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1.
2.
Steroid levels in follicular fluid (FF) obtained from stimulated ovaries in patients undergoing in vitro fertilization (IVF) were measured by capillary gas chromatography. The correlation between these levels and the maturity of the oocyte, judged from the morphology of the oocyte corona cumulus complex (OCCC) and the fertilizability of the oocytes was analysed. Oocyte maturity was associated with higher FF levels of progesterone, 17-hydroxyprogesterone, 16 alpha-hydroxyprogesterone and 20 alpha-dihydroprogesterone. Follicular fluids containing oocytes that became fertilized had significantly higher levels of 20 alpha-dihydroprogesterone and progesterone and lower levels of androstenedione. Of all the steroids determined, 20 alpha-dihydroprogesterone provides the most significant group differences. Enhanced 20 alpha-dihydrogenation in the presence of decreased 16 alpha- and 17-hydroxylation appears to be an important characteristic of the ultimate ripening stages and early luteinization, at least in stimulated cycles.  相似文献   

3.
Three approaches were investigated for improvement of in vitro maturation (IVM), in vitro fertilization (IVF), and early embryonic development in cattle. These were: 1) Selection of oocytes, 2) medium supplementation with fetal calf serum (FCS) and cow sera (DO, Dl, D10, and D20 to correspond with estrus, metestrus, diestrus, and proestrus, respectively), and 3) addition of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol-17β (E2)during maturation. Greater proportions (percentage) of oocytes initially selected for their compact cumulus cells completed IVM and IVF when compared to unselected oocytes (P < .05). Proportions (percentage) of selected oocytes that matured and cleaved after in vitro insemination according to serum type used for IVM were: FCS: 110/175 (62.9%) and 37/110 (33.6%) and DO: 130/145 (89.7%) and 52/130 (40.0%); D1 127/130 (97.7%) and 41/127 (32.3%); D10 95/98 (96.9%) and 35/95 (36.8%); D20:113/116 (97.4%) and 49/113 (43.4%). A higher proportion (P < .05) of embryos resulting from the D20 group reached four- and eight-cell stages. In FCS-supplemented maturation media with no hormones added during maturation (control), results of IVM and IVF were 157/265 (59.2%) and 39/157 (24.8%), respectively. With E2 (1 μg/ml) and FSH (5 μg/ml), comparable results were 189/215 (87.9%) and 71/189 (37.6%); with E2 (1 μg/ml) plus LH (10 μml), 280/327 (85.6%) and 111/280 (39.6%). Added hormones improved IVM results (P < .05) and, when FSH or LH was added with E2, in vitro development to four- and eight-cell stages was markedly enhanced (P < .05). Selection of oocytes, D20 serum, and added E2 and FSH or LH for IVM improved in vitro development of bovine embryos after IVF.  相似文献   

4.
The present study was designed to examine the influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos. Three experiments were conducted. In experiment 1, oocytes were classified by number of cumulus cell layers and morphology of the ooplasm as good, fair or poor. Oocytes were cultured for IVM, IVF and IVC in CR1aa medium. In experiment 2, good quality oocytes were cultured for maturation in: (1) CR1aa; (2) CR2aa; (3) TCM-199; (4) MEM or (5) RPMI-1640, and then fertilized using frozen thawed buffalo spermatozoa in CR1aa. The oocytes were cultured in the same medium used for maturation after fertilization. In experiment 3, oocytes were classified into three groups: group (1) was without gonadotropin and serve as a control; group (2) in which IVM medium was supplemented with 10microg/ml FSH and group (3) in which IVM medium was supplemented with 10IUml(-1) eCG. In all experiments, oocytes were kept at 38.5 degrees C under 5% CO(2) for IVM, IVF, IVC and examined for cleavage and embryo development rates on days 3 and 8, respectively. Good and fair quality oocytes produced a higher cleavage rate (P<0.01) than poor quality oocytes. Morula production rate was also higher (P<0.01) for good as compared with fair quality oocytes. Embryo development with poor quality oocytes was arrested at the two to sixteen cell stage. In experiment 2, the cleavage rate was higher (P<0.05) in CR1aa than CR2aa, and higher (P<0.01) than TCM-199, MEM and RPMI-1640. The numbers of morulae and blastocysts were higher (P<0.01) for oocytes cultured in CR1aa and CR2aa media than TCM-199 or MEM. In experiment 3, the addition of FSH or eCG to the maturation medium increased (P<0.01) cleavage and developmental rates of buffalo embryo compared with control medium. In conclusion, the IVM of good quality buffalo oocytes in CR1aa or CR2aa medium and the addition of FSH or eCG in maturation medium produced higher cleavage and developmental rates of IVF buffalo embryos.  相似文献   

5.
Employing a total of 3465 bovine oocytes this study was aimed at improving the efficiency of bovine embryo production under defined and undefined conditions. Following in vitro maturation (IVM) and in vitro fertilization (IVF), oocytes were allocated to various culture treatments using synthetic oviduct fluid (SOF). In our 3 experiments we showed that: 1) the addition of fetal calf serum (FCS 10% v/v) to SOF droplets after 20 to 24 h significantly improved blastocyst yields on Day 6 (21 vs 12%; P < 0.01), but not at later stages and resulted in significantly higher Day-8 blastocyst cell numbers (148 +/- 61 vs 92 +/- 35; P < 0.05); 2) the removal of bovine serum albumin (BSA) from the standard SOF medium resulted in significantly reduced blastocyst yields on Days 6, 7 and 8, respectively (17 vs 8%; 28 vs 18%; 31 vs 21%; P < 0.05); 3) the presence or absence of cumulus cells surrounding the presumptive zygote in culture in SOF had no effect on cleavage rate, percentage of 5-8 cell embryos or blastocyst yields (Day 6,7 or 8); 4) the culture of presumptive zygotes in SOF in an atmosphere of 5% CO2 in air (20% O2) resulted in significantly reduced development compared with culture in 5% CO2, 5% O2, 90% N2 in terms of blastocyst yield on Days 6, 7 and 8 and on Day 8 hatching rate, respectively (5 vs 22%; 9 vs 33%; 13 vs 48%; 50 vs 8%; P < 0.001) and 5) embryo density (1 embryo per 1 or 3 microl SOF) or replacing the culture medium every 48 h had no effect when SOF was supplemented with serum; however, under serum-free conditions, changing of the media resulted in a slightly improved Day-6 blastocyst yield such that renewal of serum-free medium mimicked the effect of serum addition.  相似文献   

6.
The present study was conducted to evaluate the influence of sperm:oocyte ratio during in vitro fertilization (IVF) of in vitro matured cumulus-intact oocytes on fertilization parameters and embryo development in pigs. In vitro matured oocytes surrounded by intact cumulus cells (COC) were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000:1, 3000:1, 4000:1, 6000:1, and 8000:1). Denuded oocytes inseminated with 2000 frozen-thawed spermatozoa:oocyte were the control group. A total of 2546 oocytes in five replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture (EC) medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The penetration rate increased in the COC groups with the sperm:oocyte ratio, reaching the highest rates with 8000:1 spermatozoa:oocyte (72.1 +/- 6.5%), similar to the control (73.5 +/- 3.5%). However, the monospermy was highest with the lower spermatozoa:oocyte rates (82.6-94.8%) and decreased drastically (P<0.05) in the COC group fertilized with 8000 sperm:oocyte (36%). The efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) showed no difference among the COC groups (20-30%) but they were significantly lower (P<0.007) than those obtained by the control group (43.7 +/- 2%). Embryo development was highest in the control group (58% for cleavage and 23% for BF) but not significantly different with the 6000 and 8000 sperm:oocyte COC groups (47 and 50% for cleavage and 19 and 17% for BF, respectively). These results indicate that the use of COC for IVF involves a drop in the efficiency of the fertilization and the necessity to increase the frozen-thawed sperm:oocyte ratio three to four times more to obtain similar embryo development to denuded oocytes.  相似文献   

7.

Background  

One of the major obstacles in using artificial insemination to manage genetics of elephant population in captivity is the large variations in semen quality among ejaculates within the same and among individuals. The objectives of this study were to determine the influences of (1) age (2) seasonality (3) and circulating testosterone (SrTest), triiodothyronine (SrT3) and tetraiodothyronine (SrT4), as well as seminal (4) testosterone (SpTest), zinc (SpZn) and protein (SpTP) on semen quality in the Asian elephant  相似文献   

8.
Domestic cat oocytes were cultured either in Waymouth MB 753/1 Medium (WAY) or in Eagle's Minimum Essential Medium (MEM) containing FSH, LH and estradiol-17beta and supplememted with one of the following: 5% fetal calf serum (FCS); 4 mg/ml bovine serum albumin (BSA); or 3 mg/ml polyvinylalcohol (PVA, a non-protein control). The oocytes were evaluated for: nuclear maturation after 48 hours of culture (in vitro maturation, IVM); fertilization and cleavage 24 to 30 hours postinsemination (in vitro fertilization, IVF); and early embryo development 48 hours postinsemination. Maturation rates were similar (P>0.05) for WAY + BSA (29.4%), MEM + BSA (46.7%) and MEM + PVA (43.3%), but were different (P<0.05) from the other treatments (range, WAY + FCS, 9.6% to WAY + PVA, 14.9%). Fertilization and cleavage rates were also similar (P>0.05) for WAY + BSA (51.4%, 30.5%), MEM + BSA (45.8%, 40.1%) and MEM + PVA (56.1%, 37.4%) and were greater (P<0.05) than all other treatments. These IVM/IVF oocytes were capable of culturing beyond 2-cells, with the highest proportion of 4- and 8- cell embryos forming in WAY and MEM media in the presence of BSA or in MEM medium containing PVA. In the domestic cat IVM/IVF system: both the type of culture medium and protein supplement influence the proportion of oocytes reaching Metaphase II; the type of protein supplement has a more significant (P<0.05) impact than medium on fertilization, cleavage and early embryo development; and nuclear maturation and fertilization in vitro can proceed in this species in the absence of supplementary protein.  相似文献   

9.
In-vitro fertilization was studied in F1 hybrids, 4 inbred and outbred strains of mice. Experiments were performed with intact ova (Series I) and denuded ova (Series II). In Series I the highest percentages of fertilization were obtained with gametes of F1 hybrids (86.7%) and of the CBA strain (87.5%). The KP, KE, and particularly the C57 strains, which in vivo give less than 100% fertilization, also gave low indices in vitro (42.9, 26.0 and 8.6% respectively). Testing gametes of these inbred strains with F1 gametes showed that spermatozoa and ova are responsible for the low percentages of fertilization. The rate of fertilization was mainly dependent on the genotype of the spermatozoon. In Series II, high percentages of fertilization (97-100%) were regularly obtained in all groups, indicating that differences between strains pertained mainly to binding of spermatozoa with the zona pellucida. The incidence of polyspermy, which did not exceed 5% in Series I and reached 58% in Series II, was dependent on the genotype of both gametes.  相似文献   

10.
Oocytes and matched samples of follicular fluid (FF) were obtained from 70 follicles of five rhesus monkeys stimulated with either pregnant mare serum gonadotropin or human menopausal gonadotropin. Follicular aspiration was performed 30-32 h after human chorionic gonadotropin administration. The concentrations of estradiol (E2), progesterone (P), testosterone (T), and dihydrotestosterone (DHT) in FF were measured. Twenty-six percent of oocytes were classified as mature (M), 41% matured in vitro (Miv), 13% were dysmature, and 20% atretic. M oocytes were associated with significantly higher levels of P and a higher P:E2 ratio. There were no differences in hormone levels associated with fertilized and nonfertilized oocytes. Thirty-five embryos developed to the six- to eight-cell stage in vitro, of which 13 exhibited optimal cleavage rates. Significantly lower levels of E2 and higher P:E2 ratios were associated with the more rapidly cleaving embryos. Proportionally more embryos showing optimal cleavage rates developed from M compared to Miv oocytes, and only embryos derived from M oocytes developed to blastocysts in culture. Optimal cleavage rates to the six- to eight-cell stage in vitro, rather than fertilization rates, are a better indicator of (subsequent) developmental capacity, and, in this study, embryonic development was closely associated with the maturity of the oocyte at recovery.  相似文献   

11.
Sperm motility is important for penetration of the zona pellucida, and this parameter has been reported to be the single most important factor determining fertilization rates. As there was no report on the relationship between sperm motility and embryo quality, we investigated the influence of sperm motility on embryo quality in 41 patients with tubal disease and/or obstruction. The patients were either unstimulated or stimulated with clomiphene or clomiphene and human menopausal gonadotrophin. Of 116 oocytes collected, 86 (74.1%) fertilised and cleaved; of these only 44 embryos had clear equal blastomeres without fragmentation (grade 3). Grade 3 embryos were equally distributed through all initial sperm motility categories, and through all categories of sperm concentration after swim-up. The ratio of motile sperm concentration in the initial semen sample to the final sperm concentration after swim-up varied from 0.5 to 67, and grade 3 embryos were distributed randomly from low to high ratios. The pregnancy rate in this series was only 14.6% per replacement. The rate of gestational sacs per embryo replaced was 7.0% (6/86); if “poor” embryos were excluded, the rate was 9.1% (6/66). The absence of correlation between sperm motility and embryo quality is discussed on morphological grounds.  相似文献   

12.
The present study was used to examine the effect of ambient temperature on the day of slaughter of buffaloes on oocyte cleavage and subsequent embryo development following in vitro fertilization (IVF)/chemical activation (parthenogenesis). A total of 601 oocytes were collected from buffaloes, which were sacrificed when the ambient temperature was >40 or ≤40 °C and the collected oocytes were matured in vitro. During each experiment about half of the matured oocytes were used for IVF whereas the remaining oocytes were subjected to one of the three chemical activation protocols viz. (i) 7% ethanol (ET) and 6-di methyl amino purine (6-DMAP), (ii) ET and cycloheximide (CHX) and (iii) ET followed by a combined treatment of 6-DMAP and CHX. Cleaved oocytes were cultured in mSOF supplemented with BSA, essential amino acids, non-essential amino acids, ITS (insulin transferrin and selenium) and l-glutamine. Low cleavage and subsequent embryo development was observed in those oocytes which were collected from buffaloes slaughtered at ambient temperature >40 °C than at ≤40 °C. There was no significant difference in cleavage rate following different chemical activations in oocytes collected from buffaloes slaughtered on the day when the maximum ambient temperature was >40 °C or ≤40 °C. These results suggest that high ambient temperature influences competence of oocytes to cleave and develop to blastocyst stage following natural activation with sperm and/or process of fertilization and subsequent embryo development.  相似文献   

13.
Resazurin is a redox dye (7-hydroxy-3H-phenoxazin-3-one-10-oxide) used for assessing potential fertility of spermatozoa and functional status of eukaryotic cells. In this study, the fertilizing capacity of spermatozoa treated with resazurin and effects of resazurin on bovine embryo development in vitro was examined. Abattoir-derived bovine oocytes were collected and subjected to in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 1, bovine oocytes (n=2767) were fertilized with spermatozoa exposed to resazurin (17.6 μg/ml) for 0, 15, 30, 60 min, respectively. There was no significant (P>0.05) difference with respect to oocyte cleavage, morula and blastocyst production between treatments. In Experiment 2, oocytes (n=1671) were treated with resazurin (1.8 μg/ml) during IVM, IVF, IVC, respectively, or during the entire IVM, IVF and IVC procedures. There was no significant (P>0.05) difference in cleavage rates. However, the proportion of embryos that developed into blastocysts, expanded and hatched blastocysts in those groups in which oocytes/embryos were treated with resazurin during IVC or IVM/IVF/IVC was significantly (P<0.05) less than those exposed to resazurin during IVM only, or during IVF only. We conclude that resazurin did not have significant adverse effects on fertilizing capability of bovine spermatozoa; however, extended treatment of embryos with resazurin may be detrimental to embryonic development.  相似文献   

14.
雷公藤多甙对小鼠卵母细胞成熟和体外受精的影响   总被引:1,自引:0,他引:1  
采用超排卵技术研究雷公藤多甙(GTW)对小鼠卵母细胞的成熟和体外受精以及脏器等的影响,GTW对小鼠卵母细胞生发泡破裂没有影响,但可以抑制卵母细胞第一极体的释放,影响卵母细胞的存活率并可降低体外受精率和超排卵的卵母细胞数量。GTW可以破坏卵母细胞成熟,降低卵母细胞的体外受精能力,影响小鼠的正常生殖功能。  相似文献   

15.
16.
This study was undertaken to compare the effects of FSH-pituitary (FSH-P), eCG, and a combination of gonadotropins containing 400 IU eCG and 200 IU hCG (PG 600) on the growth of large follicles, oocyte quality and in vitro fertilization (IVF) rate of in vitro matured (IVM) oocytes in prepubertal gilts. The ovaries were removed via midventral laparotomy 48 h (Experiment 1) or 72 h (Experiment 2) after the first injection. In Experiment 1, 30 gilts received 1 of 5 treatments: 1) saline (3 ml i.m., once, n = 6); 2) FSH-P8 (8 mg i.m., twice, with a 24-h interval, n = 6); 3) FSH-P16 (16 mg i.m., twice, with a 24-h interval, n = 6; 4) eCG (1000 IU i.m., once, n = 6); or 5) PG 600 (5 ml i.m., once, n = 6). Compared with saline, treatment with PG 600 or eCG induced significant (P < 0.05) growth of large follicles (> or = 6 mm). In Experiment 2, 16 gilts received 1 of 5 treatments: 1) saline (n = 4); 2) FSH-P8 (n = 4); 3) FSH-P16 (n = 4); 4) eCG (n = 4), or 5) PG 600 (n = 4). The same injection protocol as in Experiment 1 was used. Compared with treatment with FSH-P8 or FSH-P16, eCG increased (P<0.05) the number of large follicles. The proportion of good oocytes was increased (P<0.05) with FSH-P8 or FSH-P16 compared with treatment with eCG or PG 600. Moreover, oocytes from eCG-treated gilts had a greater (P<0.05) rate of male and female pronuclei than FSH-P or saline-treated gilts. In conclusion, treatment with FSH-P resulted in a higher proportion of oocytes with multilayer cumulus cells, whereas treatment with eCG resulted in higher pronuclear rates following in vitro fertilization in prepubertal gilts.  相似文献   

17.
The aim of the present series of experiments was to investigate the effect of the size of follicle from which the oocytes originate on their subsequent in vitro developmental ability. Ovarian follicles were isolated and grouped according to size (2–6 mm, >6 mm). Primary oocytes were carefully liberated and grouped according to morphology into one of five categories: denuded; expanded; with two or three layers of cumulus; with four or five layers; and with many (six or more) layers. Following in vitro maturation (IVM), fertilization (IVF), and culture (IVC), more oocytes with many layers of cumulus (P < 0.01, 70.2%, 73/104 vs. 46.8%, 87/186, respectively) and a higher proportion of blastocysts were obtained from follicles > 6 mm compared to 2–6 mm follicles (P < 0.01, 65.9%, 60/91 from >6 mm follicles vs. 34.3%, 34/99 from 2–6 mm follicles, respectively). Use of follicular fluid (BFF) from follicles of different sizes in the IVM medium did not significantly increase the cleavage rate or blastocyst yield compared to controls. Administration of procine folliclestimulating hormone (pFSH) to donors prior to slaughter was investigated as a possible means of increasing the number of larger sized follicles in the ovaries and, thereby, the quality of the recovered oocytes. It was found that administration of six injections of pFSH beginning 3 days prior to slaughter resulted in a significant increase (P < 0.001) in the proportion of follicles >6 mm in diameter (31.6%) compared to that in nontreated controls (6.6%) and to animals that received only four injection groups (9.4%). The blastocyst yield from oocytes originating from >6 mm follicles, whether from unstimulated or from pFSH-treated animals, was approximately double that of oocytes from 2–6 mm follicles (P < 0.01; 42.9%, 24/56 for >6 mm follicles vs. 22.8%, 21/92 for 2–6 mm follicles, respectively, for the 6 pFSH group; P > 0.05; 62.5%, 5/8 for >6 mm follicles vs. 32.8%, 22/67 for 2–6 mm follicles, respectively, for the control). © 1994 Wiley-Liss, Inc.  相似文献   

18.
Oocyte transfer is a potential method to produce offspring from valuable mares that cannot carry a pregnancy or produce embryos. From 2000 through 2004, 86 mares, 19.2 +/- 0.4 yr of age (mean +/- S.E.M.), were used as oocyte donors in a clinical program at Colorado State University. Oocytes were collected from 77% (548/710) of preovulatory follicles and during 96% (548/570) of cycles. Oocytes were collected 21.0+/-0.1h after administration of hCG to estrous donors and cultured 16.4 +/- 0.2 h prior to transfer into recipients' oviducts. At 16 and 50 d after transfer, pregnancies were detected in 201 of 504 (40%) and 159 of 504 (32%) of recipients, respectively, with an embryo-loss rate of 21% (42/201). Pregnancy rates were similar (P > 0.05) for cyclic and noncyclic recipients and for recipients inseminated with cooled, fresh or frozen semen. One or more recipients were detected pregnant at 16 and 50 d, respectively, for 80% (69/86) and 71% (61/86) of donors. More donors <20 than > or = 20 yr (mean ages +/- S.E.M. of 15.5 +/- 0.4 and 23.0 +/- 0.3 yr, respectively) tended (P = 0.1) to have one or more pregnant recipients at 50 d (36/45, 80%; 28/45, 62%, respectively). Results of the program confirm that pregnancies can consistently be obtained from older, subfertile mares using oocyte transfer.  相似文献   

19.
Influence of Griseofulvin treatment on semen quality in the dog   总被引:1,自引:0,他引:1  
Griseofulvin is used to treat dermatomycosis in many species and causes oligospermia in supra-pharmacological doses. The aim of the present study was to evaluate the effect of Griseofulvin administered at therapeutic doses upon semen quality in dogs. Four dogs were treated with Griseofulvin (25 mg/kg per day) for 30 days. Semen collections and analyses were performed before, during and for 100 days after treatment for the Griseofulvin group and 10 untreated control dogs. Semen analyses included mean percentage of forward progressively motile sperm, total sperm output, normal live sperm and normal dead sperm. There was no significant difference between control and treated dogs for each of the semen quality parameters. Therapeutic dosage of Griseofulvin had no deleterious effect upon semen quality in dogs, although this does not preclude potential embryotoxic and teratogenic effects.  相似文献   

20.
The aim of this study was to evaluate the level of asymmetrical dimethylarginine (ADMA) levels before gonadotrophine treatment and on the day of oocytes retrieval in order to determine whether ADMA can be used as a predictive marker for implantation success in in vitro fertilization (IVF) cycles. Forty-four unexplained infertile patients were included in the study. Controlled ovarian hyperstimulation was performed using the recombinant follicle-stimulating hormone (FSH) with the standard long protocol for all patients. ADMA and E2 were measured at the beginning of the ovulation induction and on oocyte retrieval day. The primary outcome was the difference in ADMA levels in implantation positive and implantation negative women. At the beginning of the ovulation induction, the mean ADMA levels were 1553 μmol/L and 1.464 μmol/L in the implantation positive and negative groups, respectively. There was no statistically significant difference between groups (p: 0.90). On the day of oocyte retrieval, the mean ADMA levels were 1173 μmol/L and 1170 μmol/L in the implantation positive and negative groups, respectively. There was no statistically significant difference between groups (p: 0.97). In conclusion, ADMA levels before gonadotrophine treatment and the day of oocytes retrieval cannot be used as a predictive marker for implantation success in IVF cycles.  相似文献   

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