Design and validation of a 90K SNP genotyping assay for the water buffalo (Bubalus bubalis)

Background: The availability of the bovine genome sequence and SNP panels has improved various genomic analyses, from exploring genetic diversity to aiding genetic selection. However, few of the SNP on the bovine chips are polymorphic in buffalo, therefore a panel of single nucleotide DNA markers exclusive for buffalo was necessary for molecular genetic analyses and to develop genomic selection approaches for water buffalo. The creation of a 90K SNP panel for river buffalo and testing in a genome wide association study for milk production is described here. Methods: The genomes of 73 buffaloes of 4 different breeds were sequenced and aligned against the bovine genome, which facilitated the identification of 22 million of sequence variants among the buffalo genomes. Based on frequencies of variants within and among buffalo breeds, and their distribution across the genome, inferred from the bovine genome sequence, 90,000 putative single nucleotide polymorphisms were selected to create an Axiom® Buffalo Genotyping Array 90K. Results: This 90K "SNP-Chip" was tested in several river buffalo populations and found to have ∼70% high quality and polymorphic SNPs. Of the 90K SNPs about 24K were also found to be polymorphic in swamp buffalo. The SNP chip was used to investigate the structure of buffalo populations, and could distinguish buffalo from different farms. A Genome Wide Association Study identified genomic regions on 5 chromosomes putatively involved in milk production. Conclusion: The 90K buffalo SNP chip described here is suitable for the analysis of the genomes of river buffalo breeds, and could be used for genetic diversity studies and potentially as a starting point for genome-assisted selection programmes. This SNP Chip could also be used to analyse swamp buffalo, but many loci are not informative and creation of a revised SNP set specific for swamp buffalo would be advised.Daniela Iamartino, Ezequiel L. Nicolazzi, Curtis P. Van Tassell, James M. Reecy, Eric R. Fritz-Waters, James E. Koltes, Stefano Biffani, Tad S. Sonstegard, Steven G. Schroeder, Paolo Ajmone-Marsan, Riccardo Negrini, Rolando Pasquariello, Paola Ramelli, Angelo Coletta, José F. Garcia, Ahmad Ali, Luigi Ramunno, Gianfranco Cosenza, Denise A.A. de Oliveira, Marcela G. Drummond, Eduardo Bastianetto, Alessandro Davassi, Ali Pirani, Fiona Brew, John L. William

Generation of Porcine and Rainbow Trout 3D Intestinal Models and Their Use to Investigate Astaxanthin Effects In Vitro

Astaxanthin (AST) is a natural compound derived from shellfish, microorganisms, and algae, with several healthy properties. For this reason, it is widely used in the diet of humans and animals, such as pigs, broilers, and fish, where its addition is related to its pigmenting properties. Moreover, AST’s ability to reduce free radicals and protect cells from oxidative damage finds application during the weaning period, when piglets are exposed to several stressors. To better elucidate the mechanisms involved, here we generate ad hoc pig and rainbow trout in vitro platforms able to mimic the intestinal mucosa. The morphology is validated through histological and molecular analysis, while functional properties of the newly generated intestinal barriers, both in porcine and rainbow trout models, are demonstrated by measuring trans-epithelial electrical resistance and analyzing permeability with fluorescein isothiocyanate–dextran. Exposure to AST induced a significant upregulation of antioxidative stress markers and a reduction in the transcription of inflammation-related interleukins. Altogether, the present findings demonstrate AST’s ability to interact with the molecular pathways controlling oxidative stress and inflammation both in the porcine and rainbow trout species and suggest AST’s positive role in prevention and health

New Stable Cell Lines Derived from the Proximal and Distal Intestine of Rainbow Trout (Oncorhynchus mykiss) Retain Several Properties Observed In Vivo

We derived two novel cell lines from rainbow trout (RT) proximal (RTpi-MI) and distal intestine (RTdi-MI) and compared them with the previously established continuous cell line RTgutGC. Intestinal stem cells, differentiating and differentiated epithelial cells, and connective cells were found in all cell lines. The cell lines formed a polarized barrier, which was not permeable to large molecules and absorbed proline and glucose. High seeding density induced their differentiation into more mature phenotypes, as indicated by the downregulation of intestinal stem cell-related genes (i.e., sox9, hopx and lgr5), whereas alkaline phosphatase activity was upregulated. Other enterocyte markers (i.e., sglt1 and pept1), however, were not regulated as expected. In all cell lines, the presence of a mixed population of epithelial and stromal cells was characterized for the first time. The expression by the stromal component of lgr5, a stem cell niche regulatory molecule, may explain why these lines proliferate stably in vitro. Although most parameters were conserved among the three cell lines, some significant differences were observed, suggesting that characteristics typical of each tract are partly conserved in vitro as well.ISSN:2073-440

Sperm fertilizing ability in vitro influences bovine blastocyst miRNA content

9 Pág.MicroRNAs (miRNAs) are small highly conserved non-coding RNA molecules that orchestrate a wide range of biological processes through post-transcriptional regulation of gene expression. During development, miRNAs play a key role in driving embryo patterning and morphogenesis in a specific and stage-dependent manner. Here, we investigated whether sperm from bulls with different fertilizing ability in vitro influence blastocyst quality and miRNA content. Results demonstrate that blastocysts obtained using sperm from high fertility sires (H group) display significantly greater cleavage and blastocyst development as well as greater transcript abundance in blastocysts for the developmental competence markers CDX2, KRT8, NANOG, OCT4, PLAC8, PTGS2, SOX17, and SOX2, compared to blastocysts generated using sperm from low fertility sires (L group). In parallel, high throughput deep sequencing and differential expression studies revealed that H blastocysts exhibit a greater miRNA content compared to L blastocysts, with hsa-miR-4755-5p and hsa-miR-548d-3p uniquely detected in the H group, and greater abundance of hsa-miR-1225-3p in the H group. Gene ontology (GO) and KEGG pathway analyses indicated that the 3 differentially expressed miRNAs identified are involved in the regulation of many biological mechanisms with a key role in aspects of early embryo development, including transcriptional regulation, cellular biosynthesis, nucleic acid metabolism, cellular differentiation, apoptosis, cytoskeleton remodeling, cell-to-cell interactions, and endocytosis. Overall, our results indicate that sperm fertilizing ability influences blastocyst developmental ability and miRNA content. In addition, we demonstrate an association between blastocyst quality and miRNA content, thus suggesting the possibility to score miRNA expression as biomarkers for improved routine embryo selection technologies to support assisted reproductive efforts.This work was supported by the Carraresi Foundation and PSR 2022.Peer reviewe

Preparation of biological scaffolds and primary intestinal epithelial cells to efficiently 3D model the fish intestinal mucosa

Tissue engineering is an elegant tool to create organs in vitro, that can help obviate the lack of organ donors in transplantation medicine and provide the opportunity of studying complex biological systems in vitro, thereby reducing the need for animal experiments. Artificial intestine models are at the core of Fish-AI, an EU FET-Open research project dedicated to the development of a 3D in vitro platform that is intended to enable the aquaculture feed industry to predict the nutritional and health value of alternative feed sources accurately and efficiently. At present, it is impossible to infer the health and nutrition value through the chemical characterization of any given feed. Therefore, each new feed must be tested through in vivo growth trials. The procedure is lengthy, expensive and requires the use of many animals. Furthermore, although this process allows for a precise evaluation of the final effect of each feed, it does not improve our basic knowledge of the cellular and molecular mechanisms determining such end-results. In turn, this lack of mechanistic knowledge severely limits the capacity to understand and predict the biological value of a single raw material and of their different combinations. The protocol described herein allows to develop the two main components essential to produce a functional platform for the efficient and reliable screening of feeds that the feed industry is currently developing for improving their health and nutritional value. It is here applied to the Rainbow Trout, but it can be fruitfully used to many other fish species

Effects of alimentary supplementation on mammary gland activity in sheep grazing on semi-natural pasture

Introduction: Grazing is a main tool for the safeguard of pasture biodiversity and the maintenance of pasture nutritional value. The increasing summer aridity trend results in the availability of a pasture with a less protein content and a high fiber content with, in addition, an increasing lignin percentage. This scenario is forecasting of farm productive loss generating the pasture abandonment risk. We attempted to search an easy and less expensive solution to improve the farm income and preserve the pasture biodiversity. Materials and Methods: A flock of 45 adult female sheep were conducted on a semi-mesophylous pasture in June, they were free to grazing until the moment of pasture maximum flowering (MxF); while until the maximum pasture dryness the animals were divided in two groups: the control group (Cnt) fed only on the pasture, while the experimental group (Exp) was also supplemented with 600 g/day/animal of corn and barley (1:1). We assessed immunohistologically and by realtime-PCR the expression of apelin (APLN) and its receptor (APNLR) in the mammary gland of sheep at the beginning and at the end of the period of differentiated diets and monitored milk production and composition of lactating animals during the trial. Results: Different immunopositivity to APLN and APLNR and significant differences in APLNR expression evidenced by realtime-PCR were observed in mammary gland between pasture maximum flowering and dryness (Cnt-Exp) samples. None difference was observed between Cnt and Exp samples. Milk production and composition showed significant differences among MxF, Cnt and Exp samples. Conclusion: APLN and APLNR expression in sheep mammary gland were not influenced by alimentary supplementation, that positively affected milk production and composition in Exp group respect to the Cnt one. Findings gave useful indication on APLN/APNLR action on sheep mammary gland; indication for flock management allowing to farm income improvement also emerged