Andrea Criscione

Sequence determination of αs1-casein isoforms from donkey by mass spectrometric methods

Four co-eluting components, with experimentally measured M(r) of 23 658, 23 786, 24 278 and 24 406 Da, were detected by reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis in the dephosphorylated casein fraction of a milk sample collected at middle lactation stage from an individual donkey belonging to the Ragusano breed. By coupling RP-HPLC, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), enzymatic digestions, MALDI-TOF MS and capillary RP-HPLC/nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) analyses, the four components were identified as donkeys alpha(s1)-CNs and their sequences completely characterized, using the known mares alpha(s1)-CN (GenBank Acc. No. AAK83668; M(r) 23750.7 Da) as reference. The proteins with M(r) of 23 786 and 23 658 Da differ in the presence of a glutamine residue at position 83 in the full-length component and present the amino acid substitutions Q(8)-->H and H(115)-->Y with respect to the mares alpha(s1)-CN. The other two components with M(r) 24 406 and 24 278 Da, which also differ in the presence of a glutamine residue at position 88 in the full-length component, show the insertion of the pentapeptide HTPRE between Leu(33) and the Glu(34). The two alpha(s1)-CNs bearing the pentapeptide insertion were named variants A (202 amino acids; M(r) 24 406) and A(1) (201 amino acids; M(r) 24 278), whereas the two alpha(s1)-CNs without the pentapeptide were named variants B (197 amino acids; M(r) 23 786) and B(1) (196 amino acids; M(r) 23 658).

Diet selection and milk production and composition in Girgentana goats with different αs1-casein genotype

In goats, alpha s1-casein polymorphism is related to different rates of protein synthesis. Two genetic variants, A and F, have been identified as strong and weak alleles based on a production of 3.5 and 0.45 g/l of alpha s1-casein per allele. The aim of the trial was to test whether goats can select their diet as a function of their genetic aptitude to produce milk at different casein levels and whether this selection can influence milk production or composition. Two groups of 8 animals, homozygous for strong (AA) or weak (FF) alleles were housed in individual pens. Using a manger subdivided into five separate containers, the goats were offered daily for 3 weeks: 1.5 kg of alfalfa pelleted hay, 0.7 kg of whole barley, 0.7 kg of whole maize, 0.7 kg of whole faba bean and 0.7 kg of pelleted sunflower cake. Total dry matter intake was similar between groups and resulted in nutrient inputs much higher than requirements. On average, goats selected 86% of maize plus barley and only 46% of faba bean plus sunflower. Indeed, AA goats selected less faba bean compared with FF goats (37.2 v. 56.7% of the available amount; P=0.01); during week 2 and week 3 they significantly increased maize selection (respectively for week 2 and week 3: 94.9 and 99.1% v. 85.3 and 87.3%) thus increasing the ratio between the high-energy feeds and the high-protein feeds (2.41 v. 1.81, P=0.023). As for true protein, the high soluble fraction (B1) and the indigestible fraction (C) were lower in the diet selected by AA goats (respectively in AA and FF groups: B1, 7.85 v. 9.23% CP, P<0.01; C, 6.07 v. 6.30% CP, P<0.001); these diet characteristics can be associated with lower losses of protein. Milk production, being similar in AA and FF groups when goats were fed with a mixed diet, significantly increased in AA group, when free-choice feeding was given (mean productions: 1198 v. 800 g/d, P<0.01). Casein content was higher in AA group than in FF group (2.70 v. 2.40%, P<0.01) whereas milk urea was higher in FF group (59.7 v. 48.8 mg/dl, P<0.01). In conclusion, when the animals were free to select their diet, their higher genetic aptitude to produce casein seemed to adjust their energy and protein dietary input in qualitative terms, thus leading to an increase in milk production and a decrease in milk urea. These results seem to demonstrate that interactions probably occurred between genetic polymorphism at the alpha s1-casein locus, diet selection and the efficiency of nutrient transformation into milk.

Grazing season and forage type influence goat milk composition and rennet coagulation properties

Two different types of pasture (cultivated and rangeland) and 2 different hay qualities (high and low quality) were examined for their effects on goat milk composition and rennet coagulation properties. Furthermore, the effect of dietary treatments in both the early and late grazing season was studied. As lactation stage is known to influence milk composition, the goats in the early and late grazing season were in the same lactation stage at the start of the experiment. The milk composition was influenced both by dietary treatment and season. Milk from goats on pasture was superior to those on hay by containing a higher content of protein and casein, and the goats on cultivated pasture had the highest milk yield. Casein composition was significantly influenced by forage treatment. Goats grazing on cultivated pasture had higher contents of αs1-casein and also of κ-casein compared with the other treatments, whereas goats grazing on rangeland had the highest content of β-casein. Factors such as milk yield, casein micelle size, αs2-casein, and calcium content were reduced in late compared with early season. More favorable rennet coagulation properties were achieved in milk from the early grazing season, with shorter firming time and higher curd firmness compared with milk from the late grazing season, but the firming time and curd firmness were not prominently influenced by forage treatment. The content of αs2-casein and calcium in the milk affected the firming time and the curd firmness positively. The influence of season and forage treatment on especially milk yield, casein content, and rennet coagulation properties is of economic importance for both the dairy industry and goat milk farmers.

Molecular characterization and genetic structure of the Nero Siciliano pig breed

Nero Siciliano is an autochthonous pig breed that is reared mainly in semi-extensive systems in northeastern Sicily. Despite its economic importance and well-appreciated meat products, this breed is currently endangered. Consequently, an analysis of intra-breed variability is a fundamental step in preserving this genetic resource and its breeding system. In this work, we used 25 microsatellite markers to examine the genetic composition of 147 unrelated Nero Siciliano pigs. The total number of alleles detected (249, 9.96 per locus) and the expected heterozygosity (0.708) indicated that this breed had a high level of genetic variability. Bayesian cluster analysis showed that the most likely number of groups into which the sample could be partitioned was nine. Based on the proportion of each individuals genome derived from ancestry, pigs with at least 70% of their genome belonging to one cluster were assigned to that cluster. The cluster size ranged from 7 to 17 (n = 108). Genetic variability in this sub-population was slightly lower than in the whole sample, genetic differentiation among clusters was moderate (FST 0.125) and the FIS value was 0.011. NeighborNet and correspondence analysis revealed two clusters as the most divergent. Molecular coancestry analysis confirmed the good within-breed variability and highlighted the clusters that retained the highest genetic diversity.

The mathematical modeling of the lactation curve for dairy traits of the donkey (Equus asinus).

In recent years, an increase in the number of donkeys farmed in Italy as a consequence of the growing demand for donkey milk for direct consumption has been observed. Some research has been carried out on jenny milk composition and on its nutritional properties, whereas milk production features are scarcely described for this species. In this work, the lactation curve shape of donkeys for milk yield and composition was investigated. A total of 453 test-day records for milk yield, fat and protein percentage, and somatic cell count of 62 lactations measured on 46 multiparous jennies of the Ragusano breed were considered. Effects of herd, age, and foaling season were assessed by using a mixed model analysis. Average and individual lactation curves were fitted using the Wood incomplete gamma function, the Cappio-Borlino modified gamma, and a third-order Legendre orthogonal polynomial model. Donkeys foaling between 6- and 10-yr-old had the highest test-day milk yield (about 1.85 kg/d). Donkeys foaling in winter and autumn had a higher daily milk yield compared with those foaling in summer and spring. Less defined results were obtained for composition traits. The general pattern of the donkey lactation curve is similar to the standard shape reported for the main dairy ruminant species, with a peak yield occurring at about 5 wk from parturition. Younger jennies tended to have lower production peaks and higher lactation persistency. Similarly to what is reported for dairy cattle, a large variability in individual patterns has been observed. No differences in goodness of fit have been observed between the models in the case of average lactation curves, whereas orthogonal polynomials were more efficient in fitting individual patterns.

Protein fraction heterogeneity in donkey’s milk analysed by proteomic methods

Abstract Donkey’s milk is often well tolerate by patients affected by cow’s milk protein allergy, probably thanks to its protein composition. This empiric evidence, confirmed by some clinical trials, needs to be better investigated. A preliminary survey on the protein fraction of donkey’s milk was carried out: fifty-six individual milk samples have been collected and analysed by IEF and SDS-PAGE. Five different IEF patterns have been identified, showing a marked heterogeneity both in casein and whey protein fractions. A single IEF pattern showed an apparent reduced amount of casein fraction highlighted by SDS. Three of the five IEF patterns have been further investigated by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS).

Mitochondrial DNA control region variation in Sanfratellano horse and two other Sicilian autochthonous breeds

Abstract Mitochondrial D-loop hypervariable region was analysed in 20 Sanfratellano and two other Sicilian autochthonous horse breeds (20 Sicilian Oriental Purebred and 20 Sicilian Indigenous) in order to investigate matrilineal genetic diversity. A total of 20 different haplotypes were identified sequencing a fragment of 397 bp; overall, haplotypes showed 31 polymorphic sites (7.8%). High diversity was detected in Sanfratellano (11 haplotypes) and Sicilian Indigenous (13 haplotypes), whereas only one haplotype was found in Sicilian Oriental Purebred. Sanfratellano sequences were compared with those belonging to the other Sicilian autochthonous horses and 118 sequences selected from the GenBank database in order to calculate the statistics of molecular diversity. Six haplotypes were exclusive of Sanfratellano which shares haplotype C, D, H, and O with the Sicilian Indigenous and haplotype U with the Sicilian Oriental Purebred; not significant differentiation was found between Sanfratellano and Sicilian Indigenous. BLAST search showed Sicilian haplotypes overlap with the database sequences but for three. Phylogenetic analysis did not show monophyletic group for Sanfratellano samples or the other breeds included in this analysis.

A genetic analysis of the Italian Salernitano horse

Salernitano (SAL) is an ancient Italian horse breed developed over the course of the ages together with Napoletano and, during the 20th century, by crossing with Thoroughbred horse lines. Excellent in hurdle jumping, this breed is currently facing a concrete risk of extinction due to the lack of appropriate management strategies. This research is the first SAL genetic characterization that aims to set up the basic knowledge for a conservation plan. A representative sample of 61 SALs was analyzed by means of a set of 16 microsatellites markers (short tandem repeats (STRs)). The sequence of hypervariable D-loop mtDNA region was also performed on a subset of 24 mares in order to study the maternal diversity and obtain a complete picture of the internal genetic variation. All the molecular data were analyzed together with those obtained from three Sicilian horse breeds investigated in a previous research (Siciliano, Sanfratellano and Sicilian Oriental Purebred). STRs markers revealed a moderate level of genetic diversity in SAL (alleles/locus 5.1, He 0.67) and confirmed the hunch of genetic erosion. Autosomal variability highlighted a very light deficit of homozygotes (FIS=-0.067). Experimental D-loop sequences were compared by multiple alignments with those retrieved from biological databases and revealed two unreported haplotypes. The phylogenetic network, which was built on mtDNA sequences, included various cosmopolitan and European horses and showed SAL haplotypes distributed among different mtDNA lineages.

Genetic diversity and relationship among the three autochthonous Sicilian donkey populations assessed by microsatellite markers

Abstract In the developed countries donkey has lost its main function as draft animal because of the mechanization in agri-culture; as a consequence donkey population was greatly reduced. According to SAVE monitoring institute, three of the eight Italian endangered donkey breeds are native of Sicily (Ragusano, Pantesco, Grigio Siciliano). Urgent safeguard plans are required. The aim of this work is to investigate the distribution of genetic diversity and the relationships among the three Sicilian autochthonous donkey breeds using a set of microsatellite markers. A total of 116 blood samples (61 Ragusano, 39 Pantesco, 16 Grigio Siciliano) were collected in 9 herds all over Sicily. Representative samples of Ragusano and Grigio donkey populations consist of unrelated individuals, whereas the sample of Pantesco represents nearly the entire studbook-registered population managed by “Ispettorato Ripartimentale delle Foreste di Erice (TP)” in the “Azienda S. Matteo”. Genomic DNA was amplified at 11 microsatellite loci (HTG10, VHL20, HTG7, HTG4, AHT5, AHT4, HMS3, HMS6, HMS7, HMS2 and HTG6), located on different chromosomes in two PCR multiplex reactions. The PCR products were mixed with GeneScan 350 ROX internal size standard and ran on an ABI Prism 377 DNA Sequencer equipped with Genescan and Genotyper softwares. MICROSATELLITES ANALYSER, FSTAT and MICROSAT softwares had been used in order to calculate the main parameters of genetic diversity and the genetic distances. Phylogeny trees were obtained using PHYLIP 3.66 and SPLITSTREE 4 4.6 software packages. Clustering analysis has been carried out using STRUCTURE 2.0 program. All microsatellites were polymorphic in each breed, generating a total of 76 alleles (from 3 to 11 in HTG4 and HTG7, respectively). The number of observed alleles was higher in Ragusano (72, 6.55 per locus), than in Grigio (55, 5 per locus) and Pantesco (45 alleli, 4.09 per locus). The number of effective alleles did not reproduce the same trend (2.62 in Grigio, 2.53 in Ragusano and 2 in Pantesco). A total of 13 private alleles (17% of the generated alleles), with frequencies always lower than 0.10, have been detected (13 in Ragusano, 2 in Pantesco, 2 in Grigio Siciliano). The genetic differentiation coefficient was low (Gst = 0.065). According to the Gst value (0.163) HMS3 was the most discriminating marker in the set, whereas HTG4 was the less one. The populations were not at genetic equilibrium, showing a significant deficit of heterozygotes. The average expected heterozygosity (He) over all population was 0.612. He was lower in Pantesco (0.500) than in Ragusano and Grigio (0.605 and 0.618, respectively). Fis value, which indicates the degree of departure from random mating, was particularly high in Pantesco (0.230), in which the sample represent the whole population, than in the other two breeds. Genetic distances estimated by different methods (Da and Ds) and the related NJ and UPGMA dendrograms, as well as the clustering analysis, agree with the evidence that Pantesco endangered breed shows the higher degree of genetic distinctness. Ragusano and Grigio result closely related (Da=0.058), probably because of their common origin: in fact before 1953 in Sicily local donkey with bay and grey coat were usually managed as a single population.

Characterization of biodiversity in six goat breeds reared in Southern Italy by means of microsatellite and SNP markers

Abstract An integrated analysis, using 20 microsatellite markers and 32 SNP markers belonging to the casein cluster has been carried out on 174 goats from 6 local goat breeds and populations from Southern Italy. Microsatellite markers provided 216 alleles (10.8 per locus; from 6.6 to 8.2 per breed). The average expected heterozygosity (He) was 0.732. Fis value (0.148) indicated a general heterozygosity deficiency. A high number of intragenic haplotypes (56) have been detected at casein loci, 25 at αS1 casein (CSN1S1), 12 at β casein (CSN2), 8 at αS2 casein (CSN1S2), 11 at κ casein (CSN3), when 1% frequency was required for each breed. The breeds with higher production and management level, fixed the lowest number of combinations at casein loci (28). Molecular data have been used to calculate genetic distances and in clustering analysis.