Pregnancies established from handmade cloned blastocysts reconstructed using skin fibroblasts in buffalo (Bubalus bubalis)

Handmade cloning (HMC), a simple, micromanipulation-free cloning technique, has been applied for the production of cloned embryos and offspring in many livestock species. The objective of the present study was to compare the effect of donor cell type on developmental competence of HMC embryos and to explore the possibility of establishing pregnancies using these embryos in buffalo. After technical optimization of the HMC procedure for in vitro development of cloned blastocysts, various donor cells were compared for their developmental efficiency. Using buffalo fetal-, newborn-, adult fibroblasts and cumulus cells, blastocyst production rates obtained from reconstructed embryos were 24.0 ± 1.8% (35/145), 33.0 ± 8.0% (56/163), 21.0 ± 9.3% (29/133) and 49.6 ± 1.9% (77/154), respectively. Blastocyst rates were higher (P < 0.05) in cumulus cell reconstructed embryos in comparison to those derived from fetal or adult fibroblasts. Pregnancy diagnosis (transrectal ultrasonography) was carried out at Day 40 of gestation. Following transfer of HMC embryos reconstructed using newborn fibroblasts 25% (2/8) buffaloes were pregnant and are at Days 201 and 94 of gestation, whereas after transfer of HMC embryos reconstructed using fetal fibroblasts, 20% (1/5) buffaloes were pregnant and are at Day 73 of gestation. In conclusion, HMC could be a simple and efficient technique for the production of cloned embryos for establishing pregnancies in buffalo.     

Hand-made cloned buffalo (Bubalus bubalis) embryos: comparison of different media and culture systems

Hand-made cloning (HMC) has proved to be an efficient alternative to the conventional micromanipulator-based technique in some domestic animal species. This study reports the development of an effective culture system for in vitro culture of zona-free cloned buffalo (Bubalus bubalis) embryos reconstructed using adult skin fibroblast cells as nucleus donor. Cleavage and blastocyst rates observed were 52 and 0% in modified Charles Rosenkrans 2 (mCR2), 61 and 4.6% in modified Synthetic Oviductal Fluid (mSOF), and 82 and 40.3% in Research Vitro Cleave (RVCL; Cook, Australia) medium, respectively. Similarly, higher blastocyst rates (24.5 +/- 4.1%) were observed when zona-free parthenotes were cultured in RVCL medium. Culturing zona-free cloned buffalo embryos on flat surfaces (FS) yielded significantly higher (p < 0.05) blastocyst rates than Well of the Wells (WOW) or microdrops (MD). Furthermore, development in WOW was found to be significantly better than MD culture. The quality of HMC blastocysts was examined using differential staining. This study establishes the application of zona-free nuclear transfer procedures for the production of hand-made cloned buffalo embryos and the development of efficient culture system and appropriate media requirements for enhancing their preimplantation development.     

High overall in vitro efficiency of porcine handmade cloning (HMC) combining partial zona digestion and oocyte trisection with sequential culture

We investigated the in vitro developmental competence of porcine embryos produced from in vitro matured (IVM) oocytes by improved HMC and parthenogenetic activation (PA). Embryos were cultured in a modified North Carolina State University (NCSU37) medium. Firstly, we compared the developmental competence between oocytes from sows and gilts by zona-intact (ZI) and zona-free (ZF) PA. Significantly higher (p < 0.05) blastocyst rates were obtained from sow oocytes (42 +/- 4% for ZF and 55 +/- 6% for ZI) than gilt oocytes (20 +/- 2% for ZF and 26 +/- 5% for ZI). Secondly, sow oocytes were used to establish the modified HMC that was based on a modified enucleation with partial zona digestion and trisection of porcine oocytes and the use of three cytoplasts and one somatic cell for embryo reconstruction. In vitro fertilization (IVF) and in parallel ZF PA were used as the control systems. After oocyte trisection, >90% of oocyte fragments were recovered, resulting in an average of 37 reconstructed embryos from 100 oocytes. Blastocyst rates of HMC, IVF, and ZF PA embryos were 17 +/- 4%, 30 +/- 6%, and 47 +/- 4%, respectively. Our results prove that HMC in pigs may result in high in vitro efficiency up until the blastocyst stage. In vivo developmental competence will be confirmed in embryo transfer experiments.     

Effects of vascular endothelial growth factor on porcine preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer

This study examined the effects of vascular endothelial growth factor (VEGF) on porcine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) at different developmental stages. Four sets of experiments were performed. In the first, supplementation of the in vitro culture medium with 5 ng/mL VEGF was suitable for porcine IVF embryo development, and the blastocyst formation rate was significantly higher than the control and other groups (57.73 ± 6.78% (5 ng/mL VEGF) vs. 43.21 ± 10.22% (control), 42.16 ± 10.24% (50 ng/mL VEGF) and 41.91 ± 11.74% (500 ng/mL VEGF); P < 0.05). The total cell number after supplementation with 5 ng/mL VEGF was significantly higher than the control and other groups (151.85 ± 39.77 (5 ng/mL VEGF) vs. 100.00 ± 34.43 (control), 91.2 ± 31.51 (50 ng/mL VEGF), and 112.53 ± 47.66 (500 ng/mL VEGF); P < 0.05). In the second experiment, when VEGF was added at different developmental stages of IVF derived embryos (early stage, days 1-3, late stage, days 4-7), the blastocyst formation rate and total cell number were significantly higher at the late stage (47.71 ± 9.13% and 131.5 ± 20.70, respectively) than in the control (34.32 ± 7.44% and 85.50 ± 20.41, respectively) and at the early stage (33.60 ± 5.78% and 86.75 ± 25.10, respectively; P < 0.05). There was no significant difference in the blastocyst development rate or total cell number between the whole culture period (days 1-7) and the late stage culture period after supplementation with 5 ng/mL VEGF (P > 0.05). In the third experiment, the cleavage rate was significantly higher when SCNT embryos were cultured with VEGF during the whole culture period than in the late stage (63.56 ± 15.52% vs. 39.72 ± 4.94%; P < 0.05), but there was no significant difference between the control and the early stage culture period (P > 0.05). The blastocyst formation rate was significantly higher at the late stage culture period with VEGF than at the early stage culture period (34.40 ± 15.06% vs. (16.07 ± 5.01%; P < 0.05). There was no significant difference in the total cell number between the groups (P > 0.05). In experiment 4, using real-time PCR, VEGF mRNA expression was detected in all the developmental stages of IVF and SCNT embryos, but the expression level varied according to the developmental stage. VEGF receptor, KDR mRNA was detected in all stages IVF and SCNT embryos. However, flt-1 mRNA was not expressed in all embryonic stages of IVF and SCNT embryos. These data suggest that VEGF supplementation at the late embryonic developmental stage might improve the developmental potential of both IVF and SCNT preimplantation porcine embryos through its receptors.     

Effect of increasing zinc sulphate concentration during in vitro maturation of bovine oocytes

The objective was to investigate the effects of supplementary zinc (Zn) during in vitro maturation (IVM) of bovine oocytes. The DNA damage in cumulus cells was low with supplemental Zn concentrations of 1.1 and 1.5 μg/mL in the IVM medium (mean ± SEM index of DNA damage was 67.52 ± 9.32, 68.52 ±13.34, 33.80 ± 4.89, and 34.65 ± 7.92 for supplementation with 0, 0.7, 1.1, and 1.5 μg/mL Zn, respectively; P < 0.01). Total glutathione concentrations did not differ following Zn supplementation of 1.1 and 1.5 μg/mL (3.7 ± 0.4 vs. 4.0 ± 0.5 pmol, respectively, in oocytes; and in cumulus cells, 0.5 ± 0.04 nmol/10⁶ cells, combined for both treatments), but were greater (P < 0.01) than supplementation with 0.7 μg/mL (1.8 ± 0.5 pmol in oocytes and 0.2 ± 0.02 nmol/10⁶ cumulus cells). Cleavage rate increased (P < 0.05) when Zn was added to the IVM medium at any concentration (67.16 ± 1.17, 73.15 ± 1.15, 74.05 ± 1.23, and 72.76 ± 0.74 for 0, 0.7, 1.1, and 1.5 μg/mL Zn). For these concentrations, subsequent embryo development to the blastocyst stage was 17.83 ± 2.15, 21.95 ± 0.95, 27.65 ± 1.61, and 30.33 ± 2.78%, highest (P < 0.01) in oocytes matured with 1.5 μg/mL Zn. There was an increase (P < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 1.1 and 1.5 μg/mL Zn relative to 0 Zn (IVM alone) and 0.7 μg/mL Zn. In conclusion, Zn during oocytes maturation significantly affected intracellular GSH content and DNA integrity of cumulus cells, and improved preimplantational embryo development. We inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Zn concentrations.     

In vitro effects of estradiol-17β, monobutyl phthalate and mono-(2-ethylhexyl) phthalate on the secretion of testosterone and insulin-like peptide 3 by interstitial cells of scrotal and retained testes in dogs

The objective was to determine the effects of estradiol-17β, monobutyl phthalate (MBP) and mono-(2-ethylhexyl) phthalate (MEHP) on testosterone and insulin-like peptide 3 (INSL3) secretions in cultured testicular interstitial cells isolated (enzymatic dispersion) from scrotal and retained testes of small-breed dogs. Suspension cultures were treated with estradiol-17β (0, 10, and 100 ng/mL), MBP (0, 0.8, and 8 mmol/L) or MEHP (0, 0.2, and 0.8 mmol/L) for 18 h, in the presence or absence of 0.1 IU/mL hCG. Testosterone (both basal and hCG-induced) and INSL3 (basal) concentrations were measured in spent medium. Effects of estradiol-17β, MBP, and MEHP on testosterone and INSL3 secretions were not affected (P > 0.15) by cell source (scrotal versus retained testis); therefore, data were combined and analyzed, and outcomes reported as percentage relative to the control. In testicular interstitial cells, basal testosterone secretion was increased (P < 0.01) by 100 ng/mL estradiol-17β (130.2 ± 10.6% of control). Among phthalates, 0.2 and 0.8 mmol/L MEHP stimulated (P < 0.01) basal testosterone secretion (135.5 ± 8.3% and 154.6 ± 12.9%, respectively). However, hCG-induced testosterone secretion was inhibited (P < 0.01) by 8 mmol/L MBP (67.7 ± 6.0%), and tended to be inhibited (P = 0.056) by 0.8 mmol/L MEHP (84.5 ± 5.6%). Basal INSL3 secretion was inhibited (P < 0.01) by 8 mmol/L MBP (73.6 ± 6.8%) and 0.8 mmol/L MEHP (76.9 ± 11.3%). In conclusion, we inferred that estradiol-17β and certain phthalate monoesters had direct effects on secretions of testosterone and INSL3 in canine testicular interstitial cells, with no significant difference between scrotal and retained testes.     

Minimally invasive transabdominal collection of preimplantation embryos from the common marmoset monkey (Callithrix jacchus)

A novel, minimally invasive, transabdominal embryo collection method (transabdominal method) was developed as an alternative to a standard abdominal incision for embryo collection in the common marmoset. The abdominal incision method was used for 304 flushes using 36 female animals, whereas the transabdominal method was used for 488 flushes using 48 females; successful embryo collection rates were 48.0% and 48.4% (P > 0.05), respectively. These techniques were successfully duplicated at another institute (German Primate Center, DPZ). At that institution, successful embryo collection rates were 88.9% and 77.8% for the abdominal incision and transabdominal methods, respectively (P > 0.05), whereas the average numbers of preimplantation embryos obtained per flush were (mean ± SD) 1.91 ± 0.35 and 1.71 ± 0.14 (P > 0.05). The transabdominal method reduced animal stress, did not require incisional wound healing, and enabled successive embryo recoveries to be done much sooner. More embryos in early developmental stages (zygotes/morulae) were recovered using the transabdominal method (76.1%) than the abdominal incision method (52.6%, P < 0.01). In contrast, recovery of arrested or abnormal embryos was not significantly different between these two methods (9.8% and 8.3%). To verify developmental ability of embryos recovered by the transabdominal method, transfer of 28 normal embryos to 14 surrogate mothers yielded a nidation rate of 57%. Five females sustained term pregnancies and eight neonates were born. This novel transabdominal method will facilitate progress in marmoset developmental biology and embryology.     

Some biochemical characteristics and preservation of epididymal camel spermatozoa (Camelus dromedarius)

Testicles were isolated from thirty five apparently healthy dromedary camels (Camelus dromedarius), aged between 5 to 18 years, in a local slaughterhouse during the rutting season. Epididymal fluid was collected from one epididymis for determination of twelve biochemical and antioxidant parameters using ELISA commercial kits. Spermatozoa were harvested from each region of the other epididymis (head, body and tail) and stored in SHOTOR^®, Green buffer^® + 20% egg yolk and INRA-96^® extenders at 5 and 30 °C. Results revealed that, in the epididymal fluid, concentrations of testosterone, glucose, albumin, total protein, cholesterol, fatty acids, iron, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were 5.19 ± 1.69 ng/mL, 3.10 ± 0.41 mmol/L, 6.26 ± 1.26 g/dL, 0.50 ± 0.07 mg/dL, 1.74 ± 0.09 mmol/L, 6.62 ± 0.81 nmol/ul, 926.20 ± 100.18 ug/dL, 51.17 ± 7.74 mIU/ml, and 143.16 ± 18.67 mIU/ml, respectively. The antioxidants activities of catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD) in the epididymal fluid were 121.55 ± 6.57 nmol/min/ml, 59.35 ± 10.98 nmol/min/ml and 0.18 ± 0.03 U/ml, respectively. Epididymal sperm motility and concentration were higher (P < 0.05) in the body and tail than the head. The viability indices of total and forward sperm motility, at 5 and 30 °C, obtained from the tail region were superior (P < 0.05) in both SHOTOR^® and INRA-96^® extenders than Green buffer extender. It may be concluded that INRA-96^® extender is the best for storing dromedary epididymal spermatozoa at 5 and 30 °C.     

Effects of exogenous melatonin on in vivo embryo viability and oocyte competence of undernourished ewes after weaning during the seasonal anestrus

This study investigated the effects of exogenous melatonin on embryo viability and oocyte competence in post-partum undernourished ewes during the seasonal anestrus. At parturition (mid-Feb), 36 adult Rasa Aragonesa ewes were assigned to one of two groups: treated (+MEL) or not treated (−MEL) with a subcutaneous implant of melatonin (Melovine®, CEVA) on the day of lambing. After 45 d of suckling, lambs were weaned, ewes were synchronized using intravaginal pessaries, and fed to provide 1.5× (Control, C) or 0.5× (Low, L) times daily maintenance requirements. Thus, ewes were divided into four groups: C−MEL, C+MEL, L−MEL, and L+MEL. At estrus (Day=0), ewes were mated. At Day 5 after estrus, embryos were recovered by mid-ventral laparotomy and classified based on their developmental stage and morphology. After embryo collection, ovaries were recovered and oocytes were classified and selected for use in in vitro fertilization (IVF). Neither diet nor melatonin treatment had a significant effect on ovulation rate and on the number of ova recovered per ewe. Melatonin treatment significantly improved the number of fertilized embryos/corpus luteum (CL) (−MEL: 0.35 ± 0.1, +MEL: 0.62 ± 0.1; P = 0.08), number of viable embryos/CL (−MEL: 0.23 ± 0.1, +MEL: 0.62 ± 0.1; P < 0.01), viability rate (−MEL: 46.6%, +MEL: 83.9%; P < 0.05), and pregnancy rate (−MEL: 26.3%, +MEL: 76.5%; P < 0.05). In particular, exogenous melatonin improved embryo viability in undernourished ewes (L−MEL: 40%, L+MEL: 100%, P < 0.01). Neither nutrition nor exogenous melatonin treatments significantly influenced the competence of oocytes during IVF. Treatment groups did not differ significantly in the number of healthy oocytes used for IVF, number of cleaved embryos, or number of blastocysts and, consequently, the groups had similar cleavage and blastocyst rates. In conclusion, melatonin treatments improved ovine embryo viability during anestrus, particularly in undernourished post-partum ewes, although the effects of melatonin did not appear to be mediated at the oocyte competence level.     

Simplified cryopreservation of porcine cloned blastocysts

Recently, a non-invasive delipation (lipid removal) method combined with ultrarapid vitrification has been used successfully for in vitro produced (IVP) porcine embryos. In the present study, this method was combined with parthenogenesis and a recent form of somatic cell nuclear transfer (SCNT) - handmade cloning (HMC) - to establish a simplified and efficient cryopreservation system for porcine cloned embryos. In Experiment 1, zonae pellucidae of oocytes were partially digested with pronase, followed by centrifugation to polarize lipid particles. Ninety percent (173/192) oocytes were successfully delipated in this way. Parthenogenetic activation (PA) after complete removal of zona resulted in similar blastocyst rates in delipated vs. control oocytes (28+/-7% vs. 28+/-5%, respectively). Subsequent vitrification of produced blastocysts with the Cryotop technique resulted in higher survival rates in the delipated group compared to the control group (85+/-6% vs. 32+/-7%, respectively; P<0.01). In Experiment 2, delipated oocytes were used for HMC with normal oocytes as control. Partial zona digestion was further applied before enucleation both in delipated and control groups, to bisect oocyte successfully. Although the blastocyst rate of reconstructed embryos was similar between groups derived from delipated vs. control oocytes (21+/-6% and 23+/-6%, respectively), after vitrification higher survival rates were achieved in the delipated groups than in controls (79+/-6% vs. 32+/-8%, respectively). Our results prove that porcine embryos produced from delipated oocytes by PA or HMC can be cryopreserved effectively by ultrarapid vitrification. Further experiments are required to assess the in vivo developmental competence of the cloned-vitrified embryos.     

Effect of co-culturing of half-destroyed and intact 4-cell mouse embryos in varying ratios on subsequent in vitro development

We studied the co-culturing effect of intact and half-destroyed 4-cell mouse embryos on blastocyst formation rate and cell counts. A laser beam was used to produce a hole and destroy an adjacent blastomere in two opposite areas of the zona in the experimental group (n = 342), and to open two opposite zonal holes in the controls (n = 318). Control and half-destroyed embryos were cultured together in varying ratios of 10:0, 7:3, 5:5, 3:7, and 0:10 (group 1-5, respectively) for 48 h in 10 μl drops of cleavage medium. They were then separated and cultured in blastocyst medium for 24 h. The results showed that half-destroyed embryos had no effect on the blastulation rates of controls (97-100%, P = 0.28). Neither was there a difference in the number of ICM (27.3 ± 6.7, 29.4 ± 9.9, 27.7 ± 9.3, 26.5 ± 6.4, in group 1-4, respectively; P = 0.491), TE (47.7 ± 18.6, 52.3 ± 13.9, 48.4 ± 19.2, 57.3 ± 12.9, in group 1-4, respectively; P = 0.101), nor total cells (75.0 ± 19.5, 81.3 ± 17.1, 76.1 ± 19.6, 83.7 ± 16.2, in group 1-4, respectively; P = 0.188) in the resulting blastocysts. However, among half-destroyed embryos, cleavage arrest decreased (58.3%, 39.6%, 17.9%, and 8.3%, in group 5 to 2, respectively; P < 0.001) and blastocyst development increased (38.3%, 58.2%, 72.6%, and 88.9%, in group 5 to 2, respectively; P < 0.001) following co-culturing with intact controls. These embryos had a higher number of ICM cells (P = 0.035), but no significant changes in TE (P = 0.262) and total cell counts (P = 0.065). The findings indicate that the co-culturing of half-destroyed with intact embryos increased the blastulation rate of the first but had no effect on the latter.     

α-Tocopherol and l-ascorbic acid increase the in vitro development of IVM/IVF swamp buffalo ( Bubalus bubalis) embryos

This study was conducted to investigate the effects of capacitating agents added at in vitro fertilization (IVF) and antioxidants supplemented during in vitro culture (IVC) on the development of buffalo embryos. In experiment I, in vitro embryo development of buffalo embryos was compared when the IVF medium was supplemented with heparin, caffeine and calcium ionophore A23187 either alone or in combination. There was no significant difference (P > 0.05) in the cleavage rates of oocytes among the treatment groups but the development rate to the blastocyst stage and the cell numbers of blastocyst in the heparin-treated group were significantly higher (P < 0.05) than that of other treatments. In experiment II, in vitro embryo development of buffalo embryos was compared when IVC medium was supplemented with either α-tocopherol (250 and 500 μM) or l-ascorbic acid (250 and 500 μM). The rate of development to the blastocyst stage of embryos cultured in medium supplemented with 250 μM α-tocopherol (33%, 41/123) and 250 μM l-ascorbic acid (31%, 38/123) was significantly higher (P < 0.05) than that of those cultured in medium alone (19%, 20/108) but not significantly different (P < 0.05) from medium supplemented with either 500 μM α-tocopherol (24%, 30/123) or 500 μM l-ascorbic acid (25%, 33/133). These results suggest that buffalo spermatozoa treated with heparin were suitable for IVF and that α-tocopherol and l-ascorbic acid added during IVC increased the rate of buffalo embryo development.     

Removal of swine zonae pellucidae with sequential exposures to pronase and acidified medium

Exposure to acidified PBS (pH 3) for 60 sec removed swine zonae pellucidae from 70.4% of 27 swine morulae, and 73.7% of these formed blastocysts in culture. Further investigations revealed that treating embryos with 0.5% pronase and acidified PBS (pH 3) for 30 sec each was more effective. Zonae were removed in 90.6% of 85 embryos (four-cell to morulae) treated. A total of 76.9% of 65 zona-free embryos and 81.6% of 38 untreated embryos formed blastocysts (P > 0.05). An additional 57 untreated and 49 zona-free embryos (morulae to blastocysts) were transferred to seven recipient sows. Four sows returned to estrus (18 to 27 days), but three others were pregnant when slaughtered at 38 to 42 days. One pregnant sow had received a combination of five zona-free and six untreated embryos and demonstrated the potential for further development of treated embryos in vivo.     

Effect of post-fusion holding time, orientation and position of somatic cell-cytoplasts during electrofusion on the development of handmade cloned embryos in buffalo (Bubalus bubalis)

The present study was conducted primarily to optimize electrofusion conditions for efficient production of zona-free nuclear transfer embryos in buffalos (Bubalus bubalis). We found that 4V AC current for proper triplet alignment and single step fusion method, using a single DC pulse of 3.36 kV/cm for 4-μs duration, produced the most convincing results for efficient reconstitution of zona-free cloned embryos. Lysis rate was very high (84.28 ± 2.59%) when triplets were in physical contact with negative electrode after applying DC current, however, cleavage rate and blastocyst rate were found to be similar when the triplets were not in physical contact with either positive or negative electrodes or when they were in physical contact with the positive electrode. Significant improvement in blastocyst production was observed when the somatic cell faced the positive electrode than when it faced the negative electrode (39.17 ± 2.74% vs. 25.91 ± 2.00%, respectively) during electrofusion. Similarly, the blastocyst rate (52.0 ± 3.4%) was found to be significantly higher when reconstructed embryos were activated 6 h post electrofusion as compared to 0, 2, 4 and 8 h (16.04 ± 6.3%; 18.36 ± 1.4%; 22.44 ± 3.7% and 30.02 ± 4.6%, respectively). This study establishes the application of zona-free nuclear transfer procedures for the production of handmade cloned buffalo embryos through optimization of electrofusion parameters and post fusion holding time for enhancing their preimplantation development.     

孕激素暴露致食蚊鱼形态雄性化的生物学效应

目的：研究不同浓度孕酮（ PRO）暴露对雌性食蚊鱼（ Gambusia affinis）的形态雄性化生物学效应。方法将性未成熟的雌性食蚊鱼个体分别静水暴露于0.5、5、50和500 nmol/L不同浓度的PRO实验组中,并设置对照组和平行组。持续暴露42 d后观察与测定食蚊鱼的体长、体重、臀鳍第3鳍条和第14,15,16椎肋骨形态雄性化变化等指标,同时观察卵巢发育状况。结果食蚊鱼暴露42 d后,与对照组相比,50和500 nmol/L实验组食蚊鱼的体长（ BL）有极显著减少（分别P＜0.01）;无论是低浓度组还是高浓度组,暴露实验后体重（ BW）都有显著性或极显著下降（P＜0.05或P＜0.01）;PRO暴露对食蚊鱼身体健康指数（CF）的影响,只有在5 nmol/L浓度时差异有显著性（P＜0.05）;500 nmol/L浓度处理后,第3臀鳍条分节数（FJ）、长度（FL）和最宽处宽度（FW）差异有显著性（P＜0.05）,表明形态雄性化变化明显;其他浓度组则无明显区别（P＞0.05）;500 nmol/L PRO实验组中,雌性食蚊鱼的第14,15,16椎肋骨的L值、D值和L：D值都出现极显著性差异（分别P＜0.01）,表明形态雄性化变化明显;组织切片观察揭示,暴露组食蚊鱼卵巢中的卵母细胞发育受到不同程度的抑制,II、III时相卵母细胞胞核发生肿胀现象。结论孕激素暴露诱导雌性食蚊鱼形态雄性化的生物效应十分明显。     

Cryopreservation of mouse embryos by vitrification: A meta-analysis

A meta-analysis of cryopreservation studies vitrifying mouse embryos was undertaken to determine the treatment effect of vitrification. Treatment by vitrification decreased embryo viability compared with controls: the odds ratio was 9.02 (CI: 3.73-21.78; P < 0.001), a 24.90% (CI: 14.88-34.91; P < 0.001) reduction in risk was associated with embryos in the control group, and for every 4.00 (CI: 3.91-4.09) embryos treated by vitrification, one does not survive. A multiple regression analysis evaluated covariates of embryo survival. For each hour increase post-hCG treatment when embryos were cryopreserved, there was a decrease of 0.36% (SEM ± 0.01) in survival (P < 0.001). The number of embryos surviving vitrification decreased 0.25% (SEM ± 0.02) per day increase in age of the female mouse (P < 0.001), whereas there was no significant difference for control group embryos. For each 1 h increase post-hCG treatment after cryopreservation when blastocysts were assessed for viability, there was a decrease of 0.13% (SEM ± 0.01) in survival. The later interval post-hCG treatment when blastocysts were assessed, the less viable they were compared with earlier blastocysts, independent of the vitrification protocol. This effect was not observed for control embryos. A high percentage of variability in the treatment effect for vitrification was likely due to underlying heterogeneity among studies. A portion of the risk associated with vitrification could be attributed to the general effects of cryopreservation. Future research should identify effects in a cryopreservation protocol specific to vitrification that affect viability of mouse embryos.     

Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts

The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P < 0.05). When electrofusion was induced 27 h after the onset of oocyte maturation, the cleavage rate (78.0%) was higher than that of electrofusion induced at 28 h (67.2%, P < 0.05), and the blastocyst yield (18.1%) was higher (P < 0.05) than that of electrofusion induced at 25 or 26 h (7.4 and 8.5%, respectively). A higher proportion of NT embryos activated at 3 h after electrofusion developed to the blastocyst stage (18.6%) in comparison with NT embryos activated at 1 h (6.0%), 2 h (8.3%), or 4 h (10.6%) after fusion (P < 0.05). No recipient was pregnant 60 d after transfer of blastocysts developed from NT embryos activated at 1 h (0/8), 2 h (0/10), or 4 h (0/9) after fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation.