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

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

Detection of fish allergen by droplet digital PCR

Fish is one of fourteen allergens that must be highlighted on the label within the ingredients list. It should be noted that the European regulation, is very restrictive to allergens with zero tolerance. Therefore it is important to establish sensitive and specific methods for detecting fish allergen. Applicability to detect and quantify fish allergen by droplet digital polymerase chain reaction (ddPCR) has been evaluated in this work. Genomic DNA of three fish species belonging to the most common fish families were analyzed. PCR primers were designed to amplify a 166 bp region of the 18S rRNA gene. Comparative studies were performed to establish the optimal primer and probe concentrations.  Annealing temperature was determined by using thermal gradient. The results have shown good applicability of the optimized 18S rRNA gene-method to detect and quantify small amounts of the target in all samples analyzed. However, validation studies are needed in order to apply ddPCR technology for routine allergens analysis.

Genotype of Melatonin Receptor MT1 (MTNR1A) and Puberty in Mediterranean Italian Buffalo

In adult buffaloes, polymorphism of the MT1 receptor gene has shown to influence the reproductive seasonality. The aim of study was to assess whether the polymorphism of the MTNR1A gene may influence puberty in Mediterranean Italian buffalo. The study was conducted using 50 prepubertal buffalo cows that at the age of 15 months were placed into the group where there was the male. Estrus detection was performed by observing estrous-behaviour and pregnancy checking by palpation per rectum and/or ultrasound between days 40 and 60 post-mating. Also of each animal dates of calving was recorded. From each buffalos a blood sample was collected and used for DNA extraction. PCR analysis was performed using 100-150 ng of DNA to amplify the second exon of the MTNRA1 gene. All PCR products were digested with 2U of enzyme HpaI to highlight the polymorphism at position 82 (characterized by a C to a T substitution) of the MTNR1A gene. Frequency of C and T alleles was respectively 0.42 and 0.58 in the analyzed population which resulted in Hardy Weinberg equilibrium. The genotypic frequency was 28% for genotype C/C, 38% for C/T and 34% for T/T. The registration of reproductive data showed that the first heat is around the age of 20 months and the first calving around 32 months. Our data show that the genotype of the MTNR1A does not influence the onset of reproductive activity in prepubertal buffalo cows

KRIT1 loss-of-function induces a chronic Nrf2-mediated adaptive homeostasis that sensitizes cells to oxidative stress: Implication for Cerebral Cavernous Malformation disease

KRIT1 (CCM1) is a disease gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease of proven genetic origin affecting 0.3â0.5% of the population. Previously, we demonstrated that KRIT1 loss-of-function is associated with altered redox homeostasis and abnormal activation of the redox-sensitive transcription factor c-Jun, which collectively result in pro-oxidative, pro-inflammatory and pro-angiogenic effects, suggesting a novel pathogenic mechanism for CCM disease and raising the possibility that KRIT1 loss-of-function exerts pleiotropic effects on multiple redox-sensitive mechanisms. To address this possibility, we investigated major redox-sensitive pathways and enzymatic systems that play critical roles in fundamental cytoprotective mechanisms of adaptive responses to oxidative stress, including the master Nrf2 antioxidant defense pathway and its downstream target Glyoxalase 1 (Glo1), a pivotal stress-responsive defense enzyme involved in cellular protection against glycative and oxidative stress through the metabolism of methylglyoxal (MG). This is a potent post-translational protein modifier that may either contribute to increased oxidative molecular damage and cellular susceptibility to apoptosis, or enhance the activity of major apoptosis-protective proteins, including heat shock proteins (Hsps), promoting cell survival. Experimental outcomes showed that KRIT1 loss-of-function induces a redox-sensitive sustained upregulation of Nrf2 and Glo1, and a drop in intracellular levels of MG-modified Hsp70 and Hsp27 proteins, leading to a chronic adaptive redox homeostasis that counteracts intrinsic oxidative stress but increases susceptibility to oxidative DNA damage and apoptosis, sensitizing cells to further oxidative challenges. While supporting and extending the pleiotropic functions of KRIT1, these findings shed new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell predisposition to oxidative damage, thus providing valuable new insights into CCM pathogenesis and novel options for the development of preventive and therapeutic strategies

Characterization of the SREBP-1 Gene Polymorphisms and Milk Traits in Dairy Sheep

The SREBP genes (Sterol Regulatory Element-Binding Proteins) are involved in the milk fat synthesis. In dairy cows some polymorphisms at the SREBP-1 gene sequence have been related with milk fat content. The aim of this study was to characterize the entire coding regions of the SREBP-1 gene in Sarda sheep breed, in order to highlight any polymorphisms and their association with milk traits. Four-hundred adult and lactating Sarda ewes were selected. Individual milk yield was recorded monthly from Day 30 to Day 150 of lactation, and fat and protein concentration were analysed. A blood sample from each ewe was taken for DNA extraction; thus, all the 19 coding exons of the SREBP-1 gene were amplified by polymerase chain reaction (PCR). Single-strand conformation polymorphism analysis (SSCP) and sequencing were used to scan mutations. Results provide, for the first time, the entire coding DNA sequence (CDS) of the SREBP-1 gene in sheep, and by sequences analysis 8 polymorphisms have been detected. The statistical analysis exhibited no relationship between polymorphisms and milk traits. The low SREBP-1 gene diversity that emerged from the present study, may be linked to the important role of this gene in the mechanism of milk fat synthesis or to the severe genetic selection performed in the Sarda sheep. However, it would be necessary to extend the study, including other breeds and other genes, in order to expanding the knowledge about the process of milk fat synthesis in dairy sheep

A Polymorphism at the melatonin receptor 1A (<i>MTNR1A</i>) gene in Sarda ewes affects fertility after AI in the spring

The effect of MTNR1A gene polymorphisms on the fertility rate after AI in Sarda sheep was evaluated in 600 lactating adult ewes. Genomic DNA was subjected to amplification of the MTNR1A gene exon II. Amplicons were digested with restriction endonuclease MnlI. Ten samples from each genotype were sequenced. A polymorphism was detected (A612G) and ewes were determined to be +/+, +/– or –/– for the allele. Allelic frequency was 0.77 for the + allele and 0.23 for the – allele. The frequency of the +/+, +/– and –/– genotypes was 68, 19 and 13%, respectively. On 16 May 2009, 60 ewes from each genotype group were synchronised using intravaginal sponges containing 40 mg fluorogestone acetate for 14 days. At sponge removal, the ewes were administered 350 IU pregnant mare’s serum gonadotropin and were then inseminated, 54–56 h later, with 400 × 106 spermatozoa. Pregnancies were confirmed 50 days after AI using transabdominal ultrasonography. Lambing dates and the number of newborn lambs were recorded within 155 days after AI. Conception and lambing rate were higher for ewes with the +/+ and +/– genotypes compared with those with the –/– genotype (P &lt; 0.01). In conclusion, there was a positive correlation between MTNR1A allele polymorphisms the reproductive response following synchronisation and AI in the spring

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