Priscila Vendramini Silva

Expression profile of genes associated with mastitis in dairy cattle

In order to characterize the expression of genes associated with immune response mechanisms to mastitis, we quantified the relative expression of the IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ and TNF- α genes in milk cells of healthy cows and cows with clinical mastitis. Total RNA was extracted from milk cells of six Black and White Holstein (BW) cows and six Gyr cows, including three animals with and three without mastitis per breed. Gene expression was analyzed by real-time PCR. IL-10 gene expression was higher in the group of BW and Gyr cows with mastitis compared to animals free of infection from both breeds (p < 0.05). It was also higher in BW Holstein animals with clinical mastitis (p < 0.001), but it was not significant when Gyr cows with and without mastitis were compared (0.05 < p < 0.10). Among healthy cows, BW Holstein animals tended to present a higher expression of all genes studied, with a significant difference for the IL-2 and IFN- γ genes (p < 0.001). For animals with mastitis no significant difference in gene expression was observed between the two breeds. These findings suggest that animals with mastitis develop a preferentially cell-mediated immune response. Further studies including larger samples are necessary to better characterize the gene expression profile in cows with mastitis.

Mapping of quantitative trait loci and confirmation of the FAT1 region on chromosome 4 in an F2 population of pigs

The objective was to map QTL on porcine chromosome 4 and to associate them with carcass and internal organ traits in an F2 population. The F1 population was produced by outbreed crossing, using two native Brazilian breed Piau boars and 18 commercial sows. A total of 617 F2 animals issued from 11 F1 boars and 54 F1 sows were typed for a total of five microsatellite markers. The data were analyzed by multiple regressions developed for the analysis of crosses between outbred lines, using the QTL Express software. Significant evidence for QTL was found for pig chromosome 4 regarding carcass and internal organ traits. All QTL were detected in the same region of the chromosome, designated FAT1.

Follicular dynamics and gene expression in granulosa cells, corpora lutea and oocytes from gilts of breeds with low and high ovulation rates.

Follicular dynamics and the expression of candidate genes using real-time polymerase chain reaction (PCR) were compared during the oestrous cycle of pig breeds with high (commercial line; n=24) and low (local Brazilian Piau; n=21) ovulation rates and prolificacy. Gilts were killed on Days 0, 4, 10 and 18 of the oestrous cycle and visible ovarian follicles were classified by follicular diameter. Recovered cumulus-oocyte complexes were classified as normal or atretic and frozen in liquid nitrogen until RNA extraction. Low ovulation rates and/or prolificacy in Piau gilts was associated with a different pattern of follicle development, with lower numbers of small follicles on Day 18, fewer large follicles on Days 0 and 18 (P≤0.05) and a higher proportion of atretic follicles on Days 0 and 18 (P≤0.05). Compared with commercial line gilts, less-prolific Piau gilts exhibited higher expression of apoptotic genes during luteolysis (CASP3 and FASL; P≤0.05), decreased expression of TGFBR2 and BAX mRNA in the corpus luteum (P≤0.05), higher expression of apoptotic genes (FAS, BCL2 and CASP8; P≤0.05) in granulosa cells and a greater abundance (P≤0.05) of genes controlling oocyte-secreted factors (GDF9, BMP15 and BMP6), suggesting underlying mechanisms controlling differences in follicular development, ovulation rate and inherent prolificacy in this pig breed.

Association between insulin-like growth factor I (IGF-I) microsatellite polymorphisms and important economic traits in pigs

Investigou-se neste trabalho a associacao entre o marcador microssatelite IGF-I em uma populacao F2 (N=459) gerada pelo acasalamento entre suinos nativos brasileiros e femeas comerciais com caracteristicas de desempenho, cortes de carcaca e qualidade da carne. A analise de associacao foi feita por meio de um modelo que incluiu genotipo, sexo e grupo como efeitos fixos e pais como efeito aleatorio. Os genotipos do IGF-I apresentaram associacao significativa com nove caracteristicas quantitativas, resultados que corroboram analises previas de QTL obtidas para essa regiao cromossomica em suinos. Efeitos aditivos de dominância, assim como a interacao genotipo × sexo, foram estimados e estao descritos neste trabalho. De acordo com os resultados obtidos, este marcador sera util nas analises de QTL na populacao analisada.

Association between leptin gene single nucleotide polymorphisms and carcass traits in pigs

Foi investigada a associacao entre os polimorfismos de base unica (SNPs) T2411C e T3266G da leptina e as caracteristicas de carcaca em suinos F2 procedentes do cruzamento de machos da raca Piau com matrizes comercias de composicao genetica Landrace, Large White e Pietrain. As analises de associacao foram feitas usando um modelo estatistico que incluia genotipo, sexo e lote como efeitos fixos, e pai e erro como efeitos aleatorios. O SNP T2411C esteve associado ao peso da copa sem pele e sem gordura (BSW), a espessura de gordura subcutânea na altura da ultima costela a 6,5 cm da linha media (P2), ao peso da paleta sem pele e sem gordura e ao peso do filezinho (SLW). O SNP T3266G foi associado a idade de abate, peso do bacon, BSW, espessura da gordura subcutânea entre a ultima e a penultima vertebra lombar, na linha media, espessura da gordura subcutânea na altura da ultima costela na linha media, P2 e peso da costela. Os genotipos combinados para ambos os SNP estiveram associados a P2, peso total da copa, rendimento de carcaca e peso do filezinho. Os SNPs analisados tem potencial para ser explorados como marcadores para composicao de carcaca em suinos.

Análise filogenética do gene da miogenina

The myogenin gene, a member of the MyoD gene family, is a regulator of the myogenesis that takes place during the embryonic development. The objective of this study was to perform a phylogenic analysis of the myogenin gene to study its evolutionary history in the domestic species that have the sequencing data deposited in the Genbank. One common method to detect a gene evolution is made by comparing the ratio of nonsynonymous nucleotide substitution by the ratio of synonymous substitutions. Values greater than one (1) means that the gene has gone through changes that made the organism more adapted to the environment. The phylogenetic trees were obtained by maximum likelihood and the synonymous and nonsynonymous substitution rates were analyzed by the parsimony method. The results point out that probably the gene suffered an adaptive evolution in the Ruminantia group, Bos Taurus and Ovis aries, after these species diverged from their common ancestral. In the other species, the gene seems to be evolved in a conservative way.

Differential expression of genes in follicular cells of swines.

The main purpose of the present study was to identify for candidate genes related to ovulation in swines. To do so, it was investigated in ovarian follicular cells through quantitative real-time PCR the differential expression of the following genes: steroidogenic acute regulator (STAR), GATA-binding protein 4 (GATA), prostaglandin F2α (PGF2α), progesterone receptor (P4R), follicle-stimulating hormone receptor (FSHR), and cytochrome P450 aromatase (CYP19). These genes encode hormone receptors (FSHR and P4R), hormone (PGF2α), steroidogenic proteins (STAR and CYP19) and transcription factor (GATA). Folicular cells were collected from sows with high and low number of piglets/litters during the follicular phase of the estrus cycle. There was difference in transcript abundance among low and high prolific sows for the STAR, GATA, PGF2α, P4R and CYP19 genes. For the FSHR gene, the fold change was not considered to be significantly different. Because in the present study only the transcript level of the above mentioned genes was analyzed, no inference can be made regarded to protein translation or activity. Therefore, gene sequence trials and other functional studies will be necessary to complement the present results, allowing a better understanding on biological complexity of these genes and their use as markers for prolificity in swines.

250 EXPRESSION PATTERN OF p53 mRNA DURING THE ESTROUS CYCLE IN SWINE

During each estrous cycle, more than 99% of the ovarian follicles undergo a degenerative process known as atresia. The p53 is an antiproliferative transcription factor that enhances the transcription rate of important genes in the apoptotic pathway. The aim of the current study was to investigate the pattern of p53 mRNA expression during estrous cycle in the pig ovary by real-time PCR technique. Sixteen prepubertal gilts (Landrace × Large White × Pietrain) were obtained from the pig farm at the Universidade Federal de Vicosa (Vicosa, MG, Brazil). The estrous cycle was synchronized with P.G. 600® (Intervet/Schering-Plough Animal Health, Millsboro, DE, USA; 400 IU eCG and 200 IU hCG). The onset of estrus was checked twice a day using a mature boar. The gilts, n = 4 per group, were slaughtered on Days 0, 6, 12, and 18 of estrous cycle. Granulosa cells from follicles were collected by vacuum aspiration and washed in PBS by centrifugation at 5.000 × g for 6 min, and the RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The ovarian cortex was stored in RNAlater (Ambion, Austin, TX, USA) and frozen at -80°C. Its RNA was extracted from 30 mg using the same Kit. For each animal in each stage, a pool of equivalent amounts of RNA from granulosa cells and ovary cortex was reverse transcribed with SuperScript III/RNaseOUT Enzyme Mix (Invitrogen Life Technologies, Carlsbad, CA, USA) to evaluate gene expression for the ovary as a whole. Quantitative real-time PCR was performed using SYBR green fluorescent detection system on ABI Prism 7300 Sequence Detection Systems (Applied Biosystems, Foster City, CA, USA). The primers were designed from swine sequences available at GenBank. The linearity of amplification for p53 mRNA was similar to the endogenous control gene, glyceraldehyde-3-phosphate dehydrogenase. Reactions were performed using 200 nM primer and 100 ng of the cDNA per reaction for both genes. The thermal cycling conditions consisted of 40 cycles of 30 s of melting at 95°C followed by 30 s of annealing and extension at 60°C. After amplification, a melting curve analysis was performed to validate the absence of non-specific products. Gene expression data were presented using the 2ACt method (Livak and Schimittgen 2001). The results of gene expression were analyzed using linear regression after transformation ln(2ACt + 1) as the dependent variable and days of estrous cycle as independent variables using the general linear model procedure (SAS Institute, Inc., Cary, NC, USA). The mRNA expression was not affected by days of estrous cycle (P = 0.86). In rats, the expression of p53 mRNA in granulosa cells has been already described. Despite the fact that no difference has been found during the estrous cycle in this study, the expression of this messenger in the pig ovary seems to be undescribed until now. In the future, for a better understanding of p53 regulation in the pig, gene expression analysis in different follicle sizes and physiological status will be presented by our group to examine the expression patterns of this gene, as well as other related ones, included in this apoptotic pathway.