Effect of culture medium and prostaglandin I(2) (PGI(2)) analogue on in vitro development of parthenogenetically activated cat oocytes

A method of reliably producing developmentally competent cat embryos in vitro is a prerequisite for study of the physiology of early development and application of assisted reproductive techniques. Oocytes were collected and then cultured in TCM-199 + 10% FBS for 4 h. The matured oocytes were activated with a 20 microsec electric pulse at 1.2 kV/mm. The activated oocytes were incubated in 2 mM of 6-dimethylaminopurine (6-DMAP) for 4 h and were then divided randomly among the treatment groups. In experiment 1, we compared the effects of three culture systems (TCM-199, CR1-aa and Tyrode's) on the in vitro development of parthenogenetically activated cat oocytes. In experiment 2, we investigated the effect of addition of Iloprost (a stable prostaglandin I(2) analogue) to Tyrode's medium on in vitro development of parthenogenetically activated oocytes. As a control, we recovered in vivo produced blastocysts and determined their average cell number. In experiment 1, the cleavage frequency of the oocytes cultured in TCM199, CR1-aa and Tyrode's media were similar (74, 72 and 83%, respectively). However, the incidence of in vitro development to the blastocyst stage was significantly higher in Tyrode's medium (20.4%) than in TCM-199 (2.4%) or CR1-aa (11.1%). Likewise, the average cell number of in vitro activated blastocysts was higher in Tyrode's than in CR1-aa or TCM-199 (106.5 +/- 45.2 vs. 68.3 +/- 25.4 and 35.0 +/- 7.7, respectively; P<0.05). In experiment 2, the percentage of parthenogenetically activated oocytes that underwent in vitro blastocyst development was significantly improved by addition of Iloprost to the culture medium (33.6 vs. 19.1%; P<0.05). The average cell number of in vivo blastocysts (909.0 +/- 226.4) was significantly higher than those of in vitro blastocysts cultured in Tyrode's medium supplemented with or without Iloprost (103.2 +/- 31.3 and 112.2 +/- 39.3, respectively; P<0.05). This result indicated that the current culture method for cat pathogenetically activated oocytes requires further improvement.     

Effect of vascular endothelial growth factor on maturation,fertilization and developmental competence of bovine oocytes

To examine the effect of Vascular Endothelial Growth Factor (VEGF) on the maturation of bovine oocytes, human recombinant VEGF(165) was used in 3 experiments. In Exp. 1, bovine cumulus oocyte complexes (COCs) were matured for 22 hr in modified Synthetic Oviduct Fluid (m-SOF) supplemented with 0 (control) or 5 ng/ml of VEGF. Maturation rate increased (P<0.05) from 78.2% in the control to 90.5% in the VEGF treated group. In Exp. 2, bovine COCs were matured in m-SOF and co-incubated with sperm in modified BO medium, each supplemented with or without 5 ng/ml VEGF. Normal fertilization rate was improved (P<0.05) from 63.0% (control) to 79.8% or 82.3% with VEGF during maturation or both maturation and fertilization. In Exp. 3, bovine COCs were matured the same way as in Exp. 1, then co-incubated with sperm for 6 hr and cultured for 162 hr in m-SOF without VEGF. Cleavage rate and development rate to the 4- to 8-cell stage were examined at 42 hr post-co-incubation and development rate to blastocyst was examined at 162 hr post-co-incubation. Cleavage, the development to the 4- to 8-cell stage and blastocyst rates (82.0%, 70.3% and 45.1%, respectively) were significantly higher (P<0.05) in the VEGF group than those in the control (67.3%, 52.5% and 33.3%, respectively). These results indicate that VEGF has a beneficial effect on the maturation of bovine oocytes.     

Resveratrol Improves the Mitochondrial Function and Fertilization Outcome of Bovine Oocytes

The aim of the present study was to address the effect of resveratrol-mediated upregulation of sirtuin 1 (SIRT1) during oocyte maturation on mitochondrial function, the developmental ability of oocytes and on mechanisms responsible for blockage of polyspermic fertilization. Oocytes collected from slaughterhouse-derived ovaries were cultured in TCM-199 medium supplemented with 10% FCS and 0 or 20 µM resveratrol (Res). We examined the effect of Res on SIRT1 expression in in vitro-matured oocytes (Exp 1); fertilization and developmental ability (Exp 2); mitochondrial DNA copy number (Mt number), ATP content and mitochondrial membrane potential in matured oocytes (Exp 3); and the time required for proteinase to dissolve the zona pellucida following in vitro fertilization (as a marker of zona pellucida hardening), as well as on the distribution of cortical granules before and after fertilization (Exp 4). In Exp 1, the 20 µM Res treatment upregulated protein expression of SIRT1 in oocytes. In Exp 2, Res treatment improved the ratio of normal fertilization and the total cell number of blastocysts. In Exp 3, Res treatment significantly increased the ATP content in matured oocytes. Additionally, Res increased the overall Mt number and mitochondrial membrane potential, but the effect was donor-dependent. In Exp 4, Res-induced zona hardening improved the distribution and exocytosis of cortical granules after in vitro fertilization. In conclusion, Res improved the quality of oocytes by improving mitochondrial quantity and quality. In addition, Res added to the maturation medium enhanced SIRT1 protein expression in oocytes and improved fertilization via reinforcement of the mechanisms responsible for blockage of polyspermic fertilization.     

Attempt at intracytoplasmic sperm injection of in vitro matured oocytes in common minke whales (Balaenoptera acutorostrata) captured during the Kushiro Coast Survey

The present study was conducted during the Kushiro Coast Survey in an attempt to produce common minke whale embryos. In Experiment 1, we attempted to determine the appropriate culture duration (30 or 40 h) for in vitro maturation (IVM) of immature oocytes using the Well of the Well method. In Experiment 2, and intracytoplasmic sperm injection (ICSI) was applied to matured oocytes from prepubertal and adult common minke whales after IVM culture (40 or 48 h), and then their embryonic development was assessed. In Experiment 1, the maturation rate of oocytes cultured for 40 h (30.4%) was significantly higher than that of oocytes cultured for 30 h (6.8%; P<0.01). In Experiment 2, a total of 35 and 46 immature oocytes derived from adult (n=2) and prepubertal (n=6) minke whales, respectively, were cultured for 40 or 48 h. The maturation rate in the oocytes from the adult whales (34.2%) tended to be higher than that of the oocytes from the prepubertal whales (19.6%), but there was no significant difference. Following ICSI, 3 out of the 10 inseminated and cultured oocytes from the adult whales cleaved (2-, 8-, and 16-cell stages); all of these oocytes had been matured for 40 in culture. However, these oocytes did not develop to further stages. Only one of the 6 oocytes derived from the prepubertal whales, IVM cultured for 40 h and inseminated, developed to the 4-cell stage. The present results indicate that a 40 h IVM culture produces significantly higher rates of in vitro maturation than a 30 h IVM culture for common minke whale oocytes. Following ICSI, some oocytes cleaved to the 16-cell stage, but no further development was observed.     

Intracytoplasmic Glutathione Levels in Heifer Oocytes Cultured in different Maturation Media and its Effect on Embryo Development

The present study was carried out to study the effect of different maturation media on embryo development of heifer oocytes and on their glutathione (GSH) synthesis during in vitro maturation (IVM). Immature heifer oocytes were matured in parallel in one of four maturation media: (i) Tissue Culture Medium (TCM)-199 supplemented with 10 ng/ml of epidermal growth factor (EGF); (i) TCM-199 supplemented with 10 ng/ml of EGF plus 1 microg/ml of FSH; (iii) TCM-199 supplemented with 10% of foetal bovine serum (FBS) and (iv) TCM-199 supplemented with 10% of FBS plus 1 microg/ml of FSH. Cow oocytes were used as control and were matured in TCM-199 supplemented with 10 ng/ml of EGF. No differences were observed in blastocyst rate among the different heifer oocyte groups (8.8, 7.5. 8.4 and 6.8%, respectively) however, the percentage of blastocysts obtained from cow oocytes was significantly higher (30%; p < 0.01) than those obtained from heifer oocytes. De novo GSH synthesis during oocyte maturation of heifer and cow oocytes was detected. No significant differences in intracytoplasmic GSH levels were observed among the experimental heifer oocyte groups or between heifer and cow oocytes both before and after IVM. In conclusion, the blastocyst yield obtained from heifer oocytes was lower than that from cow oocytes and this fact could not be explained by significant differences in intracytoplasmic GSH contents of oocytes before or after IVM.     

Effect of the in vitro maturation medium on equine oocytes: comparison of follicular fluid and oestrous mare serum

The present study evaluated the effect of supplementing the medium used to mature equine oocytes in vitro with oestrous mare serum (EMS) or horse follicular fluid (HFF). To this end, 144 ovaries were obtained from mares aged 16-21 months and transported to the laboratory in Dulbecco's phosphate buffered saline (D-PBS) at 30 degrees C. Oocytes were harvested from the ovaries by slicing, and then selected for in vitro maturation (IVM) according to the number of cumulus cell layers and the characteristics of the cytoplasm. The selected oocytes were washed three times in TCM199 medium plus HEPES (TCM-199H) or in the same medium plus glutamine (TCM-199G), then matured in vitro in six study groups established according to the in vitro maturation (IVM) treatment to see possible interactions between HEPES and glutamine on other supplements: Ten percent EMS was added to two of these media (TCM-199H+EMS and TCM-199G+EMS) and 10% HFF was added to the media in two other groups (TCM-199H+HFF and TCM-199G+HFF). IVM was performed at 38.5 degrees C for 40 h in a controlled atmosphere (5% CO2, 95% relative humidity). The findings indicate that the presence of EMS or HFF in the TCM-199H medium gives rise to the best results in terms of the proportions of oocytes reaching maturity (37.7% and 36.8%, respectively). The values obtained with EMS and HFF were statistically similar to each other but differed from the other treatments. The media containing glutamine led to the highest proportions of degenerated oocytes.     

In vitro and in vivo developmental ability of oocytes derived from porcine primordial follicles xenografted into nude mice

We evaluated the developmental ability of oocytes in porcine primordial follicles xenografted into nude mice. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the kidney capsules of ovariectomized nude mice. Forty-nine to 89 days after grafting (mean +/- SEM, 66.9 +/- 1.9 days; n = 64), the host mice showed the presence of cornified epithelial cells in their vaginal smears for the first time. The mice were then treated with 4 IU of equine chorionic gonadotropin (eCG) 60 days after first detection of vaginal cornification. Oocytes were collected from the host mice 48 h after treatment with eCG, and then matured. The maturation rates, based on the incidence of first polar body, ranged from 25.1% to 42.5%. They were then fertilized in vitro and cultured in vitro for 6 days, or transferred into estrous-synchronized recipients and recovered after 6 days. On Day 6 of culture, 15.4% of the matured oocytes had cleaved to the 2- to 8-cell stage. However, neither the embryos cultured in vitro nor those transferred and recovered developed to advanced embryonic stages, such as morulae or blastocysts. This result suggests that the developmental ability of xenografted oocytes is insufficient, even after in vitro maturation. Further strategies, such as improvement of hormonal treatment for host mice, are required to enable oocytes in xenografted ovarian tissues to acquire the cytoplasmic maturation necessary for embryonic development.     

Influences of Follicular Size on Parthenogenetic Activation and in Vitro Heat Shock on the Cytoskeleton in Cattle Oocytes

The availability of cow ovaries from the slaughterhouse has been very limited in Taiwan. To maximize the use of cow ovaries for research purposes, whole ovary dissection was performed and the developmental competence of the oocytes derived from different sizes of follicles was assessed by the rates of in vitro maturation (IVM) and parthenogenetic activation of the oocytes in Experiment 1 (Exp 1). Cumulus-oocyte complexes (COCs) derived from small (1-2 mm) and large (3-8 mm) follicles were subjected to standard IVM culture for 24 h. Mature oocytes were selected and then parthenogenetically activated using A23187 (5 microm, 5 min) or thimerosal (200 microm, 10 min) alone or combined with 6-dimethylaminopurine (2.5 mm and 3.5 h, respectively). Activation rates of the oocytes, neither from the large nor small follicles, were affected by different activation treatments (single or combined stimuli). Whereas maturation rates for the oocytes from large follicles were superior to those from small follicles in both the single (59% vs 45%) and combined treatments (76% vs 40%; p < 0.05). To understand how prolonged heat shock (HS) influences cytoskeletal configurations of mature bovine oocytes, in Experiment 2 (Exp 2), matured oocytes derived from large follicles were randomly allocated to different durations of HS treatments at 41.5 degrees C for 0 (C0h, control, n = 12), 1 (HS1h, n = 28), 2 (HS2h, n = 31), and 4 h (HS4h, n = 30). An additional control group was cultured for 4 h without HS (38.5 degrees C, 4 h, n = 35). Alterations in nuclear structures, microtubules (MTs), and microfilaments (MFs) of the oocytes were examined. Abnormalities in the chromosomes, spindle MTs and the percentages of oocytes with cytoplasmic MTs increased with time of HS treatment. The intensity of the MF distribution in the HS oocytes was also altered. Significant changes in the cytoskeleton after HS may be associated with the reduced development under hyperthermia and, perhaps, with the low pregnancy rates of the animals during hot seasons.     

Effects of partial removal of cytoplasmic lipid on survival of vitrified germinal vesicle stage pig oocytes

This study was designed to investigate whether the partial removal of cytoplasmic lipid from immature pig oocytes prior to vitrification had any positive effects on subsequent maturation, fertilization and early development. Oocytes at the germinal vesicle stage were partially freed from cumulus cells and centrifuged, and then polarized cytoplasmic lipid was removed by micromanipulation. When cultured for 44-48 h, significantly fewer of the centrifuged oocytes reached metaphase II (M-II) than did the non-centrifuged oocytes (approximately 53% vs approximately 68%, respectively); however, no further reduction in the M-II rate was observed when centrifuged oocytes were then delipated prior to culture (approximately 47%). To evaluate their sensitivity to the equilibration and vitrification solutions containing ethylene glycol, non-centrifuged, centrifuged, and delipated oocytes were cultured continuously for several minutes in those solutions, then washed and cultured further; no significant differences in the M-II rates (approximately 20-27%) were observed among the three treatment groups. When oocytes were vitrified and then warmed, significantly more delipated oocytes reached M-II in culture (approximately 15%) than did the non-delipated oocytes, whether centrifuged or not (approximately 4% in each group). When delipated, vitrified and matured oocytes were microsurgically injected with frozen-thawed spermatozoa, approximately 39% were activated and male pronucleus formation was observed in approximately 40% of activated oocytes; none developed beyond the 4-cell stage. These results show that maturation in vitro of vitrified pig oocytes can be promoted by partial removal of cytoplasmic lipid prior to vitrification and that the vitrified oocytes can be fertilized, although the embryonic development obtained in this study was limited.     

Selection of In Vitro-Matured Porcine Oocytes Based on Localization Patterns of Lipid Droplets to Evaluate Developmental Competence

Localization patterns of lipid droplets in the cytoplasm of porcine oocytes were evaluated as a novel marker for in vitro maturation (IVM) of oocytes with high developmental competence. Porcine oocytes were cultured in TCM-199, which is a complete synthetic medium, for 44 h at 38.5 C. Localization patterns were divided into 2 classes: lipid droplets localized uniformly in the whole cytoplasm (class I) and those that were centrally located (class II). After IVM in TCM-199, 60% of matured oocytes exhibited the class II pattern. To investigate the relation between the distribution of lipid droplets and the developmental rate of the oocyte, the developmental rates of class I and class II oocytes were compared after in vitro fertilization (IVF). Class II oocytes showed a significantly higher rate of blastocyst development than class I oocytes. These results suggest that porcine oocytes with high developmental competence can be selected based on the localization patterns of lipid droplets.     

Parthenogenetic activation and subsequent development of porcine oocytes activated by a combined electric pulse and butyrolactone I treatment

This study was designed to evaluate the parthenogenetic activation of porcine oocytes matured in vitro for a varied period after combined electric pulse (EP; 1500 V/cm, 100 microsec) and Butyrolactone I (BL I). After 36 h of maturation culture, the rates of activated oocytes and oocytes with two pronuclei were significantly lower than those of oocytes cultured for 42 and 48 h after EP. However, when treated by a combined EP and BL I (150 microM), these rates increased to the same level as 42 and 48 h oocytes. When oocytes cultured for 48 h and activated by a combined EP and BL I treatment were subsequently cultured in mNCSU37 medium, the rates of embryos cleaved and developed to the blastocyst stage were significantly higher than those in Whitten's medium. In contrast, when activated oocytes were cultured in mNCSU37 medium under two oxygen environments (5% vs 20% O(2)), there was no difference in the rates of cleavage, blastocyst formation and nuclear numbers per blastocyst. Our results demonstrated that the combined EP and BL I treatment of porcine oocytes matured in vitro is capable of producing high rates of good quality blastocysts when cultured in a suitable in vitro condition.     

Aspectos Cronologicos en la Maduracion Nuclear de Oocitos Bovinos Cultivados in vitro

In this study, the chronological changes in the meiotic progress of in vitro maturation of bovine oocytes was analysed. Oocytes (n = 1044) were obtained from a local abbatoir and were classified into two groups according to the presence or absence of cumulus cells. They were incubated in microdrops (5 per drop) in TCM-199 (control), TCM-199 + 10% fetal calf serum (treatment 2) and TCM-199 + 10% estrous cow serum (treatment 3). Oocytes were fixed and stained at the end of 0, 6, 12, 18, 24 and 30h of the beginning of in vitro culture, were evaluated according to the nuclear stage of maturation. These results demonstrated that the immature oocytes at the time of collection (0 h) were in the germinal vesicle stage (GV), and that the highest maturation rate was at 24 h of culture in all treatments. Serum treatments enhanced the maturation rates obtained (52.1 and 55.7%) compared to control (serum-free) medium (42.7%; P<0.05) in cumulus-cell-enclosed oocytes. In denuded oocytes, the maturation rates were lower compared to cumulus-cells-enclosed oocytes in all treatments. In conclusion, meiotic progression of bovine oocytes can be influenced by the inclusion of sera in the maturation media and by the presence of the cumulus cells.     

Ultrastructural Study of the Canine Zona Pellucida Surface During In Vitro Maturation

The aim of the present study was to compare the ultrastructure of the surface of the zona pellucida (ZP) of immature and in vitro matured dog oocytes using scanning electron microscopy (SEM). Bitch oocytes were collected after ovariohysterectomy; the ovaries were sliced and the released cumulus–oocyte complexes (COCs) were washed with phosphate buffered saline (PBS). The selected COCs were randomly allocated into three groups, two groups were processed after in vitro maturation at both 72 and 96 h and a third group was processed immediately at immature state in PBS medium. After that, oocytes were fixed, critical point dried and viewed by using SEM. The diameters of the outer holes of the ZP were measured on a total of 93 oocytes; the results were analyzed with anova . The mean diameters of holes were different between groups (p < 0.05): 0.69 ± 0.12, 1.56 ± 0.19 and 1.42 ± 0.27 μm, for immature and in vitro matured oocytes for 72 and 96 h, respectively. The difference in the hole sizes between immature and in vitro matured canine oocytes indicates that the ZP surface is related to oocyte maturity in canines.     

Nuclear and cytoplasmic maturation of bovine oocytes cultured with dbc AMP, FSH and hCG

The present investigation was undertaken to study the effect of addition of dbc AMP on bovine oocyte maturation and fertilization in vitro. The bovine oocytes isolated from 2–8 mm follicles were cultured for 26 h in TCM-199. The maturation rate (71.4 %) did not significantly increase after supplementation of the culture medium with dbc AMP (86.3 %.) or FSH + hCG (86.3 %). The in vitro fertilization rate of oocytes based on sperm penetration and presence of sperm tail in the ooplasm increased significantly in the dbc AMP (34.7 %) and the dbc AMP + FSH + hCG (33.9 %) treated groups when compared with untreated controls (17.9 %). However, dbc AMP treated oocytes were not able to secure the formation of male pronucleus 20 h after in vitro fertilization, while in oocytes matured in dbc AMP free medium both pronuclei were present in approximately 15 % of the penetrated oocytes. Also, the sperm head decondensation was blocked or slowed down by the dbc AMP treatment. It is concluded (1) that dbc AMP may improve the condition for the interaction of oocytes with spermatozoa, and (2) that the ooplasm of such dbc AMP treated oocytes apparently is not able to decandense the sperm head and transform it to the male pronucleus.     

玻璃化冷冻牛卵母细胞单精子注射后体外发育能力的研究

实验研究了不同成熟培养时间的牛卵母细胞玻璃化冷冻及胞质内单精子注射(ICSI)后的受精效果。结果表明:成熟后的新鲜牛卵母细胞按照ICSI注射方法穿刺而不注射精子组与未经穿刺的对照组相比,孤雌激活后的卵裂率、囊胚发育率及囊胚细胞数无显著差异(P>0.05);成熟培养16h(MⅠ)和23h(MⅡ)卵母细胞冷冻解冻后形态正常率均显著低于新鲜对照组(76.66%、87.33%vs100.0%)(P<0.05),冷冻解冻后二者分别成熟培养至24h,ICSI后胚胎的囊胚发育率(5.29%、14.41%)显著低于新鲜对照组(24.40%)(P<0.05);成熟培养23h与成熟培养16h的卵母细胞冷冻解冻后形态正常率及ICSI后囊胚发育率(14.41%vs5.29%)均有显著性差异(P<0.05)。实验证明,ICSI操作不会影响卵母细胞发育潜力;玻璃化冷冻影响卵母细胞解冻后形态正常率以及ICSI后胚胎的发育能力;成熟培养23h比16h的卵母细胞冷冻保存后经ICSI的胚胎发育潜力高。     

Role of acidification elicited by sialylation and sulfation of zona glycoproteins during oocyte maturation in porcine sperm-zona pellucida interactions

The porcine zona pellucida (ZP) undergoes biochemical changes during the final phase of maturation prior to fertilization. The present study was conducted to elucidate whether the acidification of ZP glycoproteins during porcine oocyte maturation influences sperm-ZP interactions. Two-dimensional gel electrophoresis clearly demonstrated that ZP acidification occurred in accordance with the sialylation and sulfation of ZP glycoproteins in oocytes matured for 44 h. The increases in the incidences of sperm penetration and polyspermy with the progress of the IVM culture period were significantly suppressed by ZP desialylation on treatment with neuraminidase as a consequence of reductions in the number of sperm bound to ZPs and the acrosome reaction (AR) in ZP-bound sperm (P<0.05). In contrast, the blocking of ZP sulfation by NaClO(3) treatment during IVM markedly reduced the incidence of polyspermy with no inhibitory effect on penetration, but the number of sperm bound to ZPs and the rate of AR-inducing sperm were decreased to the same level as in desialylated oocytes. The results indicate that ZP sulfation influences sperm-ZP interactions in a ZP sialylation-independent manner. Moreover, sialylation and sulfation were not associated with a protective proteolytic modification of the ZP matrix before fertilization. These findings suggest that ZP acidification elicited by the sialylation and sulfation of ZP glycoproteins during oocyte maturation contributes to the porcine ZP acquiring the capacity to accept sperm.     

Ultrastructural Characteristics of Non-matured and In Vitro Matured Oocytes Collected from Pre-pubertal and Adult Domestic Cat Ovaries

The present study describes the ultrastructural characteristics of cat oocytes before maturation and after 12- and 24-h in vitro maturation (IVM). Oocytes were recovered from pre-pubertal and adult queen ovaries after ovariohysterectomy and a proportion were stored in glutaraldehyde at 4°C until examination by transmission electronic microscopy (TEM). Those selected for maturation were cultured before TEM in DMEM for 12 and 24 h at 38°C in a humidified environment of 5% O₂, 5% CO₂ and 90% N₂. Specimens were divided into six groups: non-matured oocytes from pre-pubertal queens (PP0), non-matured oocytes from adult queens (A0), 12-h in vitro matured oocytes from pre-pubertal queens (PP12), 12-h in vitro matured oocytes from adult queens (A12), 24-h in vitro matured oocytes from pre-pubertal queens (PP24) and 24-h in vitro matured oocytes from adult queens (A24). Across the treatment groups, it was possible to observe differences in the thickness of the perivitelline space, the penetration of cumulus cell projections forming a junctional complex, distribution and density of small vesicles, lipid droplets, microvilli, mitochondria and cortical granules and variable degrees of development of Golgi complexes. These findings demonstrated that ultrastructural analysis of oocytes matured in vitro is a valuable tool to evaluate oocyte cytoplasmic maturation and that this IVM protocol was efficient in inducing gradual morphological changes necessary for cytoplasmic maturation of pre-pubertal and adult cat oocytes.     

Production and lambing rate of blastocysts derived from in vitro matured oocytes after gonadotropin treatment of prepubertal ewes.

The aim of this study was to evaluate the effect of gonadotropin treatment on the in vitro maturation, blastocyst production, and developmental potential to term of oocytes collected from Sardinian neonatal and prepubertal ewes at 4 to 6 wk of age. Cumulus-oocyte complexes were recovered at 24 h after withdrawal of a 1/6th size progestagenated pessary from the donors, of which each received 120 IU FSH/LH and 400 IU PMSG in a single dose 36 h before sponge removal. Treated donors produced a greater (P<.01) number of oocytes per animal (86.2 +/-7.9) compared with slaughterhouse (untreated) prepubertal ewes (55.5+/-6.1) of the same age or with treated neonatal ewes (6.1+/-0.7) 10 d old. During oocyte maturation, there were no differences in the percentage of germinal vesicle break-down (78.08 vs. 74.24), metaphase I (89.13 vs. 87.18), and metaphase II (77.91 vs. 76.38) when evaluated after 8, 14, and 24 h of maturation, respectively, between oocytes from treated and slaughterhouse (untreated) prepubertal ewes. The embryo cleavage (71.1 vs. 73.7) and blastocyst rates (22.2 vs. 19.8) were similar in the treated and the untreated prepubertal ewes after transfer of in vitro matured oocytes into ligated oviducts of temporary recipients. The in vitro viability rates of vitrified blastocysts (81.2 vs. 76.9) and the in vivo survival rates (46.1 vs. 50.0) of embryos derived from in vitro matured and in vivo fertilized oocytes showed no difference. The data suggest that gonadotropin treatment increases oocyte production per animal but has no effect on oocyte quality because embryo production and lambing rates of blastocysts derived from in vitro matured oocytes were not markedly different from those derived from untreated prepubertal ewes of the same age.     

马卵母细胞胞质内精子注射后体外发育能力的研究

本研究在非繁殖季节评估卵丘形态(松散型、致密型)、成熟培养体系(TCM 199、NCSU 23)、体外成熟时间(34、38 h)和离子霉素结合6-DMAP激活对马卵母细胞胞质内精子注射(ICSI)后体外发育能力的影响。从屠宰场采集马卵巢,获得的卵母细胞进行体外成熟,然后注射马冷冻解冻精液,统计分裂情况。试验结果表明,①马松散型卵母细胞成熟率显著高于致密型卵母细胞(P<0.05),分别为61.09%和41.24%,但ICSI后36 h分裂率无显著差异(P>0.05),分别为47.34%和44.92%;②两种培养体系对马松散型或致密型卵母细胞成熟率及ICSI后36 h分裂率无显著影响(P>0.05),但相同成熟体系培养松散型卵母细胞成熟率均显著高于致密型卵母细胞(P<0.05),然而ICSI后36 h分裂率差异不显著(P>0.05);③松散型或致密型卵母细胞在TCM 199或NCSU 23中成熟38 h成熟率均高于34 h成熟率,分别为44.43%～68.87%和34.52%～58.90%,松散型卵母细胞在TCM 199体系中成熟34 h、ICSI后激活组或对照组的分裂率显著高于成熟38 h、ICSI后激活组的分裂率(P<0.05),以及致密型卵母细胞在TCM 199体系中成熟34 h、ICSI后激活组的分裂率(P<0.05),而且显著高于松散型卵母细胞在NCSU 23体系中成熟38 h、ICSI后对照组的分裂率(P<0.05);④ICSI后用离子霉素结合6-DMAP激活对马卵母细胞ICSI后36 h分裂无显著影响(P>0.05)。因此,马松散型和致密型卵母细胞的成熟能力存在差异,TCM 199和NCSU 23成熟体系对这2种类型卵母细胞的发育能力无显著影响(P>0.05),马卵母细胞成熟38 h成熟率高于34 h成熟率,TCM 199成熟体系培养松散型卵母细胞34 h进行ICSI后的分裂率最高。离子霉素结合6-DMAP激活对TCM 199或NCSU 23体系成熟马卵母细胞ICSI后的体外发育能力无显著影响(P>0.05)。