Effects of follicle-stimulating hormone and ovarian steroids during vitro meiotic maturation on fertilization of rat oocytes

The present study examined the effects of gonadotropins and ovarian steroids during in vitro meiotic maturation of rat oocytes on their ability to undergo in vitro fertilization. Fully grown oocytes were isolated from antral follicles of immature rats and cultured as oocyte-cumulus cell complexes (OCC) under conditions in which completion of meiotic maturation occurs spontaneously. They were then exposed to spermatozoa under conditions in which oocytes matured in vivo exhibit high fertilization rates. Compared with oocytes from pregnant mare's serum gonadotropin (PMSG) or follicle-stimulating hormone (FSH)-treated rats, a simiiar proportion of the oocytes (>80%) from untreated rats underwent germinal vesicle breakdown, but such oocytes had a lower rate of fertilization (70% vs. 20%). The presence of FSH during in vitro maturation restored the fertilization rate for oocytes from untreated rats, while a cytochrome P450 inhibitor, aminoglutethimide phosphate abolished this beneficial effect of FSH. The addition of progesterone during the in vitro maturation period duplicated the beneficial effect of FSH on fertilization rate of oocytes from untreated rats; oestradiol-17β was less effective in this regard, and 5α-dihydrotestosterone was ineffective. These findings indicate that FSH and progesterone, although having no apparent effect on nuclear maturation of the oocyte, play an important role during oocyte maturation in enabling normal fertilization to occur.     

In vitro fertilization of horse follicular oocytes matured in vitro

In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 micrograms/ml heparin for 4h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 microM calcium ionophore A 23187 for 5-10 min followed by washing. After a given pretreatment, suspensions were diluted into B2 medium to a concentration of 5 x 10(6) sperm/ml and co-incubated with oocytes for 12 h or 24-48 h. In the ionophore-treated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 11 of 45 (24%) cleaved by 24-48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures.     

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.     

Developmental competence of domestic cat follicular oocytes after fertilization in vitro

Empirical evaluation of variables affecting oocyte collection, in vitro fertilization, and embryo transfer resulted in establishing a successful procedure for the artificial production of offspring in the domestic cat. Female cats were treated with pregnant mare's serum gonadotropin (PMSG, 150 IU) followed 72 or 80 h later with 100 or 200 IU human chorionic gonadotropin (hCG). After laparoscopic collection, follicular oocytes were inseminated in vitro with ejaculated, processed spermatozoa, cultured (37 degrees C, 5% CO2), and then examined for evidence of fertilization. Two- to 4-cell stage embryos were transferred to the oviducts of oocyte donors. Oocyte donor cats and naturally mated controls also were subjected to sequential laparoscopic examinations and blood sampling to assess corpora lutea (CL) function. At 24-30 h of culture, fewer (p less than 0.001) degenerate oocytes were observed in cats receiving 100 IU hCG (8.2%) compared to those receiving 200 IU (20.6%), regardless of the PMSG-hCG interval. Overall fertilization (48.1%) and cleavage (45.2%, at 30 h post-insemination) rates were greatest following an 80-h PMSG-hCG interval combined with the 100 IU hCG dose. Five of the 6 cats receiving 6 to 18 embryos became pregnant and produced from 1 to 4 kittens/litter. Gonadotropin-treated females subjected to follicular aspiration produced morphologically normal CL and circulating progesterone patterns that were qualitatively similar (p greater than 0.05) to control cats. These data indicate that domestic cat follicular oocytes are capable of fertilization in vitro, but success is dependent on both the timing and dose of the hCG stimulus. Follicles subjected to aspiration appear capable of forming normal, functional CL and the birth of live young after embryo transfer unequivocally demonstrates, for the first time, the developmental competence of in vitro-fertilized carnivore oocytes.     

Cholinergic modulation of progesterone-induced maturation of Xenopus oocytes in vitro

Mixed and muscarinic cholinergic agonists (acetylcholine, carbamylcholine, methacholine, oxotremorine, and pilocarpine) accelerated in a dose-dependent manner the progesterone-induced maturation of Xenopus laevis oocytes. None of these agonists induced oocyte maturation in the absence of progesterone. The accelerating effect of cholinergic agonists was blocked in a dose-dependent manner by specific muscarinic antagonists (atropine and scopolamine) but not by specific nicotinic antagonists (d-tubocurarine and hexamethonium). The specific nicotinic agonist, dimethylphenylpiperazine, alone induced maturation in the absence of progesterone. The optimal promoting effect of acetylcholine was observed when oocytes were exposed to acetylcholine for 30 min, 5 min after the addition of progesterone, and was markedly better than when oocytes were exposed to acetylcholine throughout their incubation with progesterone. The effect of acetylcholine was observed in both follicle-enclosed and in defolliculated oocytes, indicating that follicular cells were not the target of the cholinergic drugs.     

Ca(2+) response to cADPr during maturation and fertilization of starfish oocytes.

During the reinitiation of the meiotic cycle (maturation) induced by the hormone 1-methyladenine (1-MA), starfish oocytes undergo structural and biochemical changes in preparation for successful fertilization. Previous work has shown that the sensitivity of internal Ca(2+) stores to InsP(3) increases during maturation of the oocytes. Since Astropecten auranciacus oocytes also respond to cADPr, we have studied whether the response to cADPr also changes during maturation. We have found that the photoactivation of injected cADPr in immature oocytes immediately induces multiple patches of Ca(2+) release in the cortical region. The Ca(2+) signal then spreads from these initial points of increase to the entire cell. In mature oocytes, the uncaging of cADPr induces instead a single (or at most a dual) initial point of Ca(2+) release, which is immediately followed by the formation of a cortical Ca(2+) flash and then by the globalization of the wave and by the elevation of the fertilization envelope. External Ca(2+) plays a role in the Ca(2+) responses. Inhibition of L-type Ca(2+) channels does not affect the initial Ca(2+) release, but abolishes the cortical flash and impairs the elevation of the fertilization envelope. External Ca(2+) has other effects, as shown by the irregular appearance of the surface of oocytes incubated in Ca(2+)-free sea water. The sequence of Ca(2+) responses induced by cADPr in mature oocytes mimics those seen at fertilization, i.e., a first localized Ca(2+) increase followed by a cortical flash and by the globalization of the Ca(2+) signal. As in the case of maturation, L-type Ca(2+) channel blockers abolish the sperm induced cortical flash.     

Developmental competence of frozen-thawed yak (Bos grunniens) oocytes followed by in vitro maturation and fertilization

In the present study, we examined the ability of immature germinal vesicle (GV) and subjected to in vitro matured (MII) yak oocytes to survive after cryopreservation as well as their subsequent development following in vitro maturation and fertilization. Both GV and MII oocytes were cryopreserved by using two different vitrification solutions (VS); VS-I contained 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in TCM-199 + 20% (v/v) fetal calf serum (FCS) whereas VS-II contained 40% EG + 18% Ficoll + 0.5 M sucrose in TCM-199 + 20% FCS. The percentage of oocytes found to be morphologically normal was greater (P < 0.01) in VS-I group than in VS-II group. Rates of cleavage (30.6–42.2%) and blastocyst formation (2.9–8.9%) did not differ among groups, but were lower than in unfrozen control (55.7% and 25.4%, P < 0.01). These results show that a combination of EG and DMSO or EG, Ficoll and sucrose can be used to cryopreserve yak oocytes in French straws.     

In vitro maturation and fertilization of goat oocytes

Goat oocytes were isolated from 3-5 mm diam. follicles. The oocytes with compact cumulus mass were matured and fertilized in vitro. Three different media, viz. modified Krebs-Ringer bicarbonate, Dulbecco's and Ham's F-12 with three different additives (bovine serum albumin, BSA; follicle stimulating hormone, FSH and fetal calf serum, FCS) were tested. The three basal media gave almost similar results with Ham's F-12 being slightly better. Addition of BSA (10 mg/ml) increased the rates of maturation and penetration. FSH + BSA (2.5 micrograms/ml + 10 mg/ml) further enhanced the rates while FCS (10%) proved to be even more effective. In modified Krebs-Ringer bicarbonate and Dulbecco media with additives FCS + BSA, around 60% oocytes matured to metaphase II of which 53% were penetrated by capacitated goat spermatozoa while in F-12 medium 70% reached metaphase II and 63% were penetrated. Ham's F-12 medium with additives FCS + BSA was slightly better for maturation and penetration of goat oocytes in comparison to two other media tested.     

In vivo survival of domestic cat oocytes after vitrification, intracytoplasmic sperm injection and embryo transfer

We evaluated: (1) cleavage rate after IVF or intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification (experiment 1); and (2) fetal development after transfer of resultant ICSI-derived embryos into recipients (experiment 2). In vivo-matured cumulus-oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment. In vitro-matured oocytes were obtained by mincing ovaries (from local veterinary clinics) and placing COCs into maturation medium for 24 h. Mature oocytes were denuded and cryopreserved in a vitrification solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose. In experiment 1, for both in vivo- and in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes and after ICSI of vitrified oocytes were not different (P > 0.05). After vitrification, blastocyst development occurred only in IVF-derived, in vitro-matured oocytes. In experiment 2, 18 presumptive zygotes and four two-cell embryos derived by ICSI of vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo- and 12 in vitro-matured oocytes were transferred by laparoscopy into the oviducts of two recipients, respectively. On Day 21, there were three fetuses in one recipient and one fetus in the other. On Days 63 and 66 of gestation, four live kittens were born. In vivo viability of zygotes and/or embryos produced via ICSI of vitrified oocytes was established by birth of live kittens after transfer to recipients.     

In vitro maturation, fertilization and development of follicular oocytes from buffalo (Bubalus bubalis).

Cumulus-oocyte complexes, 5596, were cultured for 24 h in either TCM-199 or Ham's F-10 with or without gonadotrophins and supplemented with either 20% buffalo oestrous serum (BES) or fetal calf serum (FCS). The maturation rates of oocytes cultured in TCM-199 or Ham's F-10 medium supplemented with 20% BES were 47.4 +/- 17.8 and 44.8 +/- 25.6, respectively. Addition of luteinizing hormone (LH) (5 micrograms ml-1) significantly improved the maturation rate in the Ham's F-10 medium supplemented with 20% BES (76.8 +/- 18.3), but follicle-stimulating hormone (FSH) (0.5 micrograms ml-1) and oestradiol (1 microgram ml-1) failed to synergize with LH (71.7 +/- 19.5). In the TCM-199 system, LH failed to enhance the maturation rate but the addition of FSH and oestradiol significantly enhanced the proportion of mature oocytes (42.7 +/- 1.4 and 81.7 +/- 14.5, respectively; P less than 0.05). Frozen-thawed spermatozoa prepared in Bracket and Oliphant (BO) medium and treated with 5 mmol caffeine 1(-1) + 10 micrograms heparin showed a higher fertilization rate (29.8%) than those treated in Hepes-Talp and treated with 10 micrograms heparin ml-1 (19.6%). Fertilization rate was significantly improved when fresh ejaculated spermatozoa treated with 5 mmol caffeine 1-1 and 10 micrograms heparin in BO medium (50%) was used. Rate of cleavage and development were also higher when in vitro fertilization was carried out with fresh ejaculated spermatozoa treated with caffeine and heparin (34.1 and 36.8%, respectively) than with frozen-thawed spermatozoa (27.0 and 22.0%, respectively). Development rate was enhanced when fertilized ova were cultured in ligated rabbit oviduct (28.0%) than when co-cultured on oviductal cell monolayers (8.2%). The results indicate that oocytes cultured in medium supplemented with BES and gonadotrophins reveal high rates of maturation and development to the blastocyst stage after fertilization with fresh ejaculated spermatozoa.     

In vitro maturation,fertilization and development of domestic cat oocytes recovered from ovaries collected at three stages of the reproductive cycle

This study was conducted to examine the effect of the donor cat's reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 degrees C for a short period (1-6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus-oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage (P < 0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected (P > 0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower (P < 0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 degrees C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.     

Exocytosis of glycogen during maturation of amphibian oocytes

The repartition and fate of glycogen β has been followed during progesterone-induced maturation of amphibian oocytes. The use of specific staining, both at the cytological and ultrastructural level, demonstrates that glycogen tends to be extruded from the oocyte during maturation of the urodeles Pleurodeles waltlii and Ambystoma mexicanum. No such effect of the hormone is observed in Xenopus laevis, where only a slight centrifuge migration of the glycogen could be recorded. Stacks of annulate lamellae increase during the early phase of in vitro progesterone-induced maturation (2 to 9 hours after progesterone application). After germinal vesicle breakdown (about 12 hours after beginning the progesterone treatment) annulate lamellae have disappeared and numerous masses of vesicles are present in the cytoplasm of Pleurodeles and Ambystoma matured oocytes. We never observed any close relation between the annulate lamellae and these vesicles.     

In vitro maturation and fertilization of prepubertal goat oocytes

The aim of this work was to study the IVM-IVF of prepubertal goat oocytes collected from a slaughterhouse as an alternative source of oocytes to those of FSH-primed adult goats. In Experiment 1, IVM of prepubertal goat oocytes in co-culture with granulosa cells were compared with IVM in 50 microl microdrops of medium. There was no significant difference in the percentage of maturation (72.0 vs 76.9%) between the 2 groups. In Experiment 2, a low percentage of normal fertilization (24.4%) was observed for prepubertal goat oocytes matured with granulosa cells from prepubertal goats. This result was significantly lower than that obtained for ovulated (62.2%) or in vitro-matured (48.7%) oocytes from adult goats. There were no significant differences with respect to the oocytes from adult goats matured in vitro when prepubertal goat oocytes were cultured with adult goat granulosa cells (33.3%) or in microdrops (29.7%). No differences were observed among the treatments in the percentage of oocytes showing evidence of fertilization (normal fertilization + abnormal fertilization + polyspermy). In Experiment 3, it was shown that there were no differences in the percentage of normally fertilized oocytes after in vitro maturation in microdrops containing oocytes with 1 to 2 and 3 or more complete layers of cumulus cells (32.1 and 33.3% respectively). In conclusion, the ovaries of prepubertal slaughterhouse goats were found to be an economical alternative for an abundant source of oocytes for IVM-IVF research. In vitro maturation of oocytes in microdrops yielded maturation and fertilization rates comparable to those obtained with oocytes from FSH-primed adult goats. Moreover, similar maturation and fertilization rates were obtained using oocytes with 1 to 2 layers or 3 or more layers of cumulus cells.     

In vitro maturation and artificial activation of donkey oocytes

Three media were evaluated for their ability to support in vitro maturation of donkey (Equus asinus) oocytes and their development after parthenogenetic activation. The basal medium for Medium 1 (M1) and Medium 2 (M2) was M199 and DMEM/F12 respectively, whereas, Medium 3 (M3) consisted of equal parts (v/v) of M199 and DMEM/F12. All three media were supplemented with 10% (v/v) fetal calf serum, 0.01 units/mL porcine FSH, 0.01 units/mL equine LH, 200 ng/mL insulin-like growth factor 1(IGF-I), 10 μl/mL insulin-transferrin-selenium (ITS), 0.1 mg/mL taurine, 0.1 mg/mL L-cysteine, 0.05 mg/mL L-glutamine, 0.11 mg/mL sodium pyruvate, and 25 mg/mL gentamycin. There were no significant differences among the three maturation media for oocyte maturation. Maturation rate of donkey oocytes in M1 was 53% for compact (Cp) cumulus-oocyte complexes and 75% for expanded (Ex) cumulus-oocyte complexes; in M2 these were 55 and 77%, respectively; and in M3, 58 and 75%. The percentage of cleaved parthenotes and 4- or 8-cell embryos were not significantly different for oocytes matured in the various media (61 and 24% for M1; 66 and 32% for M2; and 67 and 33% for M3). Oocytes matured in M3 tended to yield a higher rate of advanced embryo development (morula) than oocytes matured in M1 (22 vs 9%; P = 0.07). In conclusion, donkey oocytes were matured and parthenogenetically activated in vitro, using methods similar to those used in the horse.     

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine (Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (−) FCs (5.2 × 10⁶ cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, −FC/S, +FC/R, and −FC/R) under 5% or 20% O₂. Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O₂ using the −FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both −FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the −FC/S culture system for promoting fertilization.     

Timing of nuclear maturation of nonstored and stored domestic cat oocytes

In this study we compared the effects of preculture storage of ovaries, IVM medium, a reduced O(2) atmosphere and duration of culture on in vitro maturation (IVM) of domestic cat oocytes. One randomly selected ovary of each pair (69 pairs) was stored in PBS at 10 degrees C for 16-24h before oocyte recovery. The second ovary from each pair was used as a nonstored control. In Experiment I, we investigated the effect of culture media (TCM 199 versus SOF) and a reduced O(2) atmosphere (a humidified gas atmosphere of either 5% CO(2) in air or 5% CO(2):5% O(2):90% N(2)) on IVM of both stored and nonstored oocytes. In the second experiment, we compared timing of nuclear maturation of both stored and nonstored oocytes cultured for 17-18, 20-21, 24-26, 28-30, 33-34 or 42-45 h before being evaluated for meiotic status. In both, Experiments I and II, the recovery rate, quality and competence for maturation of oocytes originating from stored ovaries did not differ (P>0.05) compared with nonstored. In Experiment I, neither culture medium (37.5 versus 43.2% of Metaphase II, respectively in TCM 199 versus SOF) or gas atmosphere (40.0 versus 32.5% of Metaphase II, respectively in 5% CO(2) in air versus 5% CO(2):5% O(2):90% N(2)) affected oocyte maturation. In Experiment II, the mean proportion of oocytes achieving Metaphase II within 17-18 h of culture was 36.1% and did not significantly increase (P>0.05) over time up to 28 h. The highest proportion of oocytes (67.3%) reached Metaphase II stage after 42-45 h of culture. Therefore, we conclude that two "waves" of nuclear maturation of cat oocytes can be distinguished. The first wave takes place within 26 h and it is likely that most oocytes of this wave mature by 17-18 h; the second wave occurs after 28-30 h of IVM. It can be assumed that this double wave may reflect the presence of two oocyte populations with two different degrees of "prematuration" which require different lengths of IVM.     

Nuclear maturation of domestic cat ovarian oocytes in vitro.

Using the domestic cat as a model for salvaging genetic material from rare Felidae, we collected oocytes from ovarian tissue and placed them in 1 of 3 treatments to observe time-related, meiotic changes of in vitro oocyte maturation. Oocytes obtained from ovaries collected at ovario-hysterectomy were assigned to 1 of 3 treatment groups: 1) modified Krebs-Ringer bicarbonate buffer (mKRB) + 4% BSA and 5 micrograms/ml FSH (+FSH, n = 499); 2) mKRB + 4% BSA (-FSH, n = 502); or 3) mKRB + 5% natural estrus cat serum (NE, n = 873). They were placed in the respective media in a 5% CO2 humidified environment at 38 degrees C. Beginning at 16 h, oocytes were removed at 4-h intervals through 48 h, and the meiotic status was evaluated by means of cytogenetic analysis. On the basis of chromosomal analysis, each cell was placed into one of the following categories: metaphase II (MII); metaphase I (MI); pre-MI (germinal vesicle [GV], GV breakdown, or diakinesis); degenerate or unidentifiable. The percentage of oocytes with degenerate chromatin increased over time in all culture treatments, but was always greatest (p less than 0.05) in the NE group. In the +FSH and -FSH treatments, the proportion of oocytes with nuclear material reaching MII increased with time in culture to 32 h and was equal to or greater than the proportion of oocytes with pre-MI + MI chromatin at this time interval (-FSH, 55%; +FSH, 38%).(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro fertilization of pig oocytes matured in vitro

The development of in vitro fertilization (IVF) techniques in pigs as well as in other species is of great importance because of the possible applications of this technology in different research fields. Methods of IVF vary in different incubation periods and temperatures, in the hormone concentrations used, and in the treatment of the sperm samples. It has been particularly difficult to succeed in the achievement of fertilization in the pig. In the present study we used FSH and LH concentrations of 2 IU/ml for oocyte maturation, an incubation temperature of 37°C, and dilution of spermatozoa for capacitation, and we achieved a high fertilization rate (50 to 75%) with no cases of polyspermy.     

Effect of EGF on in vitro maturation of domestic cat oocytes

The objective of this study was to evaluate the influence of different concentrations of epidermal growth factor (EGF) on in vitro maturation of domestic cat oocytes. A total of 444 cat oocytes were matured in MSOF (maturation synthetic oviductal fluid) in the presence of varying EGF concentrations: (1) MSOF (control); (2) MSOF+10 ng/mL EGF (EGF10); (3) MSOF+25 ng/mL EGF (EGF25); and (4) MSOF+50 ng/mL EGF (EGF50). After IVM, oocytes were in vitro fertilized to verify the effect of adding EGF on cytoplasmic maturation. Cleavage rate was recorded and noncleaving oocytes were stained with Hoechst 33258 and examined to determine nuclear maturation rate. Cleaved zygotes were cultured in vitro and embryo stages were evaluated on days 6 and 7. There was no difference among groups in the total number of oocytes reaching the metaphase II (MII) stage (P>0.05). The EGF25 group had the highest (P<0.01) blastocyst yield (37.5%) and developmental competence (60.9%). Cleavage rate and resulting morulae and blastocysts on day 6 for EGF25 group were higher (P<0.01) than control and EGF50 groups. Although EGF did not significantly enhance nuclear maturation rate, it had a dose-related positive effect on cytoplasmic maturation, since the oocyte's ability to cleave and reach the blastocyst stage was improved at 25 ng/mL, with intermediate improvement at 10 ng/mL, but 50 ng/mL had no significant benefit. In conclusion, the addition of EGF to the maturation medium enhanced cytoplasmic maturation of cat oocytes in vitro.