In Vitro Culture and Differentiation of Buffalo (Bubalus bubalis) Spermatogonia

The objective of this study was to develop a culture system which could support buffalo spermatogonia differentiation into spermatids in vitro. Testes from 3‐ to 5‐month‐old buffaloes were decapsulated and seminiferous tubules were enzymatically dissociated to recover spermatogonia and sertoli cells. The cells were cultured in modified Dulbecco modified Eagle medium supplemented with different concentrations of foetal bovine serum, retinol, testosterone for 2 months at 37°C. Spermatogonia and sertoli cells were identified with an antibody against c‐kit or GATA4, respectively. The viability of spermatogonia in the media supplemented with different concentrations of serum was all significantly higher (p < 0.05) compared with that in the medium without serum. A‐paired or A‐aligned spermatogonia and spermatogonial colonies (AP‐positive) were observed after 7–10 days of culture and spermatid‐like cells with a flagellum (6–8 μm) appeared after 30 days of culture. For cultured conditions, retinol could not significantly promote the formation of spermatid‐like cells (p > 0.05), whereas supplementation of testosterone could significantly promote (p < 0.05) the formation of spermatid‐like cells after 41 days of culture. The expression of the spermatid‐specific marker gene (PRM2) was identified after 30 days of culture by RT‐PCR. Yet, the transition protein 1 (TP1, a haploid makers) was not detected. Meanwhile, spermatids developed in vitro were also confirmed by Raman spectroscopy. These results suggest that buffalo spermatogonia could differentiate into spermatids in vitro based on the analysis of their morphology, PRM2 expression and Raman spectroscopy. Yet, the normality of the spermatid‐like cells was not supported by TP1 expression.     

Cryopreservation of Zona‐Free Cloned Buffalo (Bubalus Bubalis) Embryos: Slow Freezing vs Open‐Pulled Straw Vitrification

This study was carried out to compare the post‐thaw cryosurvival rate and the level of apoptosis in vitro produced zona‐free cloned buffalo blastocysts subjected to slow freezing or vitrification in open‐pulled straws (OPS). Zona‐free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re‐expansion rate following post‐thaw culture for 22–24 h. The post‐thaw re‐expansion rate was significantly (p < 0.05) higher following vitrification in OPS (71.2 ± 2.3%) compared with that after slow freezing (41.6 ± 4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen‐thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non‐cryopreserved cloned embryos (422.6 ± 67.8) and those cryopreserved by slow freezing (376.4 ± 29.3) or vitrification in OPS (422.8 ± 36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8 ± 2.0) or OPS vitrification (13.3 ± 1.8), was significantly (p < 0.05) higher than that for the control non‐cryopreserved cloned embryos (3.4 ± 0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona‐free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.     

Effects of Chemical Activation Treatment on Development of Swamp Buffalo (Bubalus bubalis) Oocytes Matured In Vitro and Fertilized by Intracytoplasmic Sperm Injection

The objective of this study was to optimize the activation protocol for buffalo oocytes after intracytoplasmic sperm injection (ICSI). The release of the second polar body (PB) at 3, 6 and 9 h after ICSI of in‐vitro matured oocytes activated either with 5 μm ionomycin (Io) or with 7% ethanol (EtOH) was preliminary examined. The highest rate of second PB extrusion occurred at 3 h of activation, and the second PB extrusion in EtOH group was significantly higher than that in Io group. Oocytes that extruded the second PB were selected and cultured either with 1.9 mm 6‐dimethylaminopurine (6‐DMAP) for 3 h or with 10 μg/ml cycloheximide (CHX) for 5 h. Significantly higher rate of oocytes formed 2 pronuclei in EtOH combined with CHX (EtOH + CHX) (62%) group compared to those of Io + CHX (42%) and EtOH + 6‐DMAP (48%) groups (p < 0.01) whereas Io + 6‐DMAP group showed intermediate value (58%). Significantly higher blastocyst formation rates were obtained in Io + 6‐DMAP (29%) and EtOH + CHX (24%) groups than in Io + CHX (6%) and EtOH + 6‐DMAP (17%) groups. Our results indicate that buffalo ICSI oocytes are effectively activated by combination treatment of Io with 6‐DMAP and EtOH with CHX resulting in the highest cleavage and blastocyst formation rates.     

Production of Wild Buffalo (Bubalus arnee) Embryos by Interspecies Somatic Cell Nuclear Transfer Using Domestic Buffalo (Bubalus bubalis) Oocytes

The objective of this study was to explore the possibility of producing wild buffalo embryos by interspecies somatic cell nuclear transfer (iSCNT) through handmade cloning using wild buffalo somatic cells and domestic buffalo (Bubalus bubalis) oocytes. Somatic cells derived from the ear skin of wild buffalo were found to express vimentin but not keratin and cytokeratin‐18, indicating that they were of fibroblast origin. The population doubling time of skin fibroblasts from wild buffalo was significantly (p < 0.05) higher, and the cell proliferation rate was significantly (p < 0.05) lower compared with that of skin fibroblasts from domestic buffalo. Neither the cleavage (92.6 ± 2.0% vs 92.8 ± 2.0%) nor the blastocyst rate (42.4 ± 2.4% vs 38.7 ± 2.8%) was significantly different between the intraspecies cloned embryos produced using skin fibroblasts from domestic buffalo and interspecies cloned embryos produced using skin fibroblasts from wild buffalo. However, the total cell number (TCN) was significantly (p < 0.05) lower (192.0 ± 25.6 vs 345.7 ± 42.2), and the apoptotic index was significantly (p < 0.05) higher (15.1 ± 3.1 vs 8.0 ± 1.4) for interspecies than that for intraspecies cloned embryos. Following vitrification in open‐pulled straws (OPS) and warming, although the cryosurvival rate of both types of cloned embryos, as indicated by their re‐expansion rate, was not significantly different (34.8 ± 1.5% vs 47.8 ± 7.8), the apoptotic index was significantly (p < 0.05) higher for vitrified–warmed interspecies than that for corresponding intraspecies cloned embryos (48.9 ± 7.2 vs 23.9 ± 2.8). The global level of H3K18ac was significantly (p < 0.05) lower in interspecies cloned embryos than that in intraspecies cloned embryos. The expression level of HDAC1, DNMT3a and CASPASE3 was significantly (p < 0.05) higher, that of P53 was significantly (p < 0.05) lower in interspecies than in intraspecies embryos, whereas that of DNMT1 was similar between the two types of embryos. In conclusion, these results demonstrate that wild buffalo embryos can be produced by iSCNT.     

Effects of FSH and LH on Steroid Production by Buffalo (Bubalus bubalis) Granulosa Cells Cultured In Vitro Under Serum‐Free Conditions

The objective of this study was to examine the effects of FSH and LH on oestradiol‐17β and progesterone production by buffalo granulosa cells cultured under serum‐free conditions. Granulosa cells (3 × 10⁵) from small (≤5 mm diameter) follicles were cultured for up to 4 days in 48‐well plates coated with 3.3 μg/cm² fibronectin in Dulbecco's modified Eagle's medium (DMEM) : nutrient mixture F‐12 Ham (1 : 1 ratio) supplemented with 10^?7 m androstenedione, 5 μg/ml human apo‐transferrin and 0.1% bovine serum albumin, in the presence or absence of FSH or LH (0, 1, 2, 4, 8, 16, 32 or 64 ng/ml each). Basal oestradiol‐17β production by granulosa cells from small follicles reduced (p < 0.01) from days 1 to 2 of culture and became undetectable by day 3 and basal progesterone production increased (p < 0.05) from day 1 through day 4 of the culture. Although there was no effect of FSH on day 1 of the culture, FSH at 2, 4, 8 and 16 ng/ml increased (p < 0.05) oestradiol‐17β production by granulosa cells from small follicles on day 2. Progesterone secretion was increased (p < 0.05) by all doses of FSH on all days of culture. All doses of LH had no effect on oestradiol‐17β or progesterone production by granulosa cells from small follicles on any day of the culture. The results of this study demonstrate a serum‐free culture system for buffalo granulosa cells and stimulatory effect of FSH but not LH on steroid hormone production by buffalo granulosa cells under these conditions.     

水牛卵母细胞冷冻保存初步研究

①用EFS30、EFS40、EDFS30、EDFS40四种玻璃化冷冻液对MⅡ期水牛卵母细胞进行毒性试验,结果表明:试验组卵母细胞形态正常率与对照组均无显著性差异(P>0.05);对卵母细胞孤雌激活后EDFS30、EDFS40组的卵裂率与对照组(75.28%)及EFS30、EFS40组差异显著(P<0.05);利用4种冷冻保护剂采用OPS法冷冻保存MⅡ期水牛卵母细胞,其中以EDFS40作为冷冻液时,卵母细胞冷冻解冻后孤雌激活卵裂率最高,达31.60%;以EDFS40作为冷冻液,比较了GMP法和OPS法的冷冻效果,结果表明GMP法冷冻效果好于OPS法。②采用不同预处理时间和平衡时间使用细管法常规冷冻G V期卵母细胞,结果表明预处理5 min、平衡15min组的形态正常率和极体排出率相对较高,分别为72.73%、27.27%。     

Assessment of DNA Damage during In Vitro Development of Buffalo (Bubalus bubalis) Embryos: Effect of Cysteamine

Comet assay was used in the present study to examine DNA damage to buffalo oocytes and embryos during in vitro culture. Embryos were produced in vitro from oocytes obtained from slaughterhouse ovaries in presence of cysteamine (IVM and IVC media supplemented with 50 and 100 μm , respectively) or in its absence (controls). Compared to controls, cysteamine supplementation increased (p < 0.01) cleavage rate and proportion of oocytes that developed to 8‐ to 16‐cell stage. The incidence of DNA damage was lower (p < 0.01) in cysteamine group than that in controls at 8‐ to 16‐ (19.3 ± 4.24 vs 72.0 ± 5.22%) but not in 2‐cell stage embryos (11.7 ± 5.63 vs 20.8 ± 5.49%) or in mature oocytes (5.3 ± 3.43 vs 10.3 ± 4.73%). The tail length, which indicates magnitude of DNA damage, was shorter (p < 0.01) in cysteamine group than in controls in mature oocytes (25.5 ± 0.5 vs 36.0 ± 0.71 pixels) and 8‐ to 16‐cell stage (49.2 ± 1.64 vs 152.7 ± 1.28 pixels) but not in 2‐cell stage embryos (36.3 ± 1.54 vs 36.4 ± 0.75 pixels). Also, exposure of oocytes/embryos to UV radiation or H₂O₂ caused extensive DNA damage. In conclusion, these results suggest that oocytes/embryos suffer from DNA damage during progress of in vitro culture, which can be partly ameliorated by cysteamine supplementation of culture media.     

Activation of Zona‐Free Buffalo (Bubalus bubalis) Oocytes by Chemical or Electrical stimulation,and Subsequent Parthenogenetic Embryo Development

Parthenogenetic activation using zona‐free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona‐free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6‐dimethylaminopurine (6‐DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona‐free oocytes when compared to zona‐intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona‐free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6‐DMAP resulted in the highest blastocyst rate.     

Buffalo (Bubalus bubalis) ES Cell–Like Cells are Capable of In Vitro Skeletal Myogenic Differentiation

When buffalo embryonic stem (ES) cell–like cells that expressed surface markers SSEA‐4, TRA‐1‐60, TRA‐1‐81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX‐1 and NUCLEOSTEMIN as confirmed by RT‐PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three‐dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF‐68 and NESTIN (ectodermal lineage), BMP‐4 and α‐skeletal actin (mesodermal lineage), and α‐fetoprotein, GATA‐4 and HNF‐4 (endodermal lineage). When these EBs were cultured on gelatin‐coated dishes, they spontaneously differentiated to several cell types such as epithelial‐ and neuron‐like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10^?8 m or 10^?7 m retinoic acid for 25 days, ES cells could be directed to form muscle cell–like cells, the identity of which was confirmed by expression of α‐actinin by immunofluorescence and of MYF‐5, MYOD and MYOGENIN genes by RT‐PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell–like cells to undergo directed differentiation to cells of skeletal myogenic lineage.     

Telomerase Activity in Swamp Buffalo (Bubalus bubalis) Oocytes and Embryos Derived from In Vitro Fertilization, Somatic Cell Nuclear Transfer and Parthenogenetic Activation

The objective of this study was to examine the telomerase activity in swamp buffalo oocytes and pre-implantation stage embryos derived from in vitro fertilization (IVF), somatic cell nuclear transfer (NT) and parthenogenetic activation (PA). Immature and mature oocytes, and embryos at the 2-4 cell, 8-16 cell, morula and blastocyst stages produced by IVF, NT and PA were collected and the telomerase activity was assayed by using a Telomerase PCR ELISA kit. Telomerase activity was detected in all developmental stages evaluated from immature oocytes to blastocyst stage embryos. Telomerase activity was detected in higher amounts in immature as compared with mature oocytes (p < 0.05). Embryos derived from NT showed a profile of telomerase activity similar to that of IVF. In IVF and NT embryos, telomerase activity was low in the 2-4 cell and 8-16 cell stages, but the activity significantly increased (p < 0.05) at the morula stage, reaching its highest level at the blastocyst stage. In PA embryos, low levels of telomerase activity were detected from the 2-4 cell to the morula stage, and the highest level of telomerase activity was found at the blastocyst stage. Telomerase activity in NT blastocysts is higher than that derived from IVF and the activity is highest in PA blastocysts. These results suggest that the successful reprogramming of telomerase activity in buffalo NT embryos follow a pattern similar to that in embryos derived from IVF and PA.     

Expression and Characterization of Constitutive Heat Shock Protein 70.1 (HSPA‐1A) Gene in In Vitro Produced and In Vivo‐Derived Buffalo (Bubalus bubalis) Embryos

Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA‐1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA‐1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo‐derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA‐1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA‐1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8–16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA‐1A revealed that it shares 91–98% identity with other mammalian sequences. It can be concluded that higher level of HSPA‐1A mRNA in IVP embryos in comparison with in vivo‐derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA‐1A gene could be used as a stress biomarker during pre‐implantation development.     

Ultrastructural Morphology and Nuclear Maturation Rates of Immature Equine Oocytes Vitrified with Different Solutions and Exposure Times

Ultrastructural morphological injuries and maturation rates were investigated in equine oocytes exposed to vitrification solutions (VS) containing synthetic ice blockers (SIBs) during different exposure times. In experiment 1, compact cumulus-oocyte complexes (COCs; n = 30) were randomly allocated to treatments: (1) fresh fixed (control); (2) VS-1 (1.4 M dimethyl sulfoxide [DMSO] + 1.8 M ethylene glycol [EG] + 1% SIB) for 3 minutes of equilibrium time and VS-2 (2.8 M DMSO + 3.6 M EG + 0.6 M sucrose + 1% SIB) for 1 minute (Eq-long); and (3) VS-1 for 1.5 minutes and VS-2 for 30 seconds (Eq-short). In experiment 2, compact (n = 248) and expanded (n = 264) COCs were evenly distributed to the following treatments: (1) immediate maturation in vitro (control); (2) vitrification using the Eq-short protocol as in experiment 1; and (3) vitrification using a stock solution containing 2.8 M formamide, 2.8 M DMSO, 2.7 M EG, 7% polyvinylpyrrolidone, and 1% SIB (Eq-short-mod). More (P < .02) oocytes with normal ultrastructural morphology were seen in fresh control and Eq-short groups than in Eq-long group. Metaphase-II (MII) rates were higher (P < .05) for oocytes with expanded cumulus than compact cumulus in the control group, and higher (P < .05) for oocytes with expanded cumulus than compact cumulus in Eq-short and Eq-short-mod groups. No difference in MII rates was detected among groups within each type of COC. In conclusion, reduction of exposure time to VS better preserved oocyte ultrastructural features, and MII rates were higher for vitrified oocytes with expanded cumulus. This study advances our knowledge on potential alternatives for vitrification of immature equine oocytes.     

Cryotop Vitrification of Buffalo (Bubalus Bubalis) In Vitro Matured Oocytes: Effects of Cryoprotectant Concentrations and Warming Procedures

The aim of the work was to evaluate the in vitro developmental competence of in vitro‐matured buffalo oocytes after Cryotop vitrification (CTV) and in vitro fertilization (IVF). To optimize parameters, two cryoprotectant (CP) concentrations and two warming–dilution procedures were applied. Oocytes were vitrified in 16.5% ethylene glycol (EG), 16.5% dimethylsulphoxide (DMSO) and 0.5 m sucrose in Groups A and C, and in higher CP concentrations (20% EG, 20% DMSO and 0.5 m sucrose) in Groups B and D. Warming was performed in 1.25 m sucrose for 1 min, then in 0.62, 0.42 and 0.31 m sucrose, 30 s each (Groups A and B), or in 0.25 m sucrose for 1 min and in 0.15 m sucrose for 5 min (Groups C and D). After warming, the oocytes were fertilized and cultured in vitro. Survival rate post‐warming was lower in Group D (83.6%) than in Groups A and B (92.4 and 92.8%, respectively), while intermediate values were found in Group C (85.7%). Survival rates at 24 h decreased in Groups C and D (52.0% and 50%, respectively) and remained high in Groups A and B (84.0% and 85.6%, respectively), thus indicating that the dilution of CP after warming is critical for buffalo oocyte cryopreservation. Similar differences were also observed in cleavage rates (42.7%, 55.3%, 28.4% and 36.3% for Groups A, B, C and D, respectively) whereas no differences in blastocyst rates were found among groups (6.4%, 7.8%, 5.9% and 6.9% for Groups A, B, C and D, respectively). Blastocyst production after IVF of vitrified oocytes proves the feasibility of CTV in buffalo species.     

Effect of Relaxin on Fertility Parameters of Frozen–Thawed Buffalo (Bubalus bubalis) Sperm

The aim of this work was to evaluate the effect of relaxin on fertility parameters of buffalo frozen/thawed sperm. Sperm were incubated in the absence of capacitating agents (negative control), with a known capacitating agent such as heparin (positive control) and with 50 and 100 ng/ml relaxin for 2 and 4 h. Sperm viability, motility, capacitation and the effect of relaxin on the fertilizing ability after heterologous IVF were evaluated. Although viability was not affected, relaxin increased (p < 0.05) sperm motility compared to the negative and positive controls both after 2 h (60.0 ± 2.0, 60.0 ± 3.1, 68.3 ± 1.7 and 69.4 ± 2.7, respectively, in negative control, positive control, 50 and 100 ng/ml relaxin) and 4 h (55.0 ± 2.5, 53.3 ± 3.0, 62.2 ± 3.0 and 65.0 ± 3.2, respectively, in negative control, positive control, 50 and 100 ng/ml relaxin) incubation. When sperm were incubated with both 100 ng/ml relaxin and heparin, a decrease (p < 0.01) of pattern A, that is low capacitation level, was observed compared to the negative control both after 2 h (54.4, 34.3 and 36.4%, respectively, in negative control, positive control and 100 ng/ml relaxin) and 4 h (51.9, 35.0 and 34.3%, respectively, in negative control, positive control and 100 ng/ml relaxin). Moreover, an increase (p < 0.01) of pattern EA, that is high capacitation level, was recorded with 100 ng/ml relaxin and heparin compared to the negative control both after 2 h (44.1, 59.3 and 57.7%, respectively, in negative control, positive control and 100 ng/ml relaxin) and after 4 h (43.0, 54.4 and 56.0%, respectively, in negative control, positive control and 100 ng/ml relaxin). Finally, relaxin increased (p < 0.01) cleavage rate compared to the negative control (57.1 ± 4.4, 72.5 ± 6.0, 71.4 ± 5.5 and 73.6 ± 2.9, respectively, in negative control, positive control, 50 and 100 ng/ml relaxin). In conclusion, relaxin has a beneficial effect on motility, capacitation and fertilizing ability of frozen–thawed buffalo sperm.     

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     

Effect of Physiologically Relevant Heat Shock on Development,Apoptosis and Expression of Some Genes in Buffalo (Bubalus bubalis) Embryos Produced In Vitro

For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8‐cell, 16‐cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8–16‐cell and blastocyst stages, relative mRNA abundance of stress‐related genes HSP 70.1 and HSP 70.2 and pro‐apoptotic genes CASPASE‐3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress‐related gene HSF1. Expression level of anti‐apoptotic genes BCL‐XL and MCL‐1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8–16‐cell and blastocyst stages. Among the genes related to embryonic development, at 8–16‐cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR‐1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress‐, apoptosis‐ and development‐related genes.     

透明质酸对牛卵母细胞体外成熟影响的研究

在改良的基础培养液中添加HA,主要探讨HA对牛卵母细胞成熟的影响。结果表明,当mTCM-199中HA浓度为3．0mg／mL时,第一极体排出率和卵裂率显著高于其他各组（P〈0．05）,初步表明3．0mg／mL是无血清培养中HA的最佳浓度。当在mTC-199中添加HA、BSA和OCS时,OCS组的第一极体排出率显著高于其他两组（P〈0．05）,分别为83．40％、84．33％和92．17％,但HA组与BSA差异不显著（P〉0．05）;卵裂率三者差异均不显著（P〉0．05）;表明在无血清培养中HA可代替BSA,但是培养的效果不如血清。HA在mTCM-199和mSOF基础培养基中,对卵母细胞的第一极体排出率和卵裂率无明显差异（P〉0．05）。表明在无血清培养下,HA在复杂和简单的培养液中都支持卵母细胞的成熟。