囊胚的发育率比对照组明显提高 ;核移植前激活的受体卵子分别在卵子体外成熟开始的第30h和 4 5h与供体细胞进行细胞融合 ,结果 ,30h组的细胞融合率和卵裂率与 4 5h组没有差异 ,但发育到 8～ 16细胞期及囊胚的发育率均比 4 5h的高 ;将供体受精卵用诺考达唑 (Nocodazole)处理后 ,进行核移植的结果 ,处理组的细胞融合率、卵裂率、发育到 8～ 16细胞期和囊胚的发育率与对照组无差异     

性控精液对奶牛体内胚胎质量、发育和移植妊娠率的影响

[目的] 研究性控精液对奶牛体内胚胎质量、胚胎发育和胚胎移植妊娠率的影响。[方法] 将144头青年奶牛随机分为对照组（63头）和试验组（81头）,使用促卵泡激素（FSH,260 mg/头）进行超排处理。对照组和试验组分别使用常规精液和性控精液输精,并对获得的体内性控胚胎进行移植,对胚胎生产、胚胎质量、胚胎发育和胚胎移植妊娠情况进行统计。[结果] 试验组供体获得的平均可用胚胎数（5.67枚）显著（P<0.05）低于对照组（6.92枚）;试验组供体获得的可用胚胎中A级胚胎比例（62.53%）、B级胚胎比例（35.29%）与对照组（A级胚胎比例66.51%、B级胚胎比例30.97%）相比差异均不显著（P>0.05）;试验组供体获得的可用胚胎中桑葚胚比例（84.10%）显著（P<0.05）高于对照组（61.24%）,囊胚比例（15.90%）显著（P<0.05）低于对照组（38.76%）;试验组的鲜胚移植妊娠率（52.41%）显著（P<0.05）低于对照组（66.13%）。[结论] 与常规精液相比,使用性控精液生产奶牛体内性控胚胎并移植后,平均可用胚胎数、可用胚胎中囊胚比例和胚胎移植妊娠率降低,可用胚胎质量未明显降低;优化性控精液使用方案和胚胎移植技术能够提高体内性控胚胎生产和胚胎移植效率。