Transporting bovine oocytes in a medium supplemented with different macromolecules and antioxidants: Effects on nuclear and cytoplasmic maturation and embryonic development in vitro.

We investigated whether supplementing the medium used to transport bovine oocytes with different macromolecules [foetal calf serum (FCS) or bovine serum albumin (BSA)] or a mixture of antioxidants (cysteine, cysteamine and catalase) affects their nuclear and cytoplasmic maturation and thereby affects their subsequent embryonic development and cryotolerance. Oocytes were transported for 6 hr in a portable incubator and then subjected to standard in vitro maturation (IVM) for 18 hr. The oocytes in the control groups were cultured (standard IVM) for 24 hr in medium containing 10% FCS (Control FCS) or 10% FCS and the antioxidant mixture (Control FCS+Antiox). The intracellular concentrations of reactive oxygen species (ROS) at the end of IVM period were lower in the oocytes subjected to simulated transport in the presence of a macromolecular supplement or the antioxidant mixture than that of the control group (FCS: 0.62 and BSA: 0.66 vs. Control FCS: 1.00, p < .05; and Transp: 0.58 and Transp Antiox: 0.70 vs. Control FCS: 1.00, p < .05). After IVM, the mitochondrial membrane potentials of the transported oocytes were lower than those of the non-transported oocytes (FCS: 0.41 and BSA: 0.57 vs. Control FCS: 1.00, p < .05; and Transp: 0.48 and Transp Antiox: 0.51 vs. Control FCS: 1.00 and Control Antiox: 0.84, p < .05). The blastocyst formation rates (36.9% average) and the re-expansion rates of vitrified-warmed blastocysts (53%, average) were unaffected (p > .05) by the treatments. In conclusion, supplementing the medium in which bovine oocytes are transported with antioxidants or different macromolecules did not affect their in vitro production of embryos or their cryotolerance.

Sperm morphometry and chromatin condensation in Nelore bulls of different ages and their effects on IVF.

The aim of this study was to evaluate the chromatin packing and sperm head morphometry of cryopreserved semen of Nelore bulls (Bos taurus indicus) of different ages. Furthermore, the influence of the degree of chromatin compaction on in vitro embryo production (IVP) was investigated. Forty bulls were divided into three groups: young (1.8-2 years), adult (3.5-7 years), and senile (8-14.3 years). The ejaculates were frozen according to standards established by the Artificial Insemination Center located in the Southeast of Brazil. Toluidine blue staining was used for simultaneous evaluation of the sperm chromatin and sperm head morphometry. Chromomycin A3 (CMA3) was applied to analyze sperm protamination and IVP for embryonic development. Spermatozoa of young bulls presented higher values for area (A, pixels), perimeter (P, pixels), and width (W, pixels) compared to adults and senile (young: A = 1848.5 ± 119.79, P = 10.23 ± 0.29, and W = 1.95 ± 0.1; adults: A = 1672.9 ± 104.46, P = 9.86 ± 0.33, and W = 1.81 ± 0.06; senile: A = 1723.1 ± 124.41, P = 9.97 ± 0.33, and W = 1.83 ± 0.09; P < 0.0001) and showed higher protamination deficiency when analyzed by CMA3 (young: 1.57 ± 0.76; adults: 1.09 ± 0.63, and senile: 0.90 ± 0.59; P < 0.05). Likewise, variables of sperm head size (A, P, and W) and protamination assessed by CMA3 showed negative correlation with age and positive correlation with ellipticity, evaluated by toluidine blue method (P < 0.05). Sperm head area was larger in spermatozoa presenting chromatin instabilities than spermatozoa without chromatin alteration (P < 0.0001). There was no difference in IVP when using semen with larger or smaller portions of spermatozoa with chromatin instabilities, indicating that the proportion of sperm with abnormal chromatin compaction (4%-16.15%) did not interfere with early embryonic development. From our results, it can be concluded that sperm of young Nelore bulls have larger heads compared to adults and senile due to reduced protamine content when evaluated by CMA3 and higher proportion of major sperm defects assessed by differential interference contrast microscopy.

Improved embryonic cryosurvival observed after in vitro supplementation with conjugated linoleic acid is related to changes in the membrane lipid profile.

The aim of this study was to evaluate the effect of supplementing serum-containing media with a mixture of cis- and trans-9,11- and -10,12-conjugated isomers of linoleic acid (CLA) during different steps of the in vitro production (IVM, IVC, or IVM + IVC) of bovine embryos on their embryonic development, cryotolerance, and lipid profile. To evaluate the impact of the CLA on membrane lipids, such as phosphatidylcholine (PC) and sphingomyelin (SM), the embryos' lipid profiles were obtained using matrix-assisted laser desorption ionization mass spectrometry. The cleavage rates (78.6%-84.8%) and blastocyst development (44.8%-51.2%) remained unaltered. The postthawing reexpansion rates were higher (P < 0.05) when the CLA was added to the IVM medium (82.6%) or to the IVM + IVC medium (83.8%) than the control (69.3%) or IVC medium (63.0%). Changes in the blastocysts' lipid profile occurred when supplementation was restricted to the IVM or IVC medium. However, the most prominent effects of the CLA on the embryonic PC and SM profiles were observed when the supplement was added to IVM + IVC media, which was an increase in the level of highly unsaturated PCs containing 36 or 38 carbons, which are likely to contain CLA residues. These results showed that the molecular mechanism resulting in the improved cryosurvival, observed with CLA supplementation during bovine embryonic in vitro production, was related to the composition of structural lipids of cellular membranes and is dependent on the treatment length. Monitoring the lipid profile of embryonic membranes may improve the CLA supplementation strategy and facilitate the development of new IVC systems to improve the cryopreservation of bovine embryos and those of other domestic species.

Efeito do flushing sobre o desempenho reprodutivo de ovelhas Morada Nova e Santa Inês submetidas à inseminação artificial em tempo fixo / The effect of flushing on the reproductive performance of Morada Nova and Santa Inês ewes submitted to fixed time artificial insemination

O objetivo do estudo foi analisar o efeito do flushing, do protocolo hormonal para sincronização de estro e da IATF sobre o desempenho reprodutivo de ovelhas Morada Nova (MN) e Santa Inês (SI). Foram utilizadas 27 ovelhas SI e 24 ovelhas MN suplementadas com concentrado (1% do peso vivo, em média) durante 75 dias de estação reprodutiva. Após 30 dias de suplementação, as ovelhas foram sincronizadas com protocolo hormonal (PH) à base de progestágeno, eCG e cloprostenol. A observação de estro foi realizada após 12, 24, 36 e 48 horas do término do PH, com o auxílio de dois rufiões, e a IATF foi realizada aproximadamente 55 horas após o final do PH. Do 20º ao 45º dia após o início do PH, realizou-se o repasse com um reproduto Dorper. O diagnóstico de prenhez foi realizado 70 dias após a IATF. Foi analisado o peso, escore da condição corporal (ECC), taxa de apresentação de estro, taxa de prenhez e prolificidade, testando-se os efeitos da raça, semana de suplementação (SS) e classe de ECC. O peso e ECC das ovelhas variaram em função da SS. Foi observada taxa de estro de 88,2%, sendo que 43,2% das ovelhas apresentaram estro até 24 horas do final do PH. A taxa de prenhez por IATF foi de 31,4% e a de prenhez após repasse foi de 50,0%, sendo que a classe de ECC interferiu nas taxas de prenhez. Obteve-se maior porcentagem de partos múltiplos de ovelhas prenhes por IATF do que por monta natural no repasse. Conclui-se que o flushing resultou em ganho de peso e aumento do ECC, o que garantiu a padronização do rebanho para a estação reprodutiva e, consequentemente, melhorou o desempenho reprodutivo. O PH utilizado aumentou a prolificidade e adiantou o início do estro; porém, não foi eficiente na sincronização de ovelhas deslanadas...

The aim of this study was to analyze the effect of flushing on the reproductive performance of Morada Nova (MN) and Santa Inês (SI) ewes submitted to fixed time artificial insemination (TAI). Twenty seven SI and 24 MN supplemented with concentrate (1% of live weight, on average), for 75 days during the breeding season. After 30 days of supplementation, ewes were synchronized with the aid of a hormonal protocol (HP) based on progesterone, eCG and cloprostenol. The estrus observation was conducted at 12, 24, 36 and 48 h after the end of HP with the aid of two ruffians. TAI was done 55 h after the end of HP. From 20 to 45 days after the beginning of the HP ewes were exposed to rams (natural breeding). The pregnancy diagnosis was evaluated 70 days after TAI. We analyzed the weight, body condition score, estrus rate, pregnancy rate and prolificacy testing the effects of race, week of supplementation and body condition score class. The weight and body conditions of ewes varied according to the week of supplementation, with higher values in the first two weeks following TAI. The estrus rate was 88.2% and 43.2% of the ewes showed estrus up to 24 hours of the end of the HP. The pregnancy rate per TAI was 31.3% and the pregnancy rate after natural breeding was 50.0%. It was observed that body condition score classes interfered in pregnancy rates. There was a higher percentage of multiple births by pregnancy by TAI than by natural breeding. It was concluded that the flushing resulted in weight gain and better body conditions ensuring the standardization of the herd for breeding season, which therefore improved reproductive performance. The HP used advanced the onset of estrus and increased prolificacy, but was inefficient in the synchronization of woolless sheep...

Heparin and penicillamine-hypotaurine-epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development.

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P<0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P<0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P>0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P>0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P>0.05) in the presence of heparin and PHE (P<0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development.

Efeito das células do cumulus e cisteamina durante o cultivo de maturação in vitro de oócitos bovinos sobre a maturação nuclear e aquisição da competência para desenvolvimento embrionário / Effects of cumulus cells and cysteamine during bovine oocyte in vitro maturation on meiosis progression and acquisition of developmental competence

Complexos cumulus-oócito (COC), oócitos desnudos (DO) e DO cocultivados com células do cumulus em suspensão (DO+CC) foram maturados in vitro (MIV) na presença ou ausência de cisteamina (50mM). Observou-se efeito benéfico da cisteamina durante o cultivo de MIV, pois a maturação nuclear no grupo COC cisteamina foi maior do que a do COC controle (P<0,05). No grupo sem a adição de cisteamina, foi observado que a ausência de CC durante o cultivo de MIV prejudicou a maturação nuclear em DO, em relação ao COC (P<0,05), todavia a cisteamina restaurou a capacidade de progressão da meiose em DO, tornando-os semelhantes aos COC (P>0,05). O acoplamento entre oócitos e CC durante MIV demonstrou ser essencial para aquisição da competência do oócito para suportar o desenvolvimento embrionário inicial, pois COC apresentaram maior porcentagem de blastocistos e eclosão quando comparados a DO e DO+CC (P<0,05). A inclusão de cisteamina no cultivo de MIV não restaurou a aquisição da competência em DO e DO+CC, que permaneceram semelhantes aos do grupo-controle (P>0,05). Conclui-se que a cisteamina no meio de MIV melhora as taxas de maturação nuclear em COC e restaura a capacidade de progressão da meiose em DO. Todavia, na concentração utilizada neste estudo, não promove efeito benéfico no desenvolvimento embrionário.

Cumulus-oocyte complexes (COC), denuded oocytes (DO) and DO co-cultured with cumulus cells in suspension (DO+CC) were in vitro matured (IVM) in the presence or absence of cysteamine (50mM). A beneficial effect of cysteamine was observed during IVM, because the nuclear maturation in the COC cysteamine group was higher than in COC control (P<0.05). In the control group, the absence of CC during IVM impaired nuclear maturation in DO when compared to COC (P<0.05), but cysteamine restored the ability of meiosis progression in DO, making them similar to COC (P>0.05). The coupling between oocytes and CC during IVM proved to be essential for the acquisition of oocyte competence to support early embryonic development, as COC had higher percentages of blastocyst and hatching when compared to DO and DO+DC (P<0.05). However, the inclusion of cysteamine in the IVM culture did not restore the acquisition of competence in DO and DO+DC, which remained similar to the control group (P>0.05). It is concluded that cysteamine in the IVM culture improves the nuclear maturation in COC and restores the progression ability of meiosis in DO. However, in the concentration used in this study, cysteamine does not promote a beneficial effect on embryo development.

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development.

The effect of melatonin during in vitro maturation (IVM) on DNA damage of cumulus cells (CCs) from bovine cumulus-oocyte complexes (COCs) and embryo development was evaluated. COCs from abattoir ovaries were cultured in maturation medium (MM) with 0.5µg/ml FSH and 5.0µg/ml LH (FSH-LH); 10(-9)M melatonin (MEL) or FSH-LH+MEL (FSH-LH-MEL). After 24h of in vitro maturation, the CCs surrounding the oocyte were subjected to DNA analysis by Comet assay. After in vitro fertilization and in vitro embryo culture, the embryo development rates were evaluated on day 2 post insemination (cleavage) and days 7-8 (blastocyst). The percentage of CCs with no DNA damage was significantly superior in MEL group (37.6±2.4) than in FSH-LH-MEL (28.0±2.4) and FSH-LH (17.8±2.41) groups. Cleavage and blastocysts rates were similar among groups. Melatonin during IVM protects the CCs from DNA damage but this effect did not influence embryo development in vitro.

Effect of antioxidants during bovine in vitro fertilization procedures on spermatozoa and embryo development.

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 microm beta-mercaptoethanol (beta-ME) and 50 microm cysteamine (Cyst)] or a pro-oxidant (5 mm buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for beta-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.

The effect of interaction between macromolecule supplement and oxygen tension on bovine oocytes and embryos cultured in vitro.

SummaryAiming to improve in vitro production of bovine embryos and to obtain supplements to replace serum for in vitro maturation (IVM), this study evaluated the effects of macromolecular supplementation of IMV medium (bovine serum albumin - BSA, polyvinyl alcohol - PVA, polyvinyl pyrrolidone - PVP, Ficoll, KnockoutSR, or fetal calf serum - FCS) and oxygen tension [5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2 (5% O2)] on oocyte maturation and embryo development. Nuclear progression to germinal vesicle breakdown, metaphase I and metaphase II stages were evaluated and overall results revealed that undefined (FCS) and semi-defined (BSA) media gave better results at 20% O2 and defined media (PVA, PVP and Ficoll) at 5% O2. Independent of macromolecule supplement, IVM at 20% O2 was considered optimal for nuclear maturation. To evaluate embryo development, oocytes matured in the previously described conditions were fertilized and cultured at the same oxygen tension used for IVM and assessed for cleavage (43.0 to 74.8%) and development to morulae (16.4 to 33.8%), blastocyst (7.7 to 52.9%) and hatched blastocyst (9.6 to 48.1%). Apart from oxygen tension, all treatments, except Knockout (22.7%), gave similar results for blastocyst development (26.5 to 38.7%). Independently of macromolecule supplement, higher development rates were obtained in an oxygen tension of 20% O2 (67.4% cleavage, 29.2% morulae, 40.8% blastocyst and 34.0% hatched blastocyst) when compared with 5% O2 (52.5, 21.8, 18.2 and 15.6%, respectively). This study indicates that BSA, PVA, PVP and Ficoll can replace serum during IVM and that the optimal atmospheric condition for in vitro production of bovine embryos is 5% CO2 and 20% O2.

Role of roscovitine and IBMX on kinetics of nuclear and cytoplasmic maturation of bovine oocytes in vitro.

The 3-isobutyl-1-methylxanthine (IBMX) is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte, and roscovitine, a purine known to specifically inhibit MPF kinase activity, maintains bovine oocytes at the germinal vesicle (GV) stage. The present study was conducted to analyze whether cytoplasmic maturation (examined by the pattern of cortical granule (CG) distribution) of bovine oocytes is improved during meiotic arrest with IBMX and roscovitine. Oocytes were matured in vitro in a 10% Knockout(SR) supplemented TCM-199 medium (Control) with either 0.5 mM IBMX or 25 microM roscovitine (ROSC). Oocytes were stained with fluorescein isothiocyanate conjugated Lens culinaris agglutinin (FITC-LCA) for CG evaluation and with Hoechst 33342 for nuclear stage assessment. At 16 h of culture, the percentage of oocytes remaining in the GV stage was higher (P < 0.05) in the ROSC group (32.41%) compared with the Control and IBMX groups (8.61% and 9.73%, respectively). At 24 h of culture, progression of meiosis to M II stage was retarded (P < 0.05) in the ROSC group (24.05%) compared to the Control (60.20%), whereas the IBMX group (33.88%) showed no significant difference to the other two groups. At 16 h of maturation, the proportion of oocytes with CG in clusters (immature cytoplasm) was similar between the groups, as was the percentage of peripheral CG (mature) at 24 h of maturation. The results of the present study demonstrated that the meiotic inhibitors IBMX and roscovitine delay the progression of nuclear maturation without affecting cytoplasmic maturation, assessed by the analysis of CG repositioning.

Fertility of male adult rats submitted to forced swimming stress

We investigated whether stress interferes with fertility during adulthood. Male Wistar rats (weighing 220 g in the beginning of the experiment) were forced to swim for 3 min in water at 32ºC daily for 15 days. Stress was assessed by the hot-plate test after the last stressing session. To assess fertility, control and stressed males (N = 15 per group) were mated with sexually mature normal females. Males were sacrificed after copulation. Stress caused by forced swimming was demonstrated by a significant increase in the latency of the pain response in the hot-plate test (14.6 ± 1.25 s for control males vs 26.0 ± 1.53 s for stressed males, P = 0.0004). No changes were observed in body weight, testicular weight, seminal vesicle weight, ventral prostate weight or gross histological features of the testes of stressed males. Similarly, no changes were observed in fertility rate, measured by counting live fetuses in the uterus of normal females mated with control and stressed males; no dead or incompletely developed fetuses were observed in the uterus of either group. In contrast, there was a statistically significant decrease in spermatid production demonstrated by histometric evaluation (154.96 ± 5.41 vs 127.02 ± 3.95 spermatids per tubular section for control and stressed rats, respectively, P = 0.001). These data demonstrate that 15 days of forced swimming stress applied to adult male rats did not impair fertility, but significantly decreased spermatid production. This suggests that the effect of stress on fertility should not be assessed before at least the time required for one cycle of spermatogenesis

Fertility of male adult rats submitted to forced swimming stress.

We investigated whether stress interferes with fertility during adulthood. Male Wistar rats (weighing 220 g in the beginning of the experiment) were forced to swim for 3 min in water at 32 degrees C daily for 15 days. Stress was assessed by the hot-plate test after the last stressing session. To assess fertility, control and stressed males (N = 15 per group) were mated with sexually mature normal females. Males were sacrificed after copulation. Stress caused by forced swimming was demonstrated by a significant increase in the latency of the pain response in the hot-plate test (14.6 +/- 1.25 s for control males vs 26.0 +/- 1.53 s for stressed males, P = 0.0004). No changes were observed in body weight, testicular weight, seminal vesicle weight, ventral prostate weight or gross histological features of the testes of stressed males. Similarly, no changes were observed in fertility rate, measured by counting live fetuses in the uterus of normal females mated with control and stressed males; no dead or incompletely developed fetuses were observed in the uterus of either group. In contrast, there was a statistically significant decrease in spermatid production demonstrated by histometric evaluation (154.96 +/- 5.41 vs 127.02 +/- 3.95 spermatids per tubular section for control and stressed rats, respectively, P = 0.001). These data demonstrate that 15 days of forced swimming stress applied to adult male rats did not impair fertility, but significantly decreased spermatid production. This suggests that the effect of stress on fertility should not be assessed before at least the time required for one cycle of spermatogenesis.

Steroidogenesis in cumulus cells of bovine cumulus-oocyte-complexes matured in vitro with BSA and different concentrations of steroids.

The present in vitro experiments were designed to evaluate the ability of bovine cumulus-oocyte-complexes (COCs) to produce steroids and also to evaluate the modulatory effects of added estradiol, progesterone and testosterone on the steroidogenic activity of COCs. Considerable estradiol accumulation was observed in the control maturation medium for in vitro maturation of bovine COCs during the 24h of maturation (P<0.05). When testosterone was added to the medium at various concentrations, a slight estradiol accumulation occurred, which, however, was lower (P<0.05) than that observed in the control medium. Slight estradiol accumulation was observed in maturation medium containing progesterone at concentrations of 2.5, 5.0 and 10.0 microg/ml, but these increases were less (P<0.05) than those observed in the control medium. However, in the presence of 1.0 microg/ml progesterone, estradiol accumulation was equal to that of the control medium (P>0.05). Progesterone accumulation (P<0.05) was observed in the control medium for in vitro maturation of bovine COCs. When estradiol was added to the maturation medium, progesterone accumulation was observed, but was significant (P<0.05) only when the medium was supplemented with the lesser concentrations of estradiol utilized in the experiment (1.0 microg/ml). The results demonstrated that (1) cumulus cells of bovine COCs are able to secrete estradiol and progesterone in culture systems for in vitro maturation, and this steroidogenesis is modulated by the steroids progesterone, testosterone and estradiol, and (2) the addition of estradiol to the in vitro maturation medium of bovine oocytes should be reviewed, since cumulus cells of COCs have been demonstrated to secrete estradiol in the maturation medium.

The effect of serum on in vitro maturation, in vitro fertilization and steroidogenesis of bovine oocytes co-cultured with granulosa cells.

The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

The effect of serum on in vitro maturation, in vitro fertilization and steroidogenesis of bovine oocytes co-cultured with granulosa cells

The influence of fetal calf serum alone (fcs) or associated with proestrous (FCS + PCS), ESTROUS (FCS + ECS) or metaestrous (FCS + MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with 9FCS + ECS and FCS + MCS resulted in higher proportions of oocytes that reached metaphase II (96.0 per cent and 93.3 per cent, respectively) and in higher proportion of embryos that reached the four- and eight-cell/morula stages (51.9 per cent and 65.6 per cent, respectively), whereas the supplementation with FCS and FCS + pcs resulted in only 79.2 per cent and 67.5 per cent, respectively, of matured oocytes nd 26.7 per cent and 34.4 per cent, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS + ECS: 10.3 ng/ml and FCS + MCS: 2.1 ng/ml), whereas the worst results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS + PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro