Correlation between the Cell Number and Diameter in Bovine Embryos Produced In Vitro

This study was designed to examine the effects of age and developmental stage of in vitro‐produced bovine embryos on the cell number of the embryos and to investigate the correlation between the cell number and diameter in the embryos. The diameter and cell number in blastocysts and expanded blastocysts collected on days 7–9 after in vitro fertilization (IVF) were examined. Although the diameters of the blastocysts collected on days 7 and 8 after IVF were smaller than those of the expanded blastocysts collected on day 9, the cell number in both types of embryos was similar. The cell numbers of the blastocysts and expanded blastocysts decreased with increasing embryo age. There were positive correlations between the cell number and diameter in bovine embryos at each stage collected on each day after IVF. However, the value of the correlation coefficient in the day‐9 expanded blastocyst group tended to be higher than that in the other groups. These results indicate that the cell number of in vitro‐produced embryos is affected by the embryonic stage and age. The diameter of the embryo may be potentially used for the viability testing of the expanded blastocysts collected on day 9 after IVF.     

Effects of (−)‐Epigallocatechin Gallate on the Motility and Penetrability of Frozen–Thawed Boar Spermatozoa Incubated in the Fertilization Medium

Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis) and is known for its antioxidant effects. The objective of the present study was to examine the effects of EGCG during in vitro fertilization (IVF) on the sperm quality and penetrability into oocytes. In the first experiment, the effects of concentration and incubation period of EGCG on the motility and penetrability of spermatozoa were examined. When frozen–thawed spermatozoa were incubated in IVF medium supplemented with 0 (control), 1, 50 and 100 μm EGCG for 1, 3 and 5 h, supplementation with 50 and 100 μm EGCG improved motility of the spermatozoa (p < 0.05), but not viability, as compared with the control group. When frozen–thawed spermatozoa were co‐incubated with in vitro‐matured (IVM) oocytes in IVF medium supplemented with 50 and 100 μm EGCG for 5 h, supplementation of EGCG had positive effects on sperm penetration rates. In the second experiment, the effects of supplementation of EGCG in IVF medium on penetrability of sperm from different boars and development of fertilized oocytes were evaluated. When frozen–thawed spermatozoa from six boars were co‐incubated with IVM oocytes in IVF medium supplemented with 50 μm EGCG, the effect of EGCG on sperm penetration and development of oocytes after fertilization was found to vary with individual boar. Our results indicate that motility and penetrability of boar spermatozoa are improved by co‐incubation with 50 μm EGCG, but the effects vary with individual boars.     

Astaxanthin Normalizes Epigenetic Modifications of Bovine Somatic Cell Cloned Embryos and Decreases the Generation of Lipid Peroxidation

Astaxanthin is an extremely common antioxidant scavenging reactive oxygen species (ROS) and blocking lipid peroxidation. This study was conducted to investigate the effects of astaxanthin supplementation on oocyte maturation, and development of bovine somatic cell nuclear transfer (SCNT) embryos. Cumulus–oocyte complexes were cultured in maturation medium with astaxanthin (0, 0.5, 1.0, or 1.5 mg/l), respectively. We found that 0.5 mg/l astaxanthin supplementation significantly increased the proportion of oocyte maturation. Oocytes cultured in 0.5 mg/l astaxanthin supplementation were used to construct SCNT embryos and further cultured with 0, 0.5, 1.0 or 1.5 mg/l astaxanthin. The results showed that the supplementation of 0.5 mg/l astaxanthin significantly improved the proportions of cleavage and blastulation, as well as the total cell number in blastocysts compared with the control group, yet this influence was not concentration dependent. Chromosomal analyses revealed that more blastomeres showed a normal chromosomal complement in 0.5 mg/l astaxanthin treatment group, which was similar to that in IVF embryos. The methylation levels located on the exon 1 of the imprinted gene H19 and IGF2, pluripotent gene OCT4 were normalized, and global DNA methylation, H3K9 and H4K12 acetylation were also improved significantly, which was comparable to that in vitro fertilization (IVF) embryos. Moreover, we also found that astaxanthin supplementation significantly decreased the level of lipid peroxidation. Our findings showed that the supplementation of 0.5 mg/l astaxanthin to oocyte maturation medium and embryo culture medium improved oocyte maturation, SCNT embryo development, increased chromosomal stability and normalized the epigenetic modifications, as well as inhibited overproduction of lipid peroxidation.     

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。     

Effect of L‐carnitine on maturation,cryo‐tolerance and embryo developmental competence of bovine oocytes

In this study, the effects of the addition of L‐carnitine in in vitro maturation (IVM) medium for bovine oocytes on their nuclear maturation and cryopreservation were investigated; they were matured in IVM medium supplemented with 0.0, 0.3, 0.6 and 1.2 mg/mL of L‐carnitine (control, 0.3, 0.6 and 1.2 groups, respectively) and some of them were vitrified by Cryotop. Moreover, the effects of L‐carnitine during in vitro fertilization (IVF) and in vitro culture (IVC) on the developmental potential and quality of IVF embryos were also examined. A significantly higher maturation rate of oocytes was obtained for 0.3 and 0.6 mg/mL groups compared with the control (P < 0.05). The blastocyst formation rate in the 0.6 group was significantly improved, whereas the rate in the 1.2 group was significantly decreased when compared with the control group (P < 0.05). No significant difference was found in embryo development between the control and the L‐carnitine group after oocyte vitrification. Supplementation of IVF and IVC media with L‐carnitine had no effect on development to the blastocyst stage of IVM oocytes treated with 0.6 mg/mL L‐carnitine. In conclusion, the supplementation of L‐carnitine during IVM of bovine oocytes improved their nuclear maturation and subsequent embryo development after IVF, but when they were vitrified the improving effects were neutralized.     

Contrasting effects of heat shock during in vitro maturation on development of in vitro‐fertilized and parthenogenetic bovine embryos

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus–oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro‐fertilized or activated with ionomycin and cultured in vitro for 192 hr post‐in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat‐shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down‐regulated the expression of AQP3 (p < .01) and up‐regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down‐regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat‐shocked oocytes.     

Effects of Addition of Tissue‐Type Plasminogen Activator in In Vitro Fertilization Medium on Bovine Embryo Development and Quality

Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue‐type plasminogen activator (t‐PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus‐oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t‐PA and/or its inhibitor epsilon‐aminocaproic acid (control, t‐PA, t‐PA+ε‐ACA, ε‐ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t‐PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε‐ACA. PAA in the treated group was significantly reduced by ε‐ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t‐PA‐modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t‐PA. In conclusion, it appears that excessive t‐PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena.     

Ameliorative Effects of Melatonin against Hydrogen Peroxide‐Induced Oxidative Stress on Boar Sperm Characteristics and Subsequent In Vitro Embryo Development

Melatonin, the major secretory product of the pineal gland, scavenges a variety of reactive oxygen and nitrogen species in vivo and in vitro, indicating that melatonin is a potent function as an antioxidant. The objective of this study was to investigate the effect of melatonin in the presence or absence of hydrogen peroxide (H₂O₂) on sperm characteristics (motility, viability, survival rate, membrane integrity, lipid peroxidation (LPO) and mitochondria activity) and also to examine the developmental rates to the blastocysts stage of porcine oocytes fertilized in vitro with semen treated with or without melatonin (100 nm ) in the presence or absence of H₂O₂ (250 μm ). The sperm were treated with melatonin in the presence or absence of H₂O₂ for 3, 6, 9 and 12 h at 37°C and then analysed for the sperm characteristics. The porcine embryos were produced by in vitro maturation and in vitro fertilization (IVM/IVF) using semen treated with or without melatonin (100 nm ) in the presence or absence of H₂O₂ (250 μm ) for 6 h. The semen characteristics, including motility, viability, survival rate, membrane integrity and mitochondria activity, were higher in the groups that were treated with melatonin in comparison to other groups, irrespective of incubation periods. Malondialdehyde levels in control, melatonin and melatonin + H₂O₂ groups were lower than H₂O₂ only group. A positive correlation was shown among motility, viability, survival rate and membrane integrity, but a negative correlation was observed between LPO and the other evaluation methods. The developmental rates to blastocysts of IVM/IVF porcine oocytes fertilized by semen treated with melatonin were significantly increased compared with any other groups, with the cell number of blastocysts shown to have a similar trend to the developmental rates. These results demonstrate that melatonin can improve the semen characteristics during in vitro storage and support the developmental ability of IVM/IVF embryos in pigs.     

Efficient Transgene Expression in IVF and Parthenogenetic Bovine Embryos by Intracytoplasmic Injection of DNA–Liposome Complexes

Transgenic animals constitute an important tool with many biotechnological applications. Although there have been advances in this field, we propose a novel method that may greatly increase the efficiency of transgenic animal production and thereby its application. This new technique consists of intracytoplasmic injection of liposomes, in bovine oocytes and zygotes, to introduce exogenous DNA. In the first experiment, we evaluated embryo development and EGFP expression in In Vitro Fertilization (IVF) embryos injected with different concentrations of exogenous DNA–liposome complexes (0.5, 5, 50, 500 ng pCX‐EGFP/μl). The highest EGFP‐embryos rates were obtained using 500 ng pCX‐EGFP/μl. In the second experiment, we evaluated embryo development and EGFP expression following the injection of DNA–liposome complexes into pre‐fertilized oocytes and presumptive zygotes, 16 and 24 h post‐fertilization. Approximately 70% of the cleaved embryos and 50% of the blastocysts expressed EGFP, when egfp–liposome was injected 16 h post‐fertilization. The percentages of positive embryos for the 24‐h post‐fertilization and pre‐fertilization groups were 30.1 and 6.3, respectively. Blastocysts that developed from injected zygotes were analysed by PCR, confirming the presence of transgene in all embryos. Finally, we examined the embryo development and EGFP expression of parthenogenetic embryos that resulted from the injection of egfp–liposome complexes into pre‐activated oocytes, and 3 and 11 h post‐activated oocytes. The group with the highest expression rate (48.4%) was the one injected 3 h post‐activation. In summary, this study reports the efficient, reproducible and fast production of IVF and parthenogenetic embryos expressing EGFP, by the intracytoplasmic injection of liposomes to introduce the foreign DNA.     

The Effect of Long‐term In Vivo Culture in Bovine Oviduct and Uterus on the Development and Cryo‐tolerance of In Vitro Produced Bovine Embryos

The objective of this study was to compare the embryo production and quality carried out entirely in vitro or partly in vitro combined with short‐ vs long‐term in vivo culture using the homologous cattle oviduct. The IVM oocytes were in vitro fertilized and cultured for 7 and 8 days (IVP‐Group), or after IVF and 2–3 days of IVC, 4–8 cell stage embryos were endoscopically transferred into oviducts of synchronized heifers (In Vivo‐Group) or IVM oocytes were co‐incubated with spermatozoa for 3–4 h and transferred into the oviducts of synchronized heifers (GIFT‐Group). Embryos of the In Vivo‐Group and the GIFT‐Group were recovered on day 7 from the oviducts and uterine horns. Embryos of all groups were either cryopreserved at day 7 (day 7 blastocysts) or cultured in vitro in CR1aa‐medium supplemented with 5% ECS for further 24 h and cryopreserved (day 8 blastocysts). The total blastocyst yield found in the in vivo cultured groups was similar to the results of the IVP‐Group. But the appearance of blastocysts was dependent on the duration of in vivo culture. The more time the embryos spent in the in vivo environment, the more blastocysts appeared at day 8. The quality of produced blastocysts assessed by cryo‐survival was also correlated to the culture conditions; the in vivo cultured embryos showed higher cryo‐tolerance. However, the duration of in vivo culture crucially influenced the cryo‐tolerance of produced blastocysts. It is concluded that tubal access is a promising tool to provide a further basis for studying embryo sensitivity to environmental changes.     

Effects of Serum-Free Culture Media on in vitro Development of Domestic Cat Embryos Following In Vitro Maturation and Fertilization

This study was conducted to determine the adequate medium for a serum‐free culture system of domestic cat embryos produced by in vitro maturation (IVM) and fertilization (IVF). Cumulusoocyte complexes recovered from cat ovaries were matured in vitro for 24 h, and then inseminated in vitro for 12 h. After insemination, the oocytes were cultured in five media [Ham's F10, Waymouth 752/1 (Waymouth), TCM199, modified Earle's balanced salt solution (MK‐1) and CR1aa], each of which contained 0.4% bovine serum albumin. There were no significant differences among the rates of fertilization of oocytes cultured in five media following IVF. The rate of oocytes/embryos developed to at least the morula stage was significantly lower (p < 0.05) in Waymouth than in MK‐1, TCM199 and CR1aa. Moreover, none of the embryos cultured in Ham's F10 and Waymouth developed to the blastocyst stage. There were no differences among the rates of development to the blastocyst stage of oocytes/embryos cultured in MK‐1, TCM199 and CR1aa. These results indicate that the type of serum‐free medium has a major impact on in vitro development of domestic cat embryos derived from IVM/IVF, and MK‐1, TCM199 and CR1aa media are suitable for in vitro culture of cat embryos in a serum‐free culture system.     

Different Culture Media Requirements of IVF and Nuclear Transfer Bovine Embryos

Important differences exist between in vitro fertilized (IVF) and nuclear transfer (NT) bovine embryos. Studies have shown that although in vitro development is comparable, post-implantation survival is greatly reduced in NT embryos. In this study, we compare serum and bovine serum albumin (BSA) supplementation during oocyte maturation and embryo culture of IVF and NT embryos. In experiment 1, oocytes and embryos were randomly distributed into different treatment groups consisting of synthetic oviductal fluid (SOF) medium supplemented with either serum, fatty acid-free BSA (FAF) or fraction V BSA during maturation and/or culture to assess IVF embryo development. In experiment 2, oocytes were matured in SOF + serum or SOF + FAF and reconstructed embryos were cultured in SOF + FAF to assess NT embryo development. Among the IVF treatment groups, a greater number of blastocysts were observed in the steer serum (SER) group (IVM and IVC in SOF + serum) on day 6; however, no significant differences were seen in blastocyst development from day 8 onwards. Hatching frequencies on days 8 and 9 were significantly greater in groups with serum, with the exception of FAF (IVM and IVC in SOF + FAF) on day 9. For the NT treatment groups, the presence of serum during IVM resulted in a higher proportion of MII oocytes and increased blastocyst development and hatching rates were compared with supplementation of FAF. These results indicate that both serum and FAF provide comparable embryo development for IVF but not for NT bovine embryos.     

In Vitro Development of Bison Embryos Using Interspecies Somatic Cell Nuclear Transfer

Interspecies somatic cell nuclear transfer (interspecies SCNT) has been explored in many domestic and non‐domestic animal species. However, problems arise during the development of these embryos, which may be related to species‐specific differences in nuclear–cytoplasmic communication. The objectives of this study were to investigate the possibility of producing bison embryos in vitro using interspecies SCNT and assess the developmental potential of these embryos. Treatment groups consisted of cattle in vitro fertilization (IVF) and cattle SCNT as controls and wood bison SCNT, plains bison SCNT and wisent SCNT as experimental groups. Cleavage and blastocyst rates were assessed, and blastocyst quality was determined using total cell number, apoptotic incidence and relative quantification of mitochondria‐related genes NRF1, MT‐CYB and TFAM. These results indicate that embryos can be produced by interspecies SCNT in all bison species/subspecies (13.34–33.54% blastocyst rates). Although increased incidence of apoptosis was observed in bison SCNT blastocysts compared to cattle SCNT controls (10.45–12.69 vs 8.76, respectively) that corresponded with significantly lower cell numbers (80–87 cells vs >100 cells, respectively), no major differences were observed in the expression of NRF1, MT‐CYB and TFAM. This study is the first to report the production of bison embryos by interspecies SCNT. Blastocyst development in all three bison species/subspecies was greater than the rates obtained in previous studies by IVF, which supports the potential role of SCNT for in vitro embryo production in this species. Yet, further investigation of developmental competence and the factors influencing blastocyst quality and viability is required.