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One main aspect of safeguard plans for livestock Autochthonous Genetic Types (AGTs) is to ensure economic sustainability of their farming. This encourages farmers to contribute to the pres- ervation of AGTs. The aim of this study was to characterize the genetic structure of four AGTs of endangered goats at the αS1, β and αS2 calcium sensitive caseins encoding genes CSN1S1, CSN2 and CSN1S2, respectively. The peculiar polymorphisms detected at these loci are associated with milk quali-quantitative charac- teristics and have been largely investigated in widespread dairy goat breeds. On the contrary, there are very few studies carried out in AGTs goats. This study was performed on 50 Neapolitan goats, typical of the Campania region, and on 61 goats which included the three breeds Monticellana, Capestrina and Grigia Ciociara, reared in Lazio region (Lazio AGTs goat). Genotyping of quantitative alleles at CSN1S1, CSN2 and CSN1S2 loci was performed according to previous studies. At the CSN1S1 locus a high variability was observed in all four AGTs. In particular, a higher frequency of the null allele N was observed in Neapolitan goats than Lazio AGTs goats (0.420 vs. 0.008). On the contrary a higher frequency of F allele, associated with a low αS1 casein content in milk, was observed in Lazio AGTs goat (0.442) than Neapolitan goat (0.120). All four AGTs do not carry the null alleles CSN1S1 01, CSN2 01 and CSN1S2 0 and the alleles associated with an intermediate content of αS1 (CSN1S1 E) and αS2(CSN1S2 D) caseins in milk. The genotyping data obtained pro- vide useful information regarding the productive aptitudes of the four AGTs studied. In fact, the high frequency of CSN1S1 N allele in Neapolitan goat, predisposes this breed to produce low-protein milk for fresh consumption while the high frequency of the allele CSN1S1 F in the Lazio AGTs goat addresses their milk to process- ing for the production of typical cheese with unique organoleptic and flavor characteristics. Developing safeguard plans that take these results into account will have a higher chance of success, with a consequent increase in the number of animals raised.

The αs2-casein is an important phosphoprotein secreted in ruminants’ milk but, nowadays, the encoding gene (CSN1S2) has not been characterized in buffaloes. In this study, we report the first characterization of buffalo CSN1S2 gene and its artiodactyla retroposons (RTs) distribution. For this purpose, we amplified and sequenced the whole gene of 2 Mediterranean river subjects eared in Campania (Italy). The gene includes 1025 bp of coding (18 exons) and 17578bp of intronic regions, 937 bp at the 5’-end and 707 bp at the 3’-end and it is characterized by 13 RTs. The first (named A) is located in the promoter region and appears to be a Bov-tA2. Further 2 elements are located in intron 1 (B and C) and show a strong similarity with L1_Art sequence. Two Bov-tA2 are located in the intron 2 (D and E). The intron eight includes a Bov-B (F), whereas the intron 12 contains a Bov-A2 (G). Five additional retroposons (H, I, L, M, N) are located in the intron 13 (Bov-tA1), 15 (Bov-A2) and 17 (Bov-tA1, Bov-B, Bov-A2). Finally, a Bov-tA2 (O) is located in the 3’ UT, near to the last exon. The observed RTs represent 19.45% of the whole gene. This figure decreases considerably in the bovine and caprine/ovine counterpart because of presence/absence of RTs observed in these species. In particular, the bovine CSN1S2 does not show the element B and C and, at the same time, includes expanded Bov-A monomers (Bov-A3 in cattle and Bov-A2, G in buffalo). Sheep and goat have a similar gene structure and, when compared to buffalo, the absence of elements C, G, I and N was noted together with the presence of an extra Bov-tA3 and of Bov-A4 in the promoter region and the intron 1, respectively. Ruminant retroposons are highly informative genetic markers that can be considered a powerful phylogenetic tool for clustering studies, animal evolutionary history, population structure and demography, rather than the setup of methods for species discrimination in meat and dairy products

The αs2-casein (CN) is a milk protein fraction encoded by the CSN1S2 gene. Previous studies on dromedary milk proteins mainly focused on the other three fractions (αs1-, β- and k-CN), and, although there is globally growing interested in camel milk, studies focusing on αs2-CN are very limited. In this respect, no information has been reported on retrotransposons (RTs). These repeats, also known as Interspersed elements (INEs), are informative genetic markers useful as phylogenetic tools for clustering, evolutionary and population structure studies, rather than for the setup of food traceability methods. Aim of this study was to identify and characterize INEs in the dromedary CSN1S2 gene. Analysis has been carried out using RepeatMasker as bioinformatic tool. Three long interspersed elements (LINEs) were found in introns 1, 10 and 11. The first and the last elements are truncated LINEs 2, respectively 50 bp and 97 bp long. Conversely, the repeat in intron 10 is 149bp long and it is a truncated LINE 1. In addition, three short interspersed elements (SINEs) have been found in introns 8, 9 and 16, with a size of 190bp, 149bp and 207 bp, respectively. The observed six repetitive elements cover 5.6% of the whole dromedary CSN1S2 gene sequence. Camel CSN1S2 gene has more INEs compared with CSN2 gene (five RTs) and they belong to the same types except for the MER5B, which was not found in CSN1S2 gene. Conversely, the CSN1S2 has less INEs compared with CSN3 (7 RTs) and is distinguished by the presence of LINE 2 type, which was not found in camels CSN3. Camel CSN1S2 also shows less INEs than the homologous bovine gene (8 RTs, 5 of them are SINEs and 3 are LINEs). These results agree with previous studies on CSN2 and CSN3 genes and confirm that camel casein genes have lower repetitive elements than cattle. This characteristic is an indication that Tylopoda diverged from Ruminantia before additional retrotransposition events took place at different times during the divergence.

The article attempts to summarize the way in which certain concepts from general semiotics and some of its applications (mainly visual semiotics and semantic analysis) can be of use to contemporary thinking around political communication. It focuses on two general tendencies within western political communication, that have been cutting across parties, leaders and nations over the last decades: a tendency to construct binary oppositions and the storytelling fashion. Indeed, semiotics can offer political communication studies something original, something which otherwise, without its contribution, would remain neglected, unconceptualized or unexpressed.

Prolactin (PRL) is one of the most versatile hormones of the pituitary gland in terms of biological actions. In particular, PRL is essential for mammary gland development, lactogenesis and milk protein gene expression. It has also considered as a potential quantitative trait locus affecting milk performance traits in dairy cattle. Aims of this study were to detect PRL gene polymorphisms and to investigate their effect in Mediterranean river buffalo milk yield. For this purpose, DNA was isolated from the blood collected from 757 unrelated lactating Mediterranean river buffaloes belonging to 14 farms located in Salerno, Caserta and Potenza provinces (Italy). Using primers designed on bubaline genome sequence we amplified and sequenced the entire PRL gene of 5 buffaloes randomly chosen. Seven-thousand one-hundred and fifty-one records for milk yield, measured monthly by the official recording system of the Italian Association of Buffalo Breeders on 1081 lactations of 544 buffaloes, were used. Association between PRL polymorphism and milk yield was investigated with a mixed linear model. The entire panel of Mediterranean river buffalo DNA samples were genotyped in outsourcing (KBiosciences, Herts, UK) for the exonic SNP. The buffalo PRL gene extends over 8.6 kb including 0.858 kb of exonic regions and 7.741 kb of intronic regions plus 361 bp of the 5′UTR. The main feature of the buffalo PRL gene is simple architecture. It contains 5 exons ranging in size from 82 (exon 1) to 306 bp (exon 5) and 4 introns from 629 bp (intron 4) to 2715 bp (intron 1). The sequence comparison of buffalo PRL gene showed 34 intronic polymorphisms (11 transversions, 22 transitions and 1 single deletions/insertions) and a transition C→T at the 108th nucleotide of the exon 5. The frequency of the PRL T108C SNP in the investigated population was 0.88 (genotype distribution: 589 T/T, 156 T/C, 12 C/C). The least-squares means for milk yield indicated a difference of 0.18 kg/day between the genotypes TT (n. subjects 432) and TC (105) or CC (7), whereas both TC and CC showed an average milk production of 8.32 kg/day. However, these differences were not significant. Therefore, this SNP appears to be selectively neutral in relation to milk yield.

Leptin is a 16 kDa protein highly expressed in the adipose tissue, in pituitary gland, brain and mammary gland. Leptin is responsible for the regulation of body weight and energy homeostasis and is one of the potential biomarker for high animal performances leading to better adaptability and productivity. In dairy and beef cattle, many studies have been reported on associations between Leptin gene (Lep) and traits like meat quality, carcass characteristics, fertility, feed intake, protein yield, energy balance and body weight. Different studies have also reported that this hormone can influence milk’s quality in dairy cattle, sheep and goats. On contrary, there have been limited research regarding association studies between SNPs at this locus and milk traits in buffalo. Lep gene in buffalo consists of three exons and two introns (GenBank:AH013754.2) and it has been mapped on chromosome 8 (BBU8q32). The aim of the present research was to identify and analyse the variability of the Lep locus in Italian Mediterranean river buffalo and to test possible associations with milk fat traits. Individual fresh milk and blood samples were collected from 766 unrelated lactating buffaloes reared in 14 farms of Southern Italy. 306 milk samples were used for fat analysis (fat content, fatty acid composition and fatty acid classes), whereas blood samples were treated for genomic DNA isolation. We sequenced the exon 3 (over 1.8 kb) of 8 buffaloes randomly chosen. Nucleotide alignment showed a synonymous transition GA at the 231th nucleotide of exon 3 that identifies a new allele named A1. The entire panel of DNA samples was genotyped in outsourcing (KBiosciences, Herts, UK). The allele frequency of the common allele (Lep A) was 0.84 (genotype distribution: 547 A/A, 196 A/A1, 23 Lep A1/A1). The statistical analysis, using a mixed linear model, showed no direct effect of Lep g.231G>A genotype on milk fatty acids composition. However, this result does not exclude the existence of an association of this SNP in other buffalo populations for other traits of economic interest.

Objective: Aim of present study was the set up of a fast and reliable protocol using species-specific markers for the quali-quantitative analysis of DNA and the detection of ruminant biological components in dairy products. For this purpose, the promoter of the gene coding for the -lactoalbumin (LALBA) was chosen as possible candidate for the presence of Short Interpersed Elements (SINEs). Methods DNA was isolated from somatic cells of 120 individual milk samples of cattle (30), Mediterranean river buffalo (30), goat (30) and sheep (30) and the gene promoter region (about 600/700 bp) of α-lactalbumin (LALBA) (from about 600 bp upstream of exon 1) has been sequenced. For the development of a single PCR protocol that allows the simultaneous identification of DNA from the four species of ruminants, the following internal primers pair were used: 5’-CACTGATCTTAAAGCTCAGGTT-3’ (forward) and 5’-TCAGAGTAGGCCACAGAAG-3’ (reverse). Results Sequencing results of LALBA gene promoter region confirmed the presence of SINEs as monomorphic “within” and variable in size “among” the selected species. Amplicon lengths were 582 bp in cattle, 592 bp in buffalo, 655 in goat and 729 bp in sheep. PCR specificity was demonstrated by the detection of trace amounts of species-specific DNA from mixed sources (0.25 ng/l). Conclusion We developed a rapid PCR protocol for the quali-quantitative analysis of DNA and the traceability of dairy products using a species-specific marker with only one pair of primers. Our results validate the proposed technique as a suitable tool for a simple and inexpensive (economic) detection of animal origin components in foodstuffs.

Lipoprotein lipase is a key enzyme for the lipid metabolism playing a fundamental role in the composition of fat in adipose tissue as well as in milk. LPL gene was poorly investigated in dairy ruminants, and it has been never studied in river buffalo. Aims of this work were to explore the genetic diversity of LPL (from exon 1 to 4) in this species; to characterize the gene promoter; and to achieve an association study with milk traits. Genomic DNA of 14 individual samples of Mediterranean Italian breed was amplified and sequenced. Thirteen consensus sequences for transcription factors were found. C/EBPα and Oct-1 are the main regulatory motifs together with LP-α, although a deeper investigation is necessary to elucidate the role of the latter element, which in buffalo (and ruminants in general) is lacking the downstream cis-acting motifs (LP-β) compared to human. A total of 11 SNPs have been detected. The SNP g.107A>G was the only polymorphism found at exon level, therefore a genotyping was achieved by PCR-RFLP in a population of 523 Mediterranean buffaloes. The allele frequency was 0.63 and 0.37 respectively for the G and A allele. This SNP was significantly associated with n3-PUFA (P

Outcome of bovine tuberculosis (bTB), an infectious disease caused by Mycobacterium bovis, is influenced by host genetic background. For this reason, polymorphism discovery association studies are a powerful tool for selective breeding helping disease control. Interleukin-10 (IL-10) is a regulatory cytokine produced by different cells and fine-tune immune response to bTB. Thus, in this research, we evaluated the role of the single nucleotide polymorphism g.4002C>T in IL-10 gene for susceptibility to bTB in Mediterranean water buffalo. We characterized 184 animals grouped in cases and controls and demonstrated that homozygous subjects TT are about 3 times more susceptible to bTB compared to CC homozygous. Indeed this polymorphism is responsible of amino acid substitution p. (Thr 175 Met) in the primary protein sequence which affects protein secondary structure. This polymorphism might represent a valid tool for marker assisted selection against bovine tuberculosis in water buffalo.

Iodine (I) is a micronutrient essential for the proper thyroid functionality in mammals. I requirement is dependent by the life stage and milk constitutes the main source of I intake for infant. For this reason, in dairy ruminants, the calibration of I is fundamental to keep the performance high. However, the effects of I supplementation on gene expression in dairy cow are not well documented. Thus, to improve knowledge in this field, we have assessed the effect of I supplementation dairy cattle using RNAsequencing. In detail, we have randomly divided 22 dairy cows into two groups: the first group (CTR, 11 animals) received a customised diet with physiological level of I2 (20 mg/day/animal of I2) while the second one (I, 11 animals) received the same diet with a I2 supplementation (20 + 65 mg/day/animal of I2) for 8 weeks (56 days). At the end of supplementation, the whole blood from both groups is been collected and the transcriptome analysis is been performed. Filtering our data using a FDR

Beta-lactoglobulin (β-lg) is the major whey protein in ruminants’ milk and in these species has been suggested to play a role also in milk fatty acid metabolism in the newborn. In cattle, sheep and goat the coding gene (BLG) has been well investigated and alleles to this locus were associated with dairy performances. In contrast, data regarding BLG polymorphism and association studies with milk composition traits are scarce in buffalo and carried out only in Indian breeds. Therefore, the aim of the present research was to identify and analyse the variability of the BLG locus in the Italian Mediterranean river buffalo and to test the possible associations between polymorphisms and milk fat traits. For this purpose, individual fresh milk and blood samples were collected from 747 unrelated lactating Mediterranean river buffaloes reared in 14 farms in the province of Salerno, Caserta and Potenza (Italy). Three-hundred and six milk samples were used for FA analysis (fat content, fatty acid composition and fatty acid classes) whereas all blood samples were treated for genomic DNA isolation. We sequenced the entire BLG gene (over 4749 bp, including 789 bp in exonic regions, from exon 1 to exon 7) plus 229 bp of the 5′UTR and 241 bp of 3′UTR of 2 Mediterranean river buffaloes randomly chosen. The sequenced BLG exons had different lengths (from 42 bp for exons 6–179 bp for exon 7) whereas intron lengths ranged from 214 bp (intron 5) to 995 bp (intron 3). Alignments of the sequences among the 2 subjects showed 33 intronic polymorphisms (11 transversions, 11 transitions, 7 deletions/ insertions and 4 inversions) and a transition C→T at the 67th nucleotide of the 5th exon. This mutation identifies a silent allele named BLG A1. Since the transition removes a Hsp92II endonuclease restriction site, the digestion of a PCR product of 353 bp, spanning the 5th exon and part of the 6th exon, allows a correct genotyping. The allele frequency of the BLG A1 allele, determined in 747 subjects was 0.70 (genotype distribution: 53 BLG A/A, 419 A1/A1, 275 A/A1). The present study suggests that no direct effect of the investigated polymorphism exists on these production traits. Although no association was found, the present work provides a starting point for additional association studies between BLG polymorphisms and other phenotypic traits in buffalo, such as milk yield.

Oxytocin is a neurohypophysial peptide released into general circulation from posterior pituitary gland. It is involved in different physiological roles, including milk ejection from the mammary gland, stimulation of uterine smooth muscle contraction during labour and affects cognitive processes, tolerance, adaptation and complex sexual and maternal behaviour. The main objective of the present research was to determine the complete donkey oxytocin-neurophysin I encoding gene (OXT) sequences and to detect genetic diversity at this locus. Using the genomic DNA as template, we sequenced and compared the whole OXT gene of 5 Italian donkeys: 2 Amiatine, 2 Ragusana and 1 Grigio Siciliano donkeys. On average the gene extends over 946 bp, composed of 517 bp of exonic regions and 429 bp of intronic regions, with an A/T and G/C content of about 27% and 73%, respectively. Furthermore, the 5’ flanking region (876 bp) was sequenced. The gene contains only three exons, ranging in size from 202 bp (exons 2) to 155 bp (exon 3), and two introns of 315 bp (intron 1) and 114 bp (intron 2). The first exon encodes a peptide leader (19 amino acids residues encoded by the nucleotides from 41 and 97), the nonapeptide hormone (from nucleotide 98 to 124), the tripeptide processing signal (GKR) (from nucleotides 125 and 133) and the first 9 of 94 residues of neurophysin I; the second exon encodes the central part of neurophysin I (67 aa), while the third exon encodes the COOH–terminal region of neurophysin I (18 aa). Among the different species of animals of species of livestock animals, the OXT donkey gene sequence showed the highest degree of similarity with the horse (98%) vs. ruminants (75%) and camelids (76%). The comparison of the sequences obtained allowed the identification of 5 SNPs: g.263A>G, g.334A>G, g.436T>C and g.304A>C in intron 1 and g.124G>A in promoter region. This last polymorphic site falls into a potential binding site for the transcription factor GATA-1 (www.generegulation. com). With this study, we provide the first contribution to the characterisation of the genomic sequence of the OXT gene and first examples of markers found at this locus in donkey. The detected polymorphisms represent a good opportunity to carry out studies focussed on the identification of significant association with the physiological processes controlled by this hormone as already performed in other species.

Fatty acid (FA) profile is a crucial factor in defining milk properties and quality. Increasing attention is placed on those FA having a positive role on human health, such as polyunsaturated FA (PUFA), oleic acid, vaccenic acid and conjugated linoleic acid (CLA). In the present work, the FA profile obtained from gas-chromatographic analysis of 703 individual milk samples (289 cows, 214 buffaloes and 200 ewes), were used to evaluate the suitability of principal component (PC) analysis (PCA) to investigate differences in milk FA profile among these species. Thirty-six FA were analyzed by PCA. The first and second PC (PC1 and PC2) accounted for 51% of the total variability (30%, 21%, for PC1, PC2, respectively). Elements of eigenvector of PC1 were positively associated with few FA, including C16:0 (0.50), the most abundant FA in milk of the 3 species (~29.3% of FA). Among those negatively correlated with PC1, some relevant FA for human health were retrieved: C18:1t11 (−0.75), CLAc9t11 (−0.79), C18:3n-3 (−0.76), EPA (−0.81) and DPA (−0.89). Negative correlations with PC2 were observed, in general, for medium chain FA (e.g., C15:0, C16:0), whereas long chain FA (e.g., C18:1 isomers, C18:2n-6 and other PUFA) exhibited positive values. Score values of PC1 and PC2 allowed the description of the relationship among animals based exclusively on the milk FA profile, revealing a clear discrimination of the 3 species: PC1 discriminated ewes milk from that of cows and buffaloes, whereas, PC2 was able to discriminate cows from buffaloes, with ewes showing intermediate values. In general, the use of PCA evidenced as a great part of variance of FA profile of ruminants (PC1) is related to the distinction between small or large ruminants, also evidencing higher amount of beneficial FA for ewes. An important part of variance (PC2) indicates as ewes milk FA has intermediate characteristics compared with cow and buffalo milk

DGAT1 has been recognised as strong functional QTL for the milk fat content in cattle. In river 10 buffalo, DGAT1 has been investigated mainly for the characterization of the gene itself and for the 11 identification of the K232A polymorphism similarly to what has been done in cattle. No 12 investigation has been carried out at transcriptomic level so far. Aim of this study was to analyse the 13 transcript profile of DGAT1 in lactating buffaloes. 14 Milk samples were collected from 8 unrelated buffalos (at the 3rd lactation and 120 days from 15 calving), reared in Piedmont Region and belonging to one farm. Total RNA was isolated from milk 16 somatic cells using TRIzol. The reverse transcription was performed using an oligo dT18, whereas 17 the PCR reaction was accomplished using the following primers 5’-ATGGGCGACCGCGGCGG-18 3’ and 5’-TCAGGTGCCGGCTGCCGG-3’, corresponding to the nucleotides 1-17 (exon 1) and 19 complementary to the base pairs 1453-1470 (exon 17) of the buffalo DGAT1 cDNA (EMBL ID: 20 DQ120929). The PCR products were cloned into pGEM-vector, and transformed into JM109 21 competent cells. White recombinant clones were randomly chosen and screened by PCR. All 22 amplicons different in size (bp) were purified and sequenced in both directions. 23 A total of 147 positive recombinant clones were analysed. The sequence analysis showed a complex 24 mRNA pattern with a total of at least 6 different transcripts. The most represented mRNA was that 25 correctly assembled (in total 86 out of 147 clones, 58.5%), 1470bp long and coding for a functional 26 protein of 489aa (amino acids). The following mRNA population was deleted of the exon 12 27 (21.09%). Despite this deletion, the mature mRNA did not undergo any frame-shift and the 28 termination codon was kept as in the normal isoform. The putative protein 474 aa long is different 29 from other predicted DGAT1 isoforms derived from the buffalo genome project. Part of the other 30 transcripts were deleted of the last 66bp of the exon 8 (7.48%). This alternative splicing is 31 consequence of the incorrect identification of a splice donor site directly in the exon 8 and it is 32 responsible for a protein isoform 22 amino acids shorter. Minor transcripts are represented by 33 mRNAs with the insertion of the intron 13 (5.44%), populations deleted of the exon 16 (4.76%) and 34 transcripts characterised by the contemporary out-splicing of the exons 6 and 7 and the insertion of 35 the intron 13 (2.72%). The investigation at DNA level will likely clarify the variability found at 36 mRNA leve

Excess thymidine is capable of synchronizing lymphocyte cultures in vitro by acting with a feed-back mechanism during the S phase of the cell cycle (Harper 2005 Methods Mol. Biol. 296, 157-166). The possibility to synchronize the embryonic growth can be a good strategy for future epigenetic studies. The present study was undertaken to test whether excess thymidine could also synchronize in vitro development of bovine embryos. Abattoir-derived cumulus-oocyte complexes (COC) of the Agerolese breed of cattle were matured in vitro using standard procedures. After maturation, COCs were transferred in drops of 300µL of IVF-TALP (25/drop) and covered with mineral oil. Frozen sperm from a bull were selected by centrifugation on a Percoll discontinuous gradient (45 to 80%). The pellet was diluted in IVF medium and added to the COC at the concentration of 1×10(6)spermmL(-1). After 18-20h of gametes co-incubation, presumptive zygotes were denuded and cultured in SOF medium containing different concentrations of thymidine (0, 300, 600, 1200, 2400µgmL(-1), final concentrations). The day after (Day 2) presumptive zygotes were washed four times in fresh SOF, classified morphologically under a stereomicroscope as not divided (n.d.), 2 cells, 3-8 cells, and 9-32 cells and cultured in standard SOF at 39°C in a humidified mixture of 5% CO(2),7% O(2), and 88% N(2). On Day 3, the embryos were again examined for the growth stage in relation to the synchronization effects. On Day 7, the embryos were evaluated for the final growth efficiency (cleavage stage and blastocyst formation). The experiment was replicated 4 times (except the 2400µgmL(-1) condition, which was replicated 2 times because of its clear toxic effect). Data were analyzed by ANOVA test. At Day 2, there were no differences between groups whatever the concentration (n.d.-2 cells: 59.7±11.2, 52.9±26.3, 56.1±14.6, 66.0±2.8, 38.0±11.3; 3-8 cells : 40.3±11.2, 47.1±26.3, 43.9±14.6, 34.0±2.8, 59.9±9.5; respectively for 300, 600, 1200, 2400, and control), while differences at Day 3 and 7 are shown in Table 1. The dosage of 300µgmL(-1) slowed embryo development without altering the developmental rate, whereas the other dosages were somewhat toxic to the zygotes, affecting the final percentage of blastocysts.