Alternatively polyadenylated calpastatin transcripts in bovine muscles / Transcriptos alternativamente poliadenilados de calpastatina en músculos de bovino

Calpastatin activity has a key role in the tenderization process that occurs during postmortem storage of meat under refrigerated conditioning. The regulation of calpastatin (CAST) expression is highly complex, the gene has four putative promoters and at least three different polyadenylation sites, and it is also alternatively spliced. We investigated the presence of alternative polyadenylation (APA) isoforms of CAST transcripts in three muscles (infraspinatus, triceps brachii and semitendinosus) of two bovine breeds (Angus and Brahman). The 3´ RACE-PCR was used to specifically amplify the different APA sites. The amplified fragments were cloned and sequenced. Sequencing confirmed the existence of three expected polyadenylation sites corresponding to short, medium and long polyadenylated transcripts. Also, transcripts with a novel APA site were found in the three muscles of both breeds. Because the same APAs isoforms were found between muscles and breeds, we could hypothesize a possible contribution to the relative abundance of different isoforms, probably in coordination with promoter preference and alternative splicing. This knowledge would be useful in the design of future experiments to analyze differential expression of CAST isoforms and their contribution to the definition of beef tenderness.

La actividad de la calpastatina tiene un rol clave en el proceso de tiernización postmortem de la carne durante su almacenamiento refrigerado. La regulación de la expresión de calpastatina (CAST) es altamente compleja; el gen tiene cuatro potenciales promotores, diferentes sitios de poliadenilación de transcriptos y también splicing alternativo. En este trabajo se investiga la presencia de isoformas de transcriptos de CAST alternativamente poliadenilados (APA) en tres músculos (infraspinatus, triceps brachii y semitendinosus) de dos razas bovinas (Angus y Brahman). Se utilizó la técnica de 3´ RACE-PCR para amplificar específicamente los diferentes sitios APA. Los fragmentos amplificados fueron clonados y secuenciados. La secuenciación confirmó la existencia de tres sitios de poliadenilación conocidos. Un nuevo sitio APA fue identificado en transcriptos de los tres músculos y en ambas razas. Dado que cualitativamente no hubo variación en la presencia de isoformas definidas por APA entre músculos y razas de terneza contrastante, podría hipotetizarse una posible contribución a la abundancia relativa de distintas isoformas, probablemente en forma coordinada con la elección de promotores y el splicing alternativo. Este nuevo conocimiento podría ser de utilidad para el diseño de experimentos de análisis de expresión diferencial de isoformas de calpastatina, para ponderar la contribución de las mismas a las variaciones en terneza de la carne.

Genetic variants in a lipid regulatory pathway as potential tools for improving the nutritional quality of grass-fed beef.

The aim of this study was to evaluate the effect of genetic variants on candidate genes corresponding to the sterol recognition element-binding protein-1 (SREBP-1) signaling pathway and stearoyl-CoA desaturases (SCD1 and SCD5) on muscle fatty acid (FA) composition of Brangus steers fattened on grass. FA profiles were measured on Longissimus lumborum muscle samples using a gas chromatography-flame ionization detection technique. A total of 43 tag single-nucleotide polymorphisms on the SCD1, SCD5, SREBP-1, SCAP, INSIG1, INSIG2, MBTPS1, MBTPS2, and SRPR genes were genotyped on 246 steers to perform a marker-trait association study. To evaluate the influence of the Indicine breed in the composite breed, additional groups of 48 Angus, 18 Hereford, 75 Hereford x Angus, and 36 Limousin x Hereford-Angus steers were also genotyped. To perform the association analysis, FA data were grouped according to the number of carbon atoms and/or number of double bonds (i.e. SFA, MUFA, PUFA, etc.). In addition, different indexes that reflect the activity of FA desaturase and elongase enzymes were calculated. SCD1 markers significantly affected C14:1/(C14:0 + C14:1) and C18:1/(C18:0 + C18:1) indexes, whereas one SNP in SCD5 was correlated with the C16:1/(C16:0 + C16:1) index. Polymorphisms in the signal recognition particle receptor (SRPR) gene were associated with all the estimated desaturase indexes. Because the evaluated markers showed no effect on total lipid content of beef, this work supports the potential utilization of these markers for the improvement of grass-fed beef without undesirable side effects.

Genetic and management factors affecting beef quality in grazing Hereford steers.

Attributes contributing to differences in beef quality of 206 Hereford steers finished on pasture were assessed. Beef quality traits evaluated were: Warner-Bratzler meat tenderness and muscle and fat color at one and seven days after slaughter and trained sensory panel traits (tenderness, juiciness, flavor, and marbling) at seven days. Molecular markers were CAPN1 316 and an SNP in exon 2 on the leptin gene (E2FB). Average daily live weight gain, ultrasound monthly backfat thickness gain and rib-eye area gain were estimated. Molecular markers effects on meat quality traits were analyzed by mixed models. Association of meat quality with post weaning growth traits was analyzed by canonical correlations. Muscle color and marbling were affected by CAPN1 316 and E2FB and Warner-Bratzler meat tenderness by the former. The results confirm that marker assisted selection for tenderness is advisable only when beef aging is a common practice. The most important sources of variation in tenderness and color of meat remained unaccounted for.

Genetic markers of body composition and carcass quality in grazing Brangus steers.

The somatotropic axis is a major regulatory pathway of energy metabolism during postnatal growth in mammals. Genes involved in this pathway influence many economically important traits. The association of selected SNPs in these genes with carcass traits was examined in grazing Brangus steers. These traits included final live weight, ultrasound backfat thickness (UBFT), rib-eye area, kidney fat weight, hot carcass weight, and intramuscular fat percentage (%IMF). Genomic DNA (N = 246) was genotyped for a panel of 15 tag SNPs located in the growth hormone receptor (GHR), insulin-like growth factor I, insulin-like growth factor-binding protein 6, pro-melanin-concentrating hormone, suppressor of cytokine signaling 2, and signal transducer and activator of transcription 6 (STAT6) genes. Allelic and haplotype frequencies were compared with those of a sample of European breeds (N = 177 steers). Two tag SNPs in the GHR affected %IMF; one of them (ss86273136) was also strongly associated with UBFT (P < 0.003). The frequency of the most favorable GHR haplotype for %IMF was lower in Brangus steers. Moreover, the haplotype carrying two unfavorable alleles was present at a frequency of 31% in this group. Four tag SNPs on STAT6 had a significant effect on UBFT. One of these, SNP ss115492467, was also associated with %IMF. The STAT6 haplotype, including all the alleles favoring UBFT, was the most abundant variant (34%) in the European cattle, while it had a frequency of 14% in the Brangus steers. The four less favorable variants (absent in the European cattle) were found at a frequency of 38% in the Brangus steers. These results support the association of GHR and STAT6 SNP with carcass traits in composite breeds, such as Brangus, under grazing conditions.

Association of a novel polymorphism in the bovine PPARGC1A gene with growth, slaughter and meat quality traits in Brangus steers.

The PPARGC1A gene (peroxysome proliferator-activated receptor-gamma coactivator 1alpha gene) controls muscle fiber type and brown adipocyte differentiation; therefore, it is a candidate gene for beef quality traits (tenderness and fat content). Two SNPs (Single Nucleotide Polymorphisms) were identified within exon 8 by multiple alignment of DNA sequences obtained from 24 bulls: a transition G/A (SNP 1181) and a transversion A/T (SNP 1299). The SNP 1181 is a novel SNP, corresponding to a non-conservative substitution (AGT/AAT) that could be the cause of amino acid substitution ((364)Serine/(364)Asparagine). A Mismatch PCR method was designed to determine genotypes of 73 bulls and 268 steers for SNP 1181. Growth, slaughter and meat quality information were available for the group of steers. Allele A of SNP 1181 was not found in Angus. In 243 steers, no significant differences (P > 0.05) were found for either final live body weight, gain in backfat thickness in Spring, kidney fat weight, kidney fat percentage, Warner-Bratzler shear force at 7 days postmortem, intramuscular fat percentage or meat colour between genotype GG and AG. This SNP could be included in breed composition and population admixture analyses because there are marked differences in allelic frequencies between Bos taurus and Bos indicus breeds.

Effect of leptin gene polymorphisms on growth, slaughter and meat quality traits of grazing Brangus steers.

Leptin is a hormone that affects the regulation of feed intake, energy balance and body composition in mammals. Several polymorphisms in the bovine leptin gene have been associated with phenotypic variance of these traits. We evaluated two known single nucleotide polymorphisms (SNPs) in the leptin gene of 253 grazing Brangus steers. Brangus is a 5/8 Angus-3/8 Brahman composite. Data were collected during two consecutive growth/fattening cycles from two farms in southeast Buenos Aires province, Argentina. One of the markers is in the promoter region of the gene (SNP1) and the other is a non-synonymous polymorphism in exon 2 (SNP2). The traits that we evaluated were live weight gain in the spring, gain in backfat thickness in the spring, final live weight, final ultrasound backfat thickness, final ultrasound rib eye area, carcass weight and length, carcass yield, kidney fat, kidney fat percentage, backfat thickness, rib eye area, and intramuscular fat percentage. Both markers affected some meat traits; though the only significant associations were of SNP1 with ultrasound rib eye area and of SNP2 with carcass yield and backfat thickness. Under the same conditions as in the present study, leptin markers could be of help only as part of a larger genotyping panel including other relevant genes.

A PCR-RFLP test to detect allelic variants of the bovine kappa-casein gene.

Point mutations in exon IV of bovine kappa-casein gene (kappaCn, CASK, CSN3) determine nine allelic variants (A, B, C, E, F, G, H, I, and A1) for the gene. These variants are associated with major differences in composition and manufacturing properties of milk (i.e., cheese yield). A PCR-RFLP test was developed in order to distinguish the different alleles. Polymorphisms are detected by digestion with the endonucleases HindIII, HaeIII, and MaeII followed by electrophoresis in agarose gels stained with ethidium bromide. Twenty eight DNA samples from different breeds of Argentina were analyzed for the A, B, and E variants. This simple PCR-RFLP test makes feasible the inclusion of kappa-casein genotypes in breeding plans.

Genotypic variability at the major histocompatibility complex (B and Rfp-Y) in Camperos broiler chickens.

Evidence for the importance of major histocompatibility complex (MHC) genotype in immunological fitness of chickens continues to accumulate. The MHC B haplotypes contribute resistance to Marek's and other diseases of economic importance. The Rfp-Y, a second cluster of MHC genes in the chicken, may also contribute to disease resistance. Nevertheless, the MHC B and Rfp-Y haplotypes segregating in broiler chickens are poorly documented. The Camperos, free-range broiler chickens developed in Argentina, provide an opportunity to evaluate MHC diversity in a genetically diverse broiler stock. Camperos are derived by cross-breeding parental stocks maintained essentially without selection since their founding. We analysed 51 DNA samples from the Camperos and their parental lines for MHC B and Rfp-Y variability by restriction fragment pattern (rfp) and SSCP typing methods for B-G, B-F (class Ia), B-Lbeta (class II) and Y-F (class Ib) diversity. We found evidence for 38 B-G genotypes. The Camperos B-G patterns were not shared with White Leghorn controls, nor were any of a limited number of Camperos B-G gene sequences identical to published B-G sequences. The SSCP assays provided evidence for the presence of at least 28 B-F and 29 B-Lbeta genotypes. When considered together B-F, B-L, and B-G patterns provide evidence for 40 Camperos B genotypes. We found even greater Rfp-Y diversity. The Rfp-Y class I-specific probe, 163/164f, revealed 44 different rfps among the 51 samples. We conclude that substantial MHC B and Rfp-Y diversity exists within broiler chickens that might be drawn upon in selecting for desirable immunological traits.