Sequencing of lipoprotein lipase gene in the Mediterranean river buffalo identified novel variants affecting gene expression.

Lipoprotein lipase (LPL) is a key enzyme for lipid metabolism, playing a fundamental role in the composition of fat in adipose tissue and milk. The LPL gene has been seldom investigated in dairy ruminants and barely studied in river buffalo (Bubalus bubalis). The aim of this work was to explore the genetic diversity of LPL and its promoter and to identify functional mutations, using a combined approach based on sequencing, dual-color electrophoretic mobility shift assay, and quantitative PCR. Thirteen consensus sequences for transcription factors were found in the promoter. Eleven SNP were detected, and the attention was focused on the SNP with potential functional effects: g.-446A>G, because the presence of G created a consensus motif for the transcription factor Sp1, and g.107A>G, which was the only exonic SNP. We developed PCR-RFLP methods for genotyping the 2 SNP and calculated the allele frequencies. A strong linkage disequilibrium (D' = 1; r2 = 0.903) was found between the 2 SNP. The dual-color electrophoretic mobility shift assay demonstrated that only genotype g.-446GG allowed the binding of the Sp1 transcription factor, resulting in overexpression of the gene (~2.5 fold), as confirmed by the quantitative PCR results. Haploinsufficiency is proposed as a regulation mechanism. This study adds further knowledge on the structure of the LPL gene and its expression in river buffalo, with potential effects on milk qualitative and quantitative production.

Genome-wide association and pathway analysis of carcass and meat quality traits in Piemontese young bulls.

A key concern in beef production is how to improve carcass and meat quality traits. Identifying the genomic regions and biological pathways that contribute to explaining variability in these traits is of great importance for selection purposes. In this study, genome wide-association (GWAS) and pathway-based analyses of carcass traits (age at slaughter (AS), carcass weight (CW), carcass daily gain (CDG), conformation score and rib-eye muscle area) and meat quality traits (pH, Warner-Bratzler shear force, purge loss, cooking loss and colour parameters (lightness, redness, yellowness, chroma, hue)) were conducted using genotype data from the 'GeneSeek Genomic Profiler Bovine LD' array in a cohort of 1166 double-muscled Piemontese beef cattle. The genome wide-association analysis was based on the GRAMMAR-GC approach and identified 37 significant single nucleotide polymorphisms (SNPs), which were associated with 12 traits (P<5 × 10-5). In particular, 14 SNPs associated with CW, CDG and AS were located at 38.57 to 38.94 Mb on Bos taurus autosome 6 and mapped within four genes, that is, Leucine Aminopeptidase 3, Family with Sequence Similarity 184 Member B, Non-SMC Condensin I Complex Subunit G and Ligand-Dependent Nuclear Receptor Corepressor-Like. Strong pairwise linkage disequilibrium was found in this region. For meat quality traits, most associations were 1 SNP per trait, except for a signal on BTA25 (at ~11.96 Mb), which was significant for four of the five meat colour parameters assessed. Gene-set enrichment analyses yielded significant results for six traits (right-sided hypergeometric test, false discovery rate <0.05). In particular, several pathways related to transmembrane transport (i.e., oxygen, calcium, ion and cation) were overrepresented for meat colour parameters. The results obtained provide useful information for genomic selection for beef production and quality in the Piemontese breed.

The single nucleotide polymorphism g.133A>C in the stearoyl CoA desaturase gene (SCD) promoter affects gene expression and quali-quantitative properties of river buffalo milk.

The stearoyl-CoA desaturase (SCD) gene has been investigated in depth in ruminants because of its effect on milk fat composition. In river buffalo, the single nucleotide polymorphism (SNP) g.133A>C in the gene promoter has been associated with milk quality and yield. However, the biological reason for such effects remains unexplored. In this study, we combined mRNA profile analysis, an electromobility shift assay, and quantitative PCR to elucidate the role of this SNP on gene transcription and its effects on milk fat traits. A preliminary genotyping of g.133A>C was carried out on a group of 303 river buffaloes to choose individuals for the downstream applications. Analysis of allele frequencies showed an increase in the minor allele C (0.25) compared with previous findings (0.16). Six animals (2 for each genotype) were chosen for cloning and 216 positive cDNA recombinant clones for SCD (72 per genotype) were analyzed by PCR. All clones showed the same length on agarose gel; therefore, random clones were chosen for sequencing. No qualitative differences were found and all gene transcripts assembled correctly. An electrophoretic mobility shift assay was performed to evaluate the binding of the transcription factor Sp1 to DNA sequences including g.133A>C. Genotype CC showed a higher binding (mean ± standard error of the mean) than genotype AA in 2 different conditions [Enzo buffer (EB), Enzo Life Science Inc., Farmingdale, NY: 201.77 ± 4.06 vs. 141.65 ± 3.77 band intensity values and Poletto buffer (PB): 95.90 ± 1.15 vs. 67.30 ± 2.14 band intensity values]. The subsequent quantitative PCR confirmed the upregulation of the CC genotype compared with the AA and AC genotypes. The association study with milk fat traits revealed a favorable effect of allele C. The heterozygous genotype had the highest values for monounsaturated fatty acids, oleic acid (C18:1 cis-9), polyunsaturated fatty acids, and odd- and branched-chain fatty acids, and the lowest values for saturated fatty acids and atherogenic and thrombogenic indices; the heterozygous genotype differed significantly from the AA genotype. The AC genotype has previously been associated with higher milk yield. Therefore, the g.133A>C SNP is a marker with dual effects and is an interesting candidate for assisted selection programs in river buffalo. These data clarified the biological role of the SNP g.133A>C in the SCD promoter and how it affects gene function, providing important knowledge on the genetic background of lipid metabolism, including the future possibility of selecting alleles with quantitatively or qualitatively favorable effects.

Transcript analysis at DGAT1 reveals different mRNA profiles in river buffaloes with extreme phenotypes for milk fat.

Buffalo DGAT1 (diacylglycerol O-acyltransferase 1) was mainly investigated for the characterization of the gene itself and for the identification of the K232A polymorphism, similar to what has been accomplished in cattle, although no information has been reported so far at the mRNA level. The importance of DGAT1 for lipid metabolism led us to investigate the transcript profiles of lactating buffaloes characterized as high (9.13 ± 0.23) and low (7.94 ± 0.29) for milk fat percentage, and to explore the genetic diversity at the RNA and DNA level. A total of 336 positive clones for the DGAT1 cDNA were analyzed by PCR and chosen for sequencing according to the differences in length. The clone assembling revealed a very complex mRNA pattern with a total of 21 transcripts differently represented in the 2 groups of animals. Apart from the correct transcript (17 exons long), the skipping of exon 12 is the most significant in terms of distribution of clones with 11.6% difference between the 2 groups, whereas a totally different mRNA profile was found in approximately 12% of clones. The sequencing of genomic DNA allowed the identification of 10 polymorphic sites at the intron level, which clarify, at least partially, the genetic events behind the production of complex mRNA. Genetic diversity was found also at the exon level. The single nucleotide polymorphism c.1053C>T represents the first example of polymorphism in a coding region for the DGAT1 in the Italian Mediterranean breed. To establish whether this polymorphism is present in other buffalo breeds, a quick method based on PCR-RFLP was set up for allelic discrimination in the Italian Mediterranean and the Romanian Murrah (200 animals in total). The alleles were equally represented in the overall population, whereas the analysis of the 2 breeds showed different frequencies, likely indicating diverse genetic structure of the 2 breeds. The T allele might be considered as the ancestral condition of the DGAT1 gene, being present in the great part of the sequenced species. These data add knowledge at the transcript and genetic levels for the buffalo DGAT1 and open the opportunity for further investigation of other genes involved in milk fat metabolism for the river buffalo, including the future possibility of selecting alleles with quantitative or qualitative favorable effects (or both).

Lipomatous muscular 'dystrophy' of Piedmontese cattle.

Lipomatous myopathy is a degenerative muscle pathology characterized by the substitution of muscle cells with adipose tissue, sporadically reported in cattle, pigs, and rarely in sheep, horses and dogs. This study investigated the pathology of this myopathy in 40 muscle samples collected from regularly slaughtered Piedmontese cattle living in Piedmont region (Italy). None of the animals showed clinical signs of muscular disease. Muscle specimens were submitted to histological and enzymatic investigations. Gross pathology revealed a different grade of infiltration of adipose tissue, involving multiple or single muscles. The most affected regions were the ventral abdomen and the shoulders, especially the cutaneous muscles and the muscles of the thoracic group. Morphological staining revealed an infiltration of adipose tissue varying in distribution and severity, changes in muscle fibre size and increased number of fibres with centrally located nuclei, suggesting muscle degeneration-regeneration. Necrosis and non-suppurative inflammatory cells were also seen. Furthermore, proliferation of connective tissue and non-specific myopathic changes were present. Chemical and physical characteristics of the affected tissue were also evaluated. The authors discuss about the aetiopathogenesis and classification of this muscle disorder whose histological lesions were similar to those reported in human dystrophies.

PCR-RFLP analysis of mitochondrial DNA in tench Tinca tinca.

Polymorphism was detected at ND1, ND6, D-loop and cyt b segments of mtDNA in 105 tench (Tinca tinca L.), using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and five composite haplotypes were identified. The diversity indices and the results of the population comparisons revealed that the identified markers provide a powerful tool for further studies on this species.

Polymorphism of the GHR gene in cattle and relationships with meat production and quality.

Growth hormone (GH) exerts its effects on growth and metabolism by interacting with a specific receptor (GHR) on the surface of the target cells. Therefore, GHR has been suggested as candidate gene for traits related to meat production in cattle. The aim of the study was to analyse the polymorphism at position 257 in exon 10 of the GHR gene and investigate relationships with 14 in vivo traits and four meat characteristics in Piemontese animals. The biallelic polymorphism already described was detected using a new PCR procedure. The statistical analysis did not show significant gene substitution effects on growth, size and meat conformation traits. As for meat characteristics, a significant gene substitution of GHR(A) over GHR(G) was observed for drip losses at day 3, with the allele GHR(A) associated with higher values. A significant dominance effect was also observed for this trait. Further investigations in other breeds will be useful for better understanding information on the effect of this GHR polymorphism.

Lack of association of GH1 and POU1F1 gene variants with meat production traits in Piemontese cattle.

Growth hormone (GH) and the Pit-1 transcription factor have been shown to be involved in the physiological mechanisms related to growth. The present study was carried out to investigate the possible association of the polymorphism at GH1 and POU1F1 loci with meat production traits in Piemontese cattle. Fourteen traits were considered, expressing growth (weight at 5, 7 and 11 months, daily gain), size [withers height (WH), trunk length (TL), chest girth (CG) at 12 months] and meat conformation [withers width (WW), shoulder muscularity (SM), loin width (LW), loin thickness (LT), thigh muscularity (TM), thigh profile (TP), bone thinness (BT)]. Data were analysed with a mixed model procedure to estimate the allele substitution and the dominance effects. The results did not provide evidence of association of GH1 and POU1F1 polymorphisms with the evaluated traits.

Polymorphism of erythrocyte malic enzyme in the goat.

Three electrophoretic variants of erythrocyte malic enzyme (ME) in goats were reported. Inheritance data indicate that they are controlled by codominant alleles. The allele frequencies in four Mediterranean populations are given.

Polymorphism of erythrocyte glucosephosphate isomerase in sheep.

An electrophoretic analysis of glucosephosphate isomerase (GPI) in seven Italian sheep populations suggests that this locus is more polymorphic than previously supposed. The observed phenotype distributions are in agreement with the hypothesis of the existence of three codominant alleles, GPI*F, GPI*S and GPI*N, GPI*S being the most frequent (0.935 divided by 1.000).

Content of alpha S1-casein and coagulation properties in goat milk.

Samples of goat milk with low and high alpha S1-casein content collected from animals of Alpine and Saanen breeds in the same stage of lactation were compared for coagulation properties (coagulation time, rate of curd formation, curd firmness) and chemical composition (total solids, ash, total protein, total casein, whey protein, fat, Ca, P, pH). Milk with low alpha S1-casein had a faster coagulation time, whereas milk with high levels produced the firmer curd associated with a better chemical composition. Within high alpha S1-casein milk, comparison between breeds showed milk from the Alpine breed had significantly better coagulation properties than that from the Saanen breed. Milk composition accounted for 27% of the variation in coagulation time, 21% of variation in cured formation rate, and 54% of variation in curd firmness.

New phenotypes of alpha s-casein in sheep.

In an electrophoretic analysis of 198 milk samples from the Massa and Biella breeds of sheep, six different alpha s-casein phenotypes were observed, of which three have not been reported previously.

Phenotypic variations in blood and milk of the Somali camel.

132 blood samples and 54 milk samples obtained from Somali camel were analysed for red blood cell antigens with the cattle reagents and for Hb, Ca, X proteins, Tf, Alb, Am, SOD, alpha-La, beta-Lg and casein systems respectively. Positive lytic reactions were obtained with the anti-B, -Q, -Q', -W, -F1 and -J reagents. No biochemical polymorphism was observed except for Hb, X protein and beta-Lg systems.