Abstract The establishment of epigenetic marks during the reprogramming window is susceptible to environmental influences, and stimuli during this critical stage can cause altered DNA methylation in offspring. In a previous study, we found that low levels of sulphur and cobalt (low S/Co) in the diet offered to oocyte donors altered the DNA methylome of bovine embryos. However, due to the extensive epigenetic reprogramming that occurs during embryogenesis, we hypothesized that the different methylation regions (DMRs) identified in the blastocysts may not maintain in adulthood. Here, we aimed to characterize DMRs previously identified in embryos, in the blood and sperm of adult progenies of two groups of heifers (low S/Co and control). We used six bulls and characterized the DNA methylation levels of KDM2A, KDM5A, KMT2D, and DOT1L genes. Our results showed that all DMRs analysed in both groups and tissues were hypermethylated unlike that noticed in the embryonic methylome profiles. These results suggest that embryo DMRs were reprogrammed during the final stages of de novo methylation during embryogenesis or later in development. Therefore, due to the highly dynamic epigenetic state during early embryonic development, we suggest that is essential to validate the DMRs found in embryos in adult individuals.

Os objetivos deste trabalho foram analisar o perfil proteico do plasma seminal ovino e identificar proteínas relacionadas com a congelabilidade do sêmen que possam ser utilizadas como marcadores para essa característica. Foram utilizados os ejaculados de cinco reprodutores, nos quais foram realizadas avaliações espermáticas e dos quais os plasmas seminais obtidos por centrifugação foram submetidos à eletroforese bidimensional em gel de poliacrilamida. Foram identificados 92 spots, considerando todos os animais analisados. A avaliação dos dados obtidos evidenciou variações significativas nos resultados das análises do sêmen dos animais e uma variabilidade no perfil proteico no plasma seminal dos carneiros. As proteínas 03 (7,9kDa; pI 6,35), 23 (13,6kDa; pI 5,01) e 31 (21,4kDa; pI 4,75) se destacaram, por apresentarem maior expressão e relações com as características espermáticas. Sugere-se que mais estudos sejam realizados para verificar se as proteínas 03, 23 e 31 podem ser utilizadas como marcadores da capacidade criopreservadora do sêmen.

The objective of this study was to analyze the protein profile of ram seminal plasma and to identify proteins associated with semen freezability, which could be used as marker for predicting this feature. Semen from five rams was used. The sperm analysis was held and the seminal plasma obtained by centrifugation was submitted to two-dimensional electrophoresis using acrylamide gel. Ninety two spots were identified considering the analyzed animals. The results showed a significant variation among sperm analysis of the animals and variability in the protein profile of the seminal plasma of the rams. The proteins 03 (7.9kDa; pI 6.35), 23 (13.6kDa; pI 5.01) e 31 (21.4kDa; pI 4.75) stood out because they showed higher expression and because of its relationship with the sperm characteristics. It is suggested more studies to verify if proteins 03, 23 and 31 could be used as markers of semen freezability.

Fourty-six non-castrated, halothane-free, male Landrace pigs were genotyped by PCR-RFLP for the Rsa I polymorphism in the PIT1 gene and classified into AA and AB genotypes. Total RNA was extracted from the pituitaries and the relative quantities of growth hormone (GH) mRNA were determined by semi-quantitative RT-PCR. Pigs with the AB genotype had higher levels of GH mRNA than those with the AA genotype (p = 0.034; Kruskal-Wallis test). This result suggests that the Rsa I polymorphism may be involved in Pit-1 protein expression or function, which in turn may influence GH transcription and expression. Thus, the Rsa I PIT1 gene polymorphism in this pig line may be used as a molecular marker to identify higher GH expression and possibly select for carcass and performance traits affected by GH.

After the advent of the genome projects, followed by the discovery of DNA polymorphisms, basic understanding of gene expression is the next focus to explain the association between polymorphisms and the level of gene expression, as well as to demonstrate the interaction among genes. Among the various techniques for the investigation of transcriptional profiling involving patterns of gene expression, quantitative PCR is the simplest analytical laboratory technique. The objective of this work was to analyze two strategies of a competitive PCR technique for the quantification of the pig growth hormone (GH) gene expression. A pair of primers was designed targeting exons 3 and 5, and two competitive PCR strategies were performed, one utilizing a specific amplicon as a competitor, and the other utilizing a low-stringency PCR amplicon as a competitor. The latter strategy proved to be easier and more efficient, offering an accessible tool that can be used in any kind of competitive reaction, facilitating the study of gene expression patterns for both genetics and diagnostics of infectious diseases.

Molecular diagnostics are performed by using DNA from different body tissues. However, it is necessary to obtain genomic DNA of good quality. Due to the impossibility of collecting blood from slaughtered animals, DNA extraction from solid tissues is necessary. The objective of this study was to describe a protocol of DNA extraction from swine skin, adipose, brain, liver, kidney and muscle tissues. We obtained high molecular weight DNA of good quality, shown by agarose gel and amplification of two DNA fragments, 605bp and 891pb, by PCR. Spectrophotometric analysis of DNA concentration showed variation among the DNA from different tissues, with the liver and adipose tissues presenting the greatest and the smallest concentration, respectively. The described protocol has proven to be advantageous due to its simplicity, quickness, affordable reagents and absence of phenol, resulting in a high molecular weight DNA of good quality from several tissues.

Dois polimorfismos (GHC e GHD) no gene que codifica para o hormônio do crescimento foram determinados em um total de 96 animais de três raças de suínos (Pietrain, Large White e Landrace), através da PCR-RFLP. As freqüências alélicas observadas para GHC foram C1 0,42, C2 0,0, C3 0,06 e C4 0,52 para Landrace; C1 0,0, C2 0,03, C3 0,14 e C4 0,83 para Large White e C1 0,02, C2 0,25, C3 0,28 e C4 0,45 para Pietrain. Para GHD, as freqüências alélicas observadas foram D1 0,69 e D2 0,31 para Landrace; D1 0,25 e D2 0,75 para Large White e D1 0,72 e D2 0,28 para Pietrain.

Two polymorphisms (GHC and GHD) in the growth hormone gene were evaluated in the ninety-six (96) animals of three breeds of pigs (Pietrain, Large White, and Landrace), through PCR-RFLP. Allele frequencies observed for the GHC polymorphism were: C1 0.42, C2 0.0, C3 0.06 and C4 0.52 for Landrace; C1 0.0, C2 0.03, C3 0.14 and C4 0.83 for Large White and C1 0.02, C2 0.25, C3 0.28 and C4 0.45 for Pietrain. The GHD polymorphism presented the following allele frequencies:. D1 0.69 and D2 0.31 for Landrace; D1 0.25 and D2 0.75 for Large White and D1 0.72 and D2 0.28 for Pietrain.