Michele Pazzola

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.

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.

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.

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.

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.

Quality traits and modeling of coagulation, curd firming, and syneresis of sheep milk of Alpine breeds fed diets supplemented with rumen-protected conjugated fatty acid

The aim of this study was to test the modeling of curd-firming (CF) measures and to compare the sheep milk of 3 Alpine breeds supplemented with or without rumen-protected conjugated linoleic acid (rpCLA). Twenty-four ewes of the Brogna, Foza, and Lamon breeds were allotted to 6 pens (2 pens/breed) and fed a diet composed of corn grain, corn silage, dried sugar beet pulp, soybean meal, wheat bran, wheat straw, and a vitamin-mineral mixture. The rpCLA supplement (12 g/d per ewe plus 4 g/d for each lamb older than 30 d) was mixed into the diet of 1 pen per sheep breed (3 pens/treatment) to provide an average of 0.945 and 0.915 g/d per ewe of the cis-9,trans-11 C18:2 and trans-10,cis-12 C18:2 conjugated linoleic acid isomers, respectively. The trial started at 38 ± 23 d after parturition, and individual morning milk samples were collected on d 16, 23, 37, 44, and 59 of the trial. Milk samples were analyzed for composition, and duplicate samples were assessed for milk coagulation properties (MCP). A total of 180 CF measures for each sample (1 every 15s) were recorded. Model parameters were the rennet coagulation time, the asymptotic potential CF, the CF instant rate constant, the syneresis instant rate constant, the maximum CF achieved within 45 min (CFmax), and the time at achievement of CFmax. The data were analyzed using a hierarchical model that considered the fixed effects of breed, diet, lamb birth, and initial days in milk, which were tested on individual ewe (random) variance; the fixed effect of sampling day, which was tested on the within-ewe sample (random) variance; and the fixed effect of instrument or cuvette position (only for MCP), which was tested on the residual (replicates within samples) variance. The local Alpine sheep breeds displayed similar milk compositions, traditional MCP, and CF modeling parameters. Supplementation with rpCLA triggered changes in milk composition and worsened MCP (e.g., delayed rennet coagulation time, slower CF instant rate constant, and a doubling of syneresis instant rate constant), but did not influence potential CF. Overall, our results indicate that rpCLA supplementation reduced the actual maximum CF (CFmax) but did not modify the interval between rennet addition and CFmax or time to CFmax.

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.

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.

Relationships between milk characteristics and somatic cell score in milk from primiparous browsing goats.

To determine milk yield and composition, total microbic count (TMC) and somatic cell count (SCC) of browsing goats throughout the first lactation, 100 goats of Sarda breed, equally distributed in four flocks (F1, F2, F3 and F4), were selected. They were exclusively fed pasture and hand-milked once daily. Individual milk samples and daily milk yield were taken from each goat at monthly intervals, from March to July. Milk samples were analyzed for: total protein, fat, lactose, urea, freezing point (FP), pH, TMC and SCC. The data was subjected to analysis of variance and to correlation matrix. On the whole, in all the flocks, milk yield showed the highest production in April and May. Fat content increased (P < 0.01) throughout the lactation. Protein content showed the lowest value (P < 0.01) in June (4.15%). Urea and pH values were fluctuating. FP was lower (P < 0.01) at the start of lactation (-0.562 Hortvet degrees). TMC log10 values were low, considering the hand milking and inadequacy of facilities on the farms. SCC increased (P < 0.01) throughout the lactation and, on the whole, SCC and TMC were not correlated.

Effect of CSN1S1 gene polymorphism and stage of lactation on milk yield and composition of extensively reared goats.

The effect of CSN1S1 genotype and lactation stage on milk yield and composition were investigated in 80 extensively reared goats. Milk yield was recorded in early, mid and late lactation and individual milk samples were collected to determine: fat, protein, lactose and casein content, pH, freezing point, somatic cell count (SCC) and total microbic mesophilic count (TMC). Relative casein composition and amino acid profile were quantified by HPLC. Fatty acid profile was measured by gas-chromatography. Genotype did not affect milk yield, while this trait was significantly affected by lactation stage (P < 0.01). CSN1S1 BB goats produced significantly higher protein and casein percentages (P < 0.05). αs1-casein (CN) was significantly higher in BB and AB goats than AF and BF, showing intermediate values in AA goats (P < 0.01). The protein percentage and the αs1 and αs2-CN fractions were not affected by lactation stage, while the casein content and the β and κ-CN significantly increased throughout lactation (P < 0.01). C4 : 0 and C6 : 0 were not affected by genotype, while C8 : 0 and C10 : 0 were higher in the AA goats than BB; most of the long chain FA were higher in BB than AA goats. MUFA and PUFA increased in late lactation. In addition, BB goats showed higher essential amino acids, resulting in an optimal composition from the nutritional point of view, when compared with AA goats. The increase of MUFA, PUFA, essential and cis-FA in late lactation indicate that the lipid composition of goats milk, with the progress of lactation, tends to improve its nutritional value.