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1.
In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.  相似文献   

2.
The present study was designed to evaluate three different in vitro fertilization (IVF) systems: a straw‐IVF system with 10 min of coincubation, a straw‐IVF system with 6‐h coincubation and the microdrop‐IVF system with 6‐h coincubation (the traditional IVF system used routinely in most of IVF laboratories) in an attempt to reduce polyspermic penetration ( Experiment 1 ). When the straw‐IVF system was tested in combination with two coincubation times, the use of 10 min of coincubation significantly increased (p < 0.001) the penetration rate and the efficiency of fertilization (67.7 ± 6.4% vs 31.9 ± 6.5% and 41.5 ± 2.5% vs 17.6 ± 2.5% for 10 min and 6 h, respectively), while there were no significant differences in the incidence of monospermy between both systems (64.3 ± 5.1% and 67.7 ± 3.4%, for 10 min and 6 h, respectively). The penetration rate in the 6‐h microdrop‐IVF system was higher (93.8 ± 3.6%; p < 0.001) compared with the 10‐min straw‐IVF system (67.7 ± 6.4%), however, monospermy was severely reduced (25.0 ± 4.3% vs 67.7 ± 3.4%, for the 6‐h microdrop‐IVF system and 10‐min straw‐IVF system, respectively). The efficiency of the IVF showed similar values between microdrop and 6‐h straw‐IVF systems, but efficiency was significantly improved (p < 0.05) when the 10‐min straw‐IVF system was used. Experiment 2 was designed to compare porcine in vitro embryo production in two IVF systems, the 6‐h microdrop‐IVF system (1000 sperm per oocyte) and 10‐min straw‐IVF system (30 000 sperm per oocyte). The blastocyst formation rates tended (p = 0.06) to be higher when the 10‐min straw‐IVF system was used compared with the 6‐h microdrop‐IVF system. In addition, the number of total cells per blastocyst increased significantly (p < 0.05) in the 10‐min straw‐IVF system. These results showed that the 10‐min straw‐IVF system is an effective way to decrease polyspermic penetration, and improve the efficiency of fertilization and the quality of blastocysts in terms of cell number per embryo.  相似文献   

3.
SHI Wen-shu  JIN Yi 《中国畜牧兽医》2017,44(12):3563-3569
This study was aimed to examine the effects of UCHL1 inhibition on porcine oocyte maturation in vitro, zona pellucida (ZP) ubiquitination and polyspermy. DAPI staining, Hoechst staining and SDS-PAGE methods were used to detect the matuation rate of porcine oocytes in vitro, the level of ubiquitination of zona pellucida (ZP) and polyspermy. The results showed that after different concentrations UCHL1 inhibitor (10, 20, 25 and 30 μmol/L, DMSO and control group) were added into maturation medium for culturing 46 h in vitro, the mature rate of control group was 86.22%, while the maturation rate of 30 μmol/L group was 15.30%, and the maturation rate of every treatment group had significant difference (P<0.05). Western blotting result showed that every group generated ubiquitin markers of ZP were about 61, 80 and 106 ku in different degree. According to the analysis of gray value, the result had significant difference (P<0.05). Conducting fertilization in vitro, the number of sperm adhered on oocyte ZP in control group was the most, the number of sperm running into oocyte was fewer, the number of sperm adhered on oocyte ZP with 30 μmol/L UCHL1 inhibitor was the fewest, and there was almost no sperm running into the oocyte. The results showed that UCHL1 inhibitor had an impact on maturation of porcine oocytes in vitro. With the higher concentration of UCHL1, the lower degree of ZP protein ubiquitinated, UCHL1 could regulate sperm attachment and polyspermy.  相似文献   

4.
史文姝  金一 《中国畜牧兽医》2017,44(12):3563-3569
试验旨在研究抑制泛素羧基末端水解酶-1（UCHL1）对猪卵母细胞体外成熟、透明带泛素化及多精入卵的影响。利用DAPI染色、Hoechst染色及SDS-PAGE等方法检测猪卵母细胞体外成熟率、透明带泛素化水平及多精入卵等情况。结果表明,添加不同浓度UCHL1抑制剂（10、20、25、30 μmol/L,二甲基亚砜（DMSO）及对照组）体外培养猪卵母细胞46 h后,对照组成熟率为86.22%,而30 μmol/L UCHL1抑制剂组的成熟率为15.30%,且各处理组间成熟率差异显著（P<0.05）;经Western blotting检测,各处理组的透明带在约61、80、106 ku处均发生不同程度的泛素标记,通过灰度值分析差异显著（P<0.05）;进行体外受精试验后,发现对照组透明带精子黏附数最多,精子入卵数较少,添加30 μmol/L UCHL1抑制剂组的透明带精子黏附数最少,几乎没有精子入卵。结果显示,UCHL1抑制剂对猪卵母细胞的体外成熟有一定影响,随着UCHL1抑制剂浓度的增加,透明带发生泛素化的程度逐渐降低,且UCHL1可调节透明带精子黏附及多精入卵。  相似文献   

5.
The high incidence of polyspermy is one of the major obstacles during in vitro fertilization (IVF) in pigs. To overcome this, we developed a novel IVF method, which involves constant rotation. Oocytes matured in vitro were mixed with spermatozoa (0.2 × 105 sperm/mL) in an IVF medium (200 μL) using a 200 μL PCR tube. This tube was then rotated at 1 rpm for 6 h at 38.5°C in a rotation mixer (experimental group). A second PCR tube was simultaneously cultured without rotation (control group). The rate of polyspermy was evaluated 12 h after insemination and was significantly (P < 0.05; 21.0% vs. 48.3%) lower in the experimental group than in the control group. Sperm penetration rate was similar in oocytes from the experimental and control groups (75.2% vs. 83.1%). However, monospermic fertilization rate of the oocytes was significantly (P < 0.05; 44.8% vs. 21.2%) higher in the experimental group than in the control group. Furthermore, the rate of blastocyst formation (30.1% vs. 20.8%) increased in the experimental group, as compared to the control group. This present system will contribute to increase the efficacy of blastocyst production through reduction of polyspermic penetration.  相似文献   

6.
Polyspermic fertilization and embryo quality are important issues for the in vitro production of pig embryos. We hypothesized that oocyte donor (prepubertal gilt vs. sow) affects polyspermy and blastocyst development in vitro and that the sexual maturity of the oocyte donor affects the response to sperm concentration in the fertilization medium. In Exp. 1, oocytes of sows and gilts were mounted and stained 12 h after insemination to provide fertilization data. In Exp. 2, putative embryos were developed in vitro to 144 h post-insemination before mounting. In both experiments, cumulus-oocyte complexes (COC) were collected from ovaries of prepubertal gilts and adult sows. Sperm were added after maturation of COC for 40 to 44 h. Sperm from two boars at 0.5 to 5.0 x 10(6) sperm/mL was used for insemination. More (P < 0.01) monospermic fertilizations were observed in oocytes derived from gilts than for oocytes from sows. There were fewer (P < 0.02) penetrated sperm per fertilized oocyte in oocytes from gilts compared with sows. There were effects of semen donor (boar) on the percentage of monospermic (P < 0.01) and polyspermic (P < 0.002) fertilizations, and on the number of penetrated sperm/fertilized oocyte (P < 0.02). In Exp. 2, cleavage and blastocyst formation was evaluated at 2 and 6 d postinsemination, respectively. More (P < 0.001) blastocysts developed from sow-derived oocytes than from gilt-derived oocytes. More (P < 0.05) total cells per blastocyst were observed in embryos from sow-derived oocytes than from gilt-derived oocytes. Semen donor affected the percentage of oocytes cleaving (P < 0.02), and a boar x sperm concentration interaction affected (P < 0.05) the incidence of blastocyt formation. Results indicate that sexual maturity of the donor is not responsible for the high incidence of polyspermy in porcine in vitro fertilization. However, blastocyst development is improved by the use of oocytes from sows rather than from prepubertal gilts.  相似文献   

7.
The aim of this study was to examine whether a morphological approach is efficient for selecting high‐quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen‐thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 105 sperms/ml). Under conditions of moderate polyspermy, 4‐cell embryos selected at 48 hr after IVF (single selection) and 8‐cell embryos selected at 79 hr after IVF from the collected 4‐cell embryos (double selection) showed high developmental competence. Likewise, 4‐ and 8‐cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4‐ and 8‐cell embryos produced under conditions of high polyspermy was significantly (p < .05) higher in comparison to moderate polyspermy conditions. These findings suggest that although high polyspermy affects the frequency of nuclear and chromosomal anomalies in porcine IVF embryos, subsequent selection based on morphological features of 4‐ and 8‐cell embryos even under high polyspermy conditions, could be an alternative option for selecting porcine IVF embryos with high development ability.  相似文献   

8.
The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm‐egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP‐intact and ZP‐free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44?0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti‐IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti‐IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP‐free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization.  相似文献   

9.
Porcine relaxin is a peptide hormone belonging to the insulin super family that has a variety of biological functions. The present experiment was designed to investigate the effects of relaxin on sperm function and on in vitro fertilization (IVF) of porcine oocytes. Porcine spermatozoa were washed, swum-up, and incubated for 1-4 h in mTALP medium supplemented with 0, 20 or 50 ng/ml porcine relaxin. Motility was determined by observing the type of forward movement of the spermatozoa, and acrosome status was evaluated by applying the triple staining technique. Immature oocytes were aspirated from antral follicles and matured in IVM medium (modified NCSU-37). Matured oocytes were co-cultured with spermatozoa in IVF medium (mTALP) supplemented with 0, 5, 10, 15 or 20 ng/ml relaxin. After 6 h of sperm-oocyte co-incubation, putative zygotes were cultured for 18 h in oocyte culture medium NCSU-37 and then assessed for the rates of monospermy, polyspermy, and male pronucleus formation after acetic orcein staining. Relaxin improved (P<0.05) sperm motility and increased the percentage of acrosome-reacted live spermatozoa during 1-4 h of incubation, although viability was not significantly improved. Significantly (P<0.05) the highest percentage of monospermic (31.7%) and lowest percentage of polyspermic (16.5%) fertilization was achieved from the sperm-oocyte co-culture group treated with 20 ng/ml relaxin as compared to other groups. The percentage of male pronucleus formation was significantly (P<0.05) greater in the 20 ng/ml relaxin-treated sperm-oocyte co-culture group than in the other groups. These results indicate that supplementation with relaxin is capable of improving sperm function and fertilization of porcine oocytes in vitro.  相似文献   

10.
A 2-year comparative study was carried out to evaluate the effect of ovary size, follicle size and oocyte quality of 3-month-old Simmental calves and the efficiency of using calf ovaries in an in vitro fertilization (IVF) programme. We evaluated the effects of different concentrations of follicle-stimulating hormone (FSH) and oestradiol-17beta (E-17beta) in the maturation medium on the in vitro development of calf oocytes into morula and blastocysts. The proportion of recovered oocytes (62.1%; 42.8%; 25.3%) and the percentage of good quality cumulus oocyte complexes (84.2%: 59.8%; 45.9%) decreased significantly (P < 0.01) with decreasing ovary size (L, M and S). The rates of two or more cells on Day 2 and of blastocysts on Day 7 and Day 9 were significantly lower (P < 0.01) for calf oocytes (61.5%; 18.9%: 15.9%) compared with those from sexually matured females (70.1%: 32.3%; 22.2%). Calf oocytes. matured in medium supplemented with 20 microg/ml or 10 microg/ml FSH plus 2 microg/ml E-17beta had higher rates of cleavage on Day 2 (64.1% and 64.7%) and blastocysts on Day 7 (24.5% and 22.4%) than the control supplemented with 10 microg/ml FSH (55.6% and 19.2%, respectively). Groups supplemented with 20 microg/ml FSH plus 2 microg/ml E-17beta and 10 mg/ml plus 4 mg/ml E-17beta showed a significantly lower developmental rate of blastocysts on Day 7 (14.6% and 14.5%). High concentrations of E-17beta (4 microg/ml) resulted in a significantly lower development of blastocysts on Day 9 (8.1%) and hatched blastocysts on Day 13 (3.5%) (P < 0.01). We conclude that the proportion of calf oocytes obtained from immature animals and their suitability for IVF are lower than those of cows. Thus, the use of oocytcs from sexually immature females would decrease the relative efficiency of IVF programmes. Supplementation with high concentrations of FSH can improve the maturation and developmental capacity of oocytes from prepubertal calves.  相似文献   

11.
Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = −0.560, p < 0.05) and in the sperm lot of moderate polyspermy (R = −0.405, p < 0.05) which suggests that penetration of excessive spermatozoa disables the oocyte cytoplasm to promote MPN formation.  相似文献   

12.
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.  相似文献   

13.
A boar sperm encapsulation technology in barium alginate has been developed to enhance reproductive performances and spermatozoa preservation time; aim of this work was to evaluate the effect of in vitro sperm encapsulation on polyspermy as a function of storage time at 18°C. A total number of 40 in vitro fertilization (IVF) tests were performed using encapsulated or diluted spermatozoa (20 IVF each treatment). Overall, 1288 in vitro matured oocytes were fertilized with spermatozoa stored at 24, 48 or 72 h at 18°C for both treatments polyspermy and normospermy, and the non‐penetration rates were assessed by optical microscopy. Results indicate a significant reduction in risk of polyspermic oocytes when spermatozoa are preserved in barium alginate membranes (incidence risk ratio: 0.766 with respect to diluted); such enhancement could be explained by lesser damage of sperm membranes achieved by encapsulation technology.  相似文献   

14.
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.  相似文献   

15.
The aim of this study was to determine how the duration of culture affects the ubiquitination of zona pellucida (ZP) proteins (ZP1, ZP2 and ZP3) during porcine oocyte maturation in vitro. We analysed the changes in ZP protein ubiquitination under three conditions: (i) during oocyte maturation from stage GV to MII; (ii) in oocytes cultured for different periods of time; and (iii) in oocytes treated with an antibody against PSMD4. Our results show that ZP1 and ZP2 are ubiquitinated at the GV stage, while ZP1, ZP2 and ZP3 are ubiquitinated at the MII stage, and band intensities for these proteins were significantly different between the GV and MII stages (p < .05). We also found that ubiquitination occurs in ZP1, ZP2 and ZP3 after cultured for 46, 52, 58 and 64 hr, and that the level of ubiquitinated ZP1 was significantly different in oocytes that were cultured for different time periods. Finally, treatment with an antibody against PSMD4 resulted in a significant decrease in ZP1 ubiquitination (p < .05), without affecting ZP2 or ZP3. The number of attached sperms per oocyte was also significantly different between control and anti‐PSMD4‐treated groups. Thus, we concluded that ZP1 and ZP2 are ubiquitinated at the GV stage, and ZP1, ZP2 and ZP3 are ubiquitinated at the MII stage. As the duration of culture increases, the ubiquitination levels of ZP proteins decrease. We also found that PSMD4 improves ZP1 ubiquitination during in vitro culture of porcine oocytes and effectively inhibits sperm–oocyte binding.  相似文献   

16.
The aim of this study was to test the effect of insulin–transferrin–selenium (ITS) and L‐ascorbic acid (AA) supplementation and the hormonal level during in vitro maturation (IVM) of small oocytes from pre‐pubertal goat on the blastocyst yield and quality. Concretely, we used four maturation media: conventional IVM medium (CM), growth medium (GM: CM+ITS+AA and low level of hormones), modified CM (mCM: CM with low level of hormones) and modified GM (mGM: CM+ITS+AA and normal level of hormones). Cumulus–oocyte complexes (COCs) were classified into two categories according to oocyte diameter: <125 μm and ≥125 μm. Large oocytes were matured 24 h in CM (Treatment A). Small oocytes were matured randomly in six experimental groups: Treatment B: 24 h in CM; Treatment C: 12 h in GM and 12 h in CM; Treatment D: 24 h in mGM; Treatment E: 12 h in mGM and 12 h in CM; Treatment F: 12 h in mCM and 12 h in CM; and Treatment G: 12 h in GM and 12 h in mGM. After IVM, oocytes were fertilized and cultured for 8 days. The blastocyst quality was assessed by the survival following vitrification/warming and the mean cell number. When different maturation media were combined, the blastocyst rate did not improve. The large oocytes produced the highest blastocysts yield. However, the culture of small oocytes in GM (53.3%) enhanced the post‐warming survival of blastocysts compared to large oocytes matured in CM (35.7%). In conclusion, IVM of pre‐pubertal goat small oocytes in GM would be useful to improve the quality of in vitro‐produced blastocysts.  相似文献   

17.
The objective of these experiments was to study bovine in vitro fertilization (IVF) conditions for blastocyst production using non-sexed sperm (Experiment 1) and sexed sperm (Experiment 2). For Experiment 1, in vitro-matured oocytes (N=707) were allocated to a 2 × 3 × 4 factorial design: time of co-incubation of gametes for fertilization (4 and 18 h), sperm dose (1, 0.33, and 0.11 × 10(6) frozen-thawed sperm/ml, and sperm source (four bulls). Pronuclear status was evaluated for a subset. Experiment 2 (N=2155 oocytes) was a 2 × 3 × 2 × 6 factorial design: sex of sperm (X and Y), sperm dose (1, 0.33, and 0.11 × 10(6) frozen-thawed sperm/ml), and sperm-sorting pressures (40 and 50 psi), replicated with sperm of six bulls. Presumptive zygotes were cultured 60 h in chemically defined medium-1 (CDM-1), and for 114 h in CDM-2. For Experiment 1, pronuclear formation, cleavage and blastocysts rates were greater for 1, and 0.33 × 10(6) than 0.11 × 10(6) sperm/ml (72 and 62 vs 42%; 89 and 81 vs 58%; and 21 and 17 vs 9%, respectively; all p<0.01); polyspermy was greater for 1, than 0.33 and 0.11 × 10(6) sperm concentrations (24 vs 2 and 0%; p<0.01). There were greater main effects (p<0.01) of pronuclear formation (69 vs 48%), polyspermy (13 vs 4%), and cleavage (63 vs 54%), at 18 than at 4 h of co-incubation of gametes (all p<0.01). For Experiment 2, cleavage and blastocyst rates were greater for 1 × 10(6) sperm/ml vs 0.33 and 0.11 (69%, 47%, and 30% cleavage and 30%, 14%, and 8% blastocysts) and 40 vs 50 psi (54% and 44% cleavage and 18% and 15% blastocysts) (p<0.01). A marked bull by fertilization sperm dose interaction was found for cleavage (p<0.05). The main conclusion was that the optimal sperm concentration for cleavage and producing blastocysts via IVF with sexed sperm was considerably higher and more variable among bulls than for unsexed sperm.  相似文献   

18.
This study was conducted to examine the effects of the reproductive cycle of donor cat on the quality of oocytes at recovery and developmental competence of oocytes after in vitro fertilization (IVF) and somatic cell nuclear transfer (NT). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular or luteal stages. After collection of oocytes, the oocytes were classified into four grades according to the morphological condition of oocyte cytoplasm and cumulus cells. A total of 16 558 oocytes were obtained from 198 ovarian pairs. The total mean numbers of oocytes and the mean numbers of oocytes with high quality (grade I) were significantly higher in ovarian pairs at the inactive stage (111.1 and 19.0 oocytes, respectively) than in ovarian pairs at the follicular stage (67.1 and 11.4 oocytes, respectively). A significant difference in the proportions of oocytes with grade I out of the total examined oocytes was observed between the follicular and luteal stages of ovaries (14.9% vs 20.2%, p < 0.05). The proportions of IVF embryos cleaved and developed to blastocysts significantly decreased with decreased quality of oocytes at recovery, irrespective of the reproductive status of ovaries. Moreover, there were no significant differences in the proportions of cleavage and development to the blastocyst stage of IVF and NT embryos among three oestrous stages of ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries has no apparent effects on the in vitro development of oocytes after IVF and NT, but the quality of oocytes at recovery influences the development of IVF embryos.  相似文献   

19.
王洋  金一 《中国畜牧兽医》2018,45(2):338-348
泛素蛋白酶体途径（ubiquitin proteasome pathway,UPP）是生物体内最主要的蛋白质降解途径,与多种疾病有关,在生物体内具有十分重要的作用,主要降解错误折叠的、多余的蛋白质。UPP是一个循环途径,其主要作用起始于泛素（ubiquitin,Ub）的激活,是受泛素激活酶（ubiquitin-activating enzyme,E1,酵母菌中是UbE1）、泛素结合酶（ubiquitin-conjugating enzyme,E2,UbE2）、泛素连接酶（ubiquitin-protein ligase,E3,UbE3）和去泛素化酶（deubiquitinating enzymes,DUBs）调控的级联过程,最终使被多聚泛素化（polyubiquitination,polyubiquitination,Poly-Ub）标记的蛋白底物在UPP的蛋白水解核心--26S蛋白酶体内被降解成寡肽,寡肽可以进入新的蛋白循环,Ub则由DUBs去除进入下一个UPP循环。UPP在海鞘类和一些哺乳类体外受精（in vitro fertilization,IVF）过程中具有十分重要的作用,能够参与精子获能和顶体反应（acrosomal reaction,AR）,促进精子顶体胞吐,在精卵结合时降解透明带（zona pellucida,ZP）蛋白,辅助精子穿透卵母细胞ZP,促进精子与卵母细胞ZP的融合,从而促使IVF的成功。通过在IVF液中添加一些UPP相关的抗体或抑制剂能够抑制IVF过程中的多精入卵,提高IVF率。作者综述了UPP的主要组成成分及其对IVF的辅助作用及相关抗体的研究进展,以期为今后研究UPP循环在生物体内的作用机制、与生物体内各种疾病的关系、在IVF过程中的作用机制及抑制多精入卵等提供一定的理论依据。  相似文献   

20.
This study was conducted to determine whether meiotic maturation could be induced in ovarian oocytes from the American brown bear (Ursus arctos), a model for gamete "rescue" techniques for endangered ursids. The bears were euthanized, and their ovaries were transported to the laboratory within 4 h. The mean ovarian size was 2.4 x 1.8 cm (range: 2.0-3.3 x 1.5-2.2 cm). The ovaries obtained from the 2 brown bears yielded 97 oocytes (48.5/female), and 88 (90.7%) of them were morphologically classified as normal quality. Oocytes were in vitro matured at 38.5 C in 5% CO2 for 24 or 48 h in TCM-199 supplemented with 10% FBS, 1 microg/ml estradiol-17beta, and 10 microg/ml FSH. In Exp. 1, morphologic evaluation of matured oocytes was conducted by measuring the diameters of oocytes with a zona pellucida (ZP) or cytoplasm without a ZP. In Exp. 2, activation was induced by applying two 20 microsec DC pulses of 2.0 kV/cm delivered by an Electro Cell Fusion Generator. The activated oocytes were cultured in TCM-199 containing 2 mM of 6-dimethylaminopurine for 4 h, in Charles Rosenkrans (CR) 1 for 3 days and the in CR2 for another 4 days. The diameters of the matured bear oocytes with a ZP and with cytoplasm without a ZP (161.8 +/- 6.0 and 135.3 +/- 7.5 microm, respectively) were significantly (P<0.05) larger than those of bovine oocytes (150.7 +/- 4.9 and 118.7 +/- 7.5 microm). The maturation rates of the bear oocytes were 17.6 and 59.4% at 24 and 48 h of in vitro maturation, the percentage of activated oocytes that developed to the 2 or 4-cell stage was 31.6%; however, no blastocysts were observed. These results indicate that bear oocytes can develop to metaphase II in an in vitro culture system and that activated oocytes can develop to the 2 or 4-cell stages.  相似文献   

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