Epigenetics in genetic improvement and animal reproduction

Epigenetics can be defined as the study of heritable changes in genetic function not related to changes in the primary sequence of the DNA molecule. The principal epigenetic phenomena are DNA methylation; posttranslational changes in histone proteins, such as methylation, acetylation, phosphorylation, ubiquitination and others; some classes of non-coding RNA; and chromatin remodelers. Depending on the combination of these modifications, the chromatin assumes a configuration that is either more open (euchromatin) or more closed (heterochromatin), thus being related to the control of genic expression. Since these epigenetic patterns are susceptible to external factors, assisted reproductive techniques (ARTs) can affect them and consequently the regulation of genic expression, given that during the critical initial periods of development the gametes and embryos are subjected to the conditions of in vitro maturation, fecundation and culture. Furthermore, research has shown that external environmental factors during gestation can influence epigenetic patterns and thus, the health and performance of the progeny. Therefore, this information can be relevant to animal improvement. Finally, it is important that in the development and adaptation of new ARTs, epigenetic alterations of gametes and embryos be avoided as much as possible. In the context of livestock production, it is essential to elucidate if and how epigenetic patterns may be involved in resistance to diseases and productive traits

Characterization of seminal plasma proteins and its relationship with quality parameters of frozen semen in ram

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

Efeito do gene receptor de prolactina sobre características quantitativas de interesse econômico em suínos

The productivity and quality increase of the animal products is coming to be of big economic interest. The prolactin (PRL) is an essencial hormone for the reproductive sucess and its receptor (PRLR) has been detected in several tissues (Kelly et al., 1991). The PRLR gene has been recently mapped to pig chromosome 16 (Vincent et al., 1997).This research was aimed at analyzing the PRLR genotypical frequency in three different swine races, Landrace, Large White and Pietrain and correlate the genotypes with characteristics of economic interest. It was analyzed 124 animals. The DNA was extracted from the pig blood and submited to PCR-RFLP technique, to the genotypes determination of the prolactin receptor gene. The statistics analysis showed that the PRLR genotypes had an effect on the daily average weight of the Landrace race (p<0,0135). The averages of EDP (Expected Difference of Progeny) in daily average weight of the Landrace race were also different in relation to the genotypes (p<0.0610), confirming the real data analysis of daily average weight increase. Attended selection methods by markers, together with traditional seletion methods can be used to increase and accelerate the improvement of economic interest characteristics in pigs, while the prolactin receptor gene (PRLR) can be used as a molecular marker for the real daily average weight increase and its EDP.O aumento da produtividade e qualidade dos produtos animais vem se tornando de grande interesse econômico. A prolactina (PRL) é um hormônio essencial para o sucesso reprodutivo e seu receptor (RPRL) tem sido detectado em vários tecidos². O gene RPRL foi recentemente mapeado em suínos no cromossomo 16(6). Este trabalho teve como objetivo analisar a frequência genotípica do RPRL em três diferentes raças de suíno, Landrace, Large White e Pietrain e correlacionar os genótipos com características de interesse. Foram analisados um total de 124 animais. O DNA foi extraído de sangue total suíno e submetido a técnica de PCR-RFLP, para determinação do genótipo do gene do receptor da prolactina. As análises estatísticas mostraram que o genótipo RPRL teve efeito sobre peso médio diário na raça Landrace (p<0,0135). As médias de DEPGMD na raça Landrace também foram diferentes em relação ao genótipo (p< 0,0610), confirmando a análise dos dados reais de Ganho de Peso Médio Diário. Métodos de seleção assistida por marcadores, juntamente com métodos de seleção tradicional poderão ser utilizados para potencializar e acelerar o melhoramento de características de interesse econômico em suínos, onde o gene do receptor de prolactina (RPRL) poderá ser utilizado como um marcador molecular para o ganho de peso médio diário real e sua DEP

DETERMINAÇÃO DE POLIMORFISMOS NO GENE DO HORMÔNIO DO CRESCIMENTO EM TRÊS POPULAÇÕES DE SUÍNOS DETERMINATION OF POLYMORPHISMS IN THE GROWTH OF THE HORMONE GENE IN THREE POPULATIONS OF PIGS

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

DNA methylation and functional characterization of the XIST gene during in vitro early embryo development in cattle

XIST, in association with the shorter ncRNA RepA, are essential for the initiation of X chromosome inactivation (XCI) in mice. The molecular mechanisms controlling XIST and RepA expression are well characterized in that specie. However, little is known in livestock. We aimed to characterize the DNA methylation status along the 5’ portion of XIST and to characterize its transcriptional profile during early development in cattle. Three genomic regions of XIST named here as promoter, RepA and DMR1 had their DNA methylation status characterized in gametes and embryos. Expression profile of XIST was evaluated, including sense and antisense transcription. Oocytes showed higher levels of methylation than spermatozoa that was demethylated. DMR1 was hypermethylated throughout oogenesis. At the 8–16-cell embryo stage DMR1 was completed demethylated. Interestingly, RepA gain methylation during oocyte maturation and was demethylated at the blastocyst stage, later than DMR1. These results suggest that DMR1 and RepA are transient differentially methylated regions in cattle. XIST RNA was detected in matured oocytes and in single cells from the 2-cell to the morula stage, confirming the presence of maternal and embryonic transcripts. Sense and antisense transcripts were detected along the XIST in blastocyst. In silico analysis identified 63 novel transcript candidates at bovine XIST locus from both the plus and minus strands. Taking together these results improve our understanding of the molecular mechanisms involved in XCI initiation in cattle. This information may be useful for the improvement of assisted reproductive technologies in livestock considering that in vitro conditions may impair epigenetic reprogramming

Protocol for extraction of genomic DNA from swine solid tissues

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

Caracterização de proteínas do plasma seminal e sua relação com parâmetros de qualidade do sêmen criopreservado em ovinos Characterization of seminal plasma proteins and its relationship with quality parameters of frozen semen in ram

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.<br>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