Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O2 compared to 5% O2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O2 tension, whereas IVD101 supported blastocyst formation only under low O2 levels but enhanced the proliferation of ICM cells.     

Culture of Bovine Embryos in Polyester Mesh Sections: The Effect of Pore Size and Oxygen Tension on In Vitro Development

The purpose of this study was to assess the feasibility of polyester mesh culture for the in vitro production of bovine embryos, as polyester mesh is an alternative way for tracking individual embryos throughout culture using time‐lapse cinematography (TLC). Bovine embryos were isolated during in vitro culture using sections of three different polyethylene terephthalate (PET) mesh products. In vitro matured and fertilized bovine oocytes were cultured in the 217 × 217, 230 × 230 or 238 × 238‐μm openings of PET mesh sections or in simple micro‐drops (control) for 7 days under either 20% or 5% O₂ tensions. No difference in embryo developmental rates was found between the culture groups in terms of cleavage, blastocyst formation and blastocyst expansion irrespective of O₂ tension. In contrast, under 20% O₂ tension, blastocysts that developed in PET mesh with 217 × 217‐μm opening had significantly higher numbers of total and trophectoderm (TE) cells than control embryos; however, the numbers and proportions of inner cell mass (ICM) cells did not differ. Under 5% O₂ tension, no difference was found among the culture groups in the numbers of total, ICM and TE cells in embryos. All three PET mesh products investigated in this study were proven to be effective to prevent embryo movement. The results demonstrate that bovine embryos can be cultured in PET mesh sections without negative side‐effects and suggest that embryo distance determined by the mesh affects embryo quality at atmospheric oxygen tension. Polyethylene terephthalate mesh with 217 × 217‐μm openings was found to be the most suitable for further application in TLC.     

注入嵌合法制作黄牛和水牛种间嵌合胚的初步研究

采用注入嵌合法初步建立了一套黄牛和水牛种间嵌合的程序与方法。采用机械剥离法或免疫外科法分离胚胎内细胞团(ICM),然后注入到已去除ICM的受体囊胚中构建形成水牛和黄牛的嵌合胚。结果发现,在用免疫外科法分离ICM时,抗血清的灭活温度从57℃升至63.5℃,ICM的获得数显著升高(0%vs100%,P<0.01),如若在分离培养液中添加6%的胎牛血清(FCS),ICM的获得数大大降低(97.6%vs0%,P<0.01)。采用免疫外科法分离得到的黄牛ICM进行水牛囊胚的ICM置换重组,重组胚的存活率与机械剥离法得到的ICM无显著差异(91.4%vs87.5%,P>0.05);但囊胚孵化率则显著提高(80%vs43.8%;P<0.05)。以上结果表明,⑴水牛和黄牛胚胎通过ICM置换获得的种间嵌合胚胎能继续发育;⑵用于黄牛ICM分离的兔抗牛抗血清需在63.5℃灭活30min,且分离需在无血清的培养液中进行;⑶通过分离ICM置换进行胚胎嵌合时,免疫外科法优于显微手术法。     

The use of embryonic stem cell derived bioactive material as a new protein supplement for the in vitro culture of bovine embryos

Embryonic stem (ES) cells are expanded versions of the inner cell mass cells that compose the early mammalian blastocyst. Components derived from ES cells may contain various bioactive materials (BM) helpful for early preimplantation embryo growth. In this study, we examined the effect of human ES cell derived BM (hES-BM) on in vitro culture of bovine embryos. When bovine parthenogenetic day 2 embryos were cultured in 10% hES-BM, a significantly higher embryo development rate (44.3%) and increased cell numbers were observed relative to control medium containing 3 mg/ml BSA (19.5%; P<0.01). Among the various concentrations (5, 10 and 15%) and days of treatment (2 or 4 days) tested, 10% hES-BM treatment for 4 days provided the best culture environment to support the growth of bovine embryos in vitro (P<0.05). Little difference was observed between 10% hES-BM and 10% FBS treatment in the examined parthenogenetic or in vitro fertilized embryos, although the hES-BM group developed at a slightly better rate. However, the ICM cell numbers were significantly higher in the hES-BM group in irrespective of embryo origin (P<0.05). In addition, the relative levels of pluripotency (Oct4, × 1.8 fold; Nanog. × 3.3 fold), embryogenesis (Stat3, × 2.8 fold; FGF4, × 18.8 fold; E-cad, × 2.0 fold) and growth (Glut5, × 2.6 fold) genes were significantly higher in the 10% hES-BM group than in the 10% FBS group (P<0.05), while the levels of other genes (Bax, Bcl2, MnSOD and Connexin43) were not different. This is the first report examining the positive effects of hES-BM on bovine embryo development in vitro. Based on our results, we conclude that hES-BM can be used as a new protein supplement for bovine preimplantation embryo development.     

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.     

Effect of short‐term exposure of cumulus–oocyte complex to 3‐morpholinosydnonimine on in vitro embryo development and gene expression in cattle

Short‐term exposure of gametes to different types of stress might induce stress tolerance in mammalian embryos. The aim of this study was to evaluate the effect of short‐term exposure of bovine mature cumulus–oocyte complex (COC) to 3‐morpholinosydnonimine (SIN‐1) on subsequent in vitro embryo development, embryo quality and relative gene expression. Matured COCs were incubated with SIN‐1 (0, 0.1, 1, 10 and 100 μM SIN‐1) for 1 hr before in vitro fertilization and zygotes were cultured until Day 7. The cleavage rate at 72 hr did not show any differences among groups. However, the blastocyst rate on Day 7 decreased with all treatments evaluated, with the embryos generated with 10 μM SIN‐1 showing the lowest embryo production rate. Embryo quality analysis did not show any differences in total cell number (TCN) or inner cell mass (ICM) among groups. Relative gene expression analysis showed a downregulation of eNOS expression and an upregulation of nNOS expression in all treatments evaluated compared to the control group. Also, a downregulation was observed in some treatments: SOD2 at 0.1 μM; SOD1 at 0.1 and 100 μM; PRDX5 at 0.1, 10 and 100 μM; and NANOG at 10 and 100 μM; and an upregulation of CDX2 expression was observed at 100 μM. The other genes (OCT4, HIF1A, HSPA1A, BCL2A and iNOS) did not show any differences in the relative gene expression. These results suggest that the short‐term exposure of mature bovine COCs to SIN‐1 does not induce stress tolerance and has no beneficial effect on bovine in vitro embryo production.     

Improved development of ICR mouse 2-cell embryos by the addition of amino acids to a serum-, phosphate-and glucose-free medium

This study was conducted to evaluate how exogenous amino acids could affect preimplantation development of ICR mouse embryos. Two-cell embryos collected from naturally mated mice were cultured in amino acid-, glucose- and phosphate-free preimplantation (P)-1 medium. In Experiments 1, 19 amino acids (aa; 1% and 0.5% of MEM essential and nonessential amino acid solutions, respectively) were added to P-1 medium supplemented with either fatty acid-free bovine serum albumin (BSA; 3 mg/mL) or human follicular fluid (hFF; 10%). Regardless of BSA or hFF addition, embryo development to the morula (84 to 86% vs. 97 to 100%) and the blastocyst (54% vs. 93 to 94%) stages was significantly (P<0.05) enhanced by the addition of aa compared with no addition. In Experiment 2, the cell number of blastomeres and inner cell mass (ICM) cells in blastocysts and the ratio of ICM cell to trophectodermal cell (TE) were evaluated after aa addition. In both BSA- and hFF-containing P-1 medium, a significant increase in total blastomere number were found after aa addition (47 to 52 vs. 62 to 63 cells) compared with no addition. However, the ICM/TE ratio was not significantly affected by aa supplementation in both media, while ICM cell number was greatly increased after aa addition in hFF-containing medium (12 vs. 17 cells). When blastocysts were further cultured up to 162 hr post-hCG injection, development to the hatched blastocyst stage was significantly promoted by aa addition (0% vs. 11 to 20%) in both BSA- and hFF-containing media. In conclusion, aa significantly promote the preimplantation development to the hatched blastocyst stage and such effect mainly exerted on supporting blastomere proliferation.     

Low Oxygen Tension and Relative Defined Culture Medium with 3, 4‐Dihydroxyflavone are Beneficial for Yak–Bovine Interspecies Somatic Cell Nuclear Transfer Embryo

With an aim to improve the efficiency of yak–bovine interspecies somatic cell nuclear transfer (iSCNT), this study investigated the effect of different culture systems on the development, quality and gene expression profile of yak–bovine iSCNT embryo. Reconstructed embryos were cultured in modified synthetic oviductal fluid (mSOF) or relative defined culture medium (RDCM) with 5% or 20% oxygen tension. Relative mRNA abundance of Oct‐4, IFNT, IGF‐2, Bax, GPX‐1, SOD‐1, CAT and GSS was analysed in blastocysts with qRT‐PCR. The blastocyst formation rate in RDCM under 5% oxygen tension was significantly higher than that under 20% oxygen tension (P < 0.05). The total cell number of blastocyst derived from RDCM with 20% oxygen tension was lower than that of other groups, whereas the group of RDCM with 5% oxygen tension showed a beneficial effect on apoptosis index and tolerance to cryopreservation (P < 0.05). However, under the same oxygen tension, the mRNA abundance of IFNT of RDCM groups was higher than that of the mSOF groups. In addition, high oxygen tension during in vitro culture (IVC) with RDCM significantly increases the mRNA expression of oxidative stress‐related genes (GPX‐1, SOD‐1, CAT and GSS) (P < 0.05). 3, 4‐Dihydroxyflavone (DHF) during high oxygen tension was able to improve the cloned blastocyst formation rate in RDCM (P < 0.05). These results for the first time showed that low oxygen tension and RDCM could improve the developmental competence and quality and alleviate the oxidative stress for yak–bovine iSCNT embryo during IVC.     

Establishment of an advanced chemically defined medium for early embryos derived from in vitro matured and fertilized bovine oocytes

A chemically defined medium would be useful for analyzing promoters or inhibitors in in vitro culture (IVC) of bovine embryos. However, an IVC system for bovine embryos in a chemically defined medium has not been fully established. The present study was carried out to establish an advanced chemically defined medium for bovine embryos that supports a high rate of embryo development to the blastocyst stage. In the first experiment, we examined the effects of addition of Medium RD (RPMI1640 and Dulbecco's MEM, 1:1 v/v) to mKSOM/aa on developmental competence. The addition of 10% RD to mKSOM/aa with BSA improved the rate of development to the blastocyst stage; however, 10% RD-mKSOM/aa with PVP, which is a chemically defined medium, caused a reduction in the percentage of hatching blastocysts. In the second experiment, embryos were cultured in the chemically defined medium of 10% RD-mKSOM/aa containing 11.7, 23.4, 46.8, 70.2 or 96.8 μM inositol. Inositol at the concentration of 70.2 μM improved the rate of development to the hatching blastocyst stage. In the third experiment, the optimal RD concentration in the IVC medium was evaluated. Embryos were cultured in the chemically defined medium supplemented with 10, 20 or 30% (v/v) RD. The rate of development to the blastocyst stage was highest with 20% RD. In the fourth experiment, the effects of N-acetylglucosamine (GlcNAc) as an IVC medium supplement on developmental competence were examined. The rate of development to the blastocyst stage with 1.0 mM GlcNAc was significantly higher than that without GlcNAc, but the rate of development with 1.2 mM GlcNAc was not different from that without GlcNAc. We also evaluated the ability of blastocysts produced in RD-mKSOM/aa to develop to normal calves after being transferred into recipients. Ten of the 16 recipients became pregnant, with 9 delivering normal calves. These results indicate that 20% RD-mKSOM/aa containing 70.2 μM myo-inositol and 1 mM GlcNAc is useful as a chemically defined medium for IVC of bovine embryos.     

Effect of Growth Factors on Bovine Blastocyst Development in a Serum-Free Medium

Shamsuddin, M.: Effect of growth factors on bovine blastocyst development in a serum-free medium. Acta vet. scand. 1994, 35, 141-147. - To investigate the effect of growth factors on pre-implantation development, bovine zygotes, produced by in vitro fertilization (IVF) of in vitro-matured (IVM) oocytes, were cultured in a serum-free medium to which the following growth factors were added one at a time: epidermal growth factor (EGF), acidic fibroblast growth factor (a-FGF), insulin-like growth factor-II (IGF-II), platelet-derived growth factor from human platelets (PDGF), and platelet-derived growth factor-AB, human, recombinant (PDGF-AB). All growth factors were added at a dose of either 10 or 50 ng/ml, except PDGF which was added at a dose of either 5 or 15 ng/ml. The control medium was TCM 199 supplemented with sodium pyruvate (0.25 mmol/1), BSA (10 mg/ml), insulin (5 μg/ml), transferrin (5 μg/ml), and sodium selenite (5 ng/ml). Embryos were cultured for 8 days (day of insemination = Day 0). The mean percentages of first cleavage on Day 2 varied from 67% to 86% and the differences between the 2 doses, or between the control and growth factor- treated groups were not significant (p≥0.13). The effects of the two doses on subsequent development up to the blastocyst stage did not differ either (p≥0.12). There was no stimulatory effect of any of the used exogenous growth factors on embryo development up to the morula or blastocyst stage on Day 7, or blastocyst stage on Day 8. Moreover, medium supplemented with PDGF had fewer blastocysts than the control (p≤0.03). The results indicate that growth factor supplementation may not necessarily increase the yield of blastocysts from bovine IVM-IVF oocytes in a serum-free medium.     

Influence of cysteine in conjunction with growth factors on the development of in vitro-produced bovine embryos

Cysteine supplementation to in vitro maturation (IVM) media of bovine oocytes increases cellular glutathione production. Beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mm cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/ml); EGF (10 ng/ml); and IGF-I + EGF (100 + 10 ng/ml). Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the maturation media (12 h C IGF-I + EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I + EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD) and manganese (Mn) SOD in embryos was assessed. No treatment effect was observed on the expression of these genes. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS.     

Addition of D-penicillamine,hypotaurine, and epinephrine (PHE) mixture to IVF medium maintains motility and longevity of bovine sperm and enhances stable production of blastocysts in vitro

The present study aimed to establish an efficient system for bovine embryo production by in vitro fertilization (IVF) that can achieve stable normal fertilization and blastocyst developmental rates in any bull without optimization of the sperm concentration in IVF medium. We examined the effects of a PHE mixture (20 μM D-penicillamine, 10 μM hypotaurine and 1 μM epinephrine), theophylline (2.5 mM), and sperm concentration (1, 2 or 5 × 10⁶ cells/ml) on fertilization and blastocyst developmental rates. High cleavage rates (78.3 to 92.4%) and blastocyst developmental rates (31.9 to 62.0%) at day 7 were obtained in the presence of PHE and theophylline in IVF medium with a sperm concentration of 2 × 10⁶ cells/ml using sperm from 9 bulls. In addition, the synergistic effect of PHE and theophylline on normal fertilization (2 pronuclei) was clarified at 12 h after IVF with a sperm concentration of 1 × 10⁶ cells/ml. Moreover, high linearity, high flagellar beat cross frequency, and low amplitude of lateral head of motile sperm were found by computer-assisted sperm analysis. In conclusion, the combination of the PHE mixture and theophylline synergistically accelerates sperm motility and sperm penetration of bovine oocytes. Theophylline activates sperm motility with increasing intracellular cAMP. However, PHE prevents an excessive increase of cAMP and maintains sperm motility without hyperactivation. When the combination of PHE and theophylline is added to IVF medium at a sperm concentration of 2 × 10⁶ cells/ml, we can achieve stable normal fertilization and blastocyst development in any bull.     

Chemical Activation with a Combination of Ionomycin and Dehydroleucodine for Production of Parthenogenetic,ICSI and Cloned Bovine Embryos

The aim of this study was to evaluate the potential of dehydroleucodine (DhL), a new drug isolated from a medicinal herb used in Argentina, for activation of bovine oocyte. Several DhL concentrations and exposure times after ionomycin (Io) treatment were tested. The optimal DhL treatment, found for parthenogenetic development, was employed to produce bovine embryos by intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). The best parthenogenic embryo developments were observed with 5 μm Io for 4 min followed by 5 μm DhL concentration and after 3‐h exposure time (52.3% cleavage; 17.4% morulae; 7.3% blastocyst; n = 109). This treatment generated no significant differences with standard Io plus 6‐dimethylaminopurine (DMAP) treatment in preimplantation embryo development. In our conditions, the embryo development reached after ICSI and SCNT assisted by the DhL treatment did not differ in terms of cleavage and blastocyst development from activation with standard Io plus DMAP treatment (p > 0.05). In conclusion, DhL utilization to activate oocytes and induce development of parthenogenotes, ICSI‐embryos or SCNT‐embryos is reported here for first time.     

Oxygen tension and medium supplements for in vitro maturation of bovine oocytes cultured individually in a chemically defined medium

The effects of adding cysteamine, EGF, and glucose as an energy substrate under low oxygen tension during in vitro maturation (IVM) were examined to find ways of improving the individual in vitro production (IVP) system in individually cultured bovine oocytes. The basic medium was mSOFaa containing 1 mg/ml polyvinyl alcohol. Immature oocytes were individually cultured in an IVM medium with 10 ng/ml EGF, 100 microM cysteamine, or EGF plus cysteamine under 20% or 5% O(2). Cleavage and blastocyst rates were significantly higher (P<0.05) in IVM culture was under 20% O(2) than in culture under 5% O(2). Under 5% O(2), neither EGF nor cysteamine improved embryonic development. The proportion of matured oocytes was significantly higher (P<0.05) in the presence of 1.5 mM glucose under 20% O(2) (68.6%), and 5.5 mM (66.7%) and 10 mM (65.5%) glucose under 5% O(2). The presence of 5.5 mM glucose significantly (P<0.05) increased the maturation rate compared with the absence of glucose, irrespective of addition of EGF and cysteamine. The addition of cysteamine alone in the maturation medium significantly (P<0.05) increased the intracellular GSH concentration in the oocytes. Also, under 5% O(2) cysteamine and/or EGF significantly (P<0.05) improved the proportions of penetrated oocytes, cleavage and blastocyst formation, which were similar levels to those of oocytes matured under 20% O(2). After vitrification, the re-expanding and hatching rates of blastocysts derived from the individual IVP system containing cysteamine under 5% O(2) were significantly (P<0.05) higher than those of blastocysts derived from the individual IVP system without cysteamine under 5% O(2) and the group IVP system under 20% O(2). The present study showed that a high glucose level (5.5 or 10 mM) was optimal in IVM culture under low (5%) oxygen tension. The addition of EGF and/or cysteamine to the maturation medium had no positive effect on nuclear maturation, but improved fertilizability, developmental competence and cryoresistance following vitrification, probably due to increased GSH synthesis during the IVM process.     

Effect of Antioxidants During Bovine In Vitro Fertilization Procedures on Spermatozoa and Embryo Development

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 μ m β-mercaptoethanol (β-ME) and 50 μ m cysteamine (Cyst)] or a pro-oxidant (5 m m buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for β-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.     

添加不同发情时期山羊血清对牛胚胎体外发育的影响

为了提高牛体外胚胎囊胚发育率,添加不同发情时期山羊血清对牛胚胎进行体外培养。分别采集发情周期D0、D2、D4和D7山羊血清（发情当天为D0）,灭活除菌备用。结果表明不同发情时期羊血清对孤紫激活胚胎孵裂率影响差异不显著,囊胚发育率分别为29.5%、32.4%、33.9%和41.3%,D7血清能显著提高囊胚发育率（P＜0.05）,且扩张和孵化胚出现较早。添加D0血清体外受精胚胎卵裂率较高,但对囊胚发育率影响差异不显著。说明孤紫激活和体外受精胚胎发育有差异,D7血清对胚胎后期发育有利。受精胚无血清培养3d后添加D7血清,囊胚发育率为42.9%,是较为理想的培养方法。     

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

The effect of melatonin during in vitro maturation (IVM) on DNA damage of cumulus cells (CCs) from bovine cumulus-oocyte complexes (COCs) and embryo development was evaluated. COCs from abattoir ovaries were cultured in maturation medium (MM) with 0.5μg/ml FSH and 5.0μg/ml LH (FSH-LH); 10(-9)M melatonin (MEL) or FSH-LH+MEL (FSH-LH-MEL). After 24h of in vitro maturation, the CCs surrounding the oocyte were subjected to DNA analysis by Comet assay. After in vitro fertilization and in vitro embryo culture, the embryo development rates were evaluated on day 2 post insemination (cleavage) and days 7-8 (blastocyst). The percentage of CCs with no DNA damage was significantly superior in MEL group (37.6±2.4) than in FSH-LH-MEL (28.0±2.4) and FSH-LH (17.8±2.41) groups. Cleavage and blastocysts rates were similar among groups. Melatonin during IVM protects the CCs from DNA damage but this effect did not influence embryo development in vitro.     

Effects of Growth Hormone on Nuclear Maturation of Ovine Oocytes and Subsequent Embryo Development

The objective of this study was to determine the effect of the presence of recombinant ovine growth hormone either alone or together with follicle stimulating hormone (FSH) during ovine oocyte in vitro maturation (IVM) on nuclear maturation and subsequent embryo development. Moreover, the effect of growth hormine (GH) on embryo development whether influenced by the presence of foetal bovine serum (FBS) was assessed. The abattoir‐derived oocytes were randomly divided into four treatment groups and cultured in maturation medium supplemented with: (i) 0.05 IU/ml FSH; (ii) 300 ng/ml roGH; (iii) FSH + roGH; and (iv) no FSH and GH (control). The percentages of germinal vesicle‐stage oocytes in GH‐treated group after 8 h of culture was significantly higher than the FSH and FSH + GH groups and lower than control (22.4%, 8.7%, 9.1%, and 32% respectively). The percentage of MII‐stage oocytes was significantly increased in the presence of GH after 16 and 24 h of culture compared to the control (44.7% and 83.1% vs 32.6% and 73.6% respectively). There was no significant synergism between GH and FSH in terms of nuclear maturation. The blastocyst rates in serum‐supplemented groups were enhanced by the presence of FSH and GH compared to the control (35.4% and 31.3 vs 11.4% respectively). Compared with either GH or FSH alone, the subsequent embryo development (blastocyst rate), however, was negatively influenced by co‐presence of both hormones (22.8%). In contrast, the corresponding values were not affected in the absence of serum. In conclusion, GH had positive effect on nuclear maturation of sheep oocytes. Moreover, the pattern of the effect of GH on embryo development was influenced by the presence of FBS during IVM.