Prediction and repeatability of milk coagulation properties and curd-firming modeling parameters of ovine milk using Fourier-transform infrared spectroscopy and Bayesian models.

The aim of this study was to apply Bayesian models to the Fourier-transform infrared spectroscopy spectra of individual sheep milk samples to derive calibration equations to predict traditional and modeled milk coagulation properties (MCP), and to assess the repeatability of MCP measures and their predictions. Data consisted of 1,002 individual milk samples collected from Sarda ewes reared in 22 farms in the region of Sardinia (Italy) for which MCP and modeled curd-firming parameters were available. Two milk samples were taken from 87 ewes and analyzed with the aim of estimating repeatability, whereas a single sample was taken from the other 915 ewes. Therefore, a total of 1,089 analyses were performed. For each sample, 2 spectra in the infrared region 5,011 to 925 cm-1 were available and averaged before data analysis. BayesB models were used to calibrate equations for each of the traits. Prediction accuracy was estimated for each trait and model using 20 replicates of a training-testing validation procedure. The repeatability of MCP measures and their predictions were also compared. The correlations between measured and predicted traits, in the external validation, were always higher than 0.5 (0.88 for rennet coagulation time). We confirmed that the most important element for finding the prediction accuracy is the repeatability of the gold standard analyses used for building calibration equations. Repeatability measures of the predicted traits were generally high (≥95%), even for those traits with moderate analytical repeatability. Our results show that Bayesian models applied to Fourier-transform infrared spectra are powerful tools for cheap and rapid prediction of important traits in ovine milk and, compared with other methods, could help in the interpretation of results.

Genetic analysis of coagulation properties, curd firming modeling, milk yield, composition, and acidity in Sarda dairy sheep.

Sheep milk is an important source of food, especially in Mediterranean countries, and is used in large part for cheese production. Milk technological traits are important for the sheep dairy industry, but research is lacking into the genetic variation of such traits. Therefore the aim of this study was to estimate the heritability of traditional milk coagulation properties and curd firmness modeled on time t (CFt) parameters, and their genetic relationships with test-day milk yield, composition (fat, protein, and casein content), and acidity in Sarda dairy sheep. Milk samples from 1,121 Sarda ewes from 23 flocks were analyzed for 5 traditional coagulation properties by lactodynamographic tests conducted for up to 60min: rennet coagulation time (min), curd-firming time (k20, min), and 3measures of curd firmness (a30, a45, and a60, mm). The 240 curd firmness observations (1 every 15 s) from each milk sample were recorded, and 4 parameters for each individual sample equation were estimated: rennet coagulation time estimated from the equation (RCTeq), the asymptotic potential curd firmness (CFP), the curd firming instant rate constant (kCF), and the syneresis instant rate constant (kSR). Two other derived traits were also calculated (CFmax, the maximum curd firmness value; and tmax, the attainment time). Multivariate analyses using Bayesian methodology were performed to estimate the genetic relationships of milk coagulation properties and CFt with the other traits; statistical inference was based on the marginal posterior distributions of the parameters of concern. The marginal posterior distribution of heritability estimates of milk yield (0.16±0.07) and composition (0.21±0.11 to 0.28±0.10) of Sarda ewes was similar to those often obtained for bovine species. The heritability of rennet coagulation time as a single point trait was also similar to that frequently obtained for cow milk (0.19±0.09), whereas the same trait calculated as an individual equation parameter exhibited larger genetic variation and a higher heritability estimate (0.32±0.11). The other curd firming and syneresis traits, whether as traditional single point observations or as individual equation parameters and derived traits, were characterized by heritability estimates lower than for coagulation time and for the corresponding bovine milk traits (0.06 to 0.14). Phenotypic and additive genetic correlations among the 11 technological traits contribute to describing the interdependencies and meanings of different traits. The additive genetic relationships of these technological traits with the single test-day milk yield and composition were variable and showed milk yield to have unfavorable effects on all measures of curd firmness (a30, a45, a60, CFP, and CFmax) and tmax, but favorable effects on both instant rate constants (kCF and kSR). Milk fat content had a positive effect on curd firmness traits, especially on those obtained from CFt equations, whereas the negative effects on both coagulation time traits were attributed to the milk protein and casein contents. Finally, in view of the estimated heritabilities and additive genetic correlations, enhancement of technological traits of sheep milk through selective breeding could be feasible in this population.

Environmental, morphological, and productive characterization of Sardinian goats and use of latent explanatory factors for population analysis.

Dairy goat farming is practiced worldwide, within a range of different farming systems. Here we investigated the effects of environmental factors and morphology on milk traits of the Sardinian goat population. Sardinian goats are currently reared in Sardinia (Italy) in a low-input context, similar to many goat farming systems, especially in developing countries. Milk and morphological traits from 1,050 Sardinian goats from 42 farms were recorded. We observed a high variability regarding morphological traits, such as coat color, ear length and direction, horn presence, and udder shape. Such variability derived partly from the unplanned repeated crossbreeding of the native Sardinian goats with exotic breeds, especially Maltese goats. The farms located in the mountains were characterized by the traditional farming system and the lowest percentage of crossbred goats. Explanatory factors analysis was used to summarize the interrelated measured milk variables. The explanatory factor related to fat, protein, and energy content of milk (the "Quality" latent variable) explained about 30% of the variance of the whole data set of measured milk traits followed by the "Hygiene" (19%), "Production" (19%), and "Acidity" (11%) factors. The "Quality" and "Hygiene" factors were not affected by any of the farm classification items, whereas "Production" and "Acidity" were affected only by altitude and size of herds, respectively, indicating the adaptation of the local goat population to different environmental conditions. The use of latent explanatory factor analysis allowed us to clearly explain the large variability of milk traits, revealing that the Sardinian goat population cannot be divided into subpopulations based on milk attitude The factors, properly integrated with genetic data, may be useful tools in future selection programs.

Variations at regulatory regions of the milk protein genes are associated with milk traits and coagulation properties in the Sarda sheep.

Regulatory variation at the ovine casein genes could have important effects on the composition and coagulation properties of milk. Herewith, we have partially resequenced the promoters and the 3'-UTR of the four casein genes in 25 Sarda sheep. Alignment of these sequences allowed us to identify a total of 29 SNPs. This level of polymorphism (one SNP every 250 bp) is remarkably high if compared with SNP densities estimated in human genic regions (approximately one SNP per bp). The 29 SNPs identified in our resequencing experiment, plus three previously reported SNPs mapping to the lactalbumin, alpha (LALBA) and ß-lactoglobulin (BLG, also known as progestagen-associated endometrial protein, PAEP) genes, were genotyped with a multiplex TaqMan Open Array Real-Time PCR assay in 760 Sarda sheep with records for milk composition and coagulation properties. Association analysis revealed the existence of significant associations of CSN1S2 and CSN3 genotypes with milk protein and casein contents. Moreover, genotypes at CSN1S1 were significantly associated with rennet coagulation time, curd firming time and curd firmness, whereas CSN2 was associated with curd firming time. These results suggest that SNPs mapping to the promoters and 3'-UTRs of ovine casein genes may exert regulatory effects on gene expression and that they could be used for improving sheep milk quality and technological traits at the population level through marker assisted selection.

Modeling of coagulation, curd firming, and syneresis of milk from Sarda ewes.

This study investigated the modeling of curd-firming (CF) over time (CF(t)) of sheep milk. Milk samples from 1,121 Sarda ewes from 23 flocks were analyzed for coagulation properties. Lactodynamographic analyses were conducted for up to 60 min, and 240 CF individual observations from each sample were recorded. Individual sample CFt equation parameters (RCT(eq), rennet coagulation time; CF(P), asymptotic potential value of curd firmness; k(CF), curd-firming instant rate constant; and k(SR), curd syneresis instant rate constant) were estimated, and the derived traits (CF(max), the point at which CF(t) attained its maximum level, and tmax, the time at which CF(max) was attained) were calculated. The incidence of noncoagulating milk samples was 0.4%. The iterative estimation procedure applied to the individual coagulation data showed a small number of not-converged samples (4.4%), which had late coagulation and an almost linear pattern of the ascending part of the CF(t) curve that caused a high value of CF(P), a low value of k(CF), and a high value of k(SR). Converged samples were classified on the basis of their CF(t) curves into no-k(SR) (18.0%), low-k(SR) (72.6%), and high-k(SR) (4.5%). A CF(t) that was growing continuously because of the lack of the syneresis process characterized the no-k(SR) samples. The high-k(SR) samples had a much larger CFP, a smaller k(CF), and an anticipation of tmax, whereas the low-k(SR) samples had a fast k(CF) and a slower k(SR). The part of the average CF(t) curves that showed an increase was similar among the 3 different syneretic groups, whereas the part that decreased was different because of the expulsion of whey from the curd. The traditional milk coagulation properties recorded within 30 min were not able to detect any appreciable differences among the 4 groups of coagulating samples, which could lead to a large underestimation of the maximum CF of all samples (if predicted by a30), with the exception of the no-k(SR) samples. Large individual variability was found and was likely caused by the effects of the dairy system, such as flock size (on CF(max), t(max), and % ewes with no-k(SR) milk), flock within flock size (representing 11 to 43% of total variance for % ewes with no-k(SR) milk and CF(max), respectively), days in milk (on all model parameters and CF(max)), parity (on RCT(eq), k(SR), and CF(max)), daily milk yield (on RCT(eq) and CF(max)), and position of the individual pendulum that significantly affected model parameters and derived traits. In conclusion, the results showed that the modeling of coagulation, curd-firming, and syneresis is a suitable tool to achieve a deeper interpretation of the coagulation and curd-firming processes of sheep milk and also to study curd syneresis.

Phenotypic factors affecting coagulation properties of milk from Sarda ewes.

In this study, milk-coagulation properties (MCP) were characterized in the Sarda sheep breed. Milk composition and MCP [rennet-coagulation time (RCT), curd-firming time [time to reach a curd firmness of 20mm (k20)], and curd firmness (a30), (a45), and (a60)] were obtained extending the lactodynamographic analysis from 30 to 60 min from a population of 1,121 ewes from 23 different farms. Managerial characteristics of farms and parity, individual daily milk yields and stage of lactation of ewes were recorded. Data were analyzed using a mixed-model procedure with fixed effects of days in milk, parity, daily milk yield, and flock size and the random effect of the flock/test day nested within flock size. Sampled farms were classified as small (<300 ewes) and medium (300 to 600 ewes), and these were kept by family operations, or as large (>600 ewes), often operated through hired workers. Daily milk yield was, on average, 1.58 ± 0.79 L/d and variability for this trait was very high. The average content of fat, protein, and casein was respectively 6.41, 5.39, and 4.20%. The class of flock size had a significant effect only on curd firmness, whereas days in milk affected RCT and k20. The flock test day, parity, and daily milk yield were important sources of variation for all MCP. The mean value of RCT (8.6 min) and the low occurrence of noncoagulating samples (0.44%) confirmed the excellent coagulation ability of sheep milk compared with cattle milk. A more rapid coagulation was observed in mid-lactating, primiparous, and high-yielding ewes. The k20 was usually reached in less than 2 min after gelation, with the most favorable values at mid lactation. The mean value of curd firmness 30 min after rennet addition (a30) was, on average, 50mm and decreased to 46 and 42 mm respectively after 45 (a45) and 60 min (a60). The decreasing value of curd-firmness traits was likely to be caused by curd syneresis and whey expulsion. The correlation between RCT and a30 was much lower than in dairy cows and about null for a45 and a60. This means that curd firmness in dairy ewes is almost independent of gelation time and this can provide specific information for this species. In conclusion, this study showed that milk from Sarda sheep is characterized by an earlier gelation, a faster increase in curd firmness with time, and greater curd firmness after 30 min compared with dairy cows. Furthermore, correlations between MCP in sheep are much lower than in bovines and some of the assumptions and interpretations related to cows cannot be applied to sheep.

Goat casein genotypes are associated with milk production traits in the Sarda breed.

The aim of the current work was to analyze, in the Sarda breed goat, genetic polymorphism within the casein genes and to assess their influence on milk traits. Genetic variants at the CSN1S1, CSN2, CSN1S2 and CSN3 gene loci were investigated using PCR-based methods, cloning and sequencing. Strong alleles prevailed at the CSN1S1 gene locus and defective alleles also were revealed. Null alleles were evidenced at each calcium-sensitive gene locus. At the CSN3 gene locus, we observed a prevalence of the CSN3 A and B alleles; the occurrence of rare alleles such as CSN3 B'', C, C', D, E and M; and the CSN3 S allele (GenBank KF644565) described here for the first time in Capra hircus. Statistical analysis showed that all genes, except CSN3, significantly influenced milk traits. The CSN1S1 BB and AB genotypes were associated with the highest percentages of protein (4.41 and 4.40 respectively) and fat (5.26 and 5.34 respectively) (P < 0.001). A relevant finding was that CSN2 and CSN1S2 genotypes affected milk protein content and yield. The polymorphism of the CSN2 gene affected milk protein percentage with the highest values recorded in the CSN2 AA goats (4.35, at P < 0.001). The CSN1S2 AC goats provided the highest fat (51.02 g/day) and protein (41.42 g/day) (P < 0.01) production. This information can be incorporated into selection schemes for the Sarda breed goat.

Sequence polymorphisms at the growth hormone GH1/GH2-N and GH2-Z gene copies and their relationship with dairy traits in domestic sheep (Ovis aries).

The purpose was to analyze the growth hormone GH1/GH2-N and GH2-Z gene copies and to assess their possible association with milk traits in Sarda sheep. Two hundred multiparous lactating ewes were monitored. The two gene copies were amplified separately and each was used as template for a nested PCR, to investigate single strand conformation polymorphism (SSCP) of the 5'UTR, exon-1, exon-5 and 3'UTR DNA regions. SSCP analysis revealed marked differences in the number of polymorphic patterns between the two genes. Sequencing revealed five nucleotide changes at the GH1/GH2-N gene. Five nucleotide changes occurred at the GH2-Z gene: one was located in exon-5 (c.556G > A) and resulted in a putative amino acid substitution G186S. All the nucleotide changes were copy-specific, except c.*30delT, which was common to both GH1/GH2-N and GH2-Z. Variability in the promoter regions of each gene might have consequences on the expression level, due to the involvement in potential transcription factor binding sites. Both gene copies influenced milk yield. A correlation with milk protein and casein content was also evidenced. These results may have implications that make them useful for future breeding strategies in dairy sheep breeding.

Association between melatonin receptor 1A (MTNR1A) gene polymorphism and the reproductive performance of Mediterranean Italian buffaloes.

A melatonin receptor 1A (MTNR1A) gene polymorphism in adult buffaloes has been reported to affect reproductive seasonality. Consequently, the aim of the present study was to assess whether this polymorphism can affect age at first conception and the interval between first and second calving in Mediterranean Italian buffaloes. The allelic frequency of the C and T alleles was 0.44 and 0.56, respectively, whereas the genotypic frequency was 26% for C/C, 40% for C/T and 34% for T/T. The average age at first mating was approximately 20 months, whereas that at calving was approximately 32 months. The largest number of calvings of primiparous buffaloes was recorded between June and October. No associations between genotype, first mating and subsequent calving date were found. The duration from first to second calving was longer in buffaloes with the C/C genotype compared with those with the T/T and C/T genotypes (P<0.01). The period of calving for buffaloes with the C/C genotype was mainly from July to September, whereas that for buffaloes with the T/T genotype was largely from March to May. The MTNR1A gene had no effect on the age of first conception in Mediterranean Italian buffaloes. Rather, the association between the T/T genotype and reproductive activity during days with a long photoperiod indicates that this polymorphism may be considered a genetic marker to identify buffaloes that are able to reproduce out of the breeding season.

Chromosomal localisation and genetic variation of the SLC11A1 gene in goats (Capra hircus).

The solute carrier family 11 member A1 (SLC11A1) gene is associated with resistance to infectious diseases. Chromosomal localisation, genomic regions corresponding to functional domains and the genetic variability of microsatellites in the 3' untranslated region (3'-UTR) of this gene were investigated in 427 goats (Capra hircus) of six breeds. Using dual colour fluorescence in situ hybridisation, SLC11A1 was localised to goat chromosome 2. Single strand conformation polymorphism was used to screen for polymorphisms in SLC11A1 exons 2, 10 and 15. There was no variation among goat breeds in the sarcoma homology 3 (SH3) binding motif, the protein kinase C phosphorylation site or the two N-linked glycosylation sites. Exon 15 exhibited variability due to the presence of two polymorphic microsatellites. Genotyping of the upstream guanine-thymine repeat (GTn) at 3'-UTR revealed eight alleles (GT11, GT12, GT14-GT19) in goats, whereas GT13 (present in cattle) was absent. Most goats carried the GT16 allele and no allele was found to be exclusive to only one breed. The coefficient of genetic differentiation value (G(ST)) was 0.084. This microsatellite appears to be an informative DNA marker for genetic linkage analysis in goats.

D-loop sequence mitochondrial DNA variability of Sarda goat and other goat breeds and populations reared in the Mediterranean area.

To provide useful knowledge on goat breed origin and history, we studied the mitochondrial DNA (mtDNA) of 69 goats from five different breeds, Camosciata delle Alpi, Maltese, Nubian, Saanen and Sarda, and one population, the Tunisian. All goats analysed displayed a moderate haplotype and nucleotide diversity. The highest was in the Sarda - the autochthonous breed reared in Sardinia. On the basis of mtDNA control region sequences, animals showed a high genetic haplotype diversity, 35 haplotypes were each represented by a single sequence and only a few haplotypes were shared among the animals. New haplotypes of goats reared in the Mediterranean area were identified and the majority of Italian goats belonged to haplogroup A. This result confirmed worldwide distribution and diversity of haplogroup A.

Polymorphism of the melatonin receptor MT1 gene and its relationship with seasonal reproductive activity in the Sarda sheep breed.

The aim was to study the polymorphisms of the melatonin receptor 1A gene (MTNR1A) and its relationship with seasonal reproduction in the Sarda sheep breed. Four-thousand multiparous ewes reared under natural photoperiod were randomly chosen. Genomic DNA was extracted and subjected to PCR for the amplification of the main part of exon II of the ovine MTNR1A gene (GenBank U14109). PCR products were subjected to restriction enzymes MnlI and RsaI and placed into +/+, +/- or -/- group for MnlI and C/C, C/T or T/T group for RsaI. Samples were cloned and sequenced. The sequences were aligned with the U14109 sequence of GenBank. Data were subjected to allelic frequency analysis and to the chi(2) test in order to evaluate the link between genotype and reproductive activity. After MnlI digestion, allelic frequency was 0.78 for allele +and 0.22 for allele -; genotype frequency of the +/+ homozygote was 68%, 20.5% for +/- and 11.5% for -/-. After RsaI, allelic frequency was 0.66 for allele C and 0.34 for allele T; genotype frequency of the C/C homozygote was 53.5%, 26% for C/T and 20.5% for T/T. The population was in Hardy-Weinberg disequilibrium both for the MnlI and RsaI. Lambing frequency of +/+ genotype ewes was higher in the period September-December while for -/- genotype in January-April (P<0.01). Lambing of C/C genotype ewes showed a higher frequency in September-December while for T/T genotype in January-April (P<0.01). Results confirmed that the polymorphism of the MTNR1A locus was also present in the Sarda with a higher incidence of the +/+ and C/C genotypes. The animals that carried one of these two gene isoforms showed a not seasonal reproductive activity with the lambing period in September-December.

Relationship between MTNR1A melatonin receptor gene polymorphism and seasonal reproduction in different goat breeds.

The reproductive activity of goats bred in temperate latitude follows a seasonal pattern, influenced by annual variation in day length. Daily variation in pineal melatonin secretion is the neuroendocrine signal recognized by animals through the link between this hormone and melatonin receptor 1a (MTNR1A). A total of 345 goats of different breeds (225 Sarda, 30 Saanen, 30 Chamois Coloured, 30 Maltese and 30 Nubian) with a kidding period in October-December or January-March were analysed to verify if a link exists between the structure of the receptor gene and reproductive activity. The main part of exon II of MTNR1A gene was amplified by PCR and then digested with MnlI and RsaI to prove the presence of restriction sites. Sequencing of 20 cloned samples and 20 purified samples permitted comparison with previously published sequences. No polymorphism was found using MnlI enzyme, as all 345 samples showed the cleavage site in position 605 and all the goats were MM genotype. However, using RsaI enzyme, some Sarda goats, showed a polymorphic site in position 53. Nine Sarda goats were R/r genotype, lacking this cleavage site only in one allele, while the other animals, both Sarda and the other breeds, presented the cleavage site in both the alleles and were thus R/R genotype. No r/r genotype was found in any of the breeds. In Sarda goats the allelic frequency was 0.98 for R allele and 0.02 for r allele; genotypic frequency was 96.00% for R/R genotype and 4.00% for R/r genotype. A strong link emerged from statistical analysis (P<0.001) between R/r genotype and reproductive activity, which was strongly influenced by photoperiod. Sequencing indicated six nucleotide changes that did not induce any amino acid change. Data showed that polymorphism was present and that it influences reproductive activity only in the Sarda breed.

Productive performance and meat quality of Mouflon×Sarda and Sarda×Sarda suckling lambs.

The objective of this study was to compare slaughtering performance, carcass characteristics, and meat quality in lambs of two different genotypes. Sixteen crossbred Mouflon×Sarda (M×S) and sixteen pure Sarda breed (S×S) suckling lambs were analysed. Chemical and fatty acid composition were determined on semitendinosus and longissimus dorsi muscles and perirenal and pelvic fats. Dressing percentage was higher (P<0.01) in M×S group and after histological dissection M×S carcasses had more muscle tissue (P<0.01) and less separable fat (P<0.05). Muscles of M×S had less cholesterol than the S×S lambs (P<0.01). Polyunsaturated/saturated and n-6/n-3 fatty acids ratios of the intramuscular lipids were optimal in both groups. Indexes of atherogenicity and of thrombogenicity in muscles varied between 0.9 and 1.1. The results indicated several good qualities in both the genotypes, but M×S lambs have some traits which could provide a higher market price.

The effect of shearing procedures on blood levels of growth hormone, cortisol and other stress haematochemical parameters in Sarda sheep.

The aim of this research was to investigate how growth hormone (GH) cortisol and some haematochemical parameters could be modified by the stress caused by the stages of shearing in Sarda breed sheep. Five groups of 10 sheep each were formed. Group A, only separated from the flock; Group B, only tied; Group C, both tied and shorn (animals in these three groups were ewe lambs shorn for the first time); Group D, adult females both tied and shorn; and Group E, adult entire males both tied and shorn (animals in these two groups had been shorn previously). Five blood samples were taken from each animal: the day before treatment (first sample); at the start of the treatment (second sample); in the middle of shearing for Groups C, D and E, 10 min after separation in Group A and 10 min after tying in Group B (third sample); at the end of treatment (fourth sample); and on the day after treatment (fifth sample). Plasma GH levels showed a decrease (P < 0.01) in Groups A, B, C and D during treatment (third and fourth samples), while Group E only at the end of shearing (fourth sample). In the third sample, the highest GH levels were recorded for Group E (P < 0.05), while it was recorded in the fourth sample for Groups A and E (P < 0.05). Cortisol levels showed a clear increase (P < 0.01) in all groups during treatment, but Group A showed a decrease in the fourth sample in comparison to the third sample. Males in the second, third and fourth sample and Group A only in the fourth sample showed lower cortisol levels when compared with the other groups (P < 0.05). Plasma glucose levels showed an increase (P < 0.01) in all groups during treatment but Groups B, C and E showed the highest values (P < 0.05). Magnesium (Mg) showed an increase in all groups in the third and fourth sample, while sodium (Na), in the same samples, only in Groups B, C and D. Potassium (K) values showed a significant decrease (P < 0.05) only in Groups C and D at the end of shearing. These results show that GH secretion is influenced by all the stress procedure: separation, tying and shearing. Shearing, even if necessary for animals, causes a significant change of the blood parameters involved in the stress response.