Marco Tazzoli

Investigation of candidate genes for glycolytic potential of porcine skeletal muscle: Association with meat quality and production traits in Italian Large White pigs

The objective of this study was to investigate the association of DNA markers in candidate genes for glycolytic potential on meat quality parameters (pH(1), pH(u), glycogen and lactate content and glycolytic potential of semimembranosus muscle) and estimated breeding values (EBVs) for average daily gain, lean cuts, back fat thickness, ham weight, and feed:gain ratio in 272 Italian Large White pigs. Three mutations in the PRKAG3 gene (T30N, G52S and I199V) were investigated as well as single nucleotide polymorphisms in two other skeletal muscle genes (PGAM2 and PKM2) involved in the glycolytic pathway. Association analysis with the PRKAG3 markers showed significant results (P<0.05) only for pH(1) (I199V, with significant additive effect) and lactate content (T30N), confirming, at least in part, the effects of this gene on meat quality traits. Significant association (P<0.05) was also observed for PGAM2 and ham weight EBV with significant additive and dominance effects. PKM2 was associated with average daily gain, lean cuts (P<0.001), back fat thickness and feed:gain ratio (P<0.05), with significant additive and/or dominance effects on these traits. PKM2 encodes for a key enzyme of the muscle glycolytic pathway and maps on porcine chromosome 7 where other studies have reported important QTL for the same traits. These data might suggest an important function of this gene in the mechanisms that produce the observed effects. The results will be important to evaluate the inclusion of some of these DNA polymorphisms in marker assisted selection programs.

Analysis of melanocortin 1 receptor (MC1R) gene polymorphisms in some cattle breeds: their usefulness and application for breed traceability and authentication of Parmigiano Reggiano cheese

Abstract In cattle, the MC1R gene has been the subject of several studies with the aim to elucidate the biology of coat colour. Then, polymorphisms of this gene have been proposed as tools for breed identification and animal products authentication. As a first step to identify breed specific DNA markers that can be used for the traceability of mono-breed dairy cattle products we investigated, using PCR-RFLP and PCR-APLP protocols, the presence and distribution of some alleles at the MC1R locus in 18 cattle breeds for a total of 1360 animals. For each of seven breeds (Italian Holstein, Italian Brown, Italian Simmental, Rendena, Jersey, Reggiana and Modenese) a large number of animals (>70) was genotyped so the obtained results can be considered with more confidence. Allele Ed was identified only in black pied cattle (Italian Holstein and Black Pied Valdostana). Allele E (this nomenclature includes all alleles except Ed, E1 and e) was observed in Italian Brown, Rendena, Jersey, Modenese, Italian Simmental, Grigio Alpina, Piedmontese, Chianina, Romagnola, Marchigiana, Swedish Red and White and Danish Red. Allele E1 was identified in Italian Brown, Rendena, Grigio Alpina, Piedmontese, Swedish Red and White and Danish Red. The recessive allele e, known to cause red coat colour, was fixed in Reggiana and almost fixed in Italian Simmental. This allele was observed also in Italian Holstein, Italian Brown, Rendena, Jersey and Modenese albeit with low frequency. Moreover, this allele was detected in Valdostana, Pezzata Rossa d’Oropa, Piedmontese, Romagnola, Swedish Red and White, Danish Red, Charoleis and Salers. In the case of the Reggiana breed, which is fixed for allele e, the MC1R locus is highly informative with respect to breeds that carry other alleles or in which allele e is at very low frequency. In theory, using the MC1R locus it is possible to identify the presence of milk from some other breeds in Parmigiano Reggiano cheese labelled as exclusively from the Reggiana breed. This possibility was practically tested by setting up protocols to extract and analyse polymorphisms of the MC1R locus in several dairy products, including Parmigiano Reggiano cheese cured for 30 months. The lower detection limit was estimated to be 5% of non expected DNA. This test can represent a first deterrent against fraud and an important tool for the valorisation and authentication of Parmigiano Reggiano cheese obtained from only Reggiana milk.

Investigation of allele frequencies of the growth hormone receptor (GHR) F279Y mutation in dairy and dual purpose cattle breeds

Abstract A major QTL for milk production traits was reported in the middle of bovine chromosome 20 and, for its map position, the growth hormone receptor (GHR) gene was considered a strong positional and functional candidate gene. A missense mutation in exon VIII (F279Y amino acid substitution) showed highly significant effects mainly on milk composition (protein percentage and fat percentage) as well as on milk yield in several dairy cattle populations. As no information about the frequency of these two GHR alleles is available in any population, we studied their distribution in dairy and dual purpose cattle breeds reared in Italy. A total of 679 animals belonging to seven cattle breeds (Italian Holstein-Friesian, n=108; Italian Brown, n=104; Italian Simmental, n=104; Jersey, n=104; Reggiana, n=108; Modenese, n=66; Rendena, n=85) were sampled. A new PCR-RFLP protocol was designed to analyse this mutation inserting an artificial restriction site for enzyme SspI. In all investigated breeds, allele F was the most frequent and ranged from 0.947 (Italian Brown and Jersey) to 0.727 (Italian Holstein-Friesian). In Rendena, Italian Simmental, Reggiana and Modenese it was 0.824, 0.909, 0.921 and 0.924, respectively. For all breeds no significant deviation from the Hardy-Weinberg equilibrium was observed. Differences in allele frequencies were statistically significant between the Italian Holstein Friesian and Rendena against all other breeds. Due to the high frequency of the putative positive allele for milk protein percentage the use of the GHR F279Y marker in marker assisted selection plans should not have a great impact on this trait in the studied breeds.

Sexing European rabbits (Oryctolagus cuniculus), European brown hares (Lepus europaeus) and mountain hares (Lepus timidus) with ZFX and ZFY loci

We describe a new method for the sex determination of tissue originating from Oryctolagus cuniculus (European rabbit), Lepus europaeus (European brown hare) and Lepus timidus (mountain hare) based on PCR‐RFLP analysis of point mutations that differentiate the ZFX and ZFY gene sequences. Among several applications, this PCR‐RFLP method could be used to investigate gender ratio and evaluate the population dynamics of these species using samples collected when sex cannot be identified.

Replacing starch with digestible fibre in growing rabbit feeding

Abstract To evaluate the effect of replacing dietary starch with digestible fibre (DF=pectin and hemicelluloses) on health status, digestive physiology, growth performance, and carcass traits, 250 hybrid rabbits weaned at 27 d were fed until slaughter (76 d) five diets with increasing DF to starch ratio (1.0 to 1.9; DF 18.9 to 22.2%; starch 19.6 to 11.5%). The digestibility of dry matter (64.7, 65.2, 66.8, 67.5 and 67.6%) and NDF (27.9, 32.2, 35.0, 40.2 and 41.5%) increased (probability of linear component of variance, L<0.001) with increasing DF to starch ratio. Final live weight and daily growth tended to decrease (L=0.06), feed intake significantly lowered (130 to 122 g/d, L=0.01) and feed conversion ratio improved (2.72 to 2.68; L<0.01). Health status, caecal fermentation and ileal mucosa traits of rabbits did not change. The feeding strategy failed in controlling the diffusion of epizootic rabbit enteropathy.

Evolution of European sea bass (Dicentrarchus labrax)freshness during storage

Abstract The study aimed to assess freshness evolution in 90 European sea bass (Dicentrarchus labrax) analyzed 2h after catch (day 0) and after 1, 2, 4, 6, and 8 days of storage at 2°C. Sea bass weighted 308±37g with average carcass yield of 89.8% and fillet yield of 48.8% slaughter weight. During storage skin colour indexes linearly changed (L<0.01) showing higher L* and lower a* and b* values. The eye liquor pH increased with storage, with both significant linear and quadratic components of variance. Fillet hardness increased from day 0 to day 4 and then decreased on day 8 (quadratic component of variance <0.01). Sensory freshness assessed by Quality Index Method showed a quadratic evolution and high correlation (R2=0.95) with days of storage.

Investigation of the Agouti gene for the identification of useful markers for coat colour association studies in domestic rabbits

Abstract In wild-type mice, it is well known that Agouti is expressed in skin where it controls the banded-hair Agouti phenotype. Molecular genetics and pharmacological studies show that mutually exclusive binding of the melanocortin 1 receptor (MC1R) by the Agouti protein or by U000300b2-melanocyte-stimulating hormone (a-MSH) signals hair-bulb melanocytes to synthesise preferentially either pheomelanin (yellow-red pigment) or eumelanin (black-brown pigment), respectively. In mice as well as in other species, loss-of-function mutations of the Agouti gene determine only the production of eumelanin while gain-of-function mutations lead to pheomelanin production. A variety of coat colours appear as a result of these alterations that show also epistatic interactions with MC1R mutations. In rabbit, classical studies have suggested the presence of three alleles at the Agouti locus: A (wild type allele), at (black and tan) and a (non-agouti). We recently showed that mutations in the rabbit MC1R gene are associated with coat colours in different breeds. We, furthermore, supposed that the MC1R gene might have partial epistatic actions over the Agouti locus. Here, in order to clarify the interactions between the MC1R and Agouti loci we studied the rabbit Agouti gene with the objective to identify mutations that could be associated or could be useful in association investigations with coat colours in domestic rabbit breeds. Total genomic DNA was extracted from peripheral blood or hair roots of rabbits belonging to breeds having different coat colours. PCR primers were designed to amplify the four exons of the Agouti gene. Sequencing of the fragment encompassing part of intron 2, exon 3 and part of intron 3 showed three single nucleotide polymorphisms (SNPs; A<T, A<G and T<C) in intron 2 and one in intron 3 (G<A). These SNPs produced two haplotypes, A-A-T-G and T-G-C-A. The first haplotype was observed in Giant Grey and Belgian Hare rabbits. The second haplotype was identified in Burgundy Fawn and Checkered Giant animals. Further studies are underway to obtain a complete characterization of the rabbit Agouti gene and to evaluate the identified markers in association studies with coat colours in this species.

Identification of mutations in the bovine KIT gene, a candidate for the Spotted locus in cattle

Abstract In mammals, abnormal migration of melanoblasts from the neural crest during embryonic development may be the reason of the pielbaldism phenotype that is a mixture of pigmented and unpigmented areas in the coat. Several cattle breeds, like for example Holstein, show the piebald spotted coat colour phenotype, that, according to crossbreeding studies, is due to a recessive allele (s), member of the allele series of the Spotted (S) locus. Dominant alleles at this locus act as suppressors of the spotted pattern and produce uniformly pigmented animals while others determine the colour-sided pattern known, for example, in the Hereford breed. The bovine v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene (KIT) gene was localized in the region of chromosome 6 where the Spotted locus was mapped. KIT plays a major role during the embryonic development in directing the migration of the melanoblasts from the neural crest. Mutations in this gene cause different coat colour patterns in mouse and human. In pigs, the KIT gene is responsible for variations at the White (I) locus. Thus, this gene can be considered a strong candidate for the Spotted locus in cattle. With the final aim to identify DNA markers associated to different coat colour pattern distributions that could eventually be used as markers for breed traceability of dairy and beef products, we sequenced the 21 bovine KIT exons in cattle breeds showing different coat colour patterns. PCR primers were designed on available bovine KIT genomic sequence in order to amplify all coding exons and parts of the intronic regions using DNA extracted from 8 animals of either Holstein, Hereford and Angus breeds. Sequencing revealed 75 mutations including 72 single nucleotide polymorphisms and 3 indels of one, three and five bp, respectively. These mutations were organized in a few haplotypes that were not observed in all studied breeds. Further investigations are in progress to evaluate the association of the identified polymorphic sites with coat colour patterns in beef and dairy cattle breeds.