OPTIMISATION OF TOTAL RNA EXTRACTION FROM BOVINE OOCYTES AND EMBRYOS FOR GENE EXPRESSION STUDIES AND EFFECTS OF CRYOPROTECTANTS ON TOTAL RNA EXTRACTION.

Gene expression is required for understanding bovine oocytes meiotic maturation as well as the potential of embryonic development. In the present study a standardized reagent protocol for total RNA extraction was designed for bovine oocytes and embryos, which is considered specific and less expensive. For such purpose oocytes (n = 795) recovered from about 80 ovaries were divided in three groups: Group 1 modified Trizol (MTP, n = 355); Group 2 Guanidinium thiocyanate protocol (GNTC, n = 140) and Group 3 Commercial Kit protocol (CKP, n = 60). Oocytes belonging to group 1 (n = 100) and 3 (n = 20) were subjected to vitrification using two cryoprotectants 1,2 propandiol (PROH) or Dimethylsulfoxide (DMSO). The 240 remaining oocytes were divided into 3 groups in which 100 were used, in fresh, for in vitro fertilization, and 140 oocytes were vitrified using PROH (n = 70) and DMSO (n = 70) as cryoprotectants, being then fertilized in vitro after thawing. Embryos were used nine days after fertilization. Gene amplification (SDHA, (GAPDH and DNMT1) was performed in oocytes, and gene quantification (DNMT1) in in vitro produced embryos at the stage of blastocyst (n = 10). Efficiency of the extraction was further compared. The purity of all samples to different protocols ranged from 1.10 to 1.25 for GNTC protocol; from 2.05 to 2.63 for the CKP and from 1.50 to 2.11 for the developed MTP, being the last one nearest to the expected purity levels for RNA samples (1.7 to 2.0). On average, for 30 fresh oocytes, from spectrophotometer readings, total RNA concentration was 127.8 ± 9.3 ng µl(-1) for MTP, against 46.4 ± 9.5 ng µl(-1) from CKP and 476 ± 12.9 ng µl(-1) for GNTC protocol. Using the MTP to evaluate RNA in 30 vitrified/thawed oocytes, resulted in a total RNA concentration of 61.3 ± 3.3 ng µl(-1) and 40.0 µ 12.4 ng µ(-1), respectively for DMSO and PROH. Regarding total RNA concentration and purity, in blastocyst stage, more purity was observed in DMSO as compared to PROH (1.8 vs 1.2) (p < 0.05). Better results were also observed on the MTP for gene amplification when compared with the other protocols. For gene quantification, the proposed protocol quantified DNMT1 gene with PCR efficiency (0.933) after normalization against GAPDH and SDHA. Amplification and quantification of genes proved specificity and efficiency of the MTP over the other protocols.

Quantitative trait loci associated with chemical composition of the chicken carcass.

Major objectives of the poultry industry are to increase meat production and to reduce carcass fatness, mainly abdominal fat. Information on growth performance and carcass composition are important for the selection of leaner meat chickens. To enhance our understanding of the genetic architecture underlying the chemical composition of chicken carcasses, an F(2) population developed from a broiler × layer cross was used to map quantitative trait loci (QTL) affecting protein, fat, water and ash contents in chicken carcasses. Two genetic models were applied in the QTL analysis: the line-cross and the half-sib models, both using the regression interval mapping method. Six significant and five suggestive QTL were mapped in the line-cross analysis, and four significant and six suggestive QTL were mapped in the half-sib analysis. A total of eleven QTL were mapped for fat (ether extract), five for protein, four for ash and one for water contents in the carcass using both analyses. No study to date has reported QTL for carcass chemical composition in chickens. Some QTL mapped here for carcass fat content match, as expected, QTL regions previously associated with abdominal fat in the same or in different populations, and novel QTL for protein, ash and water contents in the carcass are presented here. The results described here also reinforce the need for fine mapping and to perform multi-trait analyses to better understand the genetic architecture of these traits.

SNP identification and polymorphism analysis in exon 2 of the horse myostatin gene.

The myostatin gene (MSTN) belongs to the TGF-ß superfamily of secreted growth and differentiation factors and is responsible for embryonic and adult skeletal muscle development. In this study, exon 2 of the MSTN gene, which encodes part of the TGF-ß pro-peptide, was sequenced in 332 horses of 20 different breeds and compared with the horse MSTN gene sequence deposited in GenBank. The sequences obtained revealed the presence of 11 haplotypes represented by 10 variable nucleotide mutations, eight of them corresponding to amino acid sequence changes. This gene shows a high variability when compared with other genes. This might be an indication that some breeds have the same ancestry but different pressures of selection.

QTL for percentage of carcass and carcass parts in a broiler x layer cross.

An F2 experimental population, developed from a broiler layer cross, was used in a genome scan of QTL for percentage of carcass, carcass parts, shank and head. Up to 649 F2 chickens from four paternal half-sib families were genotyped with 128 genetic markers covering 22 linkage groups. Total map length was 2630 cM, covering approximately 63% of the genome. QTL interval mapping using regression methods was applied to line-cross and half-sib models. Under the line-cross model, 12 genome-wide significant QTL and 17 suggestive linkages for percentages of carcass parts, shank and head were mapped to 13 linkage groups (GGA1, 2, 3, 4, 5, 7, 8, 9, 11, 12, 14, 18 and 27). Under the paternal half-sib model, six genome-wide significant QTL and 18 suggestive linkages for percentages of carcass parts, shank and head were detected on nine chicken linkage groups (GGA1, 2, 3, 4, 5, 12, 14, 15 and 27), seven of which seemed to corroborate positions revealed by the previous model. Overall, three novel QTL of importance to the broiler industry were mapped (one significant for shank% on GGA3 and two suggestive for carcass and breast percentages on GGA14 and drums and thighs percentage on GGA15). One novel QTL for wings% was mapped to GGA3, six novel QTL (GGA1, 3, 7, 8, 9 and 27) and suggestive linkages (GGA2, 4, and 5) were mapped for head%, and suggestive linkages were identified for back% on GGA2, 11 and 12. In addition, many of the QTL mapped in this study confirmed QTL previously reported in other populations.

Quantitative trait loci associated with fatness in a broiler-layer cross.

An F(2) population established by crossing a broiler male line and a layer line was used to map quantitative trait loci (QTL) affecting abdominal fat weight, abdominal fat percentage and serum cholesterol and triglyceride concentrations. Two genetic models, the line-cross and the half-sib, were applied in the QTL analysis, both using the regression interval method. Three significant QTL and four suggestive QTL were mapped in the line-cross analysis and four significant and four suggestive QTL were mapped in the half-sib analysis. A total of five QTL were mapped for abdominal fat weight, six for abdominal fat percentage and four for triglyceride concentration in both analyses. New QTL associated with serum triglyceride concentration were mapped on GGA5, GGA23 and GG27. QTL mapped between markers LEI0029 and ADL0371 on GGA3 for abdominal fat percentage and abdominal fat weight and a suggestive QTL on GGA12 for abdominal fat percentage showed significant parent-of-origin effects. Some QTL mapped here match QTL regions mapped in previous studies using different populations, suggesting good candidate regions for fine-mapping and candidate gene searches.

Quantitative trait loci for performance traits in a broiler x layer cross.

An F(2) resource population, derived from a broiler x layer cross, was used to map quantitative trait loci (QTL) for body weights at days 1, 35 and 41, weight gain, feed intake, feed efficiency from 35 to 41 days and intestinal length. Up to 577 F(2) chickens were genotyped with 103 genetic markers covering 21 linkage groups. A preliminary QTL mapping report using this same population focused exclusively on GGA1. Regression methods were applied to line-cross and half-sib models for QTL interval mapping. Under the line-cross model, eight QTL were detected for body weight at 35 days (GGA2, 3 and 4), body weight at 41 days (GGA2, 3, 4 and 10) and intestine length (GGA4). Under the half-sib model, using sire as common parent, five QTL were detected for body weight at day 1 (GGA3 and 18), body weight at 35 days (GGA2 and 3) and body weight at 41 days (GGA3). When dam was used as common parent, seven QTL were mapped for body weight at day 1 (GGA2), body weight at day 35 (GGA2, 3 and 4) and body weight at day 41 (GGA2, 3 and 4). Growth differences in chicken lines appear to be controlled by a chronological change in a limited number of chromosomal regions.

Mapping QTLs on chicken chromosome 1 for performance and carcass traits in a broiler x layer cross.

With the objective of mapping quantitative trait loci (QTLs) for performance and carcass traits, an F2 chicken population was developed by crossing broiler and layer lines. A total of 2063 F2 chicks in 21 full-sib families were reared as broilers and slaughtered at 42 days of age. Seventeen performance and carcass traits were measured. Parental F(0) and F1 individuals were genotyped with 80 microsatellites from chicken chromosome 1 to select informative markers. Thirty-three informative markers were used for selective genotyping of F2 individuals with extreme phenotypes for body weight at 42 days of age (BW42). Based on the regions identified by selective genotyping, seven full-sib families (649 F2 chicks) were genotyped with 26 markers. Quantitative trait loci affecting body weight, feed intake, carcass weight, drums and thighs weight and abdominal fat weight were mapped to regions already identified in other populations. Quantitative trait loci for weights of gizzard, liver, lungs, heart and feet, as well as length of intestine, not previously described in the literature were mapped on chromosome 1. This F2 population can be used to identify novel QTLs and constitutes a new resource for studies of genes related to growth and carcass traits in poultry.

Identification of quantitative trait loci for carcass composition and pork quality traits in a commercial finishing cross.

A QTL study for carcass composition and meat quality traits was conducted on finisher pigs of a cross between a synthetic Piétrain/Large White boar line and a commercial sow cross. The mapping population comprised 715 individuals evaluated for a total of 30 traits related to growth and fatness (4 traits), carcass composition (11 traits), and meat quality (15 traits). Offspring of 8 sires (n = 715) were used for linkage analysis and genotyped for 73 microsatellite markers covering 14 chromosomal regions representing approximately 50% of the pig genome. The regions examined were selected based on previous studies suggesting the presence of QTL affecting carcass composition or meat quality traits. Thirty-two QTL exceeding the 5% chromosome-wise significance level were identified. Among these, 5 QTL affecting 5 different traits were significant at the 1% chromosome-wise level. The greatest significance levels were found for a QTL affecting loin weight on SSC11 and a QTL with an effect on the Japanese color scale score of the loin on SSC4. About one-third of the identified QTL were in agreement with QTL previously reported. Results showed that QTL affecting carcass composition and meat quality traits segregated within commercial lines. Use of these results for marker-assisted selection offers opportunities for improving pork quality by within-line selection.