Validação comparativa utilizando PCR quantitativo em tempo real (qPCR) e PCR convencional de sêmen bovino centrifugado em gradiente de densidade contínuo / Comparative validation using quantitative real-time PCR (qPCR) and conventional PCR of bovine semen centrifuged in continuous density gradient

The objective of the present study was to determine the sperm enrichment with X-bearing spermatozoa, after one centrifugation in a Percoll or OptiPrep continuous density gradient, using quantitative real-time polymerase chain reaction (qPCR) of sperm DNA and resultant in vitro-produced bovine embryos by PCR. Frozen/thawed sperm was layered on density gradients and the tubes were centrifuged. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Cleavage and blastocyst rates were determined through in vitro production of embryos and PCR was performed to identify the embryos' genetic sex. A difference in blastocyst rate was found in the Percoll treatment compared to OptiPrep (P<0.05). The percentage of female embryos in the Percoll and OptiPrep groups was 62.0 percent and 47.1 percent, respectively. These results were confirmed by qPCR of spermatozoa DNA and underestimation was seen only in the Percoll group. It was possible to sexing sperm using simple approach.

O objetivo do presente estudo foi determinar o enriquecimento de espermatozoides portadores do cromossomo X após a centrifugação em gradiente de densidade contínuo de Percoll ou OptiPrep, utilizando reação em cadeia da polimerase quantitativa em tempo real (qPCR) do DNA do espermatozoide e dos embriões bovinos produzidos in vitro resultantes pela PCR convencional. Espermatozoides descongelado foram depositados em gradientes de densidade e os tubos foram centrifugados. Os sobrenadantes foram gentilmente aspirados e os espermatozoides recuperados do fundo dos tubos. As taxas de clivagem e de blastocisto foram determinadas pela produção in vitro de embriões e a PCR foi realizada para a identificação genética do sexo dos embriões. Verificou-se diferença na taxa de blastocistos entre os grupos Percoll e OptiPrep (P<0,05). A porcentagem de embriões de fêmeas nos grupos Percoll e OptiPrep foi de 62,0 por cento e 47,1 por cento, respectivamente. Estes resultados foram confirmados pela qPCR do DNA de espermatozoides e uma subestimação foi observada no grupo do gradiente de densidade de Percoll. Foi possível a sexagem de espermatozoides utilizando uma metodologia simples.

Fertility Rates of Ewes Treated with Medroxyprogesterone and Injected with Equine Chorionic Gonadotropin plus Human Chorionic Gonadotropin in Anoestrous Season.

The aim of the present paper was to investigate the efficiency of the equine chorionic gonadotropin (eCG) plus human chorionic gonadotropin (hCG) associated with medroxyprogesterone acetate (MAP) to estrous ewes synchronization. Ninety Texel ewes were investigated during seasonal anoestrous. The ewes received intravaginal sponges containing MAP (60 mg) for nine days. At the time of sponges' withdrawal, the ewes were divided into three groups (G): (1) receiving 2 mL of saline i.m. (n = 30), (2) receiving eCG 400 IU i.m. (n = 30), and (3) receiving eCG 400 IU plus hCG 200 IU i.m. (n = 30). Twelve h after sponges' removal, teaser rams were used to estrus check and remained with the ewes for 96 h. The artificial insemination was made with fresh semen 10 h after estrus detection. The effect of the treatment was not significant for the estrous rates among the groups: 73%, 90%, and 86%, respectively. The main effect was observed in the pregnancy and lambing rates among the groups: 70%, 86%, 56%, and 80%, 120%, 56%, respectively. Based on these results from our study, the use of the MAP-eCG is the best choice to improve the fertility rate on ewes.