Pier Paolo Bini

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.

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.

Influence of melatonin receptor 1A gene polymorphisms on seasonal reproduction in Sarda ewes with different body condition scores and ages

In several species, circadian changes in melatonin concentrations play a key role in the photoperiodic control of seasonality. In sheep, two silent mutations in the melatonin receptor 1A gene (MTNR1A) at positions 606 and 612 of the exon II are associated with seasonal reproduction. However, in some sheep breeds, no relationships have been found between MTNR1A polymorphisms and reproductive seasonality. This lack of relationship could be due to effects of breed, body condition, age, and/or environmental conditions. Thus, the present study was conducted with the Sarda sheep breed with the aim of documenting the effect of MTNR1A gene polymorphisms on reproductive resumption and to evaluate whether such this effect was modified by differences in body condition score (BCS) and age. Six hundred three- to six-year-old multiparous ewes with BCSs between 2.5 and 3.5 were selected. Genomic DNA was extracted and subjected to PCR to amplify the ovine exon II of the MTNR1A gene. The amplicons were subjected to digestion with the restriction enzymes RsaI and MnlI to detect the T606C and A612G polymorphisms, respectively. Ewes carrying the G/G, G/A, C/C, and C/T genotypes exhibited higher fertility rates (P<0.05) and fewer numbers of days between the introduction of rams and parturition (P<0.05) than did the A/A and T/T genotypes. The data revealed that the MTNR1A gene polymorphisms influenced spring reproductive resumption in the Sarda sheep breed. Moreover, the data also indicated that, over the limited ranges evaluated in this study, BCS and age had no significant influence on reproductive activity.

Daily rhythm of blood melatonin concentrations in sheep of different ages

The aim of the study was to investigate the daily pattern of melatonin during short photoperiods in sheep of different age. Thirty Sarda sheep kept under natural light conditions and divided into three equal groups were used: Group 1 (12–18 months); Group 2 (3–4 years); Group 3 (8–9 years). Blood samples were collected every 2 h for 22 h, starting at 21 December (12:00 h) until 22 December (10:00 h) for plasma melatonin radioimmunoassay determination. Statistical significant effect of time of day and age on melatonin concentration was registered. Mesor and amplitude values were statistically lower in Group 3 than in Groups 1 and 2. This study provides the first characterization of the relationship between age and daily pattern of melatonin in sheep, highlighting a decrease in melatonin production in the older animals.

Influence of body weight on reproductive activity in Sarda female lambs

Riassunto Influenza del peso corporeo sull’attività riproduttiva delle agnelle di razza Sarda. Per la presente ricerca sono state utilizzate 300 agnelle di razza Sarda, nate tra Novembre e Dicembre, allo scopo di studiare l’effetto del peso corporeo sull’attività riproduttiva. Gli animali, allevati a fotoperiodo naturale, durante il giorno venivano tenuti al pascolo e la notte nell’ovile, dove ricevevano 300 g/capo di concentrato. Le agnelle sono state pesate il 30 Giugno, il 1° Agosto (introduzione degli arieti), e il 10 Ottobre. Dal 1° Gennaio al 15 Marzo sono stati registrati i parti, il numero e il sesso degli agnelli nati. L’attività riproduttiva ha mostrato l’apice in corrispondenza del 10 Settembre. Il peso corporeo degli animali che hanno partorito è risultato, in tutti i rilievi effettuati, superiore (P<0,01) rispetto agli animali che non hanno partorito; mentre non ha influenzato la loro prolificità. I risultati hanno evidenziato che l’inizio dell’attività riproduttiva nelle agnelle di razza Sarda è condizionato principalmente dallo sviluppo corporeo degli animali.

Can advance of first lambing induced by melatonin implants influence the next lambing time in Sarda breed sheep

Carcangiu, V., Mura, M. C., Bini, P. P., Vacca, G. M., Daga, C. and Luridiana, S. 2012. Can advance of first lambing induced by melatonin implants influence the next lambing time in Sarda breed sheep? Can. J. Anim. Sci. 92: 67-71. In adult sheep, exogenously administered melatonin from continuous slow-release implants has been shown to advance the onset of the breeding season by mimicking the stimulatory effect of short days. The aim of this study was to verify if treatment with one or two melatonin implants was effective in advancing the first conception in Sarda ewe lambs, and if this advance would also be seen in the second lambing too. In the first year, 600 ewe lambs were randomly assigned to groups M, M+M and C, each with 200 animals. On Jun. 30, group M received a single implant while group M+M received two implants. Group C was untreated. On Aug. 04, 25 rams were introduced in the groups and removed after 70 d. From these 600 animals the 420 head that lambed prior to Mar. 12 were chosen for the second year. These ewes were subdivided into two groups T1 (ewes who lambed between 2007 Jan. 01 and Feb. 10) and T2 (ewes which lambed between 2007 Feb. 11 and Mar. 12). Both in T1 and T2 the distribution of the animals in M, M+M and C group was maintained. In the first year, the treated animals lambed earlier (P<0.05) and showed higher numbers of lambed ewes at Feb. 10 (P<0.01) and at March 12 (P<0.05) compared with untreated control animals. The animals that showed an advance in the period of their first conception, also showed, in the second breeding year, an advance in the lambing time (P<0.001). This study provides evidence that the advance of first conception, obtained with melatonin treatment also influenced the reproductive activity in the following breeding season.

Genotype at the MTNR1A locus and response to melatonin treatment in Sarda lambs

Abstract With the aim to evaluate the effect of melatonin treatment and melatonin receptor 1A (MTNR1A) genotype on advance of puberty, 423 Sarda lambs were chosen. On June 26th, they were divided into three groups, each of 141 animals (groups 0, 1, and 2), on the basis of live weight. On June 30th, animals in group 1 received a single implant (18 mg melatonin), while group 2 received two implants. Group 0 was untreated. Thirty-five days after treatment (August 4th), rams were introduced and after 40 days they were removed. From January 1st to February 10th lambing dates were recorded. Genomic DNA was extracted and subjected to PCR for the amplification of exon II and then digested with 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. Data obtained were subjected to χ2 test in order to evaluate the difference in fertility among groups and the link between genotype and reproductive activity. Genotype +/+ and C/C showed the highest incidence. Treated groups showed a higher number of lambing at 10th February compared to control group (P<0.04). Melatonin treatment results more efficient in +/+ genotype.

Carcass Characteristics of Mouflon × Sarda Lambs

In dairy sheep breeding the need to find new productive and/or commercial strategies for improving farm income is becoming more and more urgent (Morand-Fehr and Boyazoglu, 1999). While in the past many things were done to optimize breeding techniques and lower production costs, today, thanks to a growing interest in high quality foodstuffs (Boyazoglu and Morand-Fehr, 2001) it is possible to produce so called “niche” products, which have a high added value. Among the different standards which determine the quality of a food, these days dietetic and organoleptic qualities are considered most important (Bonneau et al., 1996). These characteristics, combined with the link to traditions and territory and with a curtailment of supplies, could become a strong attraction for exigent consumers, who are also prepared to accept the higher costs involved in this kind of food production (Rubino et al., 1999). From this perspective, we wanted to propose a sardinian ovine crossbred, obtained from crossbreeding between sardinian wild mouflon (Ovis g. musimon) and Sarda sheep (Ovis aries). This ovine (mouflon) used to be quite widespread in the Mediterranean Basin, such that also Plinius Major (who lived in the first century A.D.) mentioned it in his Naturalis Historia. Its presence in Sardinia was documented, in the XVIII century, by the naturalists George Louis Leclerc, Compte de Buffon (L’Histoire Naturelle, 1749) and Francesco Cetti (Storia naturale di Sardegna, 1774). Still today, when the possibility exists for wild mouflon territory overlapping with that of reared sheep, it is not uncommon to notice the birth of crossbred young. In order to produce, estimate and increase the value of this product, whose natural commercial place is in the mountainous areas of Sardinia, an extensive scientific project was started. This includes the analysis of a series of parameters which begin from the study of productive traits, pass through bromatologic composition of meats, and ends with the use of an “appellation controlee” system. In this note, the results regarding carcasses dissection and tissue composition are reported.