Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos

Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA‐fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7–5.4%) of DNA‐fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.     

Effect of Lycopene on Cytoplasmic Maturation of Porcine Oocytes In Vitro

The aim of the present study was to improve cytoplasmic maturation of porcine oocytes by the addition of lycopene into in vitro maturation (IVM) media. We designed six experimental groups; IVM medium was supplemented with 10 IU/ml FSH, FSH and 10 IU/ml human chorionic gonadotrophin (hCG), or FSH and 7 μm lycopene in the first half of the IVM culture (0–22 h) followed by further culture (22–44 h) with or without hCG. The addition of lycopene into IVM media delayed the interruption of communication between an oocyte and the cumulus cells. Although meiotic competence was similar among the six groups, the glutathione level of matured oocytes was significantly higher in the lycopene‐supplemented group (9.89 pmol per oocyte) than that in other groups (7.25 and 7.81 pmol per oocyte). Fertilization rate was significantly improved in lycopene‐supplemented groups (58.3%) more than that in the group supplemented with FSH only (43.1%), whereas there were no differences in developmental competence among the groups (blastocyst rate: 20.1–29.5%). These results indicate that insufficient cytoplasmic maturation during conventional IVM resulted by disconnection of the gap junction between an oocyte and the cumulus cells in the early phase during IVM culture. We concluded that lycopene induced a prolonged sustainment of gap junctional communication between an oocyte and the cumulus cells during porcine IVM culture, which was an effective cytoplasmic maturation of porcine IVM oocytes.     

猪卵泡卵母细胞的体外成熟与体外受精

本试验对猪卵泡卵母细胞不同体外成熟培养时间、不同精子获能时间、不同精卵共孵育时间对体外受精的影响进行了研究。结果表明,体外成熟培养44 h左右,精子获能时间在1～2 h之间,精卵共孵育时间在6～8 h之间,受精后卵裂率最高。     

不同成熟时间的猪卵母细胞对体外受精的影响及孤雌发育

采用NCSU-37为体外成熟、受精、培养体系,比较不同成熟时间46h、58h和70h的猪卵母细胞对体外受精的影响和孤雌发育。结果显示,猪卵母细胞在体外成熟培养46h、58h和70h后,46h培养组的卵母细胞体外受精后的卵裂率明显低于58h和70h培养组(P<0.05),但囊胚发育率明显高于其他两组(P<0.05)。46h组的孤雌发育率明显高于其他两组(P<0.05),囊胚发育率三组之间无显著差异。表明猪卵母细胞体外成熟培养时间延长,体外受精后的囊胚发育率降低,孤雌发育率相应增加。     

Sericin Accelerates the Production of Hyaluronan and Decreases the Incidence of Polyspermy Fertilization in Bovine Oocytes During In Vitro Maturation

Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes.     

Influence of Cysteamine on In Vitro Maturation, In Vitro and In Vivo Fertilization of Equine Oocytes

The effect of cysteamine on in vitro nuclear and cytoplasmic maturation of equine oocytes collected by transvaginal ultrasound guided follicular aspiration was assessed. Oocytes were matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining, penetration rates after two different in vitro fertilization (IVF) techniques (IVF media with ionophore and Hepes buffer with heparin) and the embryo yield following oocyte intra-oviductal transfer were used as a criterion for assessing nuclear and cytoplasmic maturation, respectively. Contrary to the data described in other domestic species, there was no effect of cysteamine on in vitro nuclear maturation, IVF or in vivo embryonic development under our conditions. Ovum pick up yields (52%) and maturation rates (control group: 47% and cysteamine group: 55%) were similar to those previously reported. From 57 oocytes transferred to the oviduct in each group, the number of embryos collected was 10 (17%) in the control group and five in the cysteamine group (9%). Those two percentages were not statistically different (p > 0.05). No effect of IVF technique was seen on the success rate (6%) in each group.     

促卵泡素与促黄体素对猪卵母细胞体外成熟的影响

本试验探讨了促性腺激素（FSH、LH）对猪卵母细胞体外成熟的影响,最终找到了其合理的使用剂量。在成熟液中添加LH浓度分别为1、5、10IU／mL时,与没有添加FSH与LH的对照组（15．9％）相比,成熟率有显著提高,成熟率分别为49．1％、30．0％、36．3％。LH浓度为1IU,mL的实验组成熟率最高,显著高于对照组（P〈0．05）;然后以成熟液中只添加1IU/mLLH为对照组,试验组分别添加1、5、10IU/mLPSH。试验结果表明：添加FSH浓度为5和10IU/ml时其成熟率分别为40．1％、27．6％,低于对照组（49．0％）。添加FSH浓度为1IU／mL时其成熟率（49．1％）高于对照组（49．0％）,但差异不显著（P〉0．05）。两组试验同时得出,随着LH与FSH添加浓度的增加,成熟率呈下降趋势。通过本试验结果得出,在体外成熟培养猪卵母细胞时,各添加1IU／mL LH与FSH时,得到最佳的成熟效果。     

促卵泡素与促黄体素对猪卵母细胞体外成熟的影响

本试验探讨了促性腺激素(FSH、LH)对猪卵母细胞体外成熟的影响,最终找到了其合理的使用剂量。在成熟液中添加LH浓度分别为1、5、10 IU/mL时,与没有添加FSH与LH的对照组(15.9%)相比,成熟率有显著提高,成熟率分别为49.1%、30.0%、36.3%。LH浓度为1 IU/mL的实验组成熟率最高,显著高于对照组(p<0.05);然后以成熟液中只添加1 IU/mLLH为对照组,试验组分别添加1、5、10 IU/mLFSH。试验结果表明:添加FSH浓度为5和10 IU/ml时其成熟率分别为40.1%、27.6%,低于对照组(49.0%)。添加FSH浓度为1 IU/mL时其成熟率(49.1%)高于对照组(49.0%),但差异不显著(p>0.05)。两组试验同时得出,随着LH与FSH添加浓度的增加,成熟率呈下降趋势。通过本试验结果得出,在体外成熟培养猪卵母细胞时,各添加1 IU/mL LH与FSH时,得到最佳的成熟效果。     

Effect of Porcine and Ovine FSH on Nuclear Maturation of Pig Oocytes In Vitro

The effect of porcine or ovine FSH on the maturation rate of porcine oocytes and on the time course of meiotic progression was studied. Groups of 20 grade‐A cumulus oocyte complexes, aspirated from slaughterhouse cycling‐gilt ovaries, were cultured in vitro in 400 μl of Modified Parker's Medium supplemented with oestrous cow serum and porcine FSH (Folltropin®‐V, 0.50 mg/ml) or ovine FSH (Ovagen^TM, 0.44 iu/ml), in four‐well dishes under mineral oil, at 38.5°C, 5% CO₂ in humidified air. At the end of each 3‐h interval, from 3 to 42 h of culture, the nuclear status of oocytes was assessed microscopically (1000×), after fixation (methanol/acetic acid: 3/1) and orcein (2%) staining. Oocytes were classified as (i) immature (IMM), i.e. oocytes at germinal vesicle stage, germinal vesicle break down and prophase I, (ii) metaphase I (MI) and (iii) metaphase II (MII), i.e. oocytes at anaphase I, telophase I and metaphase II. Data were analysed using regression analysis, chi‐square and t‐test. Nuclear status was assessed in 1610 oocytes (porcine FSH: 787, ovine FSH: 823). Most of the oocytes were at MI from 24 to 33 h (porcine FSH 60.27%, ovine FSH 42.80%, p < 0.001) and at MII from 36 to 42 h (porcine FSH 80.38%, ovine FSH 67.45%, p < 0.01) of culture. Significantly higher maturation rate was observed in porcine FSH than in ovine FSH treated oocytes (86.69 ± 12.97%, 71.34 ± 9.86%, mean ± SD, p < 0.05), after 42 h of culture. In conclusion, under the specific culture conditions, porcine FSH seems to support pig oocyte maturation better than ovine FSH.     

Hanging Drop Monoculture for Selection of Optimal Antioxidants During In Vitro Maturation of Porcine Oocytes

We analysed the effect of three antioxidants that have different functional mechanisms on the in vitro maturation (IVM) of porcine oocytes. Single oocyte monoculture using the hanging drop (HD) system has some advantages such as improving analysis efficiency brought by the smaller number of samples than the number of oocytes cultured in one drop. Direct effects of ligands on single oocytes could also be detected without considering the effects of paracrine factors from other oocytes. After 22 h of pre‐culture, denuded oocytes were cultured for 22 h with 0.01 and 0.1 μg/ml of L‐carnitine (LC), lactoferrin (LF) or sulforaphane (SF) in the presence/non‐presence of oxidant stress induced by H₂O₂ supplementation to evaluate the reducing effects against oxidative stress on nuclear maturation. As a result, compared with LC and SF, LF showed effective reduction in oxidative stress at a lower concentration (0.01 μg/ml), suggesting that LF is a more effective antioxidant in porcine oocyte IVM. Additionally, LF also increased maturation rate even in culture without H₂O₂. Our results clearly suggest that the HD monoculture system is useful for screening the substances that affect porcine oocyte culture.     

Effect of the Removal of Cumulus Cells on the Nuclear Maturation, Fertilization and Development of Porcine Oocytes

The present study was conducted to investigate the effects of attachment of cumulus cells to porcine oocytes during the process of maturation and fertilization on the nuclear maturation, fertilization and subsequent development after in vitro fertilization (IVF). In the first experiment, the cumulus cells were removed from cumulus-oocyte complexes (COCs) at 0, 24 and 42 h after the onset of maturation culture and were then cultured until reaching 42 h of cultivation. In the second experiment, COCs were denuded as described in the first experiment, then fertilized and cultured for 7 days. As a control, cumulus cells were allowed to maintain attachment to the oocytes until the end of IVF. The proportion of oocytes reaching metaphase II significantly increased with the delay in the removal treatment of cumulus cells. The proportion of normal fertilization gradually increased with delay in the removal treatment of cumulus cells from COCs until the end of IVF. However, no significant difference in the proportion of normal fertilization was found between the 42-h and control groups. The removal treatment of cumulus cells in the 0- and 24-h group significantly (p < 0.05) decreased the proportion of cleaved embryos when compared with the control, and none of them developed to the blastocyst stage. The proportion of development to the blastocyst stage was significantly higher (p < 0.05) in the control group than in the 42-h group (18.1% vs 12.4%; p < 0.05). The present study indicates that the attachment of cumulus cells to the oocyte during maturation and fertilization is important to support oocyte nuclear maturation, fertilization and subsequent embryo development. Particularly, the attachment of cumulus cells to the oocyte during IVF promotes embryonic development.     

不同浓度CO2对牛卵母细胞体外成熟和受精的影响

对牛卵巢卵泡内卵母细胞在不同浓度的CO2培养箱内体外成熟、体外受精后的卵裂率及胚胎体外发育进行了研究。结果显示,含5%小牛血清(CS)的TCM-199内的卵母细胞在2%CO2条件下培养20h后,达到第二次减数分裂期(MⅡ期)的卵子数明显高于在5%CO2条件下培养的卵母细胞数(P<0.05)。两种浓度的CO2条件下培养的牛卵母细胞体外受精后的卵裂率无明显差异,2%CO2浓度下培养的卵母细胞的囊胚发育率高于5%CO2浓度下培养的卵母细胞。     

FSH 、LH对猪卵母细胞核成熟及体外成熟时间对去核效率的影响

克隆动物(核移植)的原理就是将卵裂球或体细胞核移人去核的卵母细胞或受精卵中,采用各种激活方法使之激活,发生卵裂,移植受体后产生个体。猪的体细胞克隆效率与其他动物(牛、羊)相比较低,需     

RFRP-3对猪卵母细胞体外成熟的影响

旨在研究RFRP-3对猪卵母细胞体外成熟的影响。本研究采用从屠宰场收集健康母猪的卵巢中挑取的GV期卵母细胞,随机分为3组,在培养基中分别添加0、10^-6和10^-8mol·L^-1 RFRP-3培养猪卵母细胞44 h后统计各组卵母细胞的成熟率;在后续试验中将收集到的卵母细胞随机分为2组,在培养基中分别添加0和10^-8 mol·L^-1 RFRP-3培养猪卵母细胞44 h,观察猪卵母细胞的卵丘扩展情况并计算各组的卵丘扩展指数和各组卵母细胞的成熟率;利用qRT-PCR检测卵丘扩展因子(PTGS2、HAS2、PTX3)、卵母细胞分泌因子(GDF9、BMP15)和周期蛋白相关基因(CCNB1和CDK1)的表达变化;利用ELISA试剂盒检测MPF和cAMP的含量;采用放射免疫法检测孕酮和雌激素的浓度,每组卵母细胞量不少于100枚,每个试验重复3次。结果表明,与对照组相比,添加10^-8mol·L^-1 RFRP-3培养猪卵母细胞可极显著降低卵母细胞的成熟率(P<0.01);通过显微镜观察并计算卵丘扩展指数发现,试验组中卵丘扩展无明显变化(P>0.05),但卵丘扩展因子(PTGS2、HAS2、PTX3)的表达极显著下降(P<0.01);RFRP-3可以极显著降低猪卵母细胞MPF的含量(P<0.01),对cAMP的含量无显著影响(P>0.05);添加RFRP-3可促进GDF9(P<0.01)和BMP15(P<0.05)的表达,抑制CCNB1(P<0.05)和CDK1(P<0.05)的表达;同时试验组培养基中孕酮、雌激素的浓度也极显著下降(P<0.01)。综上,RFRP-3通过调控猪卵母细胞成熟相关因子和卵丘扩展因子的表达以及类固醇激素的分泌,从而抑制卵母细胞的体外成熟。本研究为阐明RFRP-3对哺乳动物卵母细胞的调控作用奠定理论基础。     

不同因素对猪卵母细胞体外成熟培养的影响

本试验旨在探讨不同外源激素组合的添加、胰岛素—转铁蛋白—亚硒酸钠(ITS)的添加、不同培养皿、石蜡油的添加对卵母细胞体外成熟培养(IVM)的影响。结果显示:①孕马血清促性腺激素+人绒毛膜促性腺激素+促卵泡素(PMSG+HCG+FSH)组高于促卵泡素+促黄体素(FSH+LH)组和尿促性腺激素(hMG)组,相互之间差异显著(80.1%、68.3%、53.3%,P<0.05);②添加1% ITS到猪卵母细胞培养液中对卵母细胞成熟率无显著提高(P>0.05),但显著提高了孤雌激活后孤雌胚胎的卵裂率和囊胚率(63.3%、55.1%,18.7%、12.1%,P<0.05);③凹槽皿组猪卵母细胞成熟率显著高于四孔板和30 mm塑料皿组(73.3%、68.0%、68.3%,P<0.05);④石蜡油的添加对卵丘细胞扩展和卵母细胞体外成熟率均有显著提高(84.8%、69.9%,P<0.05)。结果表明培养液中添加PMSG+HCG+FSH和ITS及选用凹槽皿、成熟培养液上覆石蜡油可提高猪卵母细胞IVM效果。     

半胱氨酸对猪卵母细胞体外成熟、体外受精及GSH水平的影响

体外受精技术也称胚胎生产技术（invitro production,IVP）,包括卵母细胞的体外成熟、精子的获能、卵子的体外受精和受精卵的体外培养等几个连续的过程。半胱氨酸是GSH的前体物质,在牛体外胚胎生产过程中疏基复合物能增加细胞内GSH的合成,在受精和胚胎的开始阶段保持高浓度GSH可以增加发育率。试验的目的是评价半胱氨酸对猪卵母细胞的成熟率、受精率及成熟培养时期卵母细胞内GSH水平的影响,现报道如下。     

Phosphofructokinase and Malate Dehydrogenase Participate in the In Vitro Maturation of Porcine Oocytes

Oocyte maturation depends on the metabolic activity of cumulus–oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2‐oxoglutarate (5, 10 and 20 mm ) or hydroxymalonate (30, 60 and 100 mm ) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10^?5 and (2.54 ± 0.32) 10^?5, and for MDH, the U were (4.72 ± 0.42) 10^?5 and (4.38 ± 0.25) 10^?5 for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10^?3 and (0.94 ± 0.12) 10^?3, and for MDH (9.08 ± 0.93) 10^?3 and (1.89 ± 0.10) 10^?3 for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2‐oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.     

Energy Status Characteristics of Porcine Oocytes During In Vitro Maturation is Influenced by Their Meiotic Competence

The characteristics of energy status in porcine oocytes as related to their meiotic competence and in vitro maturation were studied. Cycling pubertal gilts in the early luteal to early follicular phases of the ovarian cycle were used as oocyte donors. The oocytes recovered from medium (MF) or small follicles (SF) were considered meiotically more or less competent, respectively. A half of oocytes from each category was matured by the standard protocol. The oocytes were examined before or after maturation by confocal microscopy, a bioluminescent cell assay and Western blotting. Four experiments, each in triplicate, were performed to assess both SF and MF oocytes in terms of metabolic units formed by mitochondria and lipids, ATP and lipid consumption and lipid droplets with adipose differentiation‐related protein (ADRP) expression. The proportion of oocytes with metabolic units, the mean ATP content and the number of lipid droplets per oocyte, and the relative number of lipid droplets with ADRP expression were significantly higher in the MF compared to SF oocytes before maturation. On the other hand, after maturation, there was an increase in the proportion of oocytes with metabolic units and the relative number of lipid droplets with ADRP expression in the SF compared to MF oocytes. In conclusion, specific differences in energy characteristics between porcine oocytes with different meiotic competence were found. Meiotically more competent oocytes are more advanced in terms of energy reserves before maturation, while meiotically less competent oocytes are more active in replenishing energy stores during maturation.     

In Vitro Fertilization and Development of Porcine Oocytes Matured in Follicular Fluid

This study was conducted to assess the fertilization and development of porcine oocytes matured in a solo follicular fluid (pFF) using different in vitro culture systems and insemination periods. Cumulus-oocyte complexes (COCs), follicular cells (FCs), and pFF were collected from the follicles of ovaries. The pFF was used as a maturation medium (MpFF) after supplementation with follicle stimulating hormone (FSH) and antibiotics. The COCs were matured in a 15 ml test tube containing 3.5 ml of MpFF with FCs (5.2 × 10⁶ cells/ml; rotating culture system) or 2 ml of MpFF without FCs in a 35-mm petri dish (static culture system) for 44 to 48 h. After maturation culture, oocytes were co-incubated with frozen-thawed spermatozoa for 5 h and then cultured for 7 days. The total mean rates of sperm penetration, normal fertilization, male pronucleus (MPN) formation, cleavage, and development to the blastocyst stage of oocytes after insemination were significantly higher (P<0.01) in the rotating culture system than in the static culture system. In conclusion, compared with the static culture system, the rotating culture system is adequate for the production of developmentally competent porcine oocytes when MpFF is used as a maturation medium.