Efeitos do tratamento crônico com isoflavonas da soja na saúde reprodutiva de coelhos machos

The aim of this study was to investigate the effects of chronic exposure to soy isoflavones concentrate on the morphology of reproductive organs, semen quality, puberty age, serum levels of testosterone and sexual behavior of male rabbits. With this purpose, pregnant female rabbits were randomly assigned to receive orally 2.5mg (ISF 2.5) or 10mg (ISF 10) of soy isoflavones/kg of body wt/day. The animals of control group were manipulated as the other groups and received placebo (corn starch). All the rabbits were maintained on a soy-and alfafa-free diet throughout the gestation and lactation. Their male offspring received the same treatments from weaning to 33 weeks of age. Chronic exposure to isoflavones did not induce statistically significant alteration in the age at puberty, semen volume, daily sperm output, sperm concentration, motility, vigor and abnormalities. Also, isoflavones exposure had no effects on serum testosterone levels or sexual behavior in any group. Histopathologic evaluation did not reveal alterations in the testis, epididymis, prostate and pro-prostate glands of the rabbits. Taken together, these results show that gestational, lactational and post-lactational exposure to soy isoflavones, in doses comparable to those found in soy-containing animal and human diets, has no adverse effects on the reproductive parameters of male rabbits

Ultrastructural and morphometric characterization of buffalo (Bubalus bubalis) ovarian preantral follicles

The main objective of the present study was to characterize buffalo preantral ovarian follicles. Parts of ovarian cortex, collected from postpubertal buffalo females that were having estrous cycles at regular intervals, were selected under stereomicroscopy and processed for optic and transmission electron microscopy. Primordial follicles were characterized as an oocyte encircled by one layer of flattened cells. The buffalo primordial follicle has a mean diameter of 35 μm and the oocyte diameter is 24.9 μm. The oocyte nucleus is relatively large and eccentric; and in the cytoplasm a large amount of mitochondria, vesicles and endoplasmic reticulum cistern, mainly of the smooth type is observed. The primordial follicles cells are rich in plasma membrane invaginations, which are observed within the cell and between the cell and the oocyte. The primary follicles (mean diameter of 41.8 μm) consist of an oocyte, with a medium diameter of 26.9 μm, surrounded by one layer of cubical granulosa cells. At this follicular stage, the beginning of zona pellucida deposition can also be seen in areas between the oocyte and follicular cells. The secondary follicles, which are surrounded by more than one layer of cubical cells, have a diameter of 53.3 μm, and the oocyte has a mean diameter of 29.4 μm. The ultrastructural analysis showed a large amount of coalescent vesicles, more evident in the oocyte periphery. The zona pellucida (ZP) is thicker at this stage and contains a large quantity of glycoproteins. In general, the ultrastructure of buffalo preantral follicles was similar to that of other mammalian species, but some differences were observed, which indicate species specific characteristics. The main differences observed were cytoplasmic vesicles quantity, mitochondria shape and inner content, ZP deposition and granulosa cell–oocyte junctions. In conclusion, the morphological differences described in this paper, could be responsible for some functional differences observed in Bubalus bubalis in vitro embryo production and follicular dynamics, when compared with Bos taurus or Bos indicus species

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

Does combining estradiol cypionate and GnRH for ovulation induction in recipient cows increase pregnancy rate after timed embryo transfer?

Estradiol cypionate (EC) or GnRH have been widely used for ovulation induction in timed embryo transfer (TET). EC administration increases the proportion of cows that show estrus, whereas GnRH promotes more synchronized ovulations. The aim of the present study was to evaluate the potential beneficial effects of combining EC and GnRH in TET. In experiment 1, no difference was observed on serum progesterone concentrations on Day 6 and 13 after GnRH treatment between GnRH and EC+GnRH groups. In experiment 2, pregnancy per embryo transfer (P/ET) did not differ (p = 0.69) between GnRH (62.8%) and EC+GnRH (58.7%) groups. In conclusion, combining EC and GnRH for ovulation induction does not increase progesterone secretion and pregnancy rate after TET in cattle

Efeitos de lipopolissacarídeos na ativação e apoptose de folículos ovarianos primordiais em novilhas

The objective of the present study was to evaluate the effect of lipopolysaccharide (LPS) administration on activation and apoptosis of primordial follicles. There was no difference in the total number of follicles as well as in the different types of follicles. Furthermore, the LPS challenge didn’t modulate the expression of genes related with ovarian reserve (HAM), oocyte survival (Survivin), activation rate (Pten, KIT, KITL1, KITL2, AKT1, SIRT1), and follicular abnormalities. Therefore, the LPS exposure with 24h interval had no effect on activation rate and primordial follicles abnormalities, and also had no effect on expression of anti-apoptotic genes and genes related with ovarian reserve, oocyte survival, activation rate, and primordial follicles abnormalities.O objetivo do presente estudo foi avaliar o efeito da administração de lipopolissacarídeo (LPS) na ativação e a apoptose de folículos primordiais. Dez novilhas saudáveis (Bos taurus taurus), com idade média de 14 meses, alojadas em sistema de confinamento e alimentadas com TMR, foram utilizadas neste experimento. Os animais foram distribuídos aleatoriamente em dois grupos: grupo LPS (LPS; n = 5), que recebeu duas injeções intravenosas de 0,5μg/kg de peso corporal de lipopolissacarídeo (Sigma Aldrich®) diluído em 2mL de solução salina (0,9% de NaCl), com intervalo de 24h; e grupo controle (CTR; n = 5), que recebeu duas injeções intravenosas de 2mL de solução salina (0,9% de NaCl), com intervalo de 24h. A primeira injeção de LPS foi realizada no d 1, e no d 5 os animais foram abatidos, os ovários foram pesados e as amostras dos ovários foram coletadas para avaliação histológica e molecular. Não houve diferença no número total de folículos, bem como nos diferentes tipos de folículos. Além disso, o desafio com LPS não modulou a expressão de genes relacionados à reserva ovariana (HAM), à sobrevivência oocitária (Survivin), à taxa de ativação (Pten, KIT, KITL1, KITL2, AKT1, SIRT1) e às anormalidades foliculares. Portanto, a exposição ao LPS com intervalo de 24h não teve efeito sobre a taxa de ativação e as anormalidades dos folículos primordiais, bem como não teve efeito sobre a expressão de genes antiapoptóticos e de genes relacionados com a reserva ovariana, a sobrevivência oocitária, a taxa de ativação e as anormalidades dos folículos primordiais

Foliculogênese, maturação nuclear e citoplasmática de oócitos bubalinos - Bubalus bubalis : uma análise estrutural

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2008.Os bubalinos apresentam grande potencial para serem utilizados tanto em propriedades com baixa como alta aplicação de tecnologia de produção, por ser uma espécie rústica e que produz principalmente leite com alto valor de mercado. Porém, devido às características inerentes a espécie, a aplicação de biotécnicas da reprodução que visam multiplicar animais de alto valor genético não tem demonstrado bons resultados. Assim sendo, o presente trabalho visou conhecer as características ultra-estruturais do desenvolvimento do gameta feminino da espécie. Para isto foram utilizados ovários e complexos cumulus-oócito (CCOs) de búfalas, devidamente processados para microscopia eletrônica de transmissão e microscopia óptica. Foi observado que os gametas e folículos pré-antrais dos bubalinos são menores que os bovinos e possuem conteúdo lipídico (que se acumula gradativamente) bastante superior a esses. Os bubalinos apresentaram menor número de folículos pré-antrais nos ovários, além disso, houve uma predominância de folículos classificados como primários em detrimento dos primordiais e secundários. Outro fator que pode impactar a aplicação de biotecnologias como a punção folicular, bem como a biosegurança é o fato da zona pelúcida dos oócitos bubalinos ser aparentemente mais frágil e conter mais fendas que a dos bovinos. A maturação citoplasmática dos bubalinos foi caracterizada pela migração de organelas para a região cortical do oócito. Somando-se a essa característica o fato que houve aumento do espaço perivitelínico e a diminuição gradativa do contato entre as células do cumulus oophorus e o oócito conforme a maturação evolui. Por fim, o presente trabalho confirmou que a possível alteração química sofrida pelos lipídeos durante o processo de maturação in vitro ocasiona uma modificação de seu aspecto ultra-estrutural no início do período de maturação. Ressalta-se o fato que também foi comprovado que, com as devidas modificações no sistema de produção in vitro de embriões, é possível obter-se embriões viáveis de búfalas, mesmo na contra estação. __________________________________________________________________________________________ ABSTRACTThe rusticity and milk production with high market value make possible to Buffalos be bred in low or highly technified production system. On the other way, some species’ inherent characteristics make difficult to apply reproductive biotechniques to multiply animals (mainly females) with high genetic potential. Considering this, the present research established the ultrastructural characteristics of female gamete development. To achieve the objective, buffalo cumulus-oocyte complexes (COCs) and ovaries were properly processed to light and transmission electron microscopy. The buffalo oocyte and preantral follicles were smaller than bovine ones and have higher lipid content (progressive accumulation). The specie also shows a lower number of preantral follicles in the ovaries, as well as, the number of primary is superior to primordial and secondary follicles. Other described factors which affects the application of ovum pick-up technique, other than the biosecurity, is the apparently fragile aspect of zona pellucida and the presence of fissures in its surface. Buffalo oocyte cytoplasmic maturation was characterized by organelle migration to cortical region. In addition, as the maturation progress, the perivetilline space increased in size and it is possible to observe a progressive reduction on granulosa cells and oocyte connections. In conclusion, the present research confirmed that the chemical modifications possibly suffered by oocyte lipids during in vitro maturation period, cause an alteration in its ultrastructural aspect. It is also important to note that is possible, with some modifications on in vitro embryo production systems, to produce buffalo embryo in seasonal anoestrous

Primordial Follicle Activation In The Ovary Of Ames Dwarf Mice

Background: The insulin receptor substrate 1 (IRS1), phosphoinositide 3-kinase (Pi3k), protein kinase B (Akt1), Forkhead Box O3a (FOXO3a) pathway is directly involved in aging and ovarian activation of follicle growth. Therefore, the aim of this work was to measure the expression of genes related to the ovarian pathway for activation of primordial follicles and FOXO3a protein phosphorylation between young and old female Ames dwarf (df/df) and normal (N) mice. Methods: For this study ovaries from N (n = 10) and df/df (n = 10) female mice were collected at 5-6 months of age and at 21-22 months of age. For immunohistochemistry ovaries from 12 month-old and df/df mice were used. Results: The expression of Irs1, Pi3k, Akt1, mammalian target of rapamycin (Mtor), suppressor of cytokine signaling -2 (Socs2), Socs3 was lower (P \u3c 0.05) in older than younger N mice and not different (P \u3e 0.05) between young and old df/df mice. The expression of Foxo3a was also lower (P \u3c 0.05) in old than younger N and df/df mice and was higher (P \u3c 0.05) in old df/df than N mice. Expression of Amh was lower (P \u3c 0.05) in old than young N and df/df mice and was higher (P = 0.0009) in df/df than N mice. Imunnostaining for p-FOXO3 was lower in df/df than N mice (P \u3c 0.001), although FOXO3 immunostaining was not different (P \u3e 0.05) between df/df and N mice. Conclusions: In sum, the present study indicates that lower expression of Irs1, Socs2, Socs3, Akt1, Pi3k, Mtor and Foxo3a mRNA in the ovaries of older mice of both genotypes is associated to a reduced ovarian activity revealed by lower expression of Amh mRNA. At the same time, ovaries of old df/df mice maintained higher expression of Foxo3a mRNA, which was associated to higher ovarian activity. We have shown that df/df females have a lower level of p-FOXO3 in oocytes from primordial/primary follicles, an important activator of follicular growth. Therefore, this study strongly indicates that Prop1df mutation causes delayed ovarian aging