Effects of canine serum collected from dogs at different estrous cycle stages on in vitro nuclear maturation of canine oocytes

Canine oocytes are ovulated at prophase of the first meiotic division and undergo maturation in the distal part of the oviduct for at least 48-72 h. Because of these differences from other domestic mammals, the efficiency of in vitro maturation (IVM) of canine oocyte is very low. The present study was conducted to evaluate the effects of canine serum on IVM of canine oocytes recovered from ovaries in various reproductive states (follicular, luteal or anestrous stages). Oocytes were recovered by mincing ovaries from bitches presented for ovariohysterectomy at various stages of the estrous cycle. Heat-inactivated canine serum was prepared with blood taken from dogs at the anestrous, estrous or diestrous stage of the estrous cycle as determined by progesterone concentration and vaginal cytology. Oocytes were cultured for 72 h in tissue culture medium (TCM)-199 supplemented with 10% canine anestrous, estrous or diestrous serum or fetal bovine serum (FBS) (experiment 1), or supplemented with 0 (control), 5%, 10% or 20% canine estrous serum (experiment 2). In experiment 1, IVM of oocytes collected at the follicular stage of the estrous cycle to metaphase II (MII) stage was higher (p < 0.05) with canine estrous serum (14.2%) than with canine anestrous (5.2%) or diestrous serum (6.3%), FBS (2.2%) or in the control (2.2%). In experiment 2, oocytes collected at the follicular stage of the estrous cycle cultured in TCM-199 with 10% canine estrous serum showed a higher maturation rate to MII stage (13.5%, p < 0.05) compared with those cultured with 5% (1.3% MII) or 20% canine estrous serum (5.1% MII) or the control (2.7% MII). In conclusion, our results demonstrate that supplementing culture medium with 10% canine estrous serum improves IVM of canine follicular stage oocytes.     

In vitro maturation and fertilization of buffalo oocytes (Bubalus bubalis ): Effects of media, hormones and sera

Media (TCM-199 and Ham's F-10); sera (fetal calf serum, FCS, and buffalo estrous serum, BES); and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol 1 ug/ml) were tested to determine the efficiency of in vitro maturation and fertilization of buffalo follicular oocytes. Immature good quality cumulus-oocyte complexes (COCs) were randomly assigned to 1 of 4 experiments. Each experiment consisted of 6 treatment groups. Oocytes cultured for 24 hours in medium (TCM-199 or Ham's F-10) containing 10% FCS or BES had a significantly higher maturation rate than those in medium alone (P < 0.05). However, the maturation rate was higher in medium supplemented with 10% FCS than with 10% BES. Addition of hormones alone or in combination with sera further improved the maturation rate, but no significant difference was observed in the maturation rate among the 3 hormone-treated groups. Immature oocytes matured in the various cultures were fertilized with frozen-thawed buffalo spermatozoa. Our findings show that hormone and/or serum supplementation of TCM-199 did not improve the fertilization rate. Supplementation of Ham's F-10 with LH alone or in combination with LH + FSH + E(2) and with FCS significantly improved the fertilization rate of oocytes while medium with FSH, E(2) or no hormones did not (P < 0.05); same media supplemented with BES resulted in lower fertilization rates both in the presence or absence of hormones. The results indicate that the culture medium has a marked effect on the fertilization rate of buffalo oocytes. Ham's F-10 + LH + FSH + E(2) supplemented with FCS was the most efficacious culture system of those studied for the in vitro maturation of buffalo oocytes.     

The influence of different types of media supplement on the meiotic maturation of bovine oocytes in vitro

This work was designed to evaluate the influence of different types of media supplements in the maturation of preovulatory oocytes in cattle. We studied 4 different supplements: serum from estrous cattle (ECS), serum from fetal calves (FCS), amniotic fluid from cattle (bAF) and follicular fluid from cattle (bFF). Preovulatory bovine oocytes recovered from ovaries of mature cows after slaughter were cultured for 24 h in TCM-199 medium containing 20% of one of these protein supplements. The percentages of oocyte maturation were significantly higher (P < 0.001) in ECS (78%), FCS (79%) and bAF (77%) than in bFF (52%). These data indicate that supplementation with ECS, FCS or bFF is adequate for maturation of bovine oocytes without addition of other compounds.     

In vitro fertilization of water buffalo follicular oocytes and their ability to cleave in vitro

Water buffalo (Murrah) oocytes were collected from ovaries obtained from a local slaughterhouse. They were classified according to the character of the cumulus cells under a stereomicroscope and then cultured in 25 mM Hepes buffered tissue culture medium-199 (TCM-199) supplemented with 5% estrous water buffalo serum in an atmosphere containing 5% CO(2) in air at 39 degrees C. After 20 to 24 hours of in vitro maturation, the oocytes were cultured at 38.5 degrees C in TCM-199 supplemented with 1% estrous water buffalo serum and in an atmosphere containing 5% CO(2) in air. Oocytes with compact and dense cumulus cells cleaved significantly further (P<0.01, 67.3%, 33 49 ) than those with fair, partially denuded oocytes with thin cumulus layers (27.5%, 25 91 ) or small remnants of cumulus cells and poor naked oocytes (3 100 ). A substantial variation in fertilization and developmental rates (16.0 to 43.8%) was observed among 4 different bulls. Late morulae were transferred nonsurgically into 14 buffalo recipients on Day 6 or 7 of their estrous cycle. One recipient was diagnosed to be pregnant by palpation per rectum on Day 60 and delivered a calf in October 1991.     

Comparison of various maturation treatments on in vitro maturation of goat oocytes and their early embryonic development and cell numbers

In the present study, comparison of 2 different culture media (Ham's F-12 and M-199) for supporting in vitro maturation of goat oocytes, and their subsequent embryonic development was evaluated in the presence or absence of sera (estrous goat serum, EGS and fetal calf serum, FCS) and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol, 1 ug/ml). Neither medium (Ham's F-12 or M-199) when supplemented with EGS and hormones showed any notable changes in the maturation rate nor in cleavage and blastocyst development. The mean cell number for blastocysts was also significantly low (P < 0.05). However, Ham's F-12 medium supplemented with FCS and hormones showed a considerable increase in the maturation rate, but subsequent embryonic development was not appreciably increased. However, maturation, cleavage and blastocyst development rates of oocytes matured in M-199 medium in combination with 10% FCS and hormones were significantly higher (P < 0.05). Mean cell number per blastocyst was also significantly increased in this latter treatment compared with that of the other groups (P < 0.05). The results thus indicated that both the culture medium and serum have a marked effect on maturation and subsequent embryonic development. Further, the results also showed that the combination of M-199 with FSH, LH and E2 supplemented with 10% FCS was the most efficacious medium for in vitro maturation and subsequent embryonic development of the media, sera and hormone combinations studied.     

In vitro production of a caprine embryo from a preantral follicle cultured in media supplemented with growth hormone

The objective was to evaluate the effects of growth hormone (GH) on the survival, growth, maturation, and fertilization of oocytes derived from caprine preantral ovarian follicles cultured in vitro. Preantral follicles were isolated from the cortex of caprine ovaries and individually cultured for 18 d in the absence (control) or presence of bovine GH at concentrations of 10 or 50 ng/mL (GH10 and GH50, respectively). Follicle development was evaluated on the basis of survival, antral cavity formation, diameter increase, and the presence of healthy cumulus-oocyte complexes and mature oocytes. After culture, oocytes were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of antrum formation after Day 6 of culture was higher in both GH10 and GH50 than in the control (81.0, 92.7, and 47.6%, respectively, P < 0.05). Percentages of grown oocytes that were acceptable for IVM were also higher (P < 0.05) in GH-treated groups than in the control (54.8, 48.8, and 11.9% for GH10, GH50, and Control). A higher percentage of oocytes in the GH50 treatment underwent meiotic resumption (50.0%), produced mature oocytes, and enabled production of an embryo after IVF than in the control group (0.0%; P < 0.05). In conclusion, GH promoted in vitro growth and maturation of goat preantral follicle oocytes and enabled production of an embryo. Furthermore, this study was apparently the first to produce a caprine embryo by in vitro fertilization of oocytes derived from preantral follicles grown in vitro.     

In vitro fertilization and cleavage of bovine oocytes matured in medium supplemented with estrous cow serum

Bovine follicular oocytes were matured in TCM 199 supplemented with: (1) fetal calf serum (FCS, 20% v/v), Luteinizing Hormone (LH, 10 ug/ml), and Estradiol-17-beta (E(2), 1 ug/ml) in Experiment 1; (2) 20% cow serum recovered at standing estrus (Experiment 2); or (3) 20% FCS (Experiment 3). Maturation, fertilization, and initial cleavage development were evaluated at 16 and 48 h after in vitro insemination. The proportions of oocytes fertilized after maturation in the presence of added hormones (78.5%, Experiment 1) or estrous serum (71.3%, Experiment 2) were significantly higher (p < 0.01) than after use of FCS alone (39.3%, Experiment 3). Cleavage of zygotes within 48 h post-insemination differed significantly (p < 0.01) between maturation treatments, 27.3%, 75.5% and 6.6% for Experiments 1, 2, and 3, respectively. Results demonstrate a beneficial influence of estrous cow serum, characterized by an elevated concentration of LH, on bovine oocyte maturation in vitro.     

In vitro maturation and fertilization of equine oocytes recovered during the breeding season

The aim of this study was to develope an efficient and reproducible procedure for in vitro maturation (IVM) and fertilization (IVF) in the horse. Cumulus-oocyte complexes (COCs) recovered from the ovaries of mares slaughtered during the breeding season were morphologically evaluated, and those showing a compact cumulus and homogeneously appearing cytoplasm were selected for culture. Effects on the maturation of estrous mare serum (EMS) versus estrous cow serum (ECS) as medium supplement were also evaluated (Experiment 1). In Experiment 2, the fertilization of in vitro matured oocytes with frozen-thawed semen separated by swim-up and treated with heparin was carried out to determine the effects on fertilization of 1) increasing sperm concentrations (1x 10(6), 5 x 10(6) and 1 x 10(7)sperm cells/ml), 2) IVM medium supplementation with EMS or ECS and 3) partial cumulus mass removal before insemination. Forty-nine percent of the collected oocytes (335 683 ) showed a compact cumulus and homogeneous ooplasm and thus were selected for culture. In Experiment 1, high nuclear maturation rates were observed in both EMS (82%,32 39 ) and ECS (87.5%,56 64 ) groups, with no statistically significant difference. In Experiment 2, the percentage of normal fertilization (2 polar bodies, 2 pronuclei and sperm tail) was similar for all 3 tested sperm concentrations (12.9%,4 31 ; 15.2%,9 59 and 15.5%,9 58 ). No advantage in using the homologous serum in IVM medium was noted in terms of fertilization (12.2%, 5 41 with EMS vs 12.9%, 4 31 for ECS). However, significantly higher fertilization rates were obtained after partial cumulus removal compared with that of oocytes fertilized with a whole cumulus (32.6%, 14 43 vs 12.2%, 5 41 ; P < 0.05). The incidence of polyspermic fertilization was low under all culture conditions (0 to 2.4%). In a replicate in which the oocytes fertilized after the cumulus removal were further cultured for 72 h two embryos, one at the 2-cell stage and the other at the 4-cell stage, could be obtained. These results indicate that, in the horse, the cumulus can be partially removed to increase the fertilization of compact-cumulus oocytes recovered during the breeding season using frozen-thawed, heparin-treated semen.     

Intracellular adenosine triphosphate and glutathione concentrations in oocytes from first estrous,multi-estrous,and testosterone-treated gilts

Cytoplasmic maturation refers to a variety of cellular changes that must occur in the oocyte in order to progress through subsequent fertilization and embryonic development. Intracellular concentrations of ATP (ATPi) or glutathione (GSHi), indicative of metabolic activity or the ability of the oocyte to form a male pronucleus and cope with cellular stress, respectively, have been used as markers of cytoplasmic maturation in vitro. In the current study, our objective was to determine if concentrations of ATPi and GSHi in oocytes recovered from three groups of gilts were associated with known differences in developmental competence within these populations. In vivo matured oocytes were surgically recovered 36-38 h after the onset of estrus from first estrous gilts, multi-estrous gilts, and multi-estrous gilts receiving testosterone (1mg/2 ml per day; day 13 to estrus, day 0=day of estrus). Concentrations of ATPi and GSHi were determined using a bioluminescent somatic cell assay kit (luciferin-luciferase reaction) and the dithiobisnitrobenzoic acid (DTNB)-glutathione reductase recycling reaction, respectively. There were no differences (P>0.05) between ATPi concentrations in oocytes from the three groups (1.52 +/- 0.10, 1.51 +/- 0.11, 1.56 +/- 0.11pmol per oocyte). In contrast, oocytes from multi-estrous gilts had higher (P<0.05) concentrations of GSHi (31.53 +/- 1.66 to 33.67 +/- 2.30 pmol per oocyte) than oocytes from first estrous gilts (25.07 +/- 0.82). Administration of testosterone did not affect (P>0.05) GSHi concentrations in oocytes from multi-estrous gilts. Differences in developmental potential between the three groups of gilts were apparently not due to different concentrations of ATPi. However, GSHi concentrations were higher in oocytes from multi-estrous gilts, suggesting that reduced developmental potential of oocytes from first-estrus gilts may be related to insufficient amounts of GSHi. The beneficial effect of exogenous testosterone on the percentage of embryos surviving early gestation does not appear to be due to increased GSHi. Of the numerous potential markers of developmental potential, two were examined in the current study, and GSHi appeared to be useful for assessing porcine oocytes.     

Influence of reproductive status on in vitro oocyte maturation in dogs

In the bitch, oocytes need 48-72 h to complete post-ovulatory maturation to the metaphase II stage in the isthmus of the oviduct, an interval similar to that found in in vitro studies. The effect of estrous cycle stage on in vitro meiotic competence of dog oocytes has been described in several studies. However, there are no reports evaluating the possible effects of pyometra or pregnancy on subsequent potential of oocytes recovered from such females to undergo in vitro maturation.In this study, immature cumulus-oocyte complexes (COCs) were recovered from fresh excised domestic dog ovaries in various reproductive states. The donor females were classified into groups based on stage of the estrous cycle: follicular (proestrus or estrus), luteal (diestrus) or anestrus or at the clinical conditions of pregnancy and pyometra. Grades 1 and 2 oocytes were cultured in vitro at 37 degrees C in TCM-199, supplemented with 25 mM Hepes/l (v/v), and with 10% heat inactived estrous cow serum (ECS), 50 microg/ml gentamicin, 2.2 mg/ml sodium carbonate, 22 microg/ml pyruvic acid, 1.0 microg/ml estradiol, 0.5 microg/ml FSH and 0.03 IU/ml hCG. The nuclear maturation rate was evaluated at 72 h of incubation under Hoechst 33342 (10 microg/ml) staining for fluorescence microscopy. There was no statistical difference in nuclear progression to the MII stage among the various reproductive states (follicular phase, 5.4%; diestrus, 4.2%; anestrus, 4.4%; pyometra, 8.1% and pregnancy, 4.7%). Resumption of meiosis was 24.6% at the follicular phase, 19.6% for diestrus, 16.4% for anestrus, 37.1% for pyometra and 29.2% for pregnancy. Positive and higher numbers of residue above the expected value were observed for the pyometra and pregnancy conditions at the metaphase/anaphase I (MI/AI) stages.Our results indicate that in vitro nuclear maturation of dogs oocytes is not influenced by the in vivo reproductive status of the female. The quality of the oocyte is a more reliable indicator of its potential for meiotic maturation in vitro than the hormonal environment of the donor female at the time of oocyte retrieval.     

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.     

Metabolism, protein content, and in vitro embryonic development of goat cumulus-oocyte complexes matured with physiological concentrations of glucose and L-lactate

No information is available concerning how the maturation environment controls the metabolism of goat oocytes. The objectives of this experiment were to: (1) Determine the concentrations of glucose, lactate, and pyruvate in caprine follicular fluid; and (2) Investigate the effects of physiological concentrations of glucose and lactate in the in vitro maturation (IVM) medium on the metabolism (glycolysis and pyruvate oxidation), protein content, and developmental competence of caprine oocytes and cumulus-oocyte complexes (COCs). Abattoir-derived COCs were matured for 18-20 hr in a defined, SOF-based medium containing 0.75, 1.5 (follicular fluid = 1.4 mM), or 3.0 mM glucose, and 3.0, 6.0 (follicular fluid = 7.1 mM), or 12.0 mM L-lactate. The protein content of oocytes and COCs was not affected (P > 0.05) by the concentration of glucose and lactate in the maturation medium. Increasing glucose and lactate decreased (P < or = 0.05) glycolytic activity of oocytes, without affecting (P > 0.05) pyruvate oxidation. In COCs, increasing glucose concentrations tended (P = 0.07) to decrease glycolysis. When metabolic activity was corrected for protein content (pmol/microg protein/3 hr), increasing glucose or lactate concentrations in the medium decreased (P < or = 0.05) pyruvate oxidation in oocytes, but increased (P < or = 0.05) pyruvate oxidation in COCs. Embryonic development (cleavage and blastocyst development, hatching, and cell number) was not affected (P > 0.05) by the glucose and lactate concentrations tested. These results indicate that concentrations of glucose and lactate in the medium have cell type-specific effects on metabolism of oocytes and COCs, but do not affect developmental competence within the range of concentrations tested.     

Effects of estradiol-17beta and progesterone supplementation on in vitro nuclear maturation of canine oocytes

Unlike in other domestic animals, in vitro maturation (IVM) of canine oocytes has had limited success. The present study investigated the effect of the estrous cycle and estradiol-17beta (E2) or progesterone (P4) supplementation on in vitro nuclear maturation of canine oocytes recovered from domestic dog ovaries in various reproductive states (follicular, luteal or anestrous stages). Oocytes were cultured in serum-free tissue culture medium (TCM)-199 supplemented with various concentrations of E2 (Exp. 1: 0, 0.1, 1.0 or 2.0 microg/ml) or P4 (Exp. 2; 0, 0.5, 1.0 or 2.0 microg/ml) for 72 h to determine the effective concentration of hormones. In Exp. 3, in order to investigate the synergistic effect of E2 and P4 supplementation, three groups of oocytes were cultured with 2 microg/ml E2 plus various concentrations of P4 (0, 0.5, 1.0 or 2.0 microg/ml). As results, the rate of maturation to metaphase II (MII) stage was significantly higher (P < 0.05) in oocytes from the follicular stage supplemented with 2 microg/ml E2 (14.7%) compared to the other groups (1.5-8.2%). Significantly higher (P < 0.05) maturation rate to MII stage was observed in oocytes from the follicular stage supplemented with 1.0 (10.0%) or 2.0 microg/ml (10.8%) P4 compared to the other groups (0-4.8%). Furthermore, more (P < 0.05) oocytes from the follicular stage supplemented with 2.0 microg/ml of E2 and P4 (16.6%) were matured to MII stage compared to oocytes from the follicular stage supplemented with 2.0 microg/ml E2 alone (10.4%) or the other groups of oocytes (0-7.8%). Interestingly, compared to 2.0 microg/ml E2 alone (10.4%), supplementation of 2 microg/ml E2 + 0.5 microg/ml P4 (3.4%) decreased the maturation of oocytes from the follicular stage to MII stage. In conclusion, the present study demonstrated that supplementation of the culture medium with E2 or P4 alone significantly increased maturation of canine oocyte to MII and that P4 supplementation with E2 further promote or decrease oocyte maturation compared to E2 alone depending on P4 concentration.     

Effect of Actin Polymerization Inhibitor During Oocyte Maturation on Parthenogenetic Embryo Development and Ploidy in Capra hircus

This study was designed to observe the effect of cytochalasin B (CCB) concentrations on ploidy and early development of parthenogenetic embryos in a caprine species. Caprine oocytes were matured in the presence of different concentrations of CCB (5, 10, 15, and 20 μg/ml) and activated by 7% ethanol followed by incubation with 2 mM DMAP. For embryos fertilized in vitro, oocytes were matured in maturation medium without CCB. The cleavage rate and further embryo development were significantly higher (P < 0.05) when oocytes were treated in this way. The percentage of embryos showed higher diploid values in 15 μg/ml CCB (83.66 ± 1.13), followed by 20 (72.22 ± 1.22), 10 (68.57 ± 1.17), and 5 μg/ml (62.00 ± 2.48). These results indicate that CCB with a concentration of 15 μg/ml in maturation medium can be used for the production of diploid parthenogenetic embryos in the caprine species.     

Glutathione content and embryo development after in vitro fertilisation of pig oocytes matured in the presence of a thiol compound and various concentrations of cysteine.

The present study examined the effect of different concentrations of cysteine in the presence of a thiol compound, beta-mercaptoethanol (BME), during in vitro maturation (IVM) of pig oocytes on cumulus expansion, nuclear maturation, intracellular glutathione (GSH) level and subsequent embryonic development after in vitro fertilisation (IVF). In experiment 1, oocytes were matured in NCSU 23 medium containing 10% porcine follicular fluid, 25 microM BME, 0.5 microgram/ml LH, 0.5 microgram/ml FSH and 0, 0.1, 0.2 or 0.4 mg/ml cysteine for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, cumulus cells were removed and a proportion of oocytes fixed to examine the rate of nuclear maturation. The remaining oocytes were co-incubated with spermatozoa for 5-6 h and putative zygotes were transferred to NCSU 23 medium containing 0.4% bovine serum albumin for 144 h. A proportion of putative zygotes were fixed 12 h after insemination to examine fertilisation parameters. In experiment 2, oocytes were matured as in experiment 1 and the GSH content was measured by a DTNB-GSSG reductase recycling assay. No mean differences among treatments were observed in nuclear maturation (78-89%). The mean differences in penetration rate (69-77%), polyspermy rate (31-40%), male pronuclear formation rate (93-96%) or mean number of sperm per oocyte (1.5-1.8) were not affected by the presence or absence of cysteine during oocyte maturation. Also no difference was observed in cleavage rates 48 h after insemination. However, compared with no addition (19%), the presence of 0.1-0.4 mg/ml cysteine during IVM increased (p < 0.001) the proportion of blastocysts (32-39%) at 144 h. In comparison with controls (5.6 pmol/oocyte), the GSH content of oocytes matured in the presence of cysteine was significantly (p < 0.001) higher (13-15 pmol/oocyte) with no mean differences among different cysteine concentrations. The results indicate that in the presence of a thiol compound, supplementation of IVM medium with cysteine can increase the GSH level and improve the developmental competence of pig oocytes following fertilisation. Further, no effect on either GSH level or embryo development was observed by increasing the levels of cysteine supplementation from 0.1 to 0.4 mg/ml.     

Effect of maturation medium on in vitro cleavage of canine oocytes fertilized with fresh and cooled homologous semen

This study evaluated the effect of three maturation media on the development of in vitro-matured and in vitro-fertilized dog oocytes. In Experiment 1 (non-comparative experiment) canine cumulus-oocyte complexes (COCs) were matured in vitro in TCM199 supplemented with estrous cow serum (10%) + gonadotropins + steroid (treatment A), TCM199 + estrous cow serum (10%) (treatment B), or TCM199 + polyvinylpyrrolidone (PVP) (4%) (treatment C). All maturation media contained a final concentration of 1 microg/ml of human somatotropin (hST). Oocytes were fertilized with fresh ejaculated sperm and development was assessed by cleavage. The objective of Experiment 2 (comparative experiment) was to compare the rates of cleavage and developmental capacity of COCs matured in vitro in same medium as in Experiment 1, and fertilized either with fresh ejaculated or with cooled extended homologous spermatozoa. In Experiments 1 and 2, oocytes fertilized with fresh semen were in vitro-matured for 48 h, while in Experiment 2 COCs fertilized with cooled semen were matured in vitro for 72 h. The results of Experiments 1 and 2 demonstrated that cleavage was not influenced by the oocyte's maturation environment. The results of Experiment 1 showed that pronucleus formation + cleavage (day 7 after IVF) was similar among treatments A, B and C (p = 0.277). Also, in Experiment 2, pronucleus formation + cleavage (day 7 after IVF) was not different for oocytes fertilized in vitro either with fresh or cooled semen and maturated in media A (p = 0.190), B (p = 0.393) or C (p = 0.687). In both experiments, the numbers of embryos that developed to the 6-8-cell stage were higher for oocytes matured in medium A and fertilized with fresh semen, when compared with numbers of oocytes matured in media B and C. Embryo development to the 6-8-cell stage of oocytes fertilized either with fresh or cooled sperm was observed in treatments A and C in Experiment 2. Cumulus cell expansion was similar among treatments in Experiment 1. In Experiment 2, cumulus cell expansion among treatments A, B and C was similar after 48 h or 72 h of IVM. In both experiments, the greatest expansion category seen was for category 2 (outer cumulus cells slightly expanded). No correlation between cumulus expansion and cleavage were observed. Polyspermy rates in oocytes matured in medium A, and fertilized with fresh sperm were not significantly different from polyspermy rates observed using media B and C, in both experiments. Our findings indicate that treatments A, B and C are similarly effective for the cleavage of dog oocytes. Furthermore, it was demonstrated that canine oocytes matured in vitro could be fertilized by homologous cooled spermatozoa and progress to cleavage.     

Evidence for the Presence of a Low Molecular Weight Factor in Sera from Various Animals that Induces in vitro Maturation of Fish Oocytes

Studies were made on whether sera from various animals or serum components could induce in vitro maturation of medaka oocytes in the absence of any exogenous hormone. When intra-follicular oocytes were preincubated in medium containing serum of some mammals or chickens, and were then washed, they matured during further incubation in the same medium without the serum. In contrast, no remarkable stimulatory effect on oocyte maturation was recognized in sera from some other mammals and from reptiles, amphibian and fish. A dialysate of rabbit serum collected using a cellulose membrane (mol. wt. retention 3,500) also induced in vitro maturation of intra-follicular oocytes. This activity was stable on treatment with proteolytic enzymes or heat-treatment. Small molecular weight fractions with this activity were obtained by filtering the dialysate through Sephadex G-10. The oocytes matured by the serum factor were capable of developing normally beyond the stage of embryonic body formation.
From these results, it is concluded that the serum of higher vertebrates contains a low molecular weight factor that stimulates in vitro nuclear and cytoplasmic maturation of intrafollicular fish oocytes.     

In vitro maturation of porcine oocytes using a defined medium and developmental capacity after intracytoplasmic sperm injection

To establish a defined in vitro maturation culture system for porcine oocytes, we examined the effects of adding cysteine (Cys) and epidermal growth factor (EGF) to the maturation medium. Furthermore, to evaluate cytoplasmic maturation, we investigated GSH concentrations and embryo development after intracytoplasmic sperm injection (ICSI). The basic media for IVM were modified TCM199 containing 10% newborn calf serum (NBCS) or 0.1% polyvinyl alcohol (PVA), supplemented with amino acids. Adding EGF (10 ng/ml) or EGF + Cys (0.57 mM) to the defined medium (0.1% PVA + amino acids) increased (P < 0.05) the rate of nuclear maturation relative to the defined medium (without these additives). After ICSI, oocytes matured in a medium supplemented with NBCS, Cys and EGF had a higher (P < 0.05) rate of pronuclear formation rate than oocytes matured in the defined IVM medium. Although there was no significant difference in cleavage rates between NBCS- and PVA-containing media supplemented with both Cys and EGF, the rate of blastocyst development was lower (P < 0.05) in the defined medium than in the NBCS-containing medium. Intracellular GSH concentrations of oocytes matured in the NBCS- and PVA-containing media supplemented with both Cys and EGF were higher (P < 0.05) than in oocytes matured in PVA alone or in oocytes before maturation. Adding Cys and EGF to a defined medium for porcine IVM improved rates of nuclear maturation and cleaved oocytes following ICSI, probably due to increased GSH concentrations. Also, embryos derived from oocytes matured in the defined medium (with the addition of Cys and EGF) developed into blastocysts after ICSI.     

The incidence of bovine diploid oocytes matured in vitro

The present work describes a cytogenetic study of in vitro matured bovine oocytes to determine the proportion of unreduced oocytes carrying the diploid number of chromosomes. Studied oocytes were derived either from a pool of oocytes collected from several different donors, or from oocytes collected separately from individual donors. In vitro maturation was performed by culturing immature oocytes for 24 h in TCM199-medium supplemented with estrous cow serum and hormones at 39 degrees C in 5% CO2. Chromosomal complement of in vitro matured oocytes was studied by Giemsa-staining and produced analyzable results in approximately 60% of the cases. The results revealed that approximately 75% of oocytes had matured to the MII stage in both groups of oocytes studied. Of these MII oocytes, 11 and 12.4% (from the oocyte pool or from individual cows, respectively) contained the diploid set of chromosomes. The occurrence of diploid MII oocytes was not quite uniform among donors: 40.5% of all cows produced one, 18.9% produced two and 2.7% (one cow) produced three diploid MII oocytes. However, a positive relationship between the number of MII oocytes in general and diploid MII oocytes among individual donors was not found. The possible factors that may lead to the formation of diploid MII oocytes observed under IVM procedures are discussed. The results of this study showed a higher incidence of diploid oocytes in cattle than previously reported.     

Efficiency of in vitro fertilization is influenced by the meiotic competence of porcine oocytes and time of their maturation

The effect of meiotic competence of oocytes and time of their maturation on the efficiency of fertilization was studied in pigs. Cycling gilts with synchronized estrous cycles were used as oocyte donors. To obtain oocytes with different meiotic competence, oocytes were recovered separately from small and medium follicles in the early, middle and late luteal or early follicular phase. They were matured for 40 h, 43 h or 47 h and fertilized by spermatozoa of a proven boar. The penetration and monospermy rates, and total efficiency of fertilization were assessed. The same data were related to the follicle size, with or without regard to the phase, and to the maturation time. Regardless of the phase and the time of maturation, the monospermy rate and total efficiency of fertilization were significantly lower for the small follicle-derived oocytes than for the medium follicle-derived oocytes (38.5±10.4% vs 63.1±7.0% and 24.7±6.3% vs 42.5±3.8%). With regard to the phase, in the small follicle-derived oocytes, the monospermy rate increased significantly (P<0.05) from the early luteal to the late luteal phase (from 25.4±2.4% to 46.4±3.9%) and remained unchanged in the early follicular phase. A similar tendency was observed in the total efficiency of fertilization. No differences were found in either of these parameters in medium follicle-derived oocytes in the late luteal and early follicular phase. With regard to the time of maturation, the total efficiency of fertilization was significantly higher (P<0.05) in the small follicle-derived oocytes matured for 47 h than in those matured for 40 h (27.7±7.4% vs. 20.5±6.1%) and in the medium follicle-derived oocytes matured for 40 h as compared with those matured for 47 h (47.1±1.9% vs. 32.7±1.1%). With regard to the phase and the time of maturation, the differences were significant only in the late luteal and early follicular phases. It can be concluded that greater meiotic competence of porcine oocytes positively influences monospermy rate and total efficiency of fertilization process. However adequate time of maturation is an important factor for oocytes with different meiotic competence to improve the IVF procedure.