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PubMed1993

In vitro maturation of equine oocytes collected by follicle aspiration and by the slicing of ovaries.

The aim of this study was to examine 2 techniques for oocyte recovery from equine ovaries at slaughter: by aspiration of follicles and by additional slicing of ovaries. The morphology and nuclear configuration of oocytes recovered with either technique, and the time course of nuclear maturation during in vitro maturation were evaluated. Recovery rates were 1.75 and 4.14 oocytes per ovary for aspiration and slicing (total 145 and 344 oocytes from 83 ovaries), respectively. The oocytes were classified according to their cumulus/ooplasm morphology into 4 groups: compact/circular(A), compact/semicircular(B), expanded(C) and others(D). The percentages of oocytes in Groups A, B, C and D were 34, 38, 25 and 3% (aspiration) and 55, 26, 17 and 3% (slicing), respectively. The proportions of oocytes with a germinal vesicle in Groups A, B, C and D were 28 29 (97%), 23 35 (66%), 11 23 (48%) and 2 4 (50%) in oocytes from aspiration and 91 100 (91%), 52 65 (80%), 15 29 (52%) and 1 2 (50%) in oocytes from slicing, respectively. Group A and B oocytes recovered by aspiration (n=212) and slicing (n=312) were cultured in TCM199 supplemented with 10% fetal bovine serum, 1 mug/ml estradiol-17beta, and 0.02 AU/ml FSH at 38.5 degrees C in 5% CO(2) in air (5 to 10 oocytes per 50- mu l microdrop). At 8, 16, 24, 32 and 40 h of culture, the oocytes were fixed and stained. There were no significant differences in the percentages of Metaphase II stage (MII)-oocytes between recovery techniques at any time points examined. The proportions of MII-oocytes were 1 42 (2%), 4 43 (9%), 21 42 (50%), 28 45 (62%), and 28 40 (70%) at the respective time point in oocytes from aspiration and 0 51 (0%), 3 54 (6%), 22 59 (37%), 43 72 (60%), and 51 76 (67%) in oocytes from slicing, respectively. In most of the oocytes, resumption of meiosis occurred between 8 and 16 h of culture. The proportions of MII-oocytes increased significantly between 16 and 24 h and between 24 and 32 h of culture.

Journal Article↗

PubMed1981

[In vitro maturation of mammalian oocytes].

A review of authors' and published data on maturation of mammalian oocytes in vitro. The following problems are considered: conditions of in vitro oocyte maturation, morphological changes of oocytes during maturation, duration and synchrony of oocyte maturation, RNA and protein synthesis during oocyte maturation, fertilization of oocytes matured in vitro. The oocytes matured in vivo and vitro were shown not to differ morphologically but their ability of subsequent development differed even if meiosis proceeded in the normal way. Upon the resumption of meiosis after the effect of LH in vivo, there appear to occur some substantial changes in the oocytes which are absent during their in vitro maturation.

Animals↗

PubMed1998

Oocyte competence for in vitro maturation is associated with histone H1 kinase activity and is influenced by estrous cycle stage in the mare.

The in vitro maturation rate of equine oocytes remains low, regardless of culture conditions. Our objective was to determine the reasons for failure of equine oocytes to resume meiosis during in vitro maturation and to ascertain the influence of the estrous cycle stage on meiotic competence. In 10 cyclic mares, 7 ultrasound-guided follicular punctures were performed alternately during the follicular phase (group DF; n = 3 punctures), at the end of the follicular phase (group EF; n = 2), and during the luteal phase (group DL; n = 2). We evaluated the competence of the oocytes for in vitro maturation and measured their maturation-promoting factor activity by histone H1 kinase assay. Puncturing once at the end of the follicular phase and once during the luteal phase, or three times during the follicular phase, yielded about 11 cumulus-oocyte complexes per 22 days. The maturation rate was different between the groups, 51% in group EF, 34% in group DL (p < 0.05), and 15% in group DF (p < 0.01), and it increased with an increase in follicular diameter (p < 0.05). After in vitro culture, the H1 kinase activity was lower in oocytes that remained in germinal vesicle or dense chromatin stages than in oocytes that reached metaphase I or metaphase II (p < 0.05). The H1 kinase activity was not different between oocytes in germinal vesicle stage after in vitro maturation and immature oocytes that were not cultured in vitro, and was higher in preovulatory oocytes that reached metaphase II in vivo than in the oocytes that reached metaphase II after in vitro maturation (p < 0.001). This is the first report on kinase activity in the equine oocyte.

Animals↗

PubMed1995

Beneficial influence of Vero cells on in vitro maturation and fertilization of bovine oocytes.

The aim of this study was to examine the effects of Vero cells and other somatic cells on in vitro maturation of bovine oocytes. Both denuded oocytes and oocytes with intact cumuli (COCs) were cultured on monolayer of Vero cells, cumulus cells and granulosa cells. The effect of gonadotropins was investigated after the addition of gonadotropins to the culture medium. The evaluation using analysis of variance revealed that removal of cumulus cells generally reduced the percentage of oocytes completing their maturation in vitro and that this effect could not be overcome by the addition of gonadotropins to the culture medium. However, in individual experiments, when oocytes were co-cultured with different monolayers of somatic cells, Vero cells were able significantly support the maturation of denuded oocytes, and their beneficial effect was further enhanced by the addition of gonadotropins (76 vs 80.9%). We did not observe a similar effect after the co-culture of oocytes with a monolayer of cumulus cells (65.3 and 53%, respectively). Granulosa cell monolayer delayed maturation in the both COCs and denuded oocytes (10.5 and 16.5%, respectively). In vitro fertilization was successful in most of the experimental groups. However, when denuded oocytes were cultured without any somatic cell support, they did not decondense the penetrated sperm head after in vitro fertilization. This study demonstrates that 1) Vero cells beneficially affect the in vitro maturation of bovine oocytes; 2) cumulus cells in the form of monolayer lose their beneficial influence on in vitro maturation of bovine oocytes; and 3) granulosa cells and FSH and LH alone (without somatic cells) do not show positive effects on in vitro maturation of bovine oocytes.

Journal Article↗

PubMedDate not supplied

The effect of bovine amniotic fluid on in vitro maturation of bovine oocytes.

Three experiments were carried out to investigate whether bovine amniotic fluid (BAF), when added as a protein supplement to the maturation medium, supports the in vitro maturation of pre-ovulatory bovine oocytes. In the first experiment, the medium was supplemented with three different concentrations of the BAF (10, 20 and 50%, respectively). In the second experiment, the effects of a combination of two protein supplements BAF plus oestrus cow serum (BAF-OCS) and BAF plus bovine follicular fluid (BAF-BFF), on the in vitro oocyte maturation rates were evaluated. In the third experiment, the effects of three different protein supplements (BAF, OCS, and BFF) on the oocyte maturation percentages were compared. Maturation rates at the metaphase II stage, following supplementation with 20% BAF (77%) and 10% BAF (68%) were significantly higher (P < 0.001 and P < 0.05, respectively) than those resulting from supplementation with 50% BAF (44%). Significant differences between the 10% BAF and 20% BAF treatments were not observed. Significant differences were, however, found between the BAF-OCS treatment (57%) and the BAF-BFF treatment (50%). The results obtained from the BAF and the OCS treatments (77 and 78%, respectively) were significantly higher (P < 0.05) than when BFF (60%) was used. It is concluded that supplementation of the in vitro maturation medium of pre-ovulatory bovine oocytes with BAF provides important levels of proteins and growth factors responsible for nuclear and cytoplasmic development.

Amniotic Fluid↗

PubMed1997

Effects of follicular fluid supplementation of in-vitro maturation medium on the fertilization and development of equine oocytes after in-vitro fertilization or intracytoplasmic sperm injection.

The aim of this study was to compare the effect of the addition of follicular fluid (FF) collected from preovulatory follicles with that of oestrous mare serum (EMS) (acting as the control) to TCM-199 medium on the in-vitro maturation, fertilization and development of equine cumulus-enclosed oocytes. Oocytes (<30 mm in diameter) were obtained from the ovaries of slaughtered mares. After in-vitro maturation in the presence of the two supplements, their fertilization, cleavage and developmental potential were compared after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) using frozen-thawed spermatozoa. Follicular fluid did not increase the maturation of oocytes to metaphase II stage compared to control. After IVF, there was no difference in fertilization rates between FF-supplemented oocytes and controls (7/87, 8.4% of oocytes showing two pronuclei with FF versus 7/116, 6% with EMS; not significant). However, after ICSI, FF-supplemented oocytes showed significantly increased normal fertilization (32/85, 37.6% of two-pronuclear oocytes) and developmental potential (15/31, 48% cleavage) compared to the control oocytes (7/47, 14.9%, P < 0.01; and 2/48, 4%, P < 0.01, respectively). Overall, ICSI resulted in increased fertilization rates compared to IVF, regardless of the presence or absence of FF (39/132, 29.5% with ICSI versus 14/203, 6.9%). These results suggest that follicular fluid supplementation may improve the maturity of equine cumulus-enclosed oocytes sufficiently for the successful use of ICSI, but not sufficiently for normal sperm-egg interaction occurring during IVF.

Animals↗

PubMed2003

Blastocyst formation rates in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection.

This study was conducted to evaluate in vivo and in vitro development of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. Oocytes were collected from slaughterhouse-derived ovaries, matured in vitro, and injected with frozen-thawed stallion sperm. In vivo development was assessed after transfer of injected oocytes to the oviducts of recipient mares. Mares were killed 7.5-8.5 days after transfer and the uterus and oviducts flushed for embryo recovery. Of 132 injected oocytes transferred, 69 (52%) were recovered; of these, 25 (36%) were blastocysts with a blastocoele and capsule. In vitro development was assessed in three culture systems. Culture of zygotes in modified Chatot, Ziomek, Bavister medium with BSA containing either 5.5 mM glucose for 7.5 days or 0.55 mM glucose for 3 days, followed by 3 mM glucose for 2 days, then 4.3 mM glucose for 2.5 days, did not result in blastocyst formation. Culture of zygotes in Dulbecco modified Eagle medium (DMEM)/F-12 with 10% fetal bovine serum with and without coculture with equine oviductal epithelial explants yielded 16% and 15% blastocyst development, respectively. Development to blastocyst was significantly lower in G1.3/2.3/BSA than in DMEM/F-12/BSA or in either medium with 10% added serum (2% vs. 18%, 18% or 20%; P < 0.05), suggesting that requirements for equine embryo development differ from those for other species. These results indicate that in vitro-matured equine oocytes are sufficiently competent to form 36% blastocysts in an optimal environment (in vivo). While we identified an in vitro culture system that provided repeatable blastocyst development without coculture, this yielded only half the rate of development achieved in vivo.

Animals↗

PubMed2006

The in vitro maturation (IVM) of human oocytes for in vitro fertilization (IVF): is it time yet to switch to IVM-IVF?

The recent study by Li et al. observed that human oocytes from patients with polycystic ovary syndrome (PCOS) matured in vitro exhibited a higher proportion of abnormal spindle structures and disturbed chromosomal configurations compared with in vivo-matured oocytes from a control group of PCOS patients. This article discusses the obstacles that must be overcome and factors that must be monitored when attempting to optimize conditions for the in vitro maturation of human oocytes, with particular attention to the strengths and weaknesses of the study by Li et al.

Animals↗

PubMed1996

Effects of follicular fluid during in vitro maturation of bovine oocytes on in vitro fertilization and early embryonic development.

In vivo, oocytes mature while bathed in preovulatory follicular fluid, so we studied maturation of oocytes in vitro in media supplemented with such fluid. Bovine oocytes were obtained from 2-to 6-mm follicles from slaughterhouse ovaries and were matured for 24 h in tissue culture medium 199 + 10% estrous cow serum supplemented with bovine follicular fluid (BFF) obtained from a preovulatory follicle either before (0 h) or 8 or 20 h after a GnRH-induced LH surge. After in vitro fertilization, oocytes matured in 10% or 40% 20-h BFF had higher cleavage rates (p < 0.05) than those matured in 10% or 40% 0-h BFF (80% and 86% vs. 46% and 51%, respectively); oocytes matured in 8-h BFF were intermediate. A greater percentage of oocytes showed cumulus expansion with 20-h BFF than with 0-h BFF (p < 0.05). In experiment 2, more (p < 0.05) embryos from oocytes matured in 20% or 40% 20-h BFF developed to blastocysts after culture of embryos compared to those matured in 20% or 40% 0-h BFF (29% and 29% vs. 7% and 14%, respectively) or those matured in no BFF (12%). Embryos from oocytes matured in a mixture of 0-h and 20-h BFF developed to the blastocyst stage similarly (27%) to those matured in 20-h BFF, suggesting that positive factors in 20-h BFF overcame any inhibitory effect of 0-h BFF. To summarize, bovine oocytes matured with BFF collected 20 h after LH surge result in higher cleavage rates after in vitro fertilization and more blastocysts than those matured in 0-h BFF. When the factors responsible are identified, they might be used to enhance maturation of oocytes in vitro.

Animals↗

PubMed1998

Evidence that Meishan and large-white hybrid preovulatory follicles may differentially affect oocyte in vitro maturation and fertilization.

Two experiments were carried out to test the hypothesis that follicles recovered from Meishan animals may provide a more favourable environment for oocyte maturation in vitro than follicles recovered from Large-White hybrid animals. In Experiment 1, all follicles > or = 4 mm were recovered from six Meishan and seven Large-White hybrid gilts in the late follicular phase and healthy oocyte cumulus complexes recovered. Cumulus oocyte complexes were randomly divided into two groups, and each group cultured for 27 or 34 h (62 and 64; 56 and 56 for Meishan and Large-White hybrid, respectively) in defined medium in the presence of either of the two largest follicle shells per animal. Subsequent examination of oocyte nuclear maturation showed that although maturation did not differ significantly between the breeds after 27 h, more (P < 0.01) Meishan oocytes co-cultured with Meishan follicles developed to metaphase II stage than Large-White hybrid oocytes co-cultured with Large-White hybrid follicles after 34 h. The next eight largest follicles per animal were cultured for 34 h to produce conditioned media. In Experiment 2, oocytes recovered from the slaughterhouse were matured for 46 h in the presence of conditioned media from Meishan (612 oocytes) or Large-White hybrid (731 oocytes) follicles, or in fresh medium in the presence of a follicle shell from slaughterhouse ovaries. Oocytes were then inseminated and 12 h later examined for penetration and male pronuclear formation. A higher (P < 0.05) percentage of oocytes cultured in Meishan follicle conditioned medium underwent sperm penetration and male pronuclear formation than oocytes cultured in conditioned media from Large-White hybrid animals. Concentration of oestradiol and progesterone in the conditioned media did not differ between the breeds (P > 0.1). In conclusion, these results suggest that (1) Meishan oocytes have advanced maturational capacity when cultured with Meishan preovulatory follicle shells and (2) differences in follicle maturation in the Meishan compared to the Large-White hybrid pig may result in an improved ability of the follicles, via conditioned media, to support oocyte maturation and fertilization in vitro.

Animals↗

PubMed1997

Successful in vitro maturation of human oocytes not exposed to human chorionic gonadotropin during ovulation induction, resulting in pregnancy.

OBJECTIVE: To report a case of successful in vitro maturation of human oocytes not exposed to hCG during ovulation induction, resulting in pregnancy after transfer of a frozen-thawed embryo resulting from intracytoplasmic sperm injection (ICSI) of the in vitro-matured human oocytes. DESIGN: Clinical study. SETTING: Hospital-based private IVF center. PATIENT(S): A couple with infertility due to severe oligospermia. INTERVENTION(S): Five immature oocytes were retrieved from a patient who failed to use hCG during ovarian stimulation and were cultured for 48 hours in B2 medium containing FSH and hCG. Five oocytes extruded the first polar body and underwent ICSI with frozen-thawed husband spermatozoa. MAIN OUTCOME MEASURE(S): In vitro maturation, fertilization after ICSI, embryo development, and pregnancy. RESULT(S): All five oocytes extruded the first polar body and were injected using ICSI. Three oocytes were fertilized, but two showed three pronuclei. The remainder, a two-pronuclei embryo subsequently cleaved and was cryopreserved. An ongoing pregnancy was obtained after-the transfer of this frozen-thawed embryo. CONCLUSION(S): Immature human oocytes at the germinal-vesicle stage that have not been exposed to hCG during ovarian stimulation can be matured in vitro and a normal pregnancy can result from ICSI of the in vitro-matured oocytes.

Adult↗

PubMed1998

Effects of glycosaminoglycans on the development of in vitro-matured and -fertilized porcine oocytes to the blastocyst stage in vitro.

We examined the effects of four glycosaminoglycans (GAGs) on the development of in vitro-matured (IVM) and -fertilized (IVF) porcine oocytes to the blastocyst stage. IVM and IVF oocytes were cultured in Whitten's medium supplemented with hyaluronic acid, chondroitin sulfate A, dermatan sulfate, or heparin at 38.5 degrees C in an atmosphere of 5% CO2 in humidified air for up to 6 days. After 2 days in culture, 28-34% of the inseminated oocytes cleaved to the 2- to 8-cell stage, and the GAGs showed no significant effect on development. After 6 days in culture, blastocysts were observed in all groups. The percentage of blastocysts was significantly higher in hyaluronic acid-supplemented medium (14%) than in dermatan sulfate-supplemented (5%), heparin-supplemented (2%), or nonsupplemented (2%) media. In addition, the percentage of blastocysts was significantly higher in chondroitin sulfate A-supplemented medium (11%) than in heparin-supplemented and nonsupplemented media, although the number of blastocysts in chondroitin sulfate A was not significantly different from that in hyaluronic acid- and dermatan sulfate-supplemented media. There were no significant differences in the mean number of nuclei per blastocyst cultured in any group. The effects of hyaluronic acid and chondroitin sulfate A on development to the blastocyst stage was examined at various concentrations. After 6 days in culture, development of IVM and IVF oocytes to the blastocyst stage was best supported in 0.5 mg/ml hyaluronic acid-supplemented (17%) and in 0.1 or 0.5 mg/ml chondroitin sulfate A-supplemented (10% or 9%, respectively) media. It is concluded from these results that hyaluronic acid and chondroitin sulfate A supported the development of porcine oocytes matured and fertilized in vitro to the blastocyst stage.

Animals↗

PubMed1998

Kinetics of nuclear maturation and protein profiles of oocytes from prepubertal and adult cattle during in vitro maturation.

The aim of this present study was to compare the kinetics of nuclear maturation between calf and cow oocytes in order to determine if there are differences between the 2 groups which could explain their disparate developmental capacity. The constitutive and neosynthetic protein patterns of cow and calf oocytes and of their corresponding cumulus cells were also compared during in vitro maturation. A total of 397 calf oocytes and 406 cow oocytes was matured in M199 + 10 ng/mL EGF. The first group of oocytes (n = 30) was immediately fixed and stained after removal from the follicle, and represent 0 h. The remaining oocytes were removed from the maturation medium at 4, 8, 12, 16, 20 and 24 h respectively. Half were denuded, fixed and stained for nuclear status; while the remainder were radiolabeled with methionine-(35S). Immediately after isolation, all the oocytes were at the GV stage. By 8 h, GVBD had occurred in most oocytes (calf: 97%; cow: 100%) and some had reached pro-metaphase I (calf: 49%; cow: 51%). By 12 h, most of the oocytes were at metaphase I (calf: 84%; cow: 94%). By 16 h, 54% of calf oocytes had reached telophase I or beyond compared with 71% of cow oocytes. This difference between the 2 groups became significant by 20 h, with 89% of cow oocytes (P < 0.05) at metaphase II and 71% of calf oocytes. By 24 h of culture, GVBD had occurred in all cases. Most oocytes completed meiosis I and were arrested at metaphase II with the first polar body extruded (calf: 72%; cow: 86%). No differences were noted in the constitutive and the neosynthetic protein profiles of cumulus cells in relation to the age of animal. Changes in neosynthetic protein patterns were observed both in cow and calf cumulus during IVM, and several proteins showed stage-specific synthesis. For the constitutive protein patterns of cow and calf oocytes, there were quantitative (38 and 40 kD) and qualitative (4, 10, 16, 17, 24, 25 and 26 kD) differences between the 2 groups. Only a few differences were observed in neosynthetic proteins between cow and calf oocytes, but there were changes in relation to nuclear status both in cow and calf oocytes. In conclusion, the difference in developmental capacity between cow and calf oocytes may be explained by a difference in the kinetics of nuclear maturation, which was significant at 20 h of culture (with 89% of cow oocytes at metaphase II and 71% of calf oocytes). At the biochemical level, our results indicate that nuclear progression during in vitro maturation of bovine oocytes is linked to changes in protein synthesis by the oocyte itself, while cumulus protein synthesis may either stimulate or modulate the process of oocyte maturation.

Aging↗

PubMed2004

[Influence of epidermal growth factor and gonadotrophin on the in vitro maturation of human oocytes].

OBJECTIVE: To study the effect of epidermal growth factor(EGF) and different concentrations of gonadotrophin (Gn) on the in vitro maturation of human oocytes. METHODS: EGF was added to the vitro culture medium in order to observe the effect of Gn combined with or without EGF on the result of in vitro maturation. The concentrations of hCG and FSH were changed respectively to observe the difference between the results. RESULTS: Adding EGF to the culture medium improved the maturation rate of oocyte significantly (P < 0.05). There was no difference between the results with different concentrations of hCG and FSH in the culture medium. CONCLUSION: EGF can improve the results of the in vitro maturation of human oocytes by increasing the maturation rate significantly. Increasing the concentration of Gn does not influence the results of in vitro maturation.

Cells, Cultured↗

PubMed2003

FSH priming improves oocyte maturation, but priming with FSH or hCG has no effect on subsequent embryonic development in an in vitro maturation program.

AIM: To determine whether maturation and subsequent blastocyst development of in vitro matured oocytes can be improved by in vivo follicle stimulating hormone (FSH) or human chorionic gonadotrophin (hCG) priming, using a mouse model. EXPERIMENTAL DESIGN: Five groups of oocytes were used: in vivo control, in vitro matured (IVM) control, IVM after 24 h in vivo priming with FSH, IVM after 48 h in vivo priming with FSH and IVM after 16 h in vivo priming with hCG. In vitro fertilization (IVF) was performed on all groups. Oocyte maturation, fertilization, blastocyst development rates and blastocyst cell numbers were assessed for all groups. RESULTS: Significant improvement in oocyte maturation was observed in the two FSH priming groups compared with the IVM control group (P<0.005 and P<0.001, respectively). There were no significant differences in fertilization between all five groups. Blastocyst development was significantly higher in the in vivo control compared to the IVM groups (P<0.001). No significant differences were observed in blastocyst cell numbers among all five groups. CONCLUSIONS: While FSH priming improves the maturation rate of IVM oocytes, FSH or hCG priming does not improve development to the blastocyst stage.

Animals↗

PubMed1998

Morphologic comparison of ovulated and in vitro-matured porcine oocytes, with particular reference to polyspermy after in vitro fertilization.

This study was conducted to evaluate morphologic differences in pig oocytes matured in vivo and in vitro, with particular reference to the potential relationship between oocyte morphology and the occurrence of polyspermy after in vitro fertilization (IVF). In vivo-matured oocytes were surgically recovered from the oviducts of gilts with ovulated follicles on day 2 of estrus, and in vitro-matured oocytes were obtained by culturing follicular oocytes in a oocyte maturation system that has resulted previously in production of live offspring following IVF. Comparisons were made of the cytoplasm density, the diameter of oocytes with or without zona pellucida (ZP), the thickness of the ZP, the size of the perivitelline space (PVS), ZP dissolution time, and cortical granule (CG) distribution before IVF, and CG exocytosis and polyspermic penetration after IVF. Oviductal oocytes have clear areas in the cytoplasm cortex, while in vitro-matured oocytes have very dense cortex. The diameter of ovulated oocytes with ZPs was significantly (P < 0.001) greater than that of in vitro-matured oocytes. However, no difference was observed in the diameter of the oocyte proper. Significantly (P < 0.001) thicker ZPs and wider PVSs were observed in the ovulated oocytes. The ZPs of ovulated oocytes were not dissolved by exposure to 0.1% pronase within 2 hr, but the ZPs of in vitro-matured oocytes were dissolved within 131.7 +/- 7.6 sec. The ZPs of ovulated oocytes, but not of in vitro-matured oocytes, were strongly labeled by a lectin from archis hypogaea that is specific for beta-D-Gal(1-3)-D-GalNAc. Polyspermy rate was significantly (P < 0.01) higher for in vitro-matured oocytes (65%) than for ovulated oocytes (28%). CGs of oviductal oocytes appeared more aggregated than those of in vitro-matured oocytes. Most of CGs were released from both groups of oocytes 6 hr after IVF regardless of whether they were polyspermic or monospermic oocytes. These results indicate that in vitro-matured and in vivo-matured pig oocytes possess equal ability to release CGs on sperm penetration. Unknown changes in the extracellular matrix and/or cytoplasm of the oocytes while in the oviduct may play an important role(s) in the establishment of a functional black to polyspermy in pig oocytes.

Animals↗

PubMed2003

Glutathione synthesis during in vitro maturation of buffalo (Bubalus bubalis) oocytes: effects of cysteamine on embryo development.

It was demonstrated that cysteamine supplementation during in vitro maturation (IVM) improves embryo development by increasing glutathione (GSH) synthesis in several species. An improved developmental competence of oocytes matured in the presence of cysteamine was also recorded in buffalo species. The purpose of this work was to investigate (1) if glutathione is de novo synthesized during in vitro maturation of buffalo oocytes, (2) if cysteamine improves buffalo embryo development via an increase in GSH synthesis, and (3) if the inhibition of glutathione synthesis by buthionine sulfoximide (BSO), in the presence or absence of cysteamine, affects subsequent embryo development and GSH synthesis.Cumulus-oocytes complexes (COCs), recovered from slaughtered animals, were matured in vitro in TCM199+10% fetal calf serum (FCS), 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17-beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 5mM BSO. Glutathione content was measured by high-performance liquid chromatography (HPLC) and fluorimetric analysis in immature oocytes and in oocytes matured in the different experimental conditions. In a second experiment, the mature oocytes were in vitro fertilized and cultured for 7 days in order to assess development to blastocysts (BLs). It was demonstrated that buffalo oocytes synthesize glutathione during in vitro maturation and that cysteamine increases glutathione synthesis. Furthermore, the promoting effects of cysteamine on embryo development and GSH synthesis were neutralized by buthionine sulfoximide. These results indicate that glutathione plays a critical role on buffalo embryo development.

Animals↗

PubMed2001

Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector.

Here we report the production of transgenic pigs that express enhanced green fluorescent protein (eGFP). Porcine oocytes were matured in vitro in a serum-free, chemically defined maturation medium, subsequently infected with a replication deficient pseudotyped retrovirus, fertilized and cultured in vitro before being transferred to a recipient female. Two litters were born from these embryo transfers; one pig from each litter was identified as transgenic and both expressed eGFP. From a tool in basic research to direct applications in production agriculture, domestic livestock capable of expressing foreign genes have many scientific applications.

Agriculture↗