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.     

Studies on MⅡ Stage Porcine Oocytes Vitrification

The study was designed to select right cryoprotectants and carrier for porcine matured oocytes to improve oocyte viability after vitrification and lay a good foundation for porcine oocytes continue development. Using 40% EG, 15% EG+15% DMSO+20% FBS, 15% EG+15% DMSO+20% SPS as cryoprotectants and taking GMP and cryotop as carrier to do porcine oocytes vitrification. The results showed that VS3 including SPS made better active of FDA (59.40%±4.93%) than VS1 （42.12%±4.08%） and VS2 (37.57%±1.21%）（P<0.05);VS3 as cryoprotectants and cryotop as carrier made better active of FDA (83.33%±3.33%) than GMP (59.40%±4.93%)(P<0.05). The data demonstrated that use SPS to instead of FBS and cryotop as carrier could significantly improve active of oocyte after vitrification.     

High survival rate of bovine oocytes matured in vitro following vitrification

Improving pregnancy rates associated with the use of cryopreserved human oocytes would be an important advance in human assisted reproductive technology (ART). Vitrification allows glasslike solidification of a solution without ice crystal formation in the living cells. We have attempted to improve the survival rates of oocytes by a vitrification technique using bovine models. In vitro matured oocytes with or without cumulus cells were vitrified with either 15.0% (v/v) ethylene glycol (EG) + 15% (v/v) dimethylsulfoxide (DMSO) + 0.5 M sucrose or 15% (v/v) EG + 15% (v/v) 1,2-propanediol (PROH) + 0.5 M sucrose, using 'Cryotop' or 'thin plastic sticker', respectively. The oocyte survival rates after vitrifying-warming, and the capacity for fertilization and embryonic development were examined in vitro. The rate of embryonic development to blastocyst was significantly higher (P<0.05) in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) PROH + 0.5 M sucrose than in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) DMSO + 0.5 M sucrose (7.4% +/- 4.1 vs. 1.7% +/- 3.0, respectively). Oocytes vitrified without cumulus cells had a higher survival rate after thawing and a superior embryonic developmental capacity compared with oocytes vitrified with cumulus cells. Prolonged pre-incubation time after thawing adversely affected the rates of embryonic cleavage and development. These results indicate that in vitro matured bovine oocytes can be vitrified successfully with the mixture of the cryoprotectants, EG + PROH, the absence of cumulus cells for vitrification does not affect oocyte survival rate after warming, and vitrified and warmed oocytes do not require pre-incubation before in vitro fertilization.     

Comparison of Ethylene Glycol and Propylene Glycol for the Vitrification of Immature Porcine Oocytes

Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P<0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P<0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC; treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P<0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development.     

Effects of cryoprotectants and cryoprotectant combinations on viability and maturation rates of Camelus dromedarius oocytes vitrified at germinal vesicle stage

Camel fertility faces many problems, which could be solved by assisted reproductive technologies (ARTs). We designed the experiment to explore the effect of different cryoprotectant concentrations and combinations on viability and maturation rates of vitrified/warmed camel oocytes. We collected ovaries directly after slaughtering from local abattoir and transported them to laboratory in a thermo‐flask containing normal physiological saline. We aspirated the oocytes from follicles, which is 2–8 mm in diameter, washed three times in TCM‐199 and then examined under stereo‐microscope for selection. We selected morphologically normal oocytes with an evenly granulated cytoplasm and a compact cumulus cell layer. We equilibrated morphologically normal oocytes in equilibration solution (ES), which is half concentration of vitrification one. After equilibration, We transported oocytes to vitrification solution using ethylene glycol (EG, 40%), dimethyl sulphoxide (DMSO, 40%) and EG 40% + DMSO 40%. The obtained results revealed that addition of EG 40% + DMSO 40% resulted in the best quality of vitrified/warmed oocytes, which is demonstrated by higher per cent survival rate (90.16%) and maturation rate (58.95%). While DMSO 40% resulted in 62.79% and 29.54%, respectively, EG 40% reported 86.11% and 53.47%, respectively. We could conclude that vitrification of immature camel oocytes by using 40% EG + 40% DMSO is suitable methods to limit drawbacks of vitrification methods, and we need further studies to assess the ability of in vitro‐produced blastocyst to develop in vivo and establish pregnancy after embryo transfer.     

Optimization of cryoprotectant treatment for the vitrification of immature cumulus-enclosed porcine oocytes: comparison of sugars,combinations of permeating cryoprotectants and equilibration regimens

Our aim was to optimize the cryoprotectant treatment for the preservation of immature porcine cumulus-oocyte complexes (COCs) by solid surface vitrification. In each experiment, the vitrification solution consisted of 50 mg/ml polyvinyl pyrrolidone, 0.3 M of the actual sugar and in total 35% (v/v) of the actual permeating cryoprotectant (pCPA) combination. After warming, the COCs were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, trehalose and sucrose were equally effective during vitrification and warming in terms of facilitating oocyte survival and subsequent embryo development. In Experiment 2, when equilibration was performed at 38.5 C in a total of 4% (v/v) pCPA for 15 min, the combination of ethylene glycol and propylene glycol (EG + PG = 1:1) was superior to EG and dimethyl sulfoxide (EG + DMSO = 1:1) in terms of oocyte survival after vitrification and the quality of resultant blastocysts. In Experiment 3, equilibration in 4% (v/v) pCPA for 15 min before vitrification was superior to that in 15% (v/v) CPA for 5 min for achievement of high survival rates irrespective of the pCPA combination used. In Experiment 4, when equilibration was performed in 4% EG + PG for 5 min, 15 min or 25 min, there was no difference in oocyte survival and subsequent embryo development after vitrification and warming; however, the developmental competence of cleaved embryos was tendentiously reduced when equilibration was performed for 25 min. In conclusion, trehalose and sucrose were equally effective in facilitating vitrification, and the optimum pCPA treatment was 5–15 min equilibration in 4% (v/v) of EG + PG followed by vitrification in 35% (v/v) EG + PG.     

High developmental rates of mouse oocytes cryopreserved by an optimized vitrification protocol: the effects of cryoprotectants, calcium and cumulus cells

Unfertilized oocytes are one of the most desired germ cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. The aim of the present study was to improve vitrification of mouse oocytes. First, the effects of calcium and cryoprotectants, dimethyl sulfoxide and ethylene glycol (EG), in vitrification medium on survival and developmental ability of vitrified oocytes were evaluated. Oocytes were vitrified by a minimal volume cooling procedure using different cryoprotectants. Most of the vitrified oocytes were morphologically normal after warming, but their fertility and development were low independently of calcium and cryoprotectants. Second, the effect of cumulus cells on ability of oocytes to be fertilized and develop in vitro was examined. The fertility and developmental ability of denuded oocytes (DOs) after IVF were reduced compared with cumulus-oocyte complexes (COCs) both in fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop to the 2-cell and blastocyst stages than those of vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate equivalent to the rate obtained with IVF using fresh COCs. Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and EG retain their developmental competence. These findings will contribute to improve oocyte vitrification in not only experimental animals but also clinical application for human infertility.     

Cryopreservation of immature buffalo oocytes: Effects of cytochalasin B pretreatment on the efficiency of cryotop and solid surface vitrification methods

The aim of the present study was to compare the efficiency of the solid surface (SSV), cryotop (CT) vitrification methods and cytochalasin B (CB) pretreatment for cryopreservation of immature buffalo oocytes. Cumulus‐oocyte complexes (COCs) were placed for 1 min in TCM199 containing 10% dimethylsulfoxide (DMSO), 10% ethylene glycol (EG), and 20% fetal bovine serum, and then transferred for 30 s to base medium containing 20% DMSO, 20% EG and 0.5 mol/L sucrose. CB pretreated ((+)CB) or non‐pretreated ((?)CB) COCs were vitrified either by SSV or CT. Surviving vitrified COCs were selected for in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of viable oocytes after vitrification in CT groups (82%) was significantly lower (P < 0.05) than that in a fresh control group (100%), but significantly higher (P < 0.05) than those in SSV groups (71–72%). Among vitrified groups, the highest maturation rate was obtained in the CT (?)CB group (32%). After IVF, the cleavage and blastocyst formation rates were similar among vitrified groups but significantly lower than those of the control group. In conclusion, a higher survival rate of oocytes after vitrification and IVM was obtained in the CT group compared with that in the SSV group, indicating the superiority of the CT method. Pretreatment with CB did not increase the viability, maturation or embryo development of vitrified oocytes.     

Effect of permeable cryoprotectant‐free vitrification on DNA fragmentation of equine oocyte–cumulus cells

DNA fragmentation of cumulus cells could be used as an indicator of oocyte vitrification success as an indirect indicator of the quality of the oocyte. This study was designed to compare the DNA fragmentation of post‐mortem equine cumulus cells before or after vitrification in the absence of permeable cryoprotectant agents. Cumulus–oocyte complexes (COCs; n = 56) were recovered from slaughterhouse ovaries and subjected to in vitro maturation (42 hr/38.2°C/5%CO₂) before (control group) or after a permeable cryoprotectant‐free vitrification method using 1 M sucrose (vitrification group). After in vitro maturation, COCs were denuded, and cumulus cells were washed and stored at ?80°C until thawing. Cumulus cell samples were processed with the chromatin dispersion test (Ovoselect, Halotech DNA, Spain). Low, high and total DNA fragmentation percentages of cumulus cells were recorded and compared between the two groups by Student's t test. Results were expressed as mean ± SEM. The vitrified group resulted in significantly higher (p < 0.05) percentages for low (16.81 ± 1.62 vs. 6.63 ± 0.77) and total (21.14 ± 1.84 vs. 12.76 ± 1.48) DNA fragmentation of cumulus cells. There were no significant differences between groups for high DNA fragmentation of cumulus cells. In conclusion, permeable cryoprotectant‐free vitrification of equine oocytes increased the total DNA fragmentation rate of cumulus cells but protected them against high DNA fragmentation rates. Further studies are needed to examine the relationship between DNA fragmentation of cumulus cells and the developmental competence of equine oocytes.     

Oocyte maturation,embryo development and gene expression following two different methods of bovine cumulus-oocyte complexes vitrification

Objective

To examine the maturational competence, embryo development and expression of genes involved in oocyte maturation and cumulus expansion (GDF9, BMP15, HAS2, TNFAIP6, FGF17 and FSHr) following two standard methods of bovine COCs vitrification.

Methods

Bovine cumulus-oocyte complexes (COCs) were aspirated from slaughtered ovaries and then distributed into three groups: non-vitrified COCs (control), vitrification 1 group (V1); vitrification was performed by 15% ethylene glycol (EG) and 15% DMSO in holding media (TCM-199 with 20% FCS); and vitrification 2 group (V2); vitrification was performed by 40% EG in holding media. After vitrification, COCs were warmed in two steps and cultured and then evaluated for nuclear maturation, embryo development and gene expressions.

Results

The mean (±SD) percentages of nuclear maturation and blastocyst/cleaved were higher in control group (79.5 ± 8.0 and 31.0 ± 5.1%) than the V1 (34.8 ± 9.1 and 4.4 ± 5.1%) and V2 (47.8 ± 11.7 and 7.1 ± 5.8%) groups (P < 0.05), respectively. Further, COCs in V2 group showed higher mean (±SD) percentages of cleavage compared to V1 group (31.8 ± 1.0 vs 21.7 ± 2.8%; P < 0.05). GDF9 and BMP15 expression levels were higher in COCs in the control than of the vitrification groups (P < 0.05). In addition, expression level of GDF9 and BMP15 was higher in V2 group than in V1group (P < 0.05). The expression of HAS2 and FGF17 in V1 group was lower (P < 0.05) than that of the V2 groups.

Conclusions

Expression of oocyte maturation genes was affected by vitrification procedure and conditions. Using EG alone for vitrification of bovine immature COCs, resulted in higher expression of GDF9, BMP15 and production of more in vitro matured and cleaved oocytes.

     

Vitrification with DAP 213 and Cryotop of Ex Situ and In Situ Feline Cumulus–Oocyte Complexes

This study was undertaken to compare cryotolerance, in terms of viability and resumption of meiosis after warming and culture (24 and 48 h), of ex situ (isolated) and in situ (enclosed in the ovarian tissue) feline cumulus–oocyte complexes (COCs) vitrified with DAP 213 (2 m DMSO, 1 m acetamide, 3 m propylene glycol) in cryotubes or Cryotop method. Ovaries were harvested from 49 pubertal queens. Of each pair of ovaries, one was dissected to release COCs randomly divided into three groups: fresh COCs (control), ex situ COCs vitrified with DAP 213 and Cryotop. The cortex of the other ovary was sectioned into small fragments (approximately 1.5 mm³) and randomly assigned to be vitrified by DAP 213 or Cryotop. After warming, ex situ and in situ (retrieved form vitrified ovarian tissue) COCs were matured in vitro. Viability of oocytes was highly preserved after warming and culture in all treatments. Proportions of oocytes surrounded by complete layers of viable cumulus cells were remarkably decreased (p < 0.00001) in both vitrification procedures compared to fresh oocytes. Resumption of meiosis occurred in all treatments. After 24 h of culture, results were similar in ex situ and in situ vitrified oocytes regardless of the vitrification protocol used (range 29–40%), albeit lower (p < 0.05) than those of fresh oocytes (65.8%). After 48 h of culture, ex situ oocytes vitrified with Cryotop achieved the rates of meiosis resumption similar to fresh oocytes (53.8% vs 67.5%; p > 0.05) and ex situ and in situ oocytes vitrified with DAP 213 showed similar rates of resumption of meiosis. These findings demonstrated that DAP 213 and Cryotop preserve the viability of ex situ and in situ oocytes, but cumulus cells are highly susceptible to vitrification. However, the capability to resume meiosis evidences that feline immature oocytes vitrified as isolated or enclosed in the ovarian cortex have comparable cryotolerance.     

Japanese Society for Animal Reproduction: award for outstanding research 2002. Cryopreservation of follicular oocytes and preimplantation embryos in cattle and horses

Factors affecting sensitivity of preimplantation embryos and follicular oocytes to cryopreservation were analyzed in the equine and bovine species. (1) Survival of equine blastocysts after two-step freezing in the presence of glycerol as the cryoprotective agent (CPA) was influenced by development of the embryonic capsule. The use of ethylene glycol (EG) with sucrose as CPAs improved the post-thaw survival of blastocysts and made it possible to transfer the embryos into recipient mares without removing the CPAs. In addition, early blastocysts cryopreserved by vitrification could develop both in vitro and in vivo when the embryos were exposed to vitrification solution in a stepwise manner. The vitrification procedure was also applied to the relatively large expanded blastocysts. (2) Bovine embryos produced in vitro have been considered to be highly sensitive to the process of cryopreservation. To solve this problem, Day-7 blastocysts produced in a serum-free system were cooled at 0.3 C/min rather than 0.6 C/min before being plunged into liquid nitrogen, resulting in no loss of the post-thaw viability. The supplementation of LAA in IVM/IVF media or IVC medium was effective in producing pronuclear-stage zygotes or morula-stage embryos relatively tolerable to freezing, respectively. (3) Transmission electron microscopic observation of immature equine oocytes showed that cellular injury occurred near the sites of gap-junctions between cumulus cells and the oocyte. In cattle, higher fertilization rates of oocytes were obtained when the oocytes were subjected to cryopreservation at an intermediate stage during IVM (GVBD for freezing, Met-I for vitrification). Vitrification of bovine Met-II oocytes in open-pulled glass capillaries, characterized by an ultra-rapid cooling rate (3,000-5,000 C/min), was found to avoid any harmful influence of vitrification and warming.     

Vitrification procedure decreases inositol 1,4,5‐trisphophate receptor expression,resulting in low fertility of pig oocytes

Although cryopreservation of mammalian oocytes is an important technology, it is well known that unfertilized oocytes, especially in pigs, are highly sensitive to low temperature and that cryopreserved oocytes show low fertility and developmental ability. The aim of the present study was to clarify why porcine in vitro matured (IVM) oocytes at the metaphase II (MII) stage showed low fertility and developmental ability after vitrification. In vitro matured cumulus oocyte complexes (COCs) were vitrified with Cryotop and then evaluated for fertility through in vitro fertilization (IVF). Although sperm‐penetrated oocytes were observed to some extent (30–40%), the rate of pronuclear formation was low (9%) and none of them progressed to the two‐cell stage. The results suggest that activation ability of cryopreserved oocytes was decreased by vitrification. We examined the localization and expression level of the type 1 inositol 1,4,5 trisphosphate receptor (IP₃R1), the channel responsible for Ca²⁺ release during IVF in porcine oocytes. Localization of IP₃R1 close to the plasma membrane and total expression level of IP₃R1 protein were both decreased by vitrification. In conclusion, our present study indicates that vitrified‐warmed porcine COCs showed a high survival rate but low fertility after IVF. This low fertility seems to be due to the decrease in IP₃R1 by the vitrification procedure.     

Effect of retinol as antioxidant on the post-thaw viability and the expression of apoptosis and developmental competence-related genes of vitrified preantral follicles in buffalo (Bubalus bubalis)

The present study evaluated the effect of supplementation of retinol in the vitrification solution on the viability, apoptosis and development-related gene expression in vitrified buffalo preantral follicles. Preantral follicles isolated from cortical slices of ovaries were randomly assigned into three groups: Group1—Control fresh preantral follicles; Group 2—Vitrification treatment (Vitrification solution 1 (VS1) –TCM-199 + 25 mM HEPES + Foetal bovine serum (FBS) 10%, Ethylene glycol (EG): 10%, Dimethyl sulphoxide (DMSO): 10%, Sucrose-0.3 M for 4 min; VS2- TCM-199 + 25 mM HEPES + FBS10%, EG:25%, DMSO: 25%, Sucrose:0.3 M for 45 s); Group3—vitrification treatment +5 μM of Retinol. Preantral follicles were placed in corresponding vitrification medium and plunged into liquid nitrogen (−196°C). After a week, the follicles were thawed and analysed for follicular viability and gene expression. There was no significant difference in the viability rates among the Group 1(Fresh preantral follicles) (91.46 ± 2.39%), Group 2 (89.59 ± 2.46%) and Group 3 (87.19 ± 4.05%). There was a significantly (p < .05) higher mRNA expression of BCL2L1, GDF-9 and BMP-15 in the vitrification + retinol group compared with the control group. There was a significantly (p < .05) higher expression of Caspase-3 and Annexin-5 in the vitrification group and Vitrification + retinol group compared with control group of follicles. It is concluded that the supplementation of 5 μM of Retinol in Vitrification solution was an efficient vitrification procedure for the vitrification of buffalo preantral follicles.     

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)。     

注射hCG后不同时间猪所排卵泡内COC的形态和卵母细胞减数分裂进程

研究了超排处理注射 h CG后不同时间猪卵丘卵母细胞复合体 (COC)的回收率和形态、卵母细胞减数分裂进程以及 COC形态与生发泡 (GV)染色质构型之间的关系。结果表明 :(1)注射 h CG后 4 h COC的回收率为 5 3.1% ,明显低于注射 h CG后 18、2 2、2 4 h(71.2 %、76 .5 %、70 .0 % ,P<0 .0 5 ) ;(2 )注射 h CG后 18、2 2、2 4 h收集的 COC分别有98.9%、98.0 %、91.4 %的卵丘已经发生扩展 ,而注射 h CG后 4 h收集的 COC则无一发生卵丘扩展 ;(3)注射 h CG后 4h,有少量卵母细胞开始恢复减数分裂 ,至 18h左右开始发生生发泡破裂 (GVBD) ,到 2 2～ 2 4 h有 5 8.4 %～ 6 0 .0 %的卵母细胞发生 GVBD;(4)外层卵丘扩展、放射冠轻微扩展的卵母细胞处于 GV- 1期的比例 (6 9.6 % )明显高于卵丘完全扩展的卵母细胞 (47.8% )和放射冠部分扩展、卵丘已经脱落的卵母细胞 (2 9.3% ) ,而后 2者发生 GVBD的比例(40 %、4 4 % )则略高于前者 (2 7% )。     

培养介质、卵丘细胞和卵泡直径对猪卵母细胞体外成熟的影响

A类卵母细胞在mTCM 199、NCSU2 3和NCSU37体系中培养 4 4～ 5 2小时后 ,成熟率分别为 76 .1%、78.1%和 6 5 .2 %。前两者差异不显著 (P >0 .0 5 ) ,但显著高于后者 (P <0 .0 5 )。卵母细胞在添加eCG和hCG的NCSU2 3体系中的成熟率 (75 .6 % )明显高于添加FSH的LH和成熟率 (6 5 .2 % ) (P <0 .0 5 )。A、B、C三类卵母细胞在NCSU2 3的成熟率分别为 73.3%、6 0 .4 %和 11.0 % ,三者间差异显著 (P <0 .0 5 )。大 (ф >6mm)、中 (ф =3～ 6mm)和小 (ф <3mm)三种卵泡中的卵母细胞在NCSU2 3中培养后 ,成熟率分别为 5 6 .2 % ,78.1%和 5 1.9% ,中等卵泡中卵母胞的体外成熟率显著高于其他两组 (P <0 .0 5 )。     

绵羊卵丘细胞对裸卵体外成熟及孤雌发育影响的研究

本研究探讨绵羊卵丘细胞对裸卵体外成熟和后期发育的影响。实验分为4组,对照组为卵丘-卵母细胞复合体(COCs);裸卵(DO)组;COCs与DO共培养组(CODO);卵丘细胞(CC)与DO共培养组(CCDO)。体外成熟培养18 h后,检测各组卵子成熟质量并进行孤雌发育研究。结果表明:CODO和CCDO组中裸卵活率显著高于DO组(P<0.05),但显著低于COCs组(P<0.05),且COCs组中卵子活率极显著高于DO组(P<0.01);在极体排出率方面,CODO、CCDO和DO3组差异不显著(P>0.05),但是它们都显著低于COCs组(P<0.05)。COCs、CODO、CCDO和DO 4组卵裂率差异均不显著(P>0.05)。然而,DO组的囊胚率显著低于CODO和CCDO组(P<0.05),极显著低于COCs组(P<0.01),但CODO和CCDO 2组中裸卵的囊胚率差异不显著(P>0.05)。总之,无论散在的卵丘细胞还是COCs均能提高裸卵体外成熟及后期发育潜力。     

血清和促性腺激素对山羊卵母细胞核体外成熟的影响

试验研究了清和促性腺激素对山羊卵丘扩展和卵母细胞核成熟的,卵母细胞与卵丘扩展的关系以及卵丘扩展与卵母细胞核成熟的关系。结果表明：（1）培养24h，添加PMSG组卵母细胞核成熟率显著高于不加激素组（P＜0．05），而培养到27h，2者成熟率之间差异变得不显著，说明添加PMSG卵母细胞核的最终成熟率没有明显影响，但加速了卵母细胞核的成熟进程；（2）M199＋BSA培养27h，卵母细胞核成熟率显著高于培养24h，而M199＋FCS培养27h与培养24h的成熟差异不显著，说明添加BSA时，卵母细胞核体外成熟速度比添加FCS的慢；（3）卵丘扩展良好与扩展不好的卵母细胞核成熟率以及第1极体形态无统计学差异，说明山羊卵母细胞的核成熟可能不依赖于卵丘扩展；（4）山羊的卵丘扩展产不依赖于卵母细胞；（5）将带壁颗粒细胞与不带壁颗粒细胞的COC分开培养发现，2者卵母细胞核成熟度无明显差异，带有壁颗粒细胞的COC卵丘扩展情况明显优于一般COC。     

Effect of Initial Cumulus Morphology on Meiotic Dynamic and Status of Mitochondria in Horse Oocytes during IVM

The aim of this investigation was to examine the chromatin configuration of the nucleus, pattern of mitochondrial aggregation and mitochondrial activity in parallel studies in the same horse oocytes. Horse oocytes recovered by ultrasound-guided follicle aspiration in vivo were classified according to two main initial cumulus morphologies as having compact or expanded cumulus. The percentage of oocytes with a diplotene meiotic configuration at the time of recovery from the follicles was highest in compact oocytes. Oocytes with expanded cumulus layers at the time of recovery matured more rapidly in vitro and reached a proportion >50% at the metaphase II stage (M 2) sooner during in vitro maturation (IVM), than did compact oocytes. The mitochondrial aggregation pattern changed from finely distributed (Type 1) through crystalline (Type 2) to an aggregated, granulated appearance (Type 3) during IVM. The pattern of mitochondrial aggregation at the time of recovery was associated with the initial cumulus morphology of the oocyte, in that compact oocytes had a higher proportion of Type 1 aggregation, whereas expanded oocytes had a higher proportion of Type 3. The fluorescence intensity of metabolic active mitochondria, measured by fluorescence intensity (Em 570) per oocyte after MitoTracker CMTM Ros orange labelling, increased in the oocytes during IVM and depended on initial cumulus investment. Oocytes with the granulated type of aggregated mitochondria Type 3 had the highest level of metabolic activity and were in more progressed stages of meiosis (A 1-M 2). Oocytes initially having expanded layers of cumulus reached significantly higher levels of mitochondrial activity after IVM than did oocytes initially having compact cumuli. During resumption of meiosis the mitochondrial activity of oocytes with initially expanded cumulus increased continuously up to M 2, whereas in oocytes from compact cumulus-oocyte complex (COC), the activity declined after A 1/T 1 stages of meiosis.