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.     

水牛卵母细胞孤雌激活及孤雌胚与体外受精胚发育比较

对MII期水牛卵母细胞进行人工诱导激活可以帮助人们间接判断体外成熟卵母细胞质量的优劣。并且，卵母细胞的充分激活也是提高核移植效率的关键因素之一。试验比较了水牛卵母细胞体外成熟时间对孤雌激活胚发育能力的影响以及不同化学激活方法对水牛卵母细胞孤雌激活效果的影响，并在相同条件下，对孤雌激活胚与体外受精胚的发育能力进行了比较。结果表明，水牛卵母细胞体外成熟27小时或30小时的囊胚发育率（19．0％或17．7％）明显高于体外成熟21小时或24小时的囊胚发育率（12．3％或13．8％）；Ion联合6-DMAP激活水牛卵母细胞的效果优于其他几组激活方法；在相同条件下，孤雌激活胚与体外受精胚的发育能力存在着差异，其中卵裂率差异不显著，但孤雌激活胚的囊胚发育率显著高于体外受精胚（13．0％vs21．7％）。     

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.     

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.     

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.     

水牛卵母细胞孤雌激活及孤雌胚与体外受精胚发育比较

比较了水牛卵母细胞体外成熟时间对孤雌激活胚发育能力的影响,以及不同化学激活方法对水牛卵母细胞孤雌激活效果的影响,并在相同条件下,对孤雌激活胚与体外受精胚的发育能力进行了比较.结果表明,水牛卵母细胞体外成熟27 h或30 h的囊胚发育率(19.0%、17.7%)明显高于体外成熟21 h或24h的囊胚发育率(12.3%、13.8%);Ion联合6-DMAP激活水牛卵母细胞的效果优于其他几组激活方法;在相同条件下,孤雌激活胚与体外受精胚的发育能力存在着差异,其中卵裂率差异不显著,但孤雌激活胚的囊胚发育率显著高于体外受精胚.     

Effects of Trichostatin A on In Vitro Development and DNA Methylation Level of the Satellite I Region of Swamp Buffalo (Bubalus bubalis) Cloned Embryos

Trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to improve the cloning efficiency in several species. This brings our attention to investigation of the effects of TSA on developmental potential of swamp buffalo cloned embryos. Swamp buffalo cloned embryos were produced by electrical pulse fusion of male swamp buffalo fibroblasts with swamp buffalo enucleated oocytes. After fusion, reconstructed oocytes were treated with 0, 25 or 50 nM TSA for 10 h. The results showed that there was no significant difference in the rates of fusion (82–85%), cleavage (79–84%) and development to the 8-cell stage (59–65%) among treatment groups. The highest developmental rates to the morula and blastocyst stages of embryos were found in the 25 nM TSA-treated group (42.7 and 30.1%, respectively). We also analyzed the DNA methylation level in the satellite I region of donor cells and in in vitro fertilized (IVF) and cloned embryos using the bisulfite DNA sequencing method. The results indicated that the DNA methylation levels in cloned embryos were significantly higher than those of IVF embryos but approximately similar to those of donor cells. Moreover, there was no significant difference in the methylation level among TSA-treated and untreated cloned embryos. Thus, TSA treatments at 25 nM for 10 h could enhance the in vitro developmental potential of swamp buffalo cloned embryos, but no beneficial effect on the DNA methylation level was observed.     

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.     

牛卵母细胞电激活研究

采用高场强单次脉冲（1600V/cm,80μs）及低场强3次脉冲（400V/cm,20μs,每次间隔20min）刺激体外成熟牛卵母细胞（成熟26h）,激活率分别为86.5%（167/193）和92.7%（101/109）,差异不显著（P>0.05）,其中多原核率分别为8.3%（16/193）和59.6%（65/109）,差异极显著（P<0.01）,但发育率差异不显著（20.6％和23.3％）。实验表明,电脉冲能显著提高牛成熟卵母细胞的激活率;多次电激会造成很大比例的多原核,但对卵母细胞的发育影响不大。     

Activation of Mouse Oocytes Following Intracytoplasmic Injection of Chicken Sperm Extract

The cytosolic factor from the sperm of a wide variety of species has been reported to induce [Ca²⁺]i increase and/or activation in oocytes. Although many species have been studied, there is no data available on the chicken sperm factor responsible for activation of oocytes. This study was aimed to verify the activation of mouse oocyte by intracytoplasmic injection of chicken sperm extract (CSE). Survival rate of oocytes without rupture or lytic degeneration following injection was greater than 80% regardless of the presence or absence of CSE. Among the intact oocytes, activation rates following injection were 27.5% (11 of 40) in the sham operation group. Injection of CSE resulted in significantly higher activation rates in the 1/10 dilution group (57.8%, 26 of 45) compared with the sham operation group (p = 0.0045). Calculated amount of injected sperm extract of 1/10 dilution of CSE was equivalent of 12 chicken sperm. When the 1/10 diluted mouse sperm extract (MSE) was injected, survival rate of injected oocytes and activation rate of the survived oocytes was 82% (41 of 50) and 78% (32 of 41), respectively. Activation rate of MSE‐injected oocytes was a little but significantly higher than that of CSE‐injected oocytes (p < 0.037). In conclusion, it suggests that CSE can activate the mouse oocyte and that oocyte activating sperm factor(s) may be common between avian and mammal.     

水牛精子提取物对猪卵母细胞的孤雌激活效果

为验证精子蛋白对卵母细胞的激活效果,为提高核移植胚胎的质量提供可行方法,通过比较不同裂解方法提取的水牛精子蛋白,不同浓度提取液显微注入猪卵母细胞,对水牛精子提取物的孤雌激活效果进行研究。结果表明:采用液氮反复冻融法和超声波破碎法预处理后提取的水牛精子提取物对猪体外成熟卵母细胞都有显著的孤雌激活效果。水牛精子蛋白浓度在0.53～5.52 mg/mL,注射剂量为2～4 pL时均可有效激活猪卵母细胞,综合效果以1.77 mg/mL较佳。     

The Epididymis of the Prepubertal Swamp Buffalo (Bubalus bubalis): Histochemistry of Phosphatases

The distribution of phosphatases was examined in the epididymis of swamp buffaloes aged 1.5-2 years, where spermatogenic activity in the testis had reached the early primary spermatocyte stage. A granular distribution of acid phosphatase was present in the luminal region of the epithelium of the efferent ductules. In the ductus epididymidis, four zones were identified and all zones showed varying degrees of acid-phosphatase activity in the epithelium, with the most pronounced activity in the apical region and stereocilia of zone II. Alkaline-phosphatase activity occurred along the basal region of the epithelium in zones I. III and IV of the ductus and in the stereocilia of zones I and II. An intense apical reaction was seen in zone II. The efferent ducts were free of this enzyme. Thus, zone II is considered the most active region, having the function of absorption and possibly steroid metabolism.     

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.     

Effect of Different Combinations of Cryoprotectants on In Vitro Maturation of Immature Buffalo (Bubalus bubalis) Oocytes Vitrified by Straw and Open‐Pulled Straw Methods

This study was designed to evaluate effects of different combinations of cryoprotectants on the ability of vitrified immature buffalo oocytes to undergo in vitro maturation. Straw and open‐pulled straw (OPS) methods for vitrification of oocytes at the germinal vesicle stage also were compared. The immature oocytes were harvested from ovaries of slaughtered animals and were divided into three groups: (i) untreated (control); (ii) exposed to cryoprotectant agents (CPAs); or (iii) cryopreserved by straw and OPS vitrification methods. The vitrification solution (VS) consisted of 6 m ethylene glycol (EG) as the standard, control vitrification treatment, and this was compared with 3 m EG + 3 m dimethyl sulfoxide (DMSO), 3 m EG + 3 m glycerol, and 3 m DMSO + 3 m glycerol. Cryoprotectants were added in two steps, with the first step concentration half that of the second (and final) step concentration. After warming, oocyte samples were matured by standard methods and then fixed and stained for nuclear evaluation. Rates of MII oocytes exposed to CPAs without vitrification were lower (54.3 ± 1.9% in EG, 47.5 ± 3.4% in EG + DMSO, 36.8 ± 1.2% in EG + glycerol and 29.9 ± 1.0% in DMSO + glycerol; p < 0.05) than for the control group (79.8 ± 1.3%). For all treatments in each vitrification experiment, results were nearly identical for straws and OPS, so all results presented are the average of these two containers. The percentages of oocytes reaching telophase‐I or metaphase‐II stages were lower in oocytes cryopreserved using all treatments when compared with control. However, among the vitrified oocytes, the highest maturation rate was seen in oocytes vitrified in EG + DMSO (41.5 ± 0.6%). Oocytes cryopreserved in all groups with glycerol had an overall low maturation rate 19.0 ± 0.6% for EG + glycerol and 17.0 ± 1.1% for DMSO + glycerol. We conclude that the function of oocytes was severely affected by both vitrification and exposure to cryoprotectants without vitrification; the best combination of cryoprotectants was EG + DMSO for vitrification of immature buffalo oocytes using either straw or OPS methods.     

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.     

Sperm Pretreatment with Dithiothreitol Increases Male Pronucleus Formation Rates After Intracytoplasmic Sperm Injection (ICSI) in Swamp Buffalo Oocytes

Failure of male pronucleus formation has hampered the success of intracytoplasmic sperm injection (ICSI) in swamp buffalo. The aim of the present study was to improve male pronucleus formation by pretreating sperm with various chemicals before ICSI. In Experiments1 and 2, sperm were treated according to one of the following protocols: (1) 0.1% Triton-X 100 (TX) for 1 min, (2) 10 µM calcium ionophore (CaI) for 20 min, (3) freezing and thawing (FT) without any cryoprotectant, or (4) no treatment (control). These sperm treatment groups then either did or did not receive additional sperm treatment with 5 mM dithiothreitol (DTT) for 20 min. Acrosomal integrity (Experiment 1) and DNA fragmentation (Experiment 2) were evaluated in the sperm before ICSI. In Experiment 3, oocytes matured in vitro were subjected to ICSI using pretreated sperm as described above and then were cultured either with or without activation. The TX- and CaI-treated sperm caused an increase in the number of acrosome-loss sperm, whereas the FT treatment and control increased the proportion of acrosome-reacted sperm (P<0.05). The DNA fragmentation did not differ among treatments (P>0.05). At 18 h post-ICSI, pronucleus (PN) formation was found only in activated oocytes. The majority of the activated ICSI oocytes contained intact sperm heads. Normal fertilization was observed in the CaI and FT treatment groups and control group when sperm were treated with DTT before ICSI. In conclusion, DTT treatment of sperm with reacted acrosomes before ICSI together with activation of the ICSI oocytes is important for successful male pronucleus formation.     

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.