Robson Carlos Antunes

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.

Association of the estrogen receptor gene Pvu II restriction polymorphism with expected progeny differences for reproductive and performance traits in swine herds in Brazil

Estrogen has an important function in swine reproduction and growth. A Pvu II restriction enzyme polymorphism has been proven to be an important genetic variation in the estrogen receptor gene (ESR) and may be considered as a candidate gene for use in pig production but there is no data regarding the prevalence of this polymorphism in the Brazilian pig population. We used DNA samples from the following three purebred pig breeds: Large White (336 females and 26 males), Landrace (304 females and 27 males) and Pietrain (125 females and 11 males). The ESR genotyping was performed using PCR-RFLP. For each breed, genotypes for the ESR gene were compared independently for expected progeny differences (EPD) in litter size (LS), average daily weight gain (DWG) (g/day) and back fat thickness (BT) as measured in mm by ultrasound. In the Large White breed, but not the other breeds, the ESR genotype was significantly (p < 0.05) associated to LS, DWG and BT. Large Whites genotyped as AA or AB had higher EPD values for the LS and BT traits compared to BB Large Whites, while AA Large Whites had higher DWG EPD values than BB Large Whites. Our results for the Large White population showed that the A allele has a beneficial effect on LS, DWG and BT expected progeny differences.

Association of a PIT1 gene polymorphism with growth hormone mRNA levels in pig pituitary glands

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.

Development of a quantitative competitive reverse transcriptase polymerase chain reaction for the quantification of growth hormone gene expression in pigs

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.

Isolation and identification of proteins from swine sperm chromatin and nuclear matrix

The aim of this study was to perform a proteomic analysis to isolate and identify proteins from the swine sperm nuclear matrix to contribute to a database of swine sperm nuclear proteins. We used prechilled diluted semen from seven boars (19 to 24 weekold) from the commercial line Landrace x Large White x Pietran. The semen was processed to separate the sperm heads and extract the chromatin and nuclear matrix for protein quantification and analysis by mass spectrometry, by LTQ Orbitrap ELITE mass spectrometer (Thermo-Finnigan) coupled to a nanoflow chromatography system (LC-MS/MS). We identified 222 different proteins in the sample; a total of 159 (71.6%) were previously described as present in the somatic or sperm nuclei of other species, 41 (18.5%) did not have a previously reported nuclear presence and 22 (9.9%) had not been characterized. The most abundant family of proteins corresponded to ribosomal (13.1%), followed by cytoskeleton (12.2%), uncharacterized (9.9%), histones (5.4%), proteasome subunits (3.6%) and heat shock (1.8%). The other proteins clustered in other families accounted for 54% of the total proteins. The protein isolation of the nuclear matrix of the swine spermatozoa was satisfactory, thus demonstrating that the protocol used was efficient. Several proteins were identified and described. However, it was not possible to identify some protein structures. Therefore, this study helps to establish a starting point for future proteomic studies comparing fertile and sub-fertile animals.

Níveis de hormônios tireoideanos circulantes, desempenho e qualidade de carcaça e carne de suínos em crescimento e terminação

The objective of this study was to compare serum concentrations of the hormones triiodothyronine (T3) and thyroxine (T4) in pigs on commercial line beginning and end of growing-finishing phase, correlating them with productive performance and quality characteristics of carcass and meat. Blood samples were collected from 48 animals at the beginning and end of growing-finishing phase by puncturing the jugular vein, around the same time (08:00 to 10:00a.m.). Measurements of serum thyroid hormones were made by enzyme-immunoassay. It was evaluated the performance and quality of carcass and meat. At the beginning and end of the housing, serum T3 concentrations were 1.85 and 1.32nmol L-1, respectively, and T4 of 100.33 and 86.53nmol L-1, respectively. There was negative correlation between T3 and low-end and average daily weight gain, final weight and backfat thickness. Inicial T4 was moderately and moderately negatively correlated with initial weight. The values of T3 and T4 in pigs early in the growing-finishing phase are larger than in termination. The thyroid hormones are associated with the initial and final weight, backfat thickness at the last lumbar, pH45min and pH24h of growing-finishing pigs.

DETERMINAÇÃO DE POLIMORFISMOS NO GENE DO HORMÔNIO DO CRESCIMENTO EM TRÊS POPULAÇÕES DE SUÍNOS

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.