Influences of melatonin treatment, melatonin receptor 1A (MTNR1A) and kisspeptin (KiSS-1) gene polymorphisms on first conception in Sarda ewe lambs.

In order to investigate if the melatonin receptor 1A (MTNR1A) and kisspeptin (KiSS-1) genes influence the reproductive response to melatonin treatment, 510 Sarda ewe lambs were divided into groups C (control) and M; Group M received one melatonin implant (18mg). After 35 days rams were introduced for 40 days and subsequent lambing dates and number of newborns were recorded. The MTNR1A gene Exon II and KiSS-1 gene Exon I were amplified and genotyped by restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism analysis. Two single nucleotide polymorphisms (SNPs; C606T and G612A) in MTNR1A and one (G1035A) in KiSS-1 were found. The most frequent genotypes were G/G (63%) and C/C (53%) for MTNR1A and G/G (92%) for KiSS-1. Treated animals showed a higher lambing rate (P<0.05) and an advanced lambing date (P<0.05) compared with controls. The three SNPs did not influence the onset of reproductive activity. The majority of the G/G animals of Group M lambed before 190 days after ram introduction (P<0.05), while in Group C a higher number of G/G animals lambed after this date. Data revealed the positive effect of melatonin treatment on the time of first conception in ewe lambs and highlighted that the G/G genotype of the MTNR1A gene is able to influence the reproductive response to melatonin treatment.

Association of pituitary specific transcription factor-1 (POU1F1) gene polymorphism with growth and biometric traits and blood metabolites in Iranian Zel and Lori-Bakhtiari sheep.

The pituitary-specific positive transcription factor 1 (POU1F1) gene has been the subject of many recent studies because of its important roles in growth and development of mammals. In this study, we investigated the single nucleotide polymorphisms (SNPs) at the third exon of POU1F1 gene and its association with growth and biometric traits and blood metabolites in two Iranian sheep breeds, Zel and Lori-Bakhtiari. Blood samples from 90 Lori-Bakhtiari and 90 Zel sheep were collected to extract DNA and the 295-bp fragment of the POU1F1 gene was amplified and the restriction fragment length polymorphism (RFLP) technique was adopted for genotyping. A SNP was identified in both Lori-Bakhtiari and Zel sheep breeds, which represents a non-synonymous single base mutation at restriction site for endonuclease AciI. The results revealed differential frequencies of alleles between the two studied breeds, where A allele was more frequent in Lori-Bakhtiari breed, while G allele was more frequent in Zel breed. When POU1F1 genotypes were tested, the animals with AA genotype had a higher weaning weight than those with GG genotype (p < 0.05), however there were not significant association between genotypes and birth weight, biometric traits (body length, body height, heart girth, thigh girth and abdominal girth) and blood metabolites (triglyceride and cholesterol) of the studied breeds (p > 0.05). These findings imply that the POU1F1 polymorphism may affect weaning weight, thus can be used as a molecular marker for this production trait.

New insights onto cardiopulmonary nematodes of dogs in Sardinia, Italy.

Dog heartworms Angiostrongylus vasorum and Dirofilaria immitis cause severe parasitological diseases; the importance of these parasitosis is growing due to their health impact on animals, the possible zoonotic implications and the recent spreading across several European countries and previously non-endemic areas. The aim of this study is to update the epidemiological scenario of cardiopulmonary nematodes A. vasorum and D. immitis in dogs of Sardinia island and to perform a morphological identification of larvae by the use of the Baermann and Knott techniques respectively and the molecular characterization of the mitochondrial cytochrome c oxidase subunit I (cox1) and the second ribosomal transcribed spacer region (ITS-2) of larvae L1 of A. vasorum. In the present study, 3.4% (5/146) of dogs resulted positive at Baermann technique for A. vasorum while 8.9% (61/684) to D. immitis. If on one side A. vasorum can be considered an emerging parasite in Sardinia, the parasitic pressure and the risk of infection for D. immitis in the island seems to be increased compared with the recent past.

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.

MTNR1A gene polymorphisms and reproductive recovery after seasonal anoestrus in different Mediterranean sheep breeds.

The aims of this study were 1) to investigate the effect of MTNR1A gene polymorphisms on reproductive performance in ewes of one Italian and two Slovenian dairy sheep breeds (Sarda, Istrian Premenka and Boska, respectively) which were located at different latitudes, and 2) to highlight if the different season of the male placement with females that was utilized in the different breeding systems in Sardinia (Italy) and Slovenia resulted in different effects of these polymorphisms on reproductive functions. Reproductively mature ewes (n = 100) from each breed were utilized to conduct the study. To evaluate the reproductive efficiency, lambing dates and number of lambs born were recorded per ewe; additionally, the duration in days from ram placement with ewes to lambing (DRPEL), litter size and the fertility rate were determined based on lambing dates. In each breed, there were eight nucleotide variations within the MTNR1A gene exon II, two of which (g.17355358 and g.17355171), respectively, resulted in a valine to isoleucine, and alanine to aspartic acid substitution, in amino acid sequence. The SNPs at position g.17355452 and g.17355458 were determined to have effects on reproductive performance. Genotypes C/C and C/T at g.17355452 in Bovska and Sarda and genotype A/A at g.17355458 in Istrian Pramenka were associated with a greater fertility and a lesser duration in days from ram placement with ewes to lambing. These findings confirmed that the nucleotide sequences of the MTNR1A gene could affect reproductive functions of Mediterranean sheep.

Improving dairy performance through molecular characterization of SREBP-1 gene in Sarda sheep breed.

This research has two aims: (i) to characterize the coding sequence of the SREBP-1 gene in dairy sheep in order to investigate possible relationships between single nucleotide polymorphisms (SNPs) and milk traits; and (ii) to investigate possible relationship between SREBP-1 gene expression and nucleotide variation. Four hundred adult and multiparous lactating Sarda breed ewes were selected from two farms. Milk samples were collected from Day 30 to Day 150 of lactation to determine the mean yield, somatic cell count, lactose, fat, and protein content of the milk. RNA was extracted from the milk samples, after which the SREBP-1 gene coding regions were amplified and sequenced to scan mutations. Whilst eight SNPs were identified, none had statistically significant association with the analysed milk traits. Moreover, the identified expression patterns were not affected by the SNP or combined genotypes. High SREBP-1 gene expression levels were found to be correlated with high milk fat content (P < 0.01), indicating the crucial role of this gene in the milk fat synthesis. In conclusion, the polymorphisms found within SREBP-1 gene exhibited no significant associations with milk traits or with individual SREBP-1 mRNA expression patterns. The findings thus suggest that this small genetic variability may derive from the selection carried out in Sarda breed to improve milk yield.

Reproductive performance in three Slovenian sheep breeds with different alleles for the MTNR1A gene.

The aim of the research was to describe the polymorphisms of the MTNR1A gene in three Slovenian sheep breeds, and determine whether these polymorphisms affect reproductive performance. A total of 100 Bovska, 110 Istrian Pramenka, and 108 Jezersko-Solcavska ewes, were used. The rams were placed with the ewes on 10 October (Bovska), and 20 September (Istrian Pramenka). At each farm, the lambing date and number of newborn lambs were recorded to evaluate the fertility rate, number of days from ram placement with ewes to lambing (DRIL), and litter size. The Jezersko-Solcavska rams were maintained with the ewes of the flock throughout the year and data related to lambing during the entire year were recorded. The DNA sequences indicated there were eight nucleotide variations and that in the position g.17355358 was always associated with that in the position g.17355452. For the Bovska breed, there was an association between the G/G genotype in the position g.17355452, and relatively greater fertility (P < 0.05) and a shorter DIRL (P < 0.05). For the Istrian Pramenka breed, the T/T genotype in the position g.17355458 was positively associated with a greater fertility (P < 0.05) and shorter DRIL (P < 0.05). For the Jezersko-Solcavska breed, ewes with the G/G genotype in the position g.17355452 lambed more when there was a decreasing photoperiod whereas the ewes with the A/A genotype lambed more (P < 0.05) during the period when there were increasing photoperiods. The different polymorphisms studied were associated with reproductive performance in the three Slovenian breeds.

Polymorphisms of the melatonin receptor 1A (MTNR1A) gene influence the age at first mating in autumn-born ram-lambs and sexual activity of adult rams in spring.

The aim of this study was to determine whether polymorphisms of the melatonin receptor 1A (MTNR1A) gene influence the age at first mating in autumn-born ram-lambs and influence the out-of-season sexual activity of adult rams. In experiment 1, 24 Rasa Aragonesa ram-lambs born in September were genotyped for their RsaI and MnlI allelic variants of the MTNR1A gene, and the date of their first mounting with ejaculation after a period of semen collection training was documented. In experiment 2, the reproductive behavior, testicle size, and plasma testosterone concentrations of 18 adult rams (6 rams for each RsaI genotype) were recorded at the beginning (March) and end (May) of the seasonal anestrus. The number of days of training to achieve the first mating with ejaculation in T/T (C/C: 85.17 ± 12.08 C/T: 86.60 ± 18.87; T/T; 26.50 ± 24.50 d; P < 0.05), and G/G ram-lambs (G/G: 51.57 ± 14.99; A/G: 95.58 ± 10.95 d; P < 0.05) was significantly fewer than it was in the other genotypes. Likewise, for the RsaI genotype, 55% of the vulva-sniffing (P < 0.001), 48% of the approaches (P < 0.01), 48% of the mountings (P < 0.05) and 49% total activities (P < 0.001) were performed by T/T rams in March, and 50% of the sexual events in May (P < 0.001). For the Mnll variant, G/G rams performed a significantly (P < 0.001) larger proportion of the vulva-sniffing (41%), approaches (46%) and total activities (40%) in March, and 52% of the vulva-sniffing (P < 0.001), 43%, of the approaches (P < 0.001), 46% of the mountings (P < 0.05), and 47% of the total activities (P < 0.001) in May. Scrotal circumference, testicular volume, and plasma testosterone concentrations did not differ significantly among genotypes. Results confirmed that the polymorphisms of the MTNR1A gene sequence can influence reproductive performance in young and adult rams. Autumn-born ram-lambs that carried the T/T or G/G genotype had an advanced ability to reproduce, and T/T or G/G adult rams exhibited the most intense reproductive behavior. Genotyping might be a useful procedure for identifying the correct and rational use of rams in modern sheep farming.

Melatonin treatment and male replacement every week on the reproductive performance in Sarda sheep breed.

The aim of this study was to highlight whether the combination of melatonin treatment and weekly male replacement could influence reproductive performances in Sarda sheep breed. In a preselected farm, on 5th March 2016, 400 lactating ewes were identified, aged 3-5 years, with body condition score (BCS) 2.5-4.0, who lambed between 20th October and 1st December 2015. The 400 chosen animals were subsequently divided into 4 groups with 100 animals in each group (M, MR, C and CR). On 20th March 2016 the animals of Group M and MR (consisting of 100 ewes each) were treated with melatonin implants and on 24th April 2016 five entire rams were introduced into each group. In groups MR and CR, males were replaced each week with other males whereas in groups M and C the introduced males were not replaced. The MR group showed the highest fertility (number of lambed ewes) compared to the other three groups (P < 0.01). Both the melatonin treated groups (M and MR) exhibited an increase in fertility greater than the controls groups C and CR (P < 0.01). However, when the two groups treated with melatonin were compared, the MR group showed a higher fertility than the M group (P < 0.01). The same can be said for the two control groups, of which the CR group showed a greater increase in fertility than the C group (P < 0.01). The mean interval of days from male introduction to lambing was lower in the treated than in the control groups (P < 0.05). The MR group presented the lowest mean interval of days from male introduction to lambing amongst all of the groups (P < 0.05). A similar trend was recorded for group CR when compared to group C (P < 0.05). In the MR and M groups the lambing peak was recorded close to 170 days after the males introduction, whereas the CR group and the C group were reported to be around 180 days and 190 days, respectively. At the 170th day from the male introduction the number of ewes lambed in the MR group was greater than that of the M group (P < 0.05) (60 vs. 42), and more than double of that of the C and CR groups (P < 0.01). In conclusion, weekly male replacement improved the reproductive activity and strengthened the effect of the pineal hormone on reproductive efficiency. Therefore male replacement either in untreated animals or in association with the melatonin implants, can be straightforwardly applied to guarantee a more efficient reproduction in sheep breeding.

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.

Melatonin treatment in spring and reproductive recovery in sheep with different body condition score and age.

With the aim to evaluate the effect of melatonin treatment on the advanced reproductive recovery in sheep with different body condition score (BCS) and age, 800 ewes were selected from two farms. These ewes (3-6 years old, multiparous and with BCS 2.5-4.0) were subdivided into two Groups (200 M and 200 C), balanced on their BCS and age. On 20 March, Group M was treated with one melatonin implant (18 mg). Group C was untreated. Males were introduced to the groups 35 days after treatment. Gestation was diagnosed between day 45 and 90 after mating by transabdominal ultrasonography. From day 150 to 190 after rams introduction, lambing date and newborns' number were recorded. The average time in days between male introduction and lambing resulted shorter in treated than in control ewes (166.4 ± 0.48 vs. 172.5 ± 0.50) (P < 0.05). At day 160 and 170 from ram introduction the fertility rate was higher in Group M than in C (P < 0.05). The overall fertility at day 190 from rams introduction showed no differences between Group M and C (337 and 339, respectively). At day 170 from male introduction the number of the 5-6 years-old lambed ewes were 2-fold higher than the youngers (P < 0.05). The animals with a BCS 3.5-4.0 had a faster response to male effect, and a shorter mean distance in days from rams introduction to lambing, compared to those scored 2.5-3.0 (166.1 ± 0.48 vs. 174.8 ± 0.51) (P < 0.05). We concluded that the ewes with BCS 3.5-4.0 and aged 5-6 years showed a better response to melatonin treatment in spring.

Day-time and night-time concentrations of airborne iron particles in the atmosphere of Madrid.

Specific measurements of iron aerosol have been made over a 70 days period at a semi-urban site of Madrid. Measurements were made with the idea of assessing the relative contributions of day-time and night-time concentrations to daily ones. Statistics of day-time, night-time, daily and survey time period concentrations are given. Time variations were studied as well, clearly showing daily cyclical pattern identified by applying time spectral analysis. This periodicity is undoubtedly due to level differences between day-time and night-time concentrations of aerosol. Day-time average level ranged from 0.23 to 5.13 micrograms/m3 over the entire survey period (70 days) in contrast with night-times ones ranging from 0.17 to 3.04 micrograms/m3. Night-time/day-time concentration ratio shows a highly consistent value, 0.430 + 0.25 (sigma) during this period. On occasion when diffusion conditions are similar, this ratio increases to 0.518 + 0.18 (sigma). Day-time, night-time and daily concentrations correlation matrix suggests that former is the best daily level indicator.

[The spectral model for characterizing gaseous pollutants in the atmosphere]. / Il modello spettrale per caratterizzare i contaminanti gassosi dell'atmosfera.

Following Tukey's indications, the Authors propose the spectral model for the analysis of the temporal series related to the main pollutants of the atmosphere. In the paper the mathematical method's development is pointed out and its theoretical bases are emphasized, in particular about the basic passages; the formulas used in practice are no. (5), (6), (7) and (8). To exemplify the spectrum evaluation, the trends of the carbon monoxide (CO), relative to the summer and winter periods, are reported in frequency.

Daily surface ozone maximum concentrations at Taliarte, Canary Islands.

Two different methods for using air quality data to estimate daily maximum concentrations along a year of surface ozone concentration in a new measurement station in Canary Islands are described. It is usually assumed that sequences of pollutant concentration show stationary behaviour. In this paper, it is shown that the application of such procedure could be useful to estimate probabilities of maximum concentration of surface ozone. Nevertheless, this method does not take into account the time structure of the original data, so that some caution should be taken into account. In this sense, an alternative procedure, assuming nonstationary behaviour of data, is developed to estimate empirically the probability distributional properties of the original series. Its application yields a substancial improve of the explanation of underlying series structure.

[Models and strategies for the control of air pollution in large urban areas]. / Modelli e strategia per controllare la contaminazione atmosferica in grandi aree urbane.

In the attempt to define the characteristics of a global approach to the problem of atmospheric pollution, the mathematical and statisticals tools currently available are presented which will allow the rationalization of both the study and the management of reduction policy. An example of a possible application of this approach is given, which presents the results obtained by modelling the data on "conventional" pollutants furnished by the experimental sampling station set up at the Istituto Superiore di Sanità (ISS) in Rome. Since the suggested approach may not do without an integrated management of date, a hypothesis for an information system is outlined as well.

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.

Characterization of the Mediterranean Italian buffaloes melatonin receptor 1A (MTNR1A) gene and its association with reproductive seasonality.

The aim of this study was to examine the polymorphism in MTNR1A gene and its relation to reproductive seasonality in Mediterranean Italian buffaloes reared in Sardinia. The mating period and calving of 100 multiparous buffalo-cows were recorded for three years (2005-2008). Genomic DNA was subjected to PCR for the amplification of the exon II, then 40 amplicons were sequenced. The obtained sequence was deposited in GeneBank database (accession number GU817415). PCR products were checked for the presence of HpaI restriction sites and assigned to genotypes "C/C", "C/T" or "T/T". Allelic frequency of C and T alleles was 0.44 and 0.56 and genotypic frequency was 26% for genotype C/C, 40% for C/T and 34% for T/T. In the three observed years the animals with C/C genotype showed the highest number of mating in the semester between August and January and their calving mainly occurred from August to September. On the other hand animals with T/T genotype showed mating mostly in the semester between February and July and calving occurred largely from March to May in all the three years. Heterozygous, in all the three years, showed about the same number of animals mated within each six-month period. The results of the present study provide for the first time a partial sequence as well as one polymorphic site of the MTNR1A receptor gene from buffaloes. Moreover our data showed an association between Single Nucleotide Polymorphism and seasonal reproductive activity in these animals.

Effect of genotype at the MTNR1A locus and melatonin treatment on first conception in Sarda ewe lambs.

A sample of 423 Sarda ewe lambs from three different farms was used to evaluate the effect of one or two melatonin implants on the time of first conception. On each farm, 141 animals were divided into three groups. On June 30 these animals received either no treatment (Group C), 18 mg melatonin (Group M1, one implant), or 18 + 18 mg melatonin (Group M2, two implants). Thirty-five days after treatment, rams were introduced in the ewe lambs flock and subsequently removed after 40 days. Lambing dates were recorded between 150 and 190 days from the first day of male introduction. Genotyping and sequencing of the MT1 exon 2 were carried out to analyze the structure and the possible influence of the MT1 receptor gene on reproductive response to melatonin treatment. Melatonin-treated animals had a higher rate of pregnancy (P < 0.05) and lambed earlier (P < 0.05) compared with untreated animals. Single nucleotide polymorphisms were found in exon II of MT1 gene at positions C606T and G612A leading to genotypes C/C, C/T or T/T and +/+, +/- and -/-, respectively. Melatonin-treated animals of +/+ genotype showed a higher number of pregnancies (P < 0.05) and lambed earlier (P < 0.05) compared to untreated animals of the same genotype. Melatonin treatment did not affect reproductive activity in any other genotype analyzed. No correlation between genotype and the time of first conception was found in untreated animals. Concluding data revealed the positive effect of melatonin treatment on the time of first conception in ewe lambs and highlighted that +/+ genotype is able to influence reproductive response to melatonin treatment.