Excess polyspermy reduces the ability of porcine oocytes to promote male pronuclear formation after in vitro fertilization

Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = −0.560, p < 0.05) and in the sperm lot of moderate polyspermy (R = −0.405, p < 0.05) which suggests that penetration of excessive spermatozoa disables the oocyte cytoplasm to promote MPN formation.     

Generation of rats from vitrified oocytes with surrounding cumulus cells via in vitro fertilization with cryopreserved sperm

The objective of this study was to evaluate fertility and full‐term development of rat vitrified oocytes after in vitro fertilization (IVF) with cryopreserved sperm. Oocytes with or without surrounding cumulus cells were vitrified with 30% ethylene glycol + 0.5 mol/L sucrose + 20% fetal calf serum by using the Cryotop method. The warmed oocytes were co‐cultured with sperm. Although the denuded/vitrified oocytes were not fertilized, some of the oocytes vitrified with cumulus cells were fertilized (32.7%) after IVF with fresh sperm. When IVF was performed with cryopreserved sperm, vitrified or fresh oocytes with cumulus cells were fertilized (62.9% or 41.1%, respectively). In addition, to confirm the full‐term development of the vitrified oocytes with surrounding cumulus cells after IVF with cryopreserved sperm, 108 vitrified oocytes with two pronuclei (2PN) were transferred into eight pseudopregnant females, and eight pups were obtained from three recipients. The present work demonstrates that vitrified rat oocytes surrounded by cumulus cells can be fertilized in vitro with cryopreserved sperm, and that 2PN embryos derived from cryopreserved gametes can develop to term. To our knowledge, this is the first report of successful generation of rat offspring derived from vitrified oocytes that were fertilized in vitro with cryopreserved sperm.     

Effects of cumulus cells on in vitro maturation of oocytes and development of cloned embryos in the pig

The purpose of this study was to investigate the role of porcine cumulus cells (CC) in oocyte maturation and somatic cell nuclear transfer (SCNT) embryo development in vitro. Denuded pig oocytes were co-cultured with CC or routinely cultured in maturation medium without a feeder layer. Porcine CC inactivated with mitomycin C or non-inactivated were used for the feeder layer in co-culture with porcine SCNT embryos to investigate comparatively the developmental competence of cloned embryos. The DNA damage aspects of apoptosis and expression pattern of genes implicated in apoptosis (Fas/FasL) as well as the mRNA expression of DNA methyltransferase (Dnmt1, Dnmt3a) of porcine SCNT embryos were also evaluated by comet assay or real-time RT-PCR, respectively. The results showed that co-culture with CC improved the extrusion rate of pbI (49.3% vs 31.5%, p<0.05) and survival rate (75.7% vs 53.3%, p<0.05) of denuded oocytes, but had no effects on blastocyst developmental rate or 2-cell-stage survival rate of in vitro fertilization embryos. Co-culture with CC inactivated by mitomycin C improved the blastocyst developmental rate (26.6% vs 13.0%, p<0.05) and decreased the apoptotic incidence (27.6% vs 46.2%, p<0.05) of porcine cloned embryos. Co-culture with inactivated CC reduced Fas and FasL mRNA expression of cloned embryos at the blastocyst stage compared with NT controls (p<0.05), but there were no differences in Dnmt1 and Dnmt3a mRNA expression among groups. Co-culture with inactivated cumulus cell monolayer significantly increased blastocyst formation and decreased the apoptotic incidence in porcine cloned embryos during in vitro development.     

Effect of vitrification procedures on the subsequent development of in vitro matured swamp buffalo oocytes following in vitro fertilization

Nowadays, the efficiency of buffalo oocytes cryopreservation is still low. The purpose of this study was to evaluate effects of two combinations of cryoprotectant agents (CPAs) and two vitrification devices for vitrification of swamp buffalo oocytes on their survival after vitrification warming, and subsequent developmental ability after in vitro fertilization. In vitro matured (IVM) oocytes were vitrified by either Cryotop (CT) or solid surface vitrification (SSV) interacting with vitrification solution A (VA) or B (VB). In the VA or VB solution exposed test, the oocytes showed similar survival rates, but decreased blastocyst rates after in vitro fertilization compared with that of untreated oocytes. After vitrification, the CT method combined with VA solution yielded a higher survival rate (91.3 ± 5.84%) of vitrified oocytes than that combined with VB solution (69.8 ± 4.19%–75.8 ± 4.55%); however, all the vitrification treatments showed lower blastocyst rates (1.1 ± 0.07%–5.2 ± 0.24%) compared with that of untreated oocytes (18.0 ± 1.09%). Our results indicated that combined vitrification treatments in this study did not improve the decreased ability of vitrified oocytes developing to the blastocyst stage.     

树鼩卵母细胞的体外成熟培养及体外受精

利用树鼩卵母细胞的成熟培养、体外受精等方法,生产树鼩的试管婴儿,期望通过此路径来实现生产转基因树鼩动物模型。使用促性腺激素进行树鼩超数排卵、TCM199完全培基对卵母细胞进行体外成熟培养、卵母细胞与体外获能的附睾精子进行体外受精、受精卵发育至桑椹胚、囊胚的实验。结果表明,卵母细胞成熟率A级76.7%、B级55.01%、C级17.39%。受精率52%,受精卵采用体细胞共培养与非共培养的分裂率分别为37.14%和9.6%。桑椹胚/囊胚发育率分别为13.57%和0,(P<0.05)。树鼩卵母细胞的成熟培养、体外受精实验方法可行,受精卵在体外能发育到囊胚阶段。     

影响黄牛小腔卵泡卵母细胞体外受精及早期胚胎发育的因素

从屠宰场收集黄牛卵巢,取皮质深层卵母细胞进行体外成熟、体外受精和早期胚胎体外培养,分析了影响其效果的因素。结果表明,在成熟培养液中添加FSH(10IU/mL)、HCG(20IU/mL)和17β-E2(1mg/L)对卵母细胞受精后早期胚胎发育能力有极显著促进作用;等量牛卵泡液(BFF)与新生牛血清(NCS)对体外受精胚胎发育效果影响不显著,以15?F为宜;颗粒细胞与输卵管上皮细胞均能显著提高卵母细胞体外成熟受精后早期胚胎的发育率,颗粒细胞 输卵管上皮细胞对克服胚胎阻滞现象效果显著。     

罗同丘细胞对小鼠卵母细胞体外成熟及其后续发育的影响

观察了卵丘细胞共培养对小鼠生发泡期部分裸露（PNO）和裸卵（NO）成熟和发育能力的影响；分别用小鼠、大鼠、猪的卵丘细胞与小鼠PNO和NO进行了共培养。检测了小鼠PNO和NO的减数分裂能力、生发泡构型、受精和胚胎发育能力。结果，PNO、NO的减数分裂能力显著（P〈0．05）低于卵丘完整复合体（COCs）的减数分裂能力。COCs中SN型卵母细胞比率显著高于PNO和NO中SN型卵母细胞比率（P〈0．05），大部分PNO和NO的卵母细胞核型为NSN型。与对照组相比，与小鼠或大鼠卵丘细胞共培养的小鼠PNO和NO的减数分裂能力没有显著提高，但是与猪卵丘细胞共培养却可以提高小鼠PNO和NO的减数分裂能力。结果表明，猪卵丘细胞能促进小鼠卵母细胞的体外成熟，但并不影响小鼠卵母细胞的受精和胚胎发育。     

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.     

Embryo development of porcine oocytes after injection with miniature pig sperm and their extracts

This study examined embryo development of porcine oocytes after microinjection of sperm extracts (SE) in porcine intracytoplasmic sperm injection (ICSI). SE was prepared from miniature pig sperm by a nonionic surfactant, and various concentrations (0.02, 0.04 and 0.08 mg/mL) of SE were injected into the matured oocytes with a first polar body. In the pronuclear stage, the rate of oocytes with two pronuclei and a second polar body (21.4%) in the sperm and SE (0.04 mg/mL) injection group was significantly higher (P < 0.05) compared to other groups. The rate of 2–4‐cell stage in sperm and SE (0.04 mg/mL) injection group was 38.1%, and it was significantly higher than that in the sperm injection group (22.9%). The rate of blastocyst stage in sperm and SE (0.04 mg/mL) injection group was 21.4%, the value was significantly higher than those in SE (0.08 mg/mL) injection group (0%), sperm injection group (5.7%), and sperm and SE (0.08 mg/mL) injection group (2.6%). These results suggest that SE induces activation of porcine oocytes and their further embryonic development, and that SE is effective for porcine ICSI.     

In vitro maturation and fertilization of prepubertal and pubertal black Bengal goat oocytes

Oocytes retrieval, in vitro maturation (IVM) and fertilization (IVF) efficiency are inevitable steps towards in vitro production of embryos. In the present study, these parameters were investigated in the ovaries of prepubertal (n = 31) and pubertal (n = 61) black Bengal goats obtained from a slaughterhouse. Nuclear maturation was evaluated upon aspiration and following IVM in TCM-199 (Earle''s salt with L-glutamine and sodium bicarbonate) for 27 h at 39℃ under 5% CO₂ in humidified air. The oocytes retrieval and efficiency (mean ± SD) per prepubertal and pubertal goats were 5.2 ± 0.6 and 6.8 ± 0.6, and 77.3 ± 0.1% and 80.5 ± 0.6%, respectively. Anaphase I - telophase I stages differed significantly (7.3 ± 0.8 vs. 2.6 ± 0.2, p < 0.05) between the two groups of goats. After IVM, the percentages of metaphase II were significantly higher (66.3 vs. 60.3, p < 0.05) in pubertal goats than in their prepubertal counterparts. The percentages of normal in vitro fertilization (IVF) in Fert-Tyrode''s albumin lactate pyruvate of pubertal goat oocytes did not differ between Percoll and swim-up sperm separation methods (36.7 ± 0.9% vs. 32.7 ± 1.3%, p > 0.05). Furthermore, sperm capacitation by heparin alone or in combination with ionomycin did not lead to a significant increase in the normal fertilization rate (34.8 ± 1.7 vs. 32.2 ± 1.5%, respectively) in the oocytes of pubertal goats. In conclusion, the ovaries of pubertal black Bengal goats obtained from the slaughterhouse could be used for in vitro embryo production. However, further optimization of the IVM and IVF techniques are necessary for satisfactory in vitro embryo production.     

单层细胞共培养对猪去卵丘卵母细胞体外成熟和孤雌发育的影响

本试验比较观察第一极体(The first polar body,PbⅠ)、Oosight imaging system观察和hocchst33342染色法对第2次减数分裂中期(Metaphase Ⅱ,MⅡ)卵母细胞判定结果的相关性分析,并探讨卵巢皮质细胞(porcine ovarian cortex cells,pOCCs)、猪输卵管上皮细胞(porcine oviductal epithelial cells,pOECs)和猪卵丘颗粒细胞(porcine cumulus cells,pCCs)等3种单层细胞体外共培养体系对猪去卵丘卵母细胞(cumulus cells denuded oocytes,Dos)体外成熟(in vitro maturation,IVM)和孤雌发育的影响。结果显示:(1)Oosight imaging system判定卵母细胞成熟的结果与hocchst33342染色法判定的结果有很强相关性(R=0.973,P<0.01,N=90);(2)pOCCs单层细胞共培养体系中猪去卵丘卵母细胞成熟率显著高于pOECs((52.5±0.30)%vs(43.8±2.18)%,P<0.05),且...     

诱导荷斯坦犊牛卵泡发育及体外受精研究

从日龄、培养条件和供体个体差异3个方面对4~9周龄犊牛JIVET(juvenile in vitro embryo transfer)技术的影响进行了初步研究。试验结果如下:不同日龄的10头母犊激素诱导后,30~40 d母犊平均获卵数为(31.8±9.75)枚,46~63 d的母犊平均获卵数为(21.8±11.67)枚,二者差异不显著(P>0.05);犊牛胚胎在3气(5%CO2,5%O2,90%N2)条件下和与颗粒细胞共培养条件(5%CO2,95%空气)下卵裂率分别为48.5%和54.1%,二者差异不显著(P>0.05),但是桑葚胚率差异显著(0%,15.2%,P<0.05);供体间个体差异很大,70%(7/10)的个体对激素诱导反应良好。     

Breed influences on in vitro development of abattoir-derived bovine oocytes

Background

There is a discrepancy in the reproductive performance between different cattle breeds. Using abattoir-derived ovaries and data base information we studied the effects of breed on in vitro fertilization and early embryo development.

Methods

The in vitro developmental competence of oocytes from cattle (n = 202) of Swedish Red (SR), Swedish Holstein (SH) and mixed beef breeds was compared, retrospectively tracing donors of abattoir-derived ovaries using a combination of the national animal databases and abattoir information. Age was significantly lower and carcass conformation score was higher in the beef breeds than in the dairy breeds.Cumulus oocyte complexes (n = 1351) were aspirated from abattoir-derived ovaries from animals of known breed (visual inspection confirmed through databases), age (databases), and abattoir information. Oocytes were matured, fertilized (frozen semen from two dairy bulls) and cultured according to conventional protocols. On day 8, blastocysts were graded and the number of nuclei determined.

Results

Cleavage rate was not different between the breeds but was significantly different between bulls. The percentage of blastocysts on day 8 was significantly higher when the oocyte donor’s breed was beef or SR than SH. There was no significant difference in blastocyst grades or stages between the breeds, but the number of nuclei in day 8 blastocysts was significantly lower in SH compared to the beef.

Conclusions

The use of abattoir-derived ovaries from animals whose background is traceable can be a valuable tool for research. Using this approach in the present study, oocyte donor breed was seen to affect early embryo development during in vitro embryo production, which may be a contributing factor to the declining fertility in some dairy breeds seen today.     

Effect of cumulus cell removal and sperm pre‐incubation with progesterone on in vitro fertilization of equine gametes in the presence of oviductal fluid or cells

In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal cells during IVF helps to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm pre‐incubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm pre‐incubation with progesterone. In experiments 1 and 2, IVF was performed in the presence of porcine oviduct epithelial cells. The removal of cumulus cells from equine oocytes after in vitro maturation tended to increase the percentage of fertilization when fresh sperm was used (1/33 vs. 4/31, p > 0.05) but had no effect when frozen sperm was used (1/32 vs. 1/32). Equine sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/14 vs. 2/18 for fresh, 1/29 vs. 1/25 for frozen). In experiments 3 and 4, IVF was performed after pre‐incubation of sperm with porcine oviductal fluid. The removal of cumulus cells tended to increase the percentage of fertilization when fresh sperm was used (1/24 vs. 3/26, p > 0.05). Sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/39 vs. 2/36 for fresh, 2/37 vs. 1/46 for frozen), but two 3–4 cell stage zygotes were obtained with fresh sperm pre‐incubated with progesterone. This is an encouraging result for the setting up of an efficient IVF procedure in equine.     

Effects of exposure to zearalenone on porcine oocytes and sperm during maturation and fertilization in vitro

The influence of acute exposure to zearalenone (ZEN) on porcine oocyte maturation, fertilization or sperm penetration ability during both in vitro maturation and fertilization was evaluated. First, oocytes were cultured in ZEN-containing (0-1000 μg/l) maturation medium and then fertilized. The oocytes maturing in vitro without ZEN were then fertilized in ZEN-containing fertilization medium. The maturation rates of oocytes and penetration ability of sperm decreased significantly in the presence of 1000 μg/l of ZEN. However, neither increases in the rates of degeneration and DNA fragmentation of oocytes nor reductions in normal and polyspermic fertilization were observed. ZEN did not affect the sperm penetration rates; however, 1000 μg/l ZEN had positive effects on normal and polyspermic fertilization rates. Therefore, it can be suggested that an acute exposure of porcine oocytes during maturation and of oocytes and sperm during fertilization to ZEN up to 1000 μg/l may not affect the fertility of the oocytes.     

受精液和受精时间对牦牛卵泡卵母细胞体外受精的影响

利用屠宰牦牛卵巢，抽取其表面2～5mm的卵泡内卵母细胞，经体外成熟后分别用BO液和改良Tyrode’S液进行体外受精研究。结果表明，BO液受精6h和改良Tyrode’s液分别受精6h和18h，牦牛体外受精卵的卵裂率差异不显著（分别为52．48％、47．67％和50．00%,P〉0．05）。它们的4细胞发育率分别为75．47％、78．05％和64．10％，8细胞发育率分别为56．60％、56．10％和48．72％，使用改良Tyrode’s液受精18h的发育率最低，与其他2组相比，差异极显著（P〈0．01）；而受精时间同为6h时，2种受精液之间发育率的差异不显著（P〉0．05）。     

猪精子及卵子质量对猪体外受精胚胎发育的影响

应用体外受精(in vitro fertilization, IVF)等生物技术可以缩短种猪培育时间,地方猪精液的冷冻保存可以解决保存和运输问题。通过对不同个体冷冻前后的精子质量、获能方式、卵子质量及多精受精率对IVF的影响进行了研究。结果显示:当精子活率从83.26%降至74.20%时,IVF的卵裂率、桑椹胚率显著下降(P0.05)。不同的获能方式对IVF胚胎的卵裂率无显著影响(P0.05)。3层(及以上)卵丘细胞的卵子IVF的卵裂率、桑椹率显著高于3层以下组(P0.05)。冷冻精子的IVF中,2-细胞、4-细胞和囊胚的发育能力显著低于新鲜精子(P0.05),受精后6～12 h的多精受精率也显著高于新鲜精子组(P0.05)。猪体外受精胚胎的发育能力受精子和卵母细胞的影响。无论使用新鲜精液还是冷冻精液的IVF胚胎,在桑椹胚期后的发育能力都会受到阻碍,多精受精可能是原因之一,但具体原因还需要更多的研究。