Effect of bovine somatotropin (bST-r), progestagen intravaginal device and temporary calf removal on estrous induction and on pregnancy rate in cows

Este trabalho teve por objetivo avaliar a eficiência de tratamentos hormonais sobre a fertilidade de vacas de corte no pós-parto com diferentes condições corporais, durante a estação de monta de outono. Setenta e três vacas pluríparas cruzadas (Hereford x Nelore) criadas extensivamente, com condição corporal entre 2 e 4, foram pesadas e distribuídas em três grupos experimentais. O grupo GSED, constituído por 25 vacas, recebeu pessário vaginal (dia 0) contendo 250mg de acetato de medroxiprogesterona e 500mg de somatotropina bovina recombinante (bST-r). Na retirada dos pessários (dia 7), as vacas receberam 0,5mg de cipionato de estradiol e procedeu-se o desmame temporário dos bezerros por 72 horas. No grupo SED, 25 vacas receberam tratamento semelhante ao grupo GSED, porém não receberam bST-r. No grupocontrole, as 23 vacas somente foram separadas dos seus bezerros por 72h. Quando da retirada dos pessários as vacas foram colocadas com touros por 30 dias. Os animais foram pesados e avaliados quanto à condição corporal no início do experimento e na retirada dos touros (dia 37). Foi constatada perda média de peso de 0,648 kg/dia e os percentuais de estro foram de 26,1%, 33,3% e 56,5%, respectivamente, para os grupos controle, SED e GSED. O diagnóstico de gestação, realizado pela palpação retal 60 dias após a retirada dos touros, indicou percentuais de prenhez de 13,0%, 8,3% e 21,7%, respectivamente, para os grupos controle, SED e GSED (P=0,16), demonstrando que os programas hormonais adotados não foram eficientes no incremento das taxas de prenhez de vacas que perdiam peso entre 50 e 70 dias após o parto. _____________________________________________________________________________________________ ABSTRACTThis study aimed to assess the efficiency of hormonal treatment in the postpartum fertility of nursing beef cows. Seventy-three cows (Hereford x Nellore) raised extensively, with body condition ranging from 2 to 4 were randomly allotted in three groups. The GSED group, with 25 cows, received an intravaginal device (ID) of 250mg of medroxiprogesteron acetate and 500mg of recombinant bovine somatotropin (bST-r; day 0). After the ID withdrawal (day 7), the cows received 0.5mg of estradiol cipionate; their calves were temporarily weaned for 72 hours. In the SED group, 25 cows received a similar treatment, however, somatotropin was not used. In the control group, 23 cows were temporarily separated from their calves for 72 hours and did not receive any hormonal treatment. The cows were put together with the bulls for 30 days just after ID withdrawal. The cows were weighted and assessed for body condition at day 0 and just after bulls withdrawal. The cows lost weight in an average of 0.648 kg/day, and 56.5%, 33.3%, and 26.1% of estrous rates were observed, respectively for the GSED, SED and control groups (GSED vs. SED, P=0.0001; GSED vs. control, P=0.0007; SED vs. control, P=0.53). The pregnancy rates were 21.7%, 8.3%, and 13.0%, respectively for the GSED, SED and control groups (P=0.16). The results showed that the hormonal program associated with temporary 72-hour calf removal did not increase conception rate of cows that loosed weight from 50 to 70 days postpartum

Ovulation induction in beef cows with different forages allowances during post partum

Este experimento teve por objetivo comparar a eficiência de um protocolo hormonal associado ao desmame por 96 horas com o desmame definitivo, em vacas em pastejo com diferentes ofertas forrageiras. Utilizaram-se 310 vacas (190 Aberdeen-Angus e 120 Charolês), entre 50 e 70 dias pós-parto, distribuídas em seis grupos. Os grupos A2, A5, B2 e B5 foram mantidos em maior (A2 e A5) e menor (B2 e B5) disponibilidade forrageira, no período parto-tratamento, e receberam (dia 0) 2mg (A2 e B2) e 5mg (A5 e B5) de benzoato de estradiol e dispositivo intravaginal com acetato de medroxiprogesterona (CIDR). Seis dias após, receberam 1.000 UI de gonadotrofina coriônica eqüina (eCG). No sétimo dia, foi retirado o CIDR e procedido ao desmame dos bezerros por 96 horas. Os grupos AD e BD foram constituídos por 52 e 48 vacas, permanecendo, respectivamente, em maior e menor disponibilidade forrageira, submetidas ao desmame definitivo dos bezerros (dia 7). Inseminaram-se as vacas que manifestaram estro entre os dias 7 e 17, sendo acasaladas, depois, até o 67ºdia. Nos dias 60 e 127 realizaram-se diagnósticos ultra-sonográficos de prenhez. Para análise dos dados, utilizou- se PROC CATMOD do pacote estatístico do SAS, não se verificando diferença (P>0,05) na prenhez entre os grupos com diferentes ofertas forrageiras. Uma nova análise das vacas, conforme seus ganhos de peso, indicou que o desmame definitivo proporciona melhor percentual de prenhez em relação ao modelo de tratamento hormonal utilizado. As vacas que perdem peso no período pós-parto respondem, ao desmame definitivo, mais lentamente do que as que ganham peso no mesmo período. __________________________________________________________________________________________ ABSTRACTThis experiment compared the efficiency of combined hormonal treatment and 96-hour calf removal with weaning in cows fed different forages allowances and with different weight gains. A total of 310 cows (190 Aberdeen Angus and 120 Charolais), 50 to 70 days postpartum, were sorted into 6 groups. Groups A2, A5, B2 and B5 were composed of 53, 49, 53 and 55 cows, respectively; the first two groups had higher forage availability, while the others had lower forage availability, in the postpartum period; groups A2 and B2 received 2mg estradiol benzoate (day zero) and the groups A5 and B5 5mg estradiol benzoate as well as an intra-vaginal device (CIDR) with progesterone. Six days later they received 1000UI equine chorionic gonadotropin (eCG). At day 7 the CIDR device was removed and the 96-hour calf removal period began. Groups AD and BD, with 52 and 48 cows and high and low forage availability respectively, in the postpartum period, were weaned on day 7. All cows that showed estrous were inseminated between day 7 and 17, and then were bred, up to day 67. Between days 60 and 127, ultrasounds diagnosis of pregnancy were performed. Data analysis was carried out using to the PROC CATMOD in the SAS statistical program. There were no significant differences in pregnancy (p>0.05) rates among groups submitted to different forage offers. Data was then analyzed according to whether the cow gamed or lost weight in the post partum period. These results indicate that weaning was more efficient than the hormonal treatment used and cows that lost weight in the have gained weight in the same period

Integrity of sperm plasm membrane, nucleus and mitochondria after freezing ram semen with ethylene glycol

Este trabalho foi conduzido com o objetivo de avaliar o sêmen ovino criopreservado com etileno glicol em relação ao glicerol, quanto à motilidade progressiva e vigor e, especialmente, quanto à integridade das membranas plasmática, nuclear e mitocondrial. Foram utilizados ejaculados de sêmen provenientes de dois carneiros da raça Ile-de-France, com padrões mínimos de volume (0,5ml), turbilhonamento (2 de 0-5), motilidade progressiva (65%), vigor (3 de 0-5), aspecto (cremoso fino, 3x106 espermatozóides/mm3) e células normais (80%). Um total de 25 pools foi dividido em duas alíquotas, as quais foram, posteriormente, congeladas com etileno glicol a 0,5M ou glicerol a 0,72M em pellets. Os parâmetros utilizados, para avaliar o desempenho dos crioprotetores, foram a motilidade progressiva, vigor espermático e integridade do acrossomo e das membranas plasmáticas dos espermatozóides. A motilidade e vigor foram aferidos no sêmen fresco, resfriado, descongelado, mantido a 38°C por 5 horas de incubação (TTL- teste de termorresistência lento) e à temperatura de 45°C por 30 minutos (TTR- teste de termorresistência rápido). Não houve diferença entre o etileno glicol e o glicerol para a avaliação da morfologia do acrossomo, motilidade progressiva e vigor. As células espermáticas criopreservadas com etileno glicol apresentaram maior integridade das membranas plasmática, nuclear e mitocondrial do espermatozóide. O etileno glicol é mais eficiente do que o glicerol para preservar a integridade das membranas espermáticas na congelação de sêmen ovino. __________________________________________________________________________________________ ABSTRACTThe aim of the present experiment was to evaluate the effect of ethylene glycol, in relation to glycerol, as a cryoprotective agent for preserving ovine spermatic cells. The semen had to present a minimal quality to be used, regarding volume (0.5 ml), wave motion (score of 2, from 0 to 5), percentage of progressively motile spermatozoa (65%), rate of progressive motility (score of 3, from 0 to 5), sperm cell concentration (3x106 cells/mm3) and normal sperm morphology (80%). Each pool of semen from ejaculates of two rams was divided into two equal subsamples. One subsample was frozen with ethylene glycol and the other with glycerol. The parameters used to evaluate the semen were sperm motility, vigor, acrossome status and membrane integrity. Sperm motility was evaluated in fresh semen, cooled semen, frozen semen, after 5 hours at 38°C or after 30 minutes at 45°C. No difference was observed between ethylene glycol and glycerol for acrossome status and sperm motility. The sperm cells that were preserved with ethylene glycol showed more integrity of the plasmatic, nuclear and mitochondrial membranes. From the viewpoint of cell membrane integrity, it can be concluded that ethylene glycol gives higher protection to the sperm cell than glycerol

Local regulation of antral follicle development and ovulation in monovulatory species

The identification of mutations in the genes encoding bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) associated with phenotypes of sterility or increased ovulation rate in sheep aroused interest in the study of the role of local factors in preantral and antral folliculogenesis in different species. An additive mutation in the BMP15 receptor, BMPR1b, which determines an increase in the ovulatory rate, has been introduced in several sheep breeds to increase the number of lambs born. Although these mutations indicate extremely relevant functions of these factors, the literature data on the regulation of the expression and function of these proteins and their receptors are very controversial, possibly due to differences in experimental models. The present review discusses the published data and preliminary results obtained by our group on the participation of local factors in the selection of the dominant follicle, ovulation, and follicular atresia in cattle, focusing on transforming growth factors beta and their receptors. The study of the expression pattern and the functionality of proteins produced by follicular cells and their receptors will allow increasing the knowledge about this local system, known to be involved in ovarian physiopathology and with the potential to promote contraception or increase the ovulation rate in mammals

Mechanical isolation of caprine preantral follicles at different reproductive stages and ovarian situation

O presente trabalho descreve os efeitos do estágio reprodutivo (animais impúberes, adultos não-gestantes e gestantes), posição anatômica do ovário (direito e esquerdo) presença de corpo lúteo funcional, peso do ovário e número de folículos antrais superficiais (FAS) sobre o número de folículos pré-antrais (FPA) isolados mecanicamente. Os FPA isolados foram divididos em 3 classes a saber: primordiais, primários e secundários. O número médio (± EP) de FPA isolados por ovário foi de 1.324 ± 193; 866 + 170 e 779 ± 139, respectivamente para ovários oriundos de animais impúberes, não-gestantes e gestantes, sendo observada diferença significativa somente entre animais impúberes e gestantes. A posição anatômica do ovário (exceto para os animais não-gestantes) e a presença ou ausência de corpo lúteo não exerceram nenhum efeito sobre o número e distribuição dos FPA isolados. De um modo geral, o número de folículos primários e secundários foi negativamente correlacionado com o peso ovariano. Já o número de folículos primordiais e secundários foi, respectivamente, positiva e negativamente correlacionado com o número de FAS. Analisando as categorias de animais isoladamente, observou-se que, nos animais impúberes, o número de folículos primordiais foi positivamente correlacionado com o número de FAZ, enquanto que o número de folículos primários e secundários foi negativamente correlacionado com o número de FAS. No tocante à categoria de animais gestantes, o número de folículos primários foi negativamente correlacionado com o peso ovariano. Dependendo da categoria de ovários estudada, foram observadas associações significativas entre o número de folículos primordiais, primários e secundários. _________________________________________________________________________________ ABSTRACTThe aïm of this study was to describe the effects of reproductive stages (pre-pubertal, non-pregnant and pregnant adult animals), ovarian anatomic position (left or right), presence of functional corpus luteum, ovarian weight and number of superficial antral follicles on the number of preantral follicles mechanically isolated. The mean number (mean ± SEM) of isolated preantral follicles per ovary was 1,324 ± 193, 866 ±170 and 779 ± 139, respectively for prepubertal, non-pregnant and pregnant adult animals. Anatomic position (except for nonpregnant animals) and the presence or absence of corpus luteum dia nof affect the number and distribution of isolated preantral follicles. In general, the number of primary and secondary follicles was negatively correlated with the ovarian weight. The numbers of primordial and secondary follicles were, respectively, positively and negatively correlated with the number of superficial antral follicles. Analyzing animal categories separately, it was observed that in prepurbetal animals, the number of primordial follicles was positively correlated with the number of superficial antral follicles. However the numbers of primary and secondary follicles were negatively correlated with the number of superficial antral follicles. In the category of pregnant animals, the number of primary follicles was negatively correlated with the ovarian weight. According to ovary category analyzed, were observed significant correlation between the number of primordial, primary and secondary isolated follicles

In vitro development of bovine preantral follicles in monolayer of ovarian cells

O presente trabalho teve como objetivos determinar a influência de células ovarianas no desenvolvimento in vitro de folículos pré-antrais, avaliar a viabilidade das células ovarianas em monocamada e a influência do soro na manutenção de folículos pré-antrais in vitro. Folículos pré-antrais (FPs) e células ovarianas foram isolados de ovários de fetos bovinos, com idade entre 6 e 8 meses de gestação, oriundos de matadouro. Células ovarianas em monocamada foram cultivadas em meio TCM-199, e a viabilidade celular, após o cultivo na presença ou ausência de FSH, foi determinada com o corante vital azul de tripan. FPs foram distribuidos em quatro tratamentos e cultivados em TCM- 199 modificado, contendo soro de novilho castrado (SNC), SNC em monocamada de células ovarianas (MCO), MCO com FSH ou meio definido com álcool polivinílico (PVA) como macromolécula. A viabilidade celular não foi afetada em conseqüência da presença ou ausência de FSH. No entanto, houve um incremento significativo no tamanho dos FPs cultivados na presença de SNC, MCO e FSH (P<0,05), o que não foi observado nos demais tratamentos. A adição de FSH ao meio de cultivo não influencia a viabilidade de células ovarianas ao longo de 20 dias. Folículos pré-antrais, com área superficial entre 4000 e 8000µm2, mas não folículos com área superficial superiores a 8000µm2, crescem in vitro na presença de células ovarianas, independentemente da presença de FSH. _________________________________________________________________________________ ABSTRACTThe 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 8000µm2 but not with size greater than 8000µm2, grow on ovarian cells in vitro, independently of FSH

Fertilization and cleavage after protein kinase c activation during bovine oocyte maturation

A formação de pró-núcleos, clivagem e perfil protéico foram avaliados após a ativação da proteína quinase C (PQC) durante a maturação de complexos cumulus-oócito (CCOs) bovinos. Visando a determinar estes efeitos da PQ-C, 1936 CCOs foram distribuídos aleatoriamente em 4 tratamentos, sendo maturados na presença de PMA (100nM phorbol 12-myristate 13-acetate, ativador da PQ-C), de 4α-PDD (100nM de 4α- phorbol 12,13-didecanoetate, forbol éster que não ativa a PQ-C; controle forbol éster), de SVE (10% de soro de vaca em estro, controle positivo) e de um controle negativo constituído pelo meio de maturação básico para os demais tratamentos, com exceção do SVE, onde não foi adicionado o álcool polivinílico (PVA). Os CCOs foram mantidos na presença de PMA por 1, 4 e 7 horas, sendo após, transferidos para o meio básico de maturação até que se completassem 24 horas de cultivo. A estimulação da PQ-C, por esses períodos, não alterou a formação de prónúcleos, porém, diminuiu a percentagem de clivagem dos zigotos. Os resultados do grupo 4α-PDD comprovam que as alterações ocorridas no grupo PMA são devido à ativação da PQ-C e não ação direta do forbol éster na célula. A análise da composição protéica demonstrou uma banda de proteína adicional no grupo SVE ao redor de 74 kilo Daltons (kDa). Os resultados desse estudo, quando associados aos dados preliminares de Western blot, indicam a importância da PQ-C na regulação da maturação, fecundação e clivagem de embriões bovinos. ________________________________________________________________________________________________ ABSTRACTIn 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), 4α- PDD (100nM de 4α-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 4α-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

Study of preantral follicle population in situ and after mechanical isolation from caprine ovaries at different reproductive stages

The purposes of this study were to estimate the population of caprine preantral follicles, and to evaluate quantitatively and qualitatively the efficiency of a specific mechanical method for the isolation of preantral follicles from mixed breed goats at different reproductive stages. On average, 37,646±4277 preantral follicles were present in goat ovaries, and 13,631±2399 preantral follicles were obtained after isolation. The number of preantral follicles isolated or in situ was not significantly affected by the reproductive stage. The mean recovery rate per ovary ([number of isolated follicles/number of in situ follicles]×100) of isolated follicles was 36.2%. The distribution of follicles in situ was 67.8% primordial, 25.8% primary and 6.4% secondary; the respective distribution after isolation was 93.8%, 5.2% and 1.0%. In this study, many polyovular follicles were also observed, mainly in prepubertal goat ovaries. Histological analysis showed that few preantral follicles were atretic in situ (4.83%±0.35) or after the isolation procedure (4.67%±0.65) in the three reproductive stages. The percentage of atretic follicles was not affected either by the mechanical method or by the reproductive stage. It is concluded that a large number of preantral follicles can be successfully isolated mechanically, with a high recovery rate and a low rate of follicular atresia, irrespective of the reproductive stage of the caprine female