Luís Fabiano Santos da Costa

Effect of fetal age and method of recovery on isolation of preantral follicles from bovine ovaries

The objective of this study was to compare enzymatic and mechanical methods, at distinct fetal ages, on isolation of different developmental stages of preantral follicles from bovine ovaries. Fetal ovaries were obtained from pregnant cattle at 150 to 270 d of gestation, and 135,521 preantral follicles at different stages of development were studied. The dissociation of ovaries with a mechanical procedure resulted in an average of 938.16 prenatral follicles. In contrast, 3,715.56 follicles were obtained when enzymatic digestion was used (P = 0.0001). Histological evaluation confirmed follicular stages and demonstrated that both mechanical and mechanical-enzymatic procedure did not affect the cellular integrity of the follicles. Granulosa cell-oocyte complexes surrounded by a basal membrane, were considered preantral follicles in this study. The ratio of different stages of isolated preantral follicles was significantly (P = 0.0001) correlated to fetal age. The earliest fetal age at which tertiary follicles were identified was at 210 d of gestation. The results confirm previous observation that follicular development and atresia are initiated during fetal development. These data provide information on methodologies to isolate intact bovine preantral follicles for investigating the control and regulation of follicular development and the growth of preantral follicles in vitro.

Follicular development and steroid concentrations in cows with different levels of fertility raised under nutritional stress

The aim of the present study was to characterize ovarian follicular development and steroid concentrations during postpartum and the estrous cycle of Brangus Ibagé cows (3/8 Nelore + 5/8 Aberdeen Angus) with different levels of fertility. Cows were classified as having high or low fertility according to the calving interval (CI). The average CI of the herd from which cows used in this study were selected was 404.6+/-5.44 and 711.2+/-20.89 days for the high and low fertility groups, respectively. Four cows of high fertility and five cows of low fertility had calves removed between 70 and 100 days after parturition. Ovarian activity was monitored daily by ultrasound for 16 days after calf removal. Days to emergency of the first follicular wave after calf removal, number of follicles with diameter >9 mm, growth rate of largest follicle, maximum diameter of largest follicle, length (days) and number of follicular waves were recorded. During this period, blood was collected daily for measurements of serum progesterone (P(4)) and estradiol (E(2)) concentrations. In another experiment, ovarian activity and P(4) and E(2) concentrations were examined during estrous cycle in five cows of high fertility and four cows of low fertility. Ovarian activity and steroid concentrations were assessed from the day prior to estrus to the 15th day of the estrous cycle (estrus = day 0). In postpartum cows of high fertility, the total number of follicles >5mm and the maximum diameter of the largest follicle were higher than in cows of low fertility (P < 0.05). Concentrations of P(4) and E(2) did not differ between groups in the postpartum cows. However, E(2) increased 5 days after calf removal (around 90 days of postpartum) in the high fertility group, followed by an increase in P(4) with average values indicating ovulation around 100 days postpartum. In cycling cows, the profile of follicular development was similar between cows of high and low fertility. There was no difference between groups for number of follicles >5mm, but the day effect was significant (P < 0.01). Plasma concentrations of P(4) and E(2) were similar in both groups. These data suggest that cows, from a population raised in the same environment have different fertility as a consequence of individual physiological characteristics.

Interação entre células do cumulus e atividade da proteína quinase C em diferentes fases da maturação nuclear de oócitos bovinos

The aim of this study was to evaluate the effect of protein kinase C (PK-C) on the meiotic resumption and progression in bovine oocyte, and to determine if the cumulus cells mediate the PK-C action in the regulation of bovine oocyte nuclear maturation. Cumulus-oocyte complexes (COC) and denuded oocytes (DO), randomly allotted to 6 treatments (T) based on the presence of an activator of PK-C (PMA) (T1 and T2), or a phorbol ester unable to activate PK-C (4aPDD-control) (T3 and T4) or a basic culture medium (T5 and T6), were cultivated for 7, 9, 12, 18 and 22 hours. The percentage of germinal vesicle breakdown (GVBD) was higher when the oocytes were cultured with PMA than in the control groups with and without cumulus cells. However, PK-C was dependent of cumulus cells to affect the progression to the stages of metaphase I (MI) and metaphase II (MII) at 12 and 18 hours of culture. At 9 and 22 hours, no difference among groups was detected. PK-C accelerates the meiotic resumption independently of the somatic cells but depends on cumulus cells for the progression to the stages of MI and MII.

Fator de crescimento derivado das plaquetas, retinol e insulina na regulação da maturação nuclear de oócitos bovinos e suas conseqüências no desenvolvimento embrionário

The aim of the present study was to determine the effect of platelet-derived growth factor (PDGF; P), insulin (I) retinol, (R) and their interactions (PI, PIR, IR and PR) on oocyte nuclear maturation (NM) and, consequent, embryonic development (ED). The basic medium for oocyte maturation in the treatments was the modified TCM-199, supplemented with PVA (control). To study the embryonic development, the oocytes were divided in three treatments, R, PIR e IR, a negative (PVA) and a positive control group (containing calf fetal serum and gonadotrophic hormones; FCSHOR). The PDGF, insulin, retinol and their interactions did not change the kinetic of the NM, in seven hours of culture (P=0.4492) but it changed after 18 hours of maturation (P<0.001) except in the treatments R and PR (P<0.001), in which the percentages of metaphase II were, respectively, 4.7% and 8.3%. These results were similar to the control group (0.0%). Considering a significant level of P<0.0001 in comparison to the control group, the higher rates of metaphase II were obtained in the presence of IR (19.0%) and PIR (21.3%). The higher rates of MII were observed when the oocytes were matured in the presence of insulin and retinol. In the embryonic development, R (18.3%), PIR (13.9%) and IR (10.6%) increased the rate of cleavage when compared to PVA group (0.0%; P<0.001). However, the oocytes were not competent enough to reach the rate obtained in the FCSHOR group (53.8%; P<0.001). In conclusion, insulin and PDGF accelerate NM and their effects are enhanced by retinol. In the embryonic development, oocytes matured in the presence of either R, IR or PIR have higher cleavage rate than PVA group but lower than those matured in the FCSHOR group.

Períodos de digestão enzimática para o resgate de folículos pré-antrais em ovários de fetos bovinos

O presente experimento foi delineado com o objetivo de determinar a influencia de diferentes periodos de digestao enzimatica nos ovarios de fetos bovinos, sobre o numero total de foliculos pre-antrais resgatados e o numero de foliculos resgatados em cada estagio de desenvolvimento. A presenca da membrana basal, envolvendo o complexo formado pelas celulas da granulosa e o oocito, foi observada atraves de um estudo histologico, o que permitiu avaliar a integridade morfologica dos foliculos pre-antrais isolados. Para isso, ovarios de fetos bovinos, entre 150 e 270 dias de gestacao, foram obtidos em frigorifico. No laboratorio, os ovarios foram seccionados em varios fragmentos com uma tesoura cirurgica e dissociados com pipetas de Pasteur, com diâmetros aproximados de 1000 e 500mm. Apos este processo, os fragmentos foram submetidos a digestao enzimatica com colagenase em uma concentracao de 200UI/ml de TCM199 modificado, por periodos de 20, 30 ou 40 minutos. Os resultados obtidos com esta tecnica permitiram determinar que o numero de foliculos pre-antrais isolados, assim como, os estagios de desen- volvimento em que esses foliculos encontravam-se no momento do isolamento, sao similares nos tres periodos de incubacao enzimatica. A estrutura morfologica desses foliculos, formada pelo oocito, celulas da granulosa e membrana basal, manteve-se intacta apos o isolamento nos tres periodos de digestao enzimatica.

Annexin II mRNA expression in bovine oocytes during follicular development

We investigated the expression of calcium-dependent phospholipid binding protein annexin-II (Ann-II) messenger RNA (mRNA) during preantral follicle development and in oocytes from antral follicles of different diameters ( 8 mm). The action of retinol on Ann-II mRNA expression in mature oocytes was also examined. Only oocytes from secondary preantral follicles expressed Ann-II mRNA and at the germinal vesicle stage expression by oocytes from follicles larger than 8 mm was significantly higher (p < 0.05) compared with oocytes from follicles smaller than 3 mm or between 5 and 8 mm. Ann-II mRNA expression by metaphase II oocytes from follicles larger than 8 mm was significantly higher (p < 0.05) than that from oocytes from follicles smaller than 3 mm, with oocytes from both these size-classes showing similar levels of Ann-II mRNA expression as oocytes recovered from 5-8 mm follicles. In the presence of retinol, Ann-II mRNA expression was higher than when retinol was absent (p < 0.05). Our data indicate that Ann-II mRNA expression is highest in competent oocytes and that retinol increases Ann-II mRNA and may be involved in the regulation of oocyte competence by decreasing the translation and/or degradation of Ann-II mRNA.

DESENVOLVIMENTO DE FOLÍCULOS PRÉ-ANTRAIS BOVINOS IN VITRO EM MONOCAMADA DE CÉLULAS OVARIANAS

The aim of the present work was to determine the influence of ovarian cells in the in vitro development of preantral follicles (PF). The viability of monolayer ovarian cells and the effect of the serum in the survive of in vitro PF was also investigated. Ovarian cells and PF were isolated from ovaries of bovine fetus between 6 and 8 months of pregnancy, obtained in a slaughterhouse. Monolayer of ovarian cells were cultured in a modified TCM-199 in the presence and absence of FSH and its viability after incubation was determined with Trypan Blue. PFs were divided in four different treatments, cultured in modified TCM-199, containing serum of castrated steer (SCS), SCS in monolayer of ovarian cells (MOC), MOC with FSH or a defined medium with polyvinyl alcohol (PVA) as macromolecule. The cellular viability was not affected by the presence or absence of FSH. However, PFs had a significant development (P<0.05) when cultivated in the presence of SNC, MCO and FSH, which was not observed in the other treatments. With these results, it was possible to conclude that FSH does not influence the viability of the ovarian cells when cultured in a monolayer for 20 days. Preantral follicles, measuring between 4000 and 8000mm2 but not with size greater than 8000mm2, grow on ovarian cells in vitro, independently of FSH.

Hepes na produção de embriões bovinos in vitro

The aim of the present study was to evaluate the range of pH changes in maturation and embryo development media, buffered with different HEPES concentrations. Initially, the effect of different concentrations of HEPES (0, 12.5 and 25.0mM) on the variation of pH in the maturation (modified TCM-199) and embryonic development (modified KSOM) media was evaluated at room temperature (25oC) and in an atmosphere of 5% CO2 in air at 39oC. In another experiment, the effect of HEPES on in vitro oocyte maturation was determined. Oocytes were maturated in TCM-199 modified either with 25.0mM of HEPES (HEPES group; n = 137) or without HEPES (control group; n = 142), performing 7 replicates and evaluating the rate of blastocyst. In this study, the medium used for fertilization was Fert-TALP while for embryo development was KSOM with 10% of fetal bovine serum with monolayer of oviduct epithelial cells. A third experiment was designed to determine the effect of HEPES on embryo development. The zygotes were divided in two groups and co-incubated with oviduct epithelial cells in modified KSOM with 10% of fetal bovine serum without HEPES (n = 95) or with 25.0mM of HEPES (n = 92). For this experiment, it was used embryos with two or more cells and the embryo development was considered from cleavage to expanded blastocyst (Bx), 7 and 9 days after insemination. The oocytes and embryos were incubated at temperature of 39oC, an atmosphere containing 5% CO2 in air and saturated humidity. The media with 25.0mM of HEPES were more efficient in minimizing the range of pH than those with 12.5mM or without HEPES. To determine the effect of HEPES during in vitro oocyte maturation, the percentage of Bl considered either the total number of oocytes or the total number of cleavages was higher in the HEPES group (21.9% or 42.9%, respectively) than those obtained in the control group (10.56% or 16.67%, respectively). When HEPES was added to embryo culture medium, the percentage of Bx (45.65%) was higher than that obtained in medium without HEPES (11.58%; p<0.01). The variability between replicates was lower when HEPES was present in embryo development medium, comparing to the medium without HEPES. Therefore, HEPES is an important compound to be present in media for oocyte maturation and embryo development, increasing the in vitro production of bovine embryos.

Fecundação e clivagem após a ativação da proteína quinase C durante a maturação de oócitos bovinos

In the present study the pronucleus formation, cleavage and protein profile after protein kinase C (PK-C) activation were evaluated during bovine oocyte maturation. A total number of 1936 bovine cumulus-oocyte complexes (COCs) were randomly distributed in 4 treatments, and matured with PMA (100nM phorbol 12-myristate 13-acetate, PK-C activator), 4a-PDD (100nM de 4a-phorbol 12,13-didecanoetate, phobol ester that do not bind on PK-C, phorbol ester control), ECS (10% of estrus cow serum, positive control) and in a negative control (basic maturation media for all treatments, except on ECS group where the polyvinyl alcohol was not added). The COCs were kept in presence of PMA for 1, 4 or 7 hours and then transferred to basic maturation media until the 24 hours maturation period was completed. The PK-C stimulation in these periods did not alter the pronucleus formation but caused a decrease in the zygotes cleavage rate. The 4a-PDD group results showed that the alterations in PMA group were caused by PK-C activation and not by direct phorbol ester action on the cell. The protein composition analysis showed an additional protein band on ECS group around 74 kilo Daltons (kDa). Altogether, these results with the preliminary Western Blot analysis, indicate an important role of PK-C on bovine oocyte maturation, fertilization and embryo cleavage.

Profile and regulation of annexin II expression during early embryogenesis in cattle

Foram estudadas a presenca de anexina II (Ann-II) durante a fase inicial do desenvolvimento embrionario bovino e sua regulacao pelo retinol e pelo fator de crescimento semelhante a insulina (IGF-I). Embrioes bovinos em diferentes estadios de desenvolvimento foram produzidos in vitro em fluido sintetico de oviduto (SOF) sem suplementacao (grupo-controle) ou suplementado com retinol (grupo retinol; 0,1ng/ml medium) ou IGF-I (grupo IGF-I; 10 ng/ml de meio). Os embrioes foram processados para extracao de mRNA, producao de cDNA e posterior analise por reacao em cadeia da polimerase (PCR) com oligonucleotideos especificos para Ann-II. Em todos os estadios de desenvolvimento embrionario, Ann-II foi detectada, exceto no estadio de 16 celulas. Os indices de blastocisto foram significativamente maiores (P<0,05) no grupo suplementado com retinol (37,8%, 45/119) durante o cultivo in vitro de embrioes (PIV) que aqueles obtidos no grupo controle (20,5%, 24/117) ou no IGF-I (25,8%, 24/93). Analise semiquantitativa da expressao de Ann-II em embrioes produzidos em meio suplementado com IGF-I ou retinol revelou uma menor expressao desse gene quando comparado com embrioes cultivados somente em SOF (P<0,05). A expressao de Ann-II nao foi diferente em embrioes cultivados na presenca de retinol e IGF-I. A presenca de retinol aumentou a producao de embrioes in vitro, e diminuiu a expressao de Ann-II em estadios iniciais do desenvolvimento embrionario bovino.