In vitro maturation of bovine cumulus-oocyte complexes in undiluted follicular fluid: effect on nuclear maturation,pronucleus formation and embryo development

Since resumption of meiosis and cytoplasmic maturation of bovine oocytes takes place in close association with follicular fluid, it would be logical to assume that this might be a perfect maturation medium. To test the hypothesis, abattoir-derived cumulus-oocyte complexes (COCs) were in vitro matured in undiluted (i) mixed follicular fluid (FF) from 3 to 15 mm follicles from abattoir ovaries, (ii) preovulatory follicular fluid (POF) from the dominant follicle from a cyclic unstimulated heifer, (iii) preovulatory follicular fluid (OPU) from synchronised and superovulated heifers 60 h after prostaglandin and 20 h after GnRH treatment, and in (iv) TCM-199 with 5% serum. Subsequent to IVM, the COC were subjected to IVF and IVC, and embryo development was followed until the blastocyst stage at Day 8 after insemination. The MII rates in the TCM-199 (69%), POF (69%) and OPU (72%) groups were not different from each other but different from the FF (41%) group (P<0.05). In spite of the high MII rates, none of the follicular fluids supported embryo development: the FF, POF and OPU blastocyst rates were alike (3%, 3%, 2%) and different (P<0.05) from the rates in the TCM-199 (19%). During IVM in follicular fluids but not in TCM-199, the expanded cumulus masses became trapped in a coagulum. Although it could be prevented by the presence of heparin during IVM, it did not improve the blastocyst rates. In conclusion, undiluted preovulatory follicular fluids supported nuclear maturation but not further embryonic development as judged by the high MII and low blastocyst rates.     

Effect of follicle-stimulating hormone on nuclear and cytoplasmic maturation of sow oocytes in vitro

A series of experiments were conducted to evaluate the effects of FSH supplementation during IVM on porcine oocyte nuclear maturation, and subsequent fertilization, cleavage and embryo development. Cumulus-oocyte complexes (COCs) were cultured 40 h without FSH (control), 40 h with FSH (FSH 0-40 h), or 20 h with FSH followed by a 20-h culture period without FSH (FSH 0-20 h). Nuclear stage of oocytes was assessed at intervals from 12 to 40 h of IVM. Furthermore, oocytes were in vitro fertilized, fixed and stained to determine normally fertilized and polyspermic oocytes. Additionally, COCs were matured with FSH, fertilized and zygotes cultured in NCSU-23. The percentage of cleaved embryos and blastocysts were determined and the number of nuclei was counted. The presence of FSH during the first 20 h of IVM retarded germinal vesicle breakdown. After 40 h of culture 84, 67 and 58% MII oocytes were observed in the FSH 0-20 h, FSH 0-40 h and control groups, respectively. After IVF, penetration rates were similar at 27, 26 and 29%, while the proportion of polyspermic oocytes was 7, 19 and 11% of penetrated oocytes for control, FSH 0-40 and FSH 0-20 h groups, respectively. Cleavage and blastocyst rates differed among treatments (21, 29 and 38%, and 7, 15 and 20% for control, FSH 0-40 and FSH 0-20 h groups, respectively). No differences in blastocyst cell number were found among groups. Blastocyst rates, based on number of cleaved embryos, were 51 and 52% for the FSH 0-40 and FSH 0-20 h groups, which differed significantly from the control group (31%). The results indicate that FSH has a stimulatory effect on nuclear and cytoplasmic maturation of sow oocytes. Addition of FSH for the first 20 h of culture was most beneficial, based on cleavage and blastocyst development rates.     

Preovulatory follicular fluid during in vitro maturation decreases polyspermic fertilization of cumulus-intact porcine oocytes in vitro maturation of porcine oocytes

Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P<0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos.     

Effect of sheep and human follicular fluid on the maturation of sheep oocytes in vitro

This study examines the effect of sheep and human follicular fluid on the in vitro maturation (IVM) of sheep follicular oocytes. Oocyte cumulus complexes recovered post mortem were matured for 24 to 26 h at 38.6 degrees C, 5% CO(2) in air, in TCM-199 bicarbonate medium supplemented with 20% fetal calf serum (FCS) and, where stated, with maturation hormones, including FSH (5.0 ug/ml), LH (5.0 ug/ml) and estradiol (1 ug/ml), or with sheep follicular fluid recovered from large (>5mm) or small (2 to 5mm) ovarian follicles post mortem, or with human periovular follicular fluid obtained during routine IVF procedures. The matured oocytes were then denuded, and their maturation stage and developmental capacity were assessed by in vitro fertilization (IVF) and culture (IVC). It was found that inclusion of sheep or human follicular fluid or hormone supplements in the IVM media more than doubled the number of oocytes completing maturation (FCS alone 33%, compared with 76.2% for maturation hormones, 84.2% for fluid from large and 69.6% for fluid from small sheep follicles and 82.6% for human follicular fluid), and significantly increased fertilization rates (FCS alone 51.6%, compared with 71.9% for maturation hormones, 78.4% for fluid from the large and 75.7% for fluid from small sheep follicles and 73.1% for human follicular fluid) without discernible adverse effects on the development of the cleaving embryos to the morula or blastocyst stage in culture. Omission of FCS and supplements from the IVM medium resulted in a marked reduction (56%) in the number of oocytes maturing. This reduction could be offset to a large part, but not completely, by inclusion of human follicular fluid or human follicular fluid plus LH (5 ug/ml) in the medium. The results of this study show that addition of sheep or human follicular fluid to maturation medium can enhance rather than inhibit the maturation and fertilizability of sheep follicular oocytes in vitro.     

Pure preovulatory follicular fluid promotes in vitro maturation of in vivo aspirated equine oocytes

In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those aspirated again 8 days after the initial follicular aspiration, and (4) high follicular concentrations of meiosis activating sterols (MAS) are beneficial for in vitro maturation of equine oocytes. During estrus, 19 pony mares were treated with 25 mg CEG. After 24 h (Al) and again after 8 days (A2), all follicles >4mm were aspirated and incubated individually for 30 h in the following culture media: standard culture medium (SM), preovulatory follicular fluid collected before CEG containing low MAS concentrations (FF1), preovulatory follicular fluid collected before CEG containing high MAS concentrations (FF2) or preovulatory follicular fluid collected 35 h after administration of CEG containing low MAS concentrations (FF3). Cumulus expansion rate was significantly affected by culture medium. The overall nuclear maturation rate was significantly higher for oocytes collected at A1 (67%) than for oocytes collected at A2 (30%). For oocytes collected at A1, the maturation rates were 71% (FF1), 61% (FF2), 79% (FF3) and 56% (SM). An electrophoretic protein analysis of the culture media revealed the presence of a 200-kDa protein in FF3. The results demonstrate that (1) equine oocytes can be matured during culture in pure equine preovulatory follicular fluid, (2) preovulatory follicular fluid collected after gonadotrophin-priming seems superior in supporting in vitro maturation than standard culture medium, (3) oocytes aspirated 8 days after a previous aspiration are less competent for in vitro maturation than oocytes recovered during the initial aspiration, and (4) the regulation of meiotic resumption during in vitro culture of equine oocytes might be related to the presence of a 200-kDa protein.     

Factors influencing resumption of meiotic maturation and cumulus expansion of porcine oocyte-cumulus cell complexes in vitro

The present study was undertaken to examine effects of various combinations of epidermal growth factor (EGF), transforming growth factor-b?₁ (TGF-b?₁), follicle-stimulating hormone (FSH), luteinizing hormone (LH), androstenedione (A₄), and estradiol-17b? (E₂) on meiotic maturation and cumulus expansion in the pig using an in vitro model system. Oocyte-cumulus cell complexes (OCC) were cultured in the media containing the abovementioned agents for 24 hr and were observed for germinal vesicle breakdown (GVBD), indicative of initiation of meiotic maturation, and for expansion of their cumulus cells. Treatment with EGF significantly increased (P < 0.05) incidence of GVBD, with maximal stimulation occurring at 1 ng/ml (55% vs. 12% in the control). Concentrations of EGF as low as 100 pg/ml significantly stimulated GVBD over control (37% vs. 12%). Addition of EGF (1 ng/ml) and FSH (1.5 μg/ml) together and LH (2 μg/ml) and FSH (1.5 μg/ml) together resulted in significantly higher (P < 0.01) GVBD levels than were observed in response to EGF, FSH, or LH alone. Addition of E₂ (1 μg/ml) had no effect by itself but significantly decreased the incidence of GVBD in the presence of FSH and of LH + FSH. Addition of A₄ (1 μg/ml) significantly reduced the percentage of oocytes undergoing GVBD when added alone or with FSH. Although both EGF and LH stimulated cumulus expansion, FSH was more effective in stimulating cumulus expansion than EGF or LH. TGF-b?₁ had no effect on GVBD or cumulus expansion. These studies indicate that these hormones may have differing roles in oocyte maturation and that their interactions may be part of an intricate system regulating the maturation of oocytes during follicular development in vivo. © 1993 Wiley-Liss, Inc.     

Effects of bovine follicular fluid on maturation of bovine oocytes

Three experiments were conducted to determine the effects of follicular fluid and media on bovine oocyte maturation. Experiments 1 and 3 test the effects of follicular fluid obtained at different times after the LH surge on bovine oocyte maturation in vitro, while Experiment 2 was designed to compare TALP and Medium 199 as serum-free maturation media. Bovine follicular fluid (BFF) was obtained from preovulatory follicles either before (0 h BFF) or at 4, 8, 12 or 20 h after a GnRH-induced LH surge. Oocytes were obtained from follicles 1 to 6 mm in diameter from ovaries retrieved from a slaughterhouse. In Experiment 1, both 0 h and 4 h BFF inhibited resumption of meiosis, whereas BFF collected at 8, 12 and 20 h did not. When oocytes were cultured in media that contained equal portions of 0 and 8 h BFF, meiosis was not inhibited. In Experiment 2, Medium 199 supplemented with bovine serum albumin (BSA) was superior to Tyrode's medium with albumin, lactate and pyruvate for oocyte maturation. In Experiment 3, a higher percentage (P<0.05) of oocytes cultured for 18 h in 40% 20 h BFF in Medium 199 reached Metaphase-II (64%) than those cultured in 0 h BFF (41%) or control medium (39%). There was a transient meiotic arrest due to 0 h BFF as evidenced by the higher percentage of oocytes with germinal vesicles at 8 h of incubation (35% with 0 h vs 20% with 20 h; P<0.05). Furthermore, expansion of cumulus cells was induced in 8 and 20 h BFF, but not 0 h BFF.     

Effect of homologous preovulatory follicular fluid on in vitro maturation of equine cumulus-oocyte complexes

The objective of this study was to test the hypothesis that incubating equine cumulus-oocyte complexes (COCs) in medium containing 50% or 100% homologous preovulatory follicular fluid would improve cumulus expansion and nuclear maturation. Oocytes were incubated in one of three media: 1) supplemented TCM-199 (control), 2) 50% (v/v) follicular fluid in control medium or 3) 100% follicular fluid. Cumulus expansion was evaluated subjectively, and nuclear maturation was evaluated by staining oocytes with Hoechst 33258. The hypothesis that incubating COCs in medium containing follicular fluid would improve cumulus expansion was supported. More (P < 0.05) compact COCs incubated in 50% or 100% follicular fluid developed a moderately to completely expanded cumulus after 24 and 36 h of incubation and more (P < 0.05) expanded COCs incubated in 100% follicular fluid developed a moderately to completely expanded cumulus after 36 h of incubation compared to control medium. The hypothesis that incubating COCs in medium containing follicular fluid would improve nuclear maturation was not supported. Although more (P < 0.05) compact COCs incubated in 50% follicular fluid reached polar body-stage compared to those in control medium, the nuclear maturation rate in the control medium was lower than it was when the same medium was used in a preliminary experiment (described in main text); therefore, the apparent superiority of 50% follicular fluid must be interpreted cautiously. Based on these results, future studies are warranted to further address the value of adding preovulatory follicular fluid to equine IVM culture systems.     

Lysophosphatidic acid stimulates nuclear and cytoplasmic maturation of golden hamster immature oocytes in vitro via cumulus cells

Lysophosphatidic acid (LPA), a member of the phospholipid autacoid family, is induced in incubated human follicular fluid by lysophospholipase D. It is well known that LPA functions as a growth factor and the hypothesis that LPA in human follicular fluid takes a part in meiosis of oocytes is quite plausible. We studied the effects of LPA on the maturation of golden hamster immature oocytes in vitro. Hamster oocytes with a germinal vesicle were cultured in Tyrode's albumin lactate pyruvate (TALP) medium with 10(-5) M LPA, 10 ng/ml epidermal growth factor (EGF), 30 ng/ml insulin-like growth factor-1, 1 ng/ml tumor growth factor-alpha or 1 ng/ml basic fibroblast growth factor. The nuclear maturation rates in the LPA and EGF groups were significantly higher than in the control group and the other growth factors did not show any stimulatory effect (LPA group; 74.3% [75/101], EGF group; 82.4% [89/108] vs. control group; 60.2% [59/98], p < 0.05, p < 0.01, respectively). When the cells of cumulus were removed, EGF and LPA did not increase the nuclear maturation rates. Cotreatment EGF and LPA did not significantly enhance the stimulatory effect observed with LPA alone on maturation in vitro. The penetration rate determined by the zona-free hamster oocyte test was significantly higher in the LPA group than in the control group (26.7% vs. 13.2%, p < 0.05) and was comparable with that of oocytes matured in vivo. In conclusion, LPA stimulates the nuclear and cytoplasmic maturation of hamster immature oocytes via cumulus cells.     

Effects of harvest methods of bovine oocytes co-cultured with follicular hemisections in vitro on nuclear maturation

This study was undertaken to evaluate the in vitro response of bovine cumulus-oocyte complexes (COC) to follicular cells based on 2 COC harvest periods (1 and 6 h after slaughter) and 2 harvest methods (puncturing and mincing). In Experiment 1, the nuclear maturation stage of oocytes was analyzed following harvest and again prior to in vitro culture. More than 95% of the oocytes were at the germinal vesicle stage (GV stage). In Experiment 2, COC were cultured in vitro inside follicular hemisections for 24 h and the percentage of oocytes at the GV stage was the same regardless of the harvest period. In Experiment 3, COC were harvested 6 h following slaughter using the 2 methods and then cultured for 24 h inside follicular hemisections; again, the percentage of oocytes at the GV stage was not statistically different (P>0.05). In a follow up experiment, the culture medium was supplemented with LH (5.0 microg/ml) and the percentage of oocytes maintained at the GV stage did not decrease. These results indicate that COC response to inhibitory factor(s) produced by follicular cells is not influenced by harvest time, harvest method or LH supplementation.     

In vitro maturation and fertilization of follicular oocytes in cattle

This work aims towards developing research concerning the improvement of animal reproduction, embryo development and genetic engineering. In our laboratory, an attempt has been made to standardize in vitro conditions able to optimally support bovine oocyte maturation and fertilization in order to yield viable embryos. Ovaries from cows and heifers, obtained from local slaughter-house, were used for recovery of oocytes from antral follicles. Cumulus-oocyte complexes were statically cultured for 24h at 39 degrees C in medium TCM 199 supplemented with fetal calf serum inactivated, hormones, glucose and granulosa cells under a 5% CO2 and 95% humidity atmosphere. A first group of oocytes was used for fixing and staining procedure for evidence of in vitro maturation. After culture 69.4% (77/111) of oocytes reached full maturation showing cumulus expansion, first polar body extrusion and the 2nd metaphase plate. A 2nd group was used for in vitro fertilization. In vitro semen capacitation was obtained with swim-up system (8.9) with separation of high motility fraction in Talp Hepes medium. Oocytes and spermatozoa were coincubated for 18-20h in Talp medium at 39 degrees C with 5% CO2 and 95% humidity. At the end of culture stereoscope and microscope observations were made for evidence of fertilization. After IVF 67.4% (58/86) resulted fertilized. Most of them showed two pronuclei and residual sperm tail. In few cases oocytes with 1 pronucleus and the swollen sperm head or with syngamy or polyspermic were found. In these experiments high percentages of in vitro matured and in vitro fertilized oocytes have been obtained. These bovine zygotes can be considered an essential step to develop new technologies in cattle breeding.     

Effects of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro

Porcine follicular oocytes, collected from antral follicles (2–5 mm in diameter) of gilt ovaries, were matured in vitro with or without porcine follicular fluid (pFF), gonadotrophins (GTH) or fetal calf serum (FCS) for 48 hours at 37°C under 5% CO₂ in air, and their ability of male pronucleus (mPN) formation was examined after in vitro fertilization. Formation of mPN was observed in 38.6% of penetrated oocytes matured in modified Krebs-Ringer bicarbonate solution (TYH) 18 hours after insemination. The addition of GTH into the maturation medium did not improve the proportion of mPN-formed oocytes (20–30%). In contrast, the mPN formation rate elevated significantly (59.5%) when the oocytes were cultured with pFF, and the addition of follicle-stimulating hormone (FSH) enhanced this pFF action (the rate became 81.0%). In the presence of FSH, significant pFF effect was observable at the concentration of 5%, and its efficiency was elevated with the increase of pFF concentration. When the oocytes were matured with FCS, the mPN formation rate was unchanged or decreased rather than improved (0–25%). These results suggest that pFF, but not FCS, have substance(s) stimulating the ability of mPN formation in porcine oocytes.     

In vitro maturation and early developmental capacity of bovine oocytes cultured in pure follicular fluid and supplementation with follicular wall

Mammalian oocytes mature in follicular fluid (FF), surrounded by follicular cells. In the present study, in vitro maturation of bovine oocytes cultured in FF from dominant follicles 15-17mm in diameter (with various forms of heat pretreatment) and supplementation with follicular wall from follicles 3-5mm in diameter (FW1) were examined. Heat pretreatment of FF was as follows: (1) no treatment (FF1); (2) 56 degrees C for 30min (FF2); and (3) 100 degrees C for 20s (FF3). After IVM in FF1, oocytes underwent IVF and IVC and embryo development was assessed (up to the morula stage). The rate of oocyte maturation was decreased in pure FF1 versus control (44.5% versus 62.8%, P<0.001). In the control medium, FW1 did not significantly affect nuclear maturation. By contrast, the addition of FW1 to FF1 increased the rate of matured oocytes approximately two-fold (85.9% versus 45.6%, P<0.001). Furthermore, the maturation rate in the FF+FW1 system declined (from 85.9 to 71.0%, P<0.001), whereas that in the FF system increased (from 45.6 to 71.6%, P<0.001) with increased temperature of the FF treatment. Supplementation of the control medium with FW1 increased the yield of morulae (42.6% versus 13.7%, P<0.001). However, the stimulatory effect of FW1 on the morula rate was much higher in pure FF1 (72.5% versus 31.7%, P<0.001). These findings indicated, for the first time, the stimulatory impact of FW1 on in vitro maturation and early developmental capacity of bovine oocytes cultured in pure FF from dominant follicles. We also inferred that bovine FF constituents affecting bovine oocyte maturation and the meiosis-promoting ability of the FW were heat-labile.     

Influence of hyaluronan accumulation during cumulus expansion on in vitro porcine oocyte maturation

During oocyte maturation, the cumulus-oocyte complexes (COCs) expand dramatically. This phenomenon, which is known as cumulus expansion, is the result of the synthesis and accumulation of hyaluronan in the extracellular space between cumulus cells. The purpose of this study was to investigate the effect of 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of hyaluronan synthesis, on cumulus expansion during in vitro porcine oocyte maturation and hyaluronan accumulation within COCs. Further, this study aimed to examine the influence of hyaluronan accumulation within COCs on the rate of oocyte maturation. Cumulus expansion was observed during in vitro maturation. However, the addition of DON to the maturation medium significantly inhibited cumulus expansion. The total inhibition of hyaluronan accumulation within COCs was observed with the use of confocal microscopy. Moreover, a positive correlation between the area of cumulus expansion and the rate of oocyte maturation was observed. These results demonstrate that the hyaluronan accumulation within the COCs during oocyte maturation affects oocyte maturation. On the basis of these results, we propose that hyaluronan accumulation within the COCs during cumulus expansion is a necessary step in the porcine oocyte maturation process.     

Effects of epidermal growth factor and hormones on granulosa expansion and nuclear maturation of dog oocytes in vitro

Gonadotropins, steroids and growth factors stimulate or inhibit cumulus expansion, nuclear maturation, or both, of most mammalian oocytes in vitro. The objective was to evaluate the effects of epidermal growth factor (EGF) and various hormone combinations on in vitro granulosa/cumulus (G-C) expansion and nuclear maturation of domestic dog oocytes derived from advanced preantral and early antral follicles. Follicles were collected after enzymatic digestion of ovarian tissue and cultured for 66 h in F-12/DME with 20% fetal bovine serum, 2mM glutamine and 1% antibiotic-antimycotic (Control). Treatments comprised the following groups; each was cultured both with and without EGF (5 ng/mL): Control, FSH (0.5 microg/mL), LH (5 microg/mL), estradiol-17beta (E2, 1 microg/mL), FSH+LH, and FSH+LH+E2. Granulosa/cumulus expansion was scored on a scale of 0 (no expansion) to +3 (maximum expansion). The interaction between EGF and hormone treatment affected (P=0.011) maximum G-C expansion. With the exception of the E2 group, EGF increased (P<0.05) the proportion of oocytes exhibiting +3 expansion. The synergism of E2 with FSH+LH enhanced maximum G-C expansion; compared to all other treatments, the greatest expansion was observed in the FSH+LH+E2+EGF group (83.5+/-3.5%). When cultured in EGF alone, oocytes failed to reach metaphase I-II (MI-MII) stages. The interaction between EGF and hormone treatment tended (P=0.089) to increase the proportion of oocytes resuming or completing nuclear maturation (GVBD-MII). In addition, supplementing culture media with hormones increased (P=0.010) the GVBD-MII rate. Therefore, EGF in combination with FSH and LH enhanced G-C expansion of cultured canine oocytes, with no significant effect on the proportion of oocytes derived from advanced preantral and early antral follicles that reached MI-MII.     

Heparin effect on in vitro nuclear maturation of bovine oocytes

Oocytes undergo numerous biochemical and morphological changes during their development from preantral to preovulatory phases. In vitro studies have suggested several compounds that might induce oocyte maturation. Heparin is a natural component of ooplasm, follicular fluid and uterine fluid and previous studies indicated that it might act as a chromatin maturation factor in bovine oocytes. We tested this hypothesis in vitro by timing germinal vesicle breakdown (GVBD) and first polar body (PB) formation without any other natural or introduced factors that might influence the rate of oocyte maturation. We also determined if these oocytes could be fertilized. Bovine oocytes were incubated in a salt medium and TCM 199 supplemented with different concentrations of heparin for 24 h at 37.5 degrees C in a humidified atmosphere of 5% CO2. With 1.0 and 6.5 mg/ml heparin, the time of GVBD was reduced from 4.7+/-1.1 h to about 1.5 h and the time of first PB formation was reduced from 22.0+/-1.1 h to 9.0-11.0 h in salt medium. In TCM 199, only 6.5 mg/ml heparin significantly reduced the time of PB formation. In both incubation media, 1.0 and 6.5 mg/ml heparin induced GVBD, extrusion of the first PB and formation of the metaphase II nucleus. Moreover, heparin did not interfere with the fertilization of oocytes matured in TCM 199. Based on the results, we propose that heparin plays an important role in the rearrangement of the oocyte chromatin and acts as an oocyte maturation factor.     

Origin of bovine follicular fluid and its effect during in vitro maturation on the developmental competence of bovine oocytes

Protein supplementation during in vitro maturation can profoundly affect both the rate and overall efficiency of the maturation procedure. The present study was conducted to assess the ability of different concentrations (1, 5, and 10%) of bovine follicular fluid (bFF) to support in vitro maturation of oocytes and subsequent developmental capacity. The bFF was derived either from competent follicles ( > 8 mm) obtained by transvaginal recovery following superovulation or from a pool of small follicles (2-5 mm) from abbatoir-derived ovaries. Bovine oocytes were cultured for 24 h in synthetic oviduct fluid medium (m-SOF) supplemented with polyvinylpyrrolidone. Following fertilization and embryo culture, more oocytes (P < 0.05) reached the blastocyst stage when oocytes were cultured with 5% bFF from competent follicles (41 +/- 3.7%) compared with bFF derived from small follicles (16 +/- 2.9%). Estradiol and recombinant human follicle stimulating hormone added to the competent bFF during maturation acted in synergy to increase blastocyst production rate (P < 0.05); this blastocyst production rate (57 +/- 1.2%) was higher than those obtained with the addition of these two hormones to bFF derived from small follicles (26 +/- 2.9%). The quality of blastocysts obtained was reflected by inner cell mass (51.30 +/- 3.5 and 25.50 +/- 3.7) and trophectoderm cell numbers (99.72 +/- 2.5 and 94.80 +/- 4.7) for bFF from competent and small follicles, respectively. In conclusion, follicular fluid originating from competent follicles increased the developmental competence of abbatoir-derived oocytes.     

Low oxygen tension during in vitro maturation of porcine follicular oocytes improves parthenogenetic activation and subsequent development to the blastocyst stage

To establish a reliable in vitro maturation system for activation and subsequent development as nuclear recipients for the effective production of pig clones, we assessed maturation, activation and parthenogenetic development in response to the following: (1) type of immature oocytes (cumulus-oocyte complexes (COCs) or parietal granulosa plus cumulus-oocyte complexes (GCOCs)); (2) oxygen (O(2)) tension (5 or 20%); and (3) maturation period (36-60 h). The rate of nuclear maturation to metaphase-II (M-II) in the GCOC group (73.0 +/- 3.1%) was higher than that in the COC group (P < 0.05, 60.6 +/- 3.5%), but the rates did not differ between the 5 and 20% O(2) tension groups. M-II rate increased (P < 0.05) to about 70% after 42 h and then remained constant until 60 h of culture. When oocytes were matured under 5% O(2) tension and stimulated, the rate of normal oocyte activation (a female pronucleus formation and emission of the second polar body) was higher (P < 0.05, 38.5 +/- 3.9%) than when oocytes were matured under 20% O(2) tension (24.5 +/- 3.9%). On the other hand, the rate of normal activation was not significantly different between the COC and GCOC groups, and the highest (P < 0.05) normal activation rate was obtained in oocytes cultured for 48 and 54 h (48.4 +/- 5.5% and 47.9 +/- 8.2%, respectively). When COC and GCOC matured for 48 h under 5 and 20% O(2) tension were stimulated and subsequently cultured in vitro for 6 days, the rate of blastocyst formation did not differ between the oocyte types nor between the O(2) tension groups. However, blastocyst quality, as measured by mean total cell number, was significantly higher in the 5% O(2) group (P < 0.05, 34.6 +/- 2.0 for COC; 33.8 +/- 1.8 for GCOC) compared with the 20% O(2) group (25.9 +/- 1.8 for COC; 27.0 +/- 2.0 for GCOC). In conclusion, low O(2) tension (5%) during in vitro maturation of porcine oocytes promoted their ability to be activated normally and improved the quality of parthenogenetic blastocysts developed in vitro in modified NCSU-37 solutions. This knowledge may be applicable for preparation of in vitro matured oocytes with good quality as recipient oocytes for generating pig clones.