In vitro maturation and fertilization of goat oocytes

Follicular cumulus-enclosed goat oocytes were matured in vitro in the presence of granulosa cells, follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol-17beta. While 86% of the oocytes from follicles 2 to 6 mm in diameter achieved meiotic maturation, only 24% of the oocytes from follicles 1 to 2 mm in diameter progressed to Metaphase II. Exposure of follicle-enclosed cumulus-oocyte complexes to 20 degrees C prior to culture resulted in 11.5% of the oocytes exhibiting abnormal meiotic spindle. This indicated that immature goat oocytes are particularly sensitive to temperature. Ejaculated spermatozoa were capacitated according to the technique previously proposed for ram sperm (1). The fertilization rates of ovulated and mechanically denuded in vitro-matured oocytes were 85 and 82.8%, respectively; 59.7% of ovulated and 57.1% of in vitro-matured oocytes were normally fertilized, as shown by the presence of both the female and the male pronucleus as well as by the remnants of the sperm tail in the ooplasm, 17 hours after insemination. Polyspermy was the main abnormality detected, and it affected almost 20% of the inseminated oocytes. The cleavage rate (two to fourcell stage) 41 hours after insemination of in vitro-matured and fertilized oocytes was 58%.     

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 fertilization of goat oocytes

Experiments were carried out to achieve fertilization (IVF) and initial embryonic development of goat oocytes in vitro. Oocyte/cumulus complexes were recovered from large follicles (greater than 7 mm) of hormonally treated doses and from 1-6-mm follicles of ovaries from hormonally superstimulated and nontreated goats. Three different sperm treatment/IVF media were used: defined medium (Brackett and Oliphant, Biol Reprod 1975; 12:260-274) with modifications (mDM); TALP (Bavister and Yanagimachi, Biol Reprod 1977; 16:228-237), as modified by Parrish et al. (Theriogenology 1986; 25:591-600), i.e. modified TALP (mTALP); and HEPES-buffered M199 with modifications (mH-M199). Immature oocytes (from 1-6 mm, small antral follicles) were cultured for in vitro maturation (IVM) in M199 buffered with bicarbonate and with modifications including supplementation with 20% (v/v) goat serum (mB-M199) with either (a) 100 micrograms LH/ml, (b) 5 micrograms FSH/ml, or (c) no added gonadotropin control. Insemination of (in vivo or in vitro) matured oocytes was performed with swim-up separated and heparin-treated freshly ejaculated sperm; additionally, caffeine was included in the mDM treatment. Use of mDM yielded better results than mTALP or mH-M199 (p less than .05). Results with oocytes after IVM were significantly better than those obtained with oocytes matured in vivo (68.4% vs. 45.5%, p less than 0.05). Presence of LH or FSH during oocyte maturation improved both the IVM and IVF results over those of the control (p less than 0.05). The highest proportion of fertilized oocytes (fertilization rate) was achieved by combining the use of mDM for sperm and IVF with IVM in the presence of LH.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro maturation and fertilization of goat oocytes frozen at the germinal vesicle stage

The ability of frozen immature goat oocytes to undergo in vitro maturation (IVM) and fertilization (IVF) was investigated. Fully grown germinal vesicle stage (GV-stage) goat oocytes were submitted to different variables of cryopreservation: 1) exposure to propanediol before maturation but without freezing to detect the level of damage attributable to propanediol alone, 2) removal of cumulus cells to mimic damage attributable to osmotic stress during cryoprotectant exposure or freezing procedure, and 3) rapid freezing with propanediol. Maturation and fertilization rates were 82.1, 71, 65.3 and 23.7% and 71.2, 40, 58.4 and 23.1% for control, exposed, denuded and frozen oocytes, respectively. These results indicate that freezing sticto sensu (i.e., cooling and warming phases) have detrimental effects on IVM of GV-stage oocytes, whereas the reduced IVF rates of post-thaw matured oocytes are imputable to a cryoprotectant effect.     

Mitochondrial organization in prepubertal goat oocytes during in vitro maturation and fertilization

The aim of this study was to evaluate mitochondrial distribution during in vitro maturation (at 0, 15, 20, and 27 hr of IVM) and fertilization of prepubertal goat oocytes compared to mitochondrial distribution of ovulated and in vitro fertilized oocytes from adult goats. Oocytes from prepubertal goats were recovered from a slaughterhouse and were matured in M199 with hormones and serum for 27 hr. Ovulated oocytes were collected from gonadotrophin-treated Murciana goats. Frozen-thawed spermatozoa were selected by centrifugation in Percoll gradient and were capacitated in DMH with 20% steer serum for 1 hr. Ovulated and IVM-oocytes were inseminated in DMH medium with steer serum and calcium lactate for 20 hr. Oocytes and presumptive zygotes were stained with Mitotraker Green FM and observed under a confocal laser scanning microscope. Ultrastructural morphology of oocytes and presumptive zygotes were analyzed by transmission electron microscopy (TEM). Prepubertal goat oocytes at germinal vesicle stage (GV) presented mitochondria localized in the cortical and perinuclear region. IVM-oocytes at metaphase II presented mitochondria peripheral polarized to the region opposite were the metaphase spindle is positioned and within the polar body. Ovulated oocytes presented peripheral mitochondria distribution and mitochondrial aggregation around the MII spindle. At 20 hr post-insemination, mitochondria were distributed around the two synchronous pronuclei (2PN rpar; in zygotes ovulated oocytes whereas in prepubertal 2PN-zygotes mitochondria presented a peripheral polarized distribution. Images by TEM detected that immature prepubertal goat oocytes that are less electrodense and present fewer cristae than in vitro matured prepubertal goat oocytes; these are characterized by being associated to swollen vesicles. Mol. Reprod. Dev. 73: 617-626, 2006 (c) 2006 Wiley-Liss, Inc.     

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.     

In vitro fertilization of intact sheep and cattle oocytes with goat spermatozoa

In an earlier study we reported on the fertilization of in vitro-matured bovine oocytes by ram spermatozoa. The present results extend those observations and demonstrate the penetration of bovine and ovine oocytes matured in culture by goat spermatozoa. Freshly ejaculated and in vitro capacitated goat spermatozoa penetrated 37.1% of bovine and 48.1% of ovine intact oocytes. Monospermic fertilization was detected in about 90% of the oocytes of both species, and the development of pronuclei followed a developmental pattern similar to that of homologous fertilization.     

In vitro maturation, in vitro fertilization and embryonic development of canine oocytes

In this study we have investigated the efficiency of in vitro maturation (IVM) as a basic way to study the development of canine oocytes after in vitro fertilization (IVF). We decided, therefore, to perform two-part experiments. Firstly, experiment I compared the effects of TCM199 without fetal bovine serum (FBS) with TCM199 supplemented with 5% FBS on the in vitro nuclear maturation rate of canine oocytes. For the efficiency of meiotic development to the metaphase II (MII) stage, we found that 4.7% (4/64) of all oocytes grown in TCM199 without FBS developed to the MII stage compared with only 1.7% (1/59) of those grown in TCM199 with 5% FBS for 48 h. Therefore, FBS did not increase in vitro nuclear maturation. In experiment II, the cleavage rate of canine oocytes used for IVF was investigated following heparin treatment. Canine oocytes were fertilized in four groups: Fert-TALP medium without heparin (Fert I) or Fert-TALP medium supplemented with 10, 20 or 30 microg/ml heparin (Fert II, Fert III, Fert IV, respectively). Oocytes that were grown for 24 h in Fert I following fertilization showed the highest rate of all of the groups, 6.5% (5/77) and developed to the early morula stage. Markedly, the oocytes cultured in Fert I for 24 h following insemination had a higher rate of embryonic development than other groups. We can assert that, unlike findings in other mammals, heparin treatment in canine IVF does not increase the efficiency of the fertilization rate and is therefore not an important factor.     

Influence of the collection technique of prepubertal goat oocytes on in vitro maturation and fertilization

Prepubertal goat ovaries obtained from a slaughterhouse were used to study the influence of the oocyte collection technique (dissection, aspiration and slicing) on the number of oocytes recovered and their capacity for maturation and fertilization in vitro. The oocytes were recovered using 3 techniques, were selected for culture and were classified according to the number of cumulus cell layers. The numbers of oocytes selected per ovary were 1.71, 1.27 and 6.05 for dissection, aspiration and slicing, respectively. The percentages of maturation obtained for slicing (56.9%) were lower than those obtained for dissection and aspiration (69.3 and 72.0%, respectively). The proportion of oocytes with the most cumulus cell layers (complete cumulus) was greatest for oocytes recovered by dissection, but this had no influence on their capacity for nuclear maturation. The total percentage of fertilization was similar for oocytes obtained by dissection and by slicing, but the latter yielded a lower percentage of normal fertilization (29.1 vs 18.2%). Of the oocytes obtained by slicing, no difference was observed in the fertilization rate between oocytes with a partial cumulus and a complete cumulus. The decrease in maturation time from 27 to 25.5 and 24 h did not improve the results for fertilization but caused a decrease in the percentage of nuclear maturation. In conclusion, the recovery of oocytes using the slicing technique yielded more oocytes per ovary than dissection or aspiration, although the in vitro fertilization capacity of oocytes obtained by the slicing method was lower than for oocytes obtained by dissection.     

In vitro maturation and fertilization of swamp buffalo oocytes and their subsequent development

The present experiment was carried out to evaluate the maturation, fertilization and subsequent embryo culture of swamp buffalo oocytes in vitro. The oocytes (n=273) were collected and morphologically graded based on the structure of cumulus-oocyte complexes as Grade 1 (compact, n=81), Grade 2 (expanded, n=70), Grade 3 (partially denuded, n=65) or Grade 4 (completely denuded, n=57). More than 60% of the in vitro matured oocytes co-cultured with capacitated spermatozoa demonstrated evidence of fertilization or cleavage to the 2-cell stage when either Grade 1 or 2 oocytes were used. The percentage of fertilized oocytes undergoing 2-cell stage cleavages from Grade 3 (53%) and Grade 4 (46%) groups was significantly lower (P<0.01) than that observed in the Grade 1 (64%) and Grade 2 (68%) groups. Development to the 6 to 8 cell stage substantiated fertilization of Grade 1 and 2 oocytes. These results demonstrated that swamp buffalo oocytes are capable of maturing in vitro, forming embryos, and developing at least to the 8-cell stage in culture medium alone.     

In vitro maturation, fertilization and early cleavage rates of bovine fetal oocytes

The in vitro developmental competence of oocytes harvested from 3 to 6 mm follicles from ovaries of 7.5 months to term fetuses and adult cows was compared. Cumulus oocyte complexes (COCs) were washed and placed in 200 microl droplets of maturation medium 199, supplemented with 10 microg/ml FSH, 10 microg/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM Hepes, and 10% fetal bovine serum (FBS) under oil and incubated for 24 h at 39 degrees C and 5% CO2. Matured oocytes were exposed to frozen-thawed TALP swim-up, heparin-capacitated sperm (20 h, 39 degrees C, 5% CO2). Presumptive zygotes were cultured in medium 199 containing 8 mg/ml BSA-V, 100 IU/ml penicillin G, 75 microg/ml streptomycin, and 10 mM Hepes (48 h, 39 degrees C, 5% CO2). Oocytes/embryos were fixed, stained with DAPI, and evaluated under fluorescent microscopy to assess maturation, fertilization, and subsequent embryonic development. There was a difference (P<0.05) between fetal and adult cow oocytes for in vitro maturation (IVM; 80.1% versus 92.0%), fertilization (69.3% versus 79.9%), and cleavage rates (36.7% versus 49.9%), respectively. Poor IVM, fertilization and embryonic development of fetal oocytes may be due to a higher incidence of blockage at germinal vesicle (GV) and metaphase-I (M-I) stage after IVM (12.0% versus 2.3% for fetal versus adult oocytes, respectively, P<0.05). Although the IVF results with fetal oocytes are poorer than with adult cow oocytes, they were still high enough to be considered for use in research and when death of the dam and/or fetus is pre-mature or sudden.     

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.     

In vitro maturation of horse oocytes

Ovaries collected from slaughtered mares of unknown reproductive history were transported to the laboratory, and their oocytes were recovered and cultured in modified TCM 199 supplemented with 20% horse serum and additional granulosa cells. To characterize the ovaries, the size and number of follicles were counted. To determine the time required for nuclear maturation, oocytes were fixed either after 18 h (n=23), 24 h (n=50), or 30 h (n=33) of culture. After co-culture with granulosa cells most oocytes reached metaphase II (M II) by 30 h (72.7%). After 24 h of maturation only 56.0% of the cultured oocytes had reached metaphase II (M II).     

In Vitro maturation and fertilization of domestic cat follicular oocytes

The time course and conditions necessary for oocyte maturation and subsequent fertilization in vitro were studied in the domestic cat. Darkly pigmented oocytes surrounded by cumulus cells and a tight corona radiata were collected from ovaries removed at ovariohysterectomy. After culture in Eagle's minimum essential medium, oocytes were evaluated for nuclear maturation by analyzing chromosomal spreads. Oocytes achieved metaphase II after intervals of 40–48 hr of in vitro incubation. The incidence of maturation was enhanced (P<0.05) when oocytes were recovered from inactive (54%) or follicular (56%) stage donors compared to those recovered from luteal phase (29%) or pregnant (35%) cats. The proportion of oocytes successfully maturing in vitro in medium containing no hormone supplementation (37%) was less (P<0.01) than counterparts cultured in follicle-stimulating hormone (FSH) only (48%) or FSH and luteinizing hormone (LH) (54%). The efficiency of maturation was not influenced (P >0.05) by either maintenance/transport temperature (4°C vs. 22°C) or delaying recovery of oocytes from antral follicles (2–8 hr vs. 24–32 hr). Approximately 36% of the in vitro matured oocytes cocultured with spermatozoa demonstrated evidence of fertilization; however, there appeared to be a critical development period for maximizing the incidence of fertilization. These results demonstrate that domestic cat antral oocytes are capable of maturing in vitro, and maturation is influenced by the reproductive status of the donor and the presence of gonadotropins in the culture medium. These oocytes are capable of forming embryos and developing to at least the 16-cell stage in vitro.     

In vitro maturation, fertilization and culture to blastocysts of bovine oocytes in protein-free media

This study examined the role of protein supplementation at the various steps of the in vitro production of bovine embryos derived from two different morphological categories of COC. The basic medium was TCM 199 and was supplemented with hormones during maturation in vitro and either estrous cow serum (ECS), bovine serum albumin (BSA) at various concentrations or polyvinyl-alcohol (PVA). Fertilization in vitro was carried out using frozen-thawed semen or one bull in Fert-talp containing heparin, hypotaurin and epinephrine and either 6 mg/ml BSA or 1 mg/ml PVA. In vitro culture up to the blastocyst stage was performed in TCM 199 supplemented with either ECS, BSA or PVA. The first experiment investigated the influence of different medium-supplements (ECS, BSA or PVA) on nuclear maturation and revealed no significant differences among treatment groups nor between categories of COC (63.9% to 74.9% and 48.9% to 77.0%, respectively). The time course of in vitro fertilization was elucidated in Experiment 2 in medium supplemented with either protein or PVA during maturation and fertilization. Penetration was not affected (70.9% to 79.3% penetration 12 h after onset of oocyte-sperm-co-incubation), but formation of pronuclei was decreased (P < 0.05) 12 and 19 h after onset of oocyte-sperm-co-incubation and was retarded in medium supplemented with PVA (12 h: 63.8 vs 21.4 %; 19 h: 57.5 vs 20.8 %, respectively) while cleavage was not affected. In Experiment 3, six treatment groups were formed in which the two different morphological categories of cumulus-oocyte-complexes (COC) were incubated in basic medium supplemented with 1) ECS during maturation and embryo culture and BSA during fertilization; 2) PVA during maturation and embryo culture, fertilization medium with PVA; 3) PVA during maturation and embryo culture, fertilization medium with BSA; 4) BSA (1 mg/ml) during maturation, fertilization and embryo culture; 5) BSA (6 mg/ml) during maturation, fertilization and embryo culture; and 6) BSA (10 mg/ml) during maturation, fertilization and embryo culture. The rates of cleavage and the development to morulae or blastocysts did not differ (P > 0.05) among treatment groups and between both categories of COC and were showing a high degree of variability (cleavage 54.0% to 65.1% and 41.3% to 55.7%, respectively; morulae 25.3% to 53.0% and 26.0% to 51.2%, respectively; blastocysts 5.4% to 24.7% and 0.6% to 20.3%, respectively). Parthenogenetic activation only rarely occurred in medium containing PVA throughout all steps of in vitro production of bovine embryos (Experiment 4) and led to early cleavage stages (8%), but no development to morula- or blastocyst-stages was observed. It is concluded that 1) formation of pronuclei was retarded in medium lacking protein-supplementation, indicating that BSA is required for regular fertilization in vitro and 2) under our experimental conditions, protein-supplementation is not necessary for maturation and development up to the blastocyst stage in vitro.     

In vitro maturation and fertilization of oocytes from Norwegian semi-domestic reindeer (Rangifer tarandus )

Reindeer oocytes were submitted to in vitro maturation, fertilization and culture (IVM,IVF,IVC) using the established procedures for bovine in vitro embryo production. The study was conducted outside the main breeding season. Semen was collected from epididymides immediately after slaughter, and was diluted in Tris-fructose-citric acid extender containing 6% glycerol and 20% egg-yolk and then frozen in liquid nitrogen. Following 24 h of maturation, cumulus expansion was complete, and 71% of the oocytes reached Metaphase II (MII), with extrusion of the first polar body. Of the remaining oocytes, 22% were at the germinal vesicle stage (GV), 2% at diakinesis and 5% at Metaphase I (MI). The percentages of fertilization and cleavage were 36.0 and 31.8%, respectively. Two of the fertilized oocytes developed to the morula stage after 7 d of culture.     

In vitro fertilization of Siberian hamster oocytes

Siberian hamsters were superovulated and various media were tested in an effort to fertilize the recovered oocytes in vitro. The highest percentage of fertilized ova was achieved by using a modified Tyrode's medium, designated MT (Bavister, J. Reprod. Fertil., 18:544-545, '69), previously formulated to fertilize Syrian hamster ova in vitro. Spermatozoa incubated in this medium in a concentrated state overnight (14 hr) and then diluted (1 hr) fertilized 39% of the ova. Similar results (40%) were obtained with this medium by adding 20% human follicular fluid to fresh concentrated sperm for 30 min and then diluting the sperm for 2-3 hr prior to the addition of ova. Ova fertilized in vitro cleaved to the two-cell stage but failed to develop any further in culture. Two-cell embryos recovered from mated hamsters and cultured did not undergo additional cleavage. Four-cell embryos collected from mated females and cultured cleaved to the six- to eight-cell stage and stopped. Techniques and media used for fertilizing large numbers of Syrian and Chinese hamster ova in vitro will have to be modified to achieve the same degree of success in the Siberian hamster.     

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.