DETERMINAÇÃO DO SEXO DE EMBRIÕES BOVINOS PRODUZIDOS IN VITRO: UMA REVISÃO DE MÉTODOS COM ÊNFASE PARA A PCR

The development of new animal reproduction biotechniques has been provided the obtaining of genetic improvement in cattle herds around the world. Recently, the ascension of the in vitro production (IVP) of bovine embryos provided important development in the technology of bovine embryos. Although being a consolidated technique, some studies show disproportion between the sexes of calves born by means of IVP, being the percentage of males higher than the theoretically expected rate of 50% for each sex. This inconvenient opened the perspective for the development of methods for sex determination for the embryos before the transference, once the production of females is desirable in economic terms. This revision will approach the advantages and disadvantages of the invasive and non-invasive methods developed for the sex determination in bovine embryos, with emphasis for PCR assay that is the technique that displays the best results.O desenvolvimento de novas biotécnicas em reprodução animal tem proporcionado incrementos significativos no padrão genético de bovinos em todo o mundo. A produção in vitro (PIV) de embriões bovinos ocasionou um salto de qualidade na tecnologia da produção de embriões. Embora a PIV seja uma técnica consolidada, alguns estudos têm demonstrado uma desproporção no sexo de embriões nascidos, onde a porcentagem de embriões machos tem sido maior do que a teoricamente esperada de 50% para cada um dos sexos. Esse inconveniente abriu a perspectiva para o desenvolvimento de métodos para a determinação do sexo de embriões bovinos antes da transferência, uma vez que os produtos do sexo feminino são economicamente mais valorizados. A presente revisão abordará as vantagens e desvantagens dos principais métodos invasivos e não-invasivos, avaliados quanto ao seu potencial de determinação do sexo de embriões bovinos, com ênfase para a PCR que é a técnica que tem apresentado os melhores resultados

Effect Of Fixative Type And Fixation Time On The Morphology Of Equine Preantral Ovarian Follicles

The aim of this study was to investigate the efficacy of the tissue fixatives Bouin, Carnoy and 10% Formaldehyde in equine ovarian fragments. Ovaries (n=4) from mares of mixed breeds were obtained at a local slaughterhouse and transported at 20 oC in a thermo container. Immediately after collection, the ovaries were washed with a modified PBS solution (CultilabR, Campinas-SP, Brazil) and divided into nine fragments with approximately 5x5x1 mm, removed from the parenchyma of each ovary. The ovarian fragments were then immersed in three different fixatives, Bouin (B) Carnoy (C) or 10% Formaldehyde (F) for 6, 12 or 24 hours. Each fragment was individually immersed in a 20 mL tube containing 20 times the volume of fixative solution. After this period, the fragments were held in 70% ethanol for 24 hours. Each procedure was performed in four replicates. For histological analysis, the specimens were dehydrated in increasing concentrations of alcohol, submitted to diaphanization in xylol and embedded in paraffin. Serial sections of 5 'Êm were made with the use of a rotating microtome (LeicaR type, Wetzlar, Germany), followed by slide mounting and staining with periodic acid-Schiff (PAS) and hematoxylin. A total of 540 slides with 1,620 sections were evaluated, which contained 465 preantral follicles that were classified as normal or degenerated. Follicles were considered as degenerated when presented at least one of the following aspects: cytoplasm retraction, pyknotic nucleus, cytoplasmic vacuoles, displacement of granulosa cells and/or disruption of the basal membrane. A logistic regression test was used for statistical analysis, and differences were considered significant when P<0.05. The Carnoy fixative, when used for 24 hours, provided the best conditions of morphological integrity (53.3%; 32/60) compared to all others, and the use of Boiun for 24 hours was considered the worst treatment (19.1%; 9/47). The other treatments lead to the following results: C12h 50% (30/60), C6 H 40% (24/60), F24h 37.8% (17/45), F12h 35.1% (13/37), F6h 32% (16/50), B12h 30.5% (18/59) and B6h 24.4% (11/45). Therefore, we suggest that fixation of equine ovarian tissue with Carnoy for 24 hours is the most suitable protocol for morphological preservation of pre-antral follicles.37124325

Association of recombinant bovine somatotropin (rBST) with equine chorionic gonadotropin (eCG) on antral follicle count and oocyte production in Holstein and Tabapuã heifers

The objective of this study was to investigate whether the use of rbST and eCG prior to ultrasound-guided follicular aspiration (OPU) improves oocyte yield and quality in Tabapuã and Holstein heifers. The study was conducted in two phases, 20 days apart, in a change-over design. The dominant follicle was ablated two days (D-2) before two treatments: stimulation (6 Holstein and 8 Tabapuã), 500 mg of rbST (Boostin®) on D0 and 500 IU of eCG (Folligon) on D2; and control (6 Holstein and 8 Tabapuã), in which heifers received injections of the excipient. Heifers were aspirated on D4. Oocytes were subjected to a well established commercial in vitro embryo production protocol (Vitrogen®) and embryos were evaluated seven days after fertilization. There was an effect from the interaction of treatment and breed, so that hormonal stimulation increased antral follicle count (2-8 mm) in Tabapuã (29.9±2.6 to 41.4±2.6), but not in Holstein heifers (14.4±2.6 to 15.5±2.6). Tabapuã heifers had higher mean antral follicle count than Holsteins (35.6±1.8 vs. 15.0±2.1). The number of viable oocytes was not increased by stimulation in Tabapuã (from 4.7±1.0 to 5.2±1.1 in control and simulation, respectively) or in Holstein heifers (1.3±1.9 to 2.0±1.6 in control and simulation, respectively). There was no difference in the percentage of heifers with more than five viable oocytes in the group treated (33 vs 27%). The number of blastocysts was not affected by treatment (1.75 vs. 1.00 in hormonal stimulation and control, respectively). The increase in antral follicle count in the stimulated Tabapuã heifers did not reflect upon oocyte yield. The differential breed response to the hormonal treatment underscores the need for additional tests, especially for the Holstein breed, in order to enhance OPU efficiency

IMPACTO DO HERPESVÍRUS BOVINO 1 E DO VÍRUS DA DIARRÉIA VIRAL BOVINA NA TRANSFERÊNCIA DE EMBRIÕES

A transferência de embriões bovinos é uma biotécnica da reprodução animal, amplamente difundida no Brasil, que tem possibilitado a produção de embriões com alto potencial genético. Considerando a difusão da técnica e o grande número de embriões gerados e distribuídos, no aspecto sanitário, devem ser avaliados os riscos da disseminação de agentes infecciosos da esfera reprodutiva. O herpesvírus bovino 1, responsável pela rinotraqueíte infecciosa bovina, e o vírus da diarréia viral bovina, causador da diarréia viral bovina, são dois vírus capazes de infectar embriões por meio das fêmeas doadoras e/ou do material biológico relacionado à técnica.The bovine embryo transfer is an animal reproduction biotechnique widely diffused in Brazil that has been allowing the production of embryos with high genetic potential. Considering the technique diffusion and the high number of produced and distributed embryos, in the sanitary aspect its use in wide scale can contribute with risks of infectious diseases dissemination. Bovine herpesvirus 1 and bovine viral diarrhea virus, respectively, the etiological agents of the infectious bovine rhinotracheitis and bovine viral diarrhea are two potential viruses able to infect embryos through donator females and/or biological materials related to the technique