Genetic variability detected at the lactoferrin locus (LTF) in the Italian Mediterranean river buffalo

Lactoferrin (LTF) is multi-functional protein belonging to the whey protein fractions of the milk. The gene LTF encoding for such protein is considered a potential candidate for body measurement, milk composition and yield. This study reports on the genetic variability at LTF locus in the Italian Mediterranean river buffalo and its possible association with milk yield. Eleven polymorphic sites were found in the DNA fragment spanning the exons 15-16. In particular, the intron 15 was extremely polymorphic with 9 SNPs detected, whereas the remaining 2 SNPs were exonic mutations (g.88G>A at the exon 15 and g.1351G>A at the exon 16) and both synonymous. The genotyping of the informative samples evidenced 3 haplotypes, whose frequencies were 0.6; 0.3 and 0.1 respectively, whereas the analysis of the exonic SNPs showed a perfect condition of linkage disequilibrium (g.88A/g.1351G and g.88G/g.1351A). The association study carried out by using the SNP g.88G>A showed that buffalo LTF gene has no statistically significant influence on daily milk yield. This study adds knowledge to the genetic variability of a species less investigated than the other ruminant species, that may serve as a useful tool for large-scale screening of buffalo populations

Analysis of meiotic segregation by triple-color fish on both total and motile sperm fractions in a t(1p;18) river buffalo bull

Chromosomal aberrations are relatively frequent pathologies in both humans and animals. Among them, translocations present a specific meiotic segregation pattern able to give a higher percentage of unbalanced gametes that can induce fertility problems. In this study, the meiotic segregation patterns of 1p, 1q and 18 Bubalus bubalis chromosomes were analyzed in both total sperm fraction and motile sperm fraction of a t(1p;18) carrier and a control bulls by triple-color FISH analysis with a pool of specific BAC probes. The frequencies of each total sperm fraction products in the carrier resulting from alternate, adjacent I, adjacent II and 3:1 segregation were 39%, 20%, 1% and 38%, respectively. On the other hand, the frequencies of each motile sperm fraction products in the carrier resulting from alternate, adjacent I, adjacent II and 3:1 segregation were 93%, 5%, 0% and 2%, respectively. The frequencies of normal sperms in the carrier were 27% and 69% in total sperm fraction and motile sperm fraction, respectively. The frequencies detected in motile sperm fraction were also validated by comparison with bull's progeny. To our knowledge, this is the first report on the meiotic segregation patterns in motile sperm fractions of B. bubalis bull carrying a chromosomal translocation. These data suggest that translocation has a very limited effect on aneuploidy in the gametes, and therefore, on the reproductive abilities of the bull

Molecular cloning, promoter analysis and SNP identification of Italian Nicastrese and Saanen lactoferrin gene

Lactoferrin (Lf) is an iron-binding glycoprotein found in exocrine secretions including milk. High levels of lactoferrin may have a role in the prevention of microbial infection of the mammary gland. In this report we sequenced and characterized goat lactoferrin cDNA and its promoter region in two different breeds of goat. The complete cDNA comprised 2356 nucleotides, including 38bp at the 5'-UTR and 194bp at the 3'-UTR. The open reading frame is 2127bp long and it encodes a mature protein of 689 aminoacids. A total of 19 nucleotide differences, 11 of them being responsible for 8 aminoacid changes, were identified through the comparison with French, Korean and Tibetan goat lactoferrin cDNAs. About 1700bp of the lactoferrin gene promoter were sequenced. Sequence analysis revealed a non-canonical TATA box, multiple SP1/GC elements, and other putative binding sites for transcription factors, such as NF-kappaB, STAT3 and AP2. Two SNPs were identified, one of which would seem to create a new putative AP2 consensus sequence. The presence of an additional AP2 binding site could be associated with quantitative differences of such protein fraction, which could enhance all the activities related to such protein, and improve mammary gland defence against bacterial infections

Mediterranean River Buffalo CSN1S1 gene: search for polymorphisms and association studies.

The aim of the present work was to study the variability at CSN1S1 locus of the Italian Mediterranean river buffalo and to investigate possible allele effects on milk yield and its composition. Effects of parity, calving season and month of production were also evaluated. Three SNPs were detected. The first mutation, located at position 89 of 17th exon (c.628C>T), is responsible for the amino acid change (p.Ser178Leu). The other two polymorphisms, detected at the positions 144 (c.882G>A) and 239 (c.977A>G) of 19th exon respectively, are silent (3’ UTR). Associations between the CSN1S1 genotypes and milk production traits were investigated using 4,122 test day records of 503 lactations from 175 buffalo cows. Milk yield, fat and protein percentages were analyzed using a mixed linear model. A significant association between the c.628C>T SNP and the protein percentage was found. In particular, the CC genotype showed an average value of about 0.04% higher than the CT and TT genotypes. The allele substitution effect of the cytosine into the thymine was -0.014, with a quite low (0.3%) protein percentage (PP) contribution on total phenotypic variance. A large dominance effect was detected. Furthermore, a characterization of the CSN1S1 transcripts and a method based on MboI-ACRS-PCR for a rapid genotyping of c.628C>T were provided

DNA polymerase alpha inhibition by aphidicolin and fragile site expression in prometaphase chromosomes of the Italian Mediterranean River Buffalo (Bubalus bubalis, 2n=50)

The present study reports on the expression and localization of "fragile sites" (FS) on prometaphase chromosomes of two groups of river buffaloes (Bubalus bubalis, 2n=50; Mediterranean Italian breed), reared in two different farms, with the aim to characterize chromosome fragility in this species. Totally, 400 aphidicolin induced breakages were identified and localized on the standardized ideogram of the river buffalo karyotype. Preliminary results can be synthesized as follows: (a) aphidicolin showed a remarkable decondensing effect on chromosome structure, enabling further studies at high resolution level; (b) the chromosomal expression of the breakages was not different in the two groups of animals; (c) the most fragile chromosomes were the inactive-X, chromosomes 9, 8 and active-X, showing 42, 32, 31 and 30 breakages, respectively; (d) the breaks were localized in the RBG-negative bands (corresponding to eterochromatic regions) or at the band-interband regions; (e) the chromosomal distribution of the break sites was not random and only partially related to chromosome length. The study is in progress to determine the relative incidence of the fragile sites at chromosomal band level, in order to construct a 'fragile-site map' of river buffalo, which could be utilized for genetic improvement programs of the species