Glycohistochemical investigation on canine and feline zonae pellucidae of preantral and antral oocytes

Glycoconjugate modifications were analysed in the zona pellucida during development of oocytes in dog and cat using conventional histochemical staining methods with or without previous carbohydrate digestion. A series of lectins combined with desulphation and sialic acid degradation were applied. No differences were observed between dog and cat follicles using conventional histochemical staining methods. In both species, the zona pellucida and follicular fluid/intercellular matrix strongly reacted with PAS and high iron diamine stain (HID) and reacted moderately with low iron diamine stain (LID). Treatment with testicular hyaluronidase, chondroitinase ABC, chondroitinase AC and chondroitinase B treatment diminished HID and LID positivity of follicular fluid and intercellular matrix. Lectins that gave the most intense staining of the zona pellucida of both species were SBA, PNA, RCA-I, GSA-IB4 and WGA, indicating the presence of beta-D-GalNAc, D-Gal and GlcNAc residues. Sulpho- and asulpho-carbohydrates were identified in terminal and/or subterminal positions linked to sialic acid residues. In conclusion, the results indicate that glycosaminoglycans are not present in the zona pellucida of both species. Differences were observed in carbohydrate residues and in their spatial distribution, depending on species and developmental stage of the follicles. The similarity in lectin affinity between ooplasm and zona pellucida of oocytes present in follicles at different stages of development confirm the involvement of oocytes in zona pellucida production

Glycoconjugates in small antral ovarian follicles of the river buffalo (Bubalus bubalis L.)

Types and distribution patterns of glycoconjugates in antral ovarian follicles were investigated in the buffalo, using periodic-acid Schiff (PAS), high iron diamine (HID), low ion diamine (LID) and lectin histochemical staining methods. HID and LID staining procedures were preceded in some cases by digestion with testicular hyaluronidase, Streptomyces hyaluronidase, chondroitinase ABC and heparitinase (heparinase III). Lectin staining was performed with the use of 12 horseradish peroxidase (HRP) lectin conjugates. Some lectin staining procedures were preceded by neuraminidase digestion and saponification. Large amounts of isomeric chondroitin sulphates and a minor quantity of heparan sulphate and hyaluronic acid and/or chondroitin were found in follicular fluid. Lectin staining of buffalo follicular fluid revealed glycoconjugates with different glucidic determinants such as beta-N-acetylgalactosamine, beta-galactose-(1-3)-N-acetylgalactosamine, beta-galactose-(1-4)-N-acetylglucosamine, N-acetylglucosamine, alpha-fucose and alpha-glucose/alpha-mannose, and sialic acid residues. Glycosaminoglycans were absent in the zona pellucida of oocytes in small antral follicles. Acidic glycoconjugates in the zona pellucida were caused by sulphated groups and sialic acid residues. Our data show few internal glucidic residues, such as N-acetylglucosamine in the buffalo zona pellucida but many subterminal beta-N-acetylgalactosamine, alpha- and beta-galactose determinants masked by sialic acids. These findings demonstrate that buffalo follicular fluid has a very heterogeneous composition that is similar to that found in small and large bovine follicles. No differences in composition of the follicular fluid were observed in the follicles examined

Chromosomal analysis of embryos produced by artificially inseminated superovulated cattle

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Application of the cDNA-AFLP method for studying gene expression in Fibrobacter succinogenes S85 exposed to 134.2 kHz electromagnetic field

AbstractMany biological effects related to the exposure of cells and tissues to electromagnetic fields have been reported in the literature, including those influencing DNAs and RNAs structure and ..

Exercise-induced up-regulation of MMP-1 and IL-8 genes in endurance horses

<p>Abstract</p> <p>Background</p> <p>The stress response is a critical factor in the training of equine athletes; it is important for performance and for protection of the animal against physio-pathological disorders.</p> <p>In this study, the molecular mechanisms involved in the response to acute and strenuous exercise were investigated using peripheral blood mononuclear cells (PBMCs).</p> <p>Results</p> <p>Quantitative real-time PCR (qRT-PCR) was used to detect modifications in transcription levels of the genes for matrix metalloproteinase-1 (<it>MMP-1</it>) and interleukin 8 (<it>IL-8</it>), which were derived from previous genome-wide expression analysis. Significant up-regulation of these two genes was found in 10 horses that had completed a race of 90–120 km in a time-course experimental design.</p> <p>Conclusion</p> <p>These results suggest that <it>MMP-1 </it>and <it>IL-8 </it>are both involved in the exercise-induced stress response, and this represents a starting point from which to understand the adaptive responses to this phenomenon.</p

Exercise induced stress in horses: Selection of the most stable reference genes for quantitative RT-PCR normalization

<p>Abstract</p> <p>Background</p> <p>Adequate stress response is a critical factor during athlete horses' training and is central to our capacity to obtain better performances while safeguarding animal welfare.</p> <p>In order to investigate the molecular mechanisms underlying this process, several studies have been conducted that take advantage of microarray and quantitative real-time PCR (qRT-PCR) technologies to analyse the expression of candidate genes involved in the cellular stress response.</p> <p>Appropriate application of qRT-PCR, however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability.</p> <p>Results</p> <p>The expression of nine potential reference genes was evaluated in lymphocytes of ten endurance horses during strenuous exercise. These genes were tested by qRT-PCR and ranked according to the stability of their expression using three different methods (implemented in <it>geNorm</it>, <it>NormFinder </it>and <it>BestKeeper</it>). Succinate dehydrogenase complex subunit A (<it>SDHA</it>) and hypoxanthine phosphoribosyltransferase (<it>HPRT</it>) always ranked as the two most stably expressed genes. On the other hand, glyceraldehyde-3-phosphate dehydrogenase (<it>GAPDH</it>), transferrin receptor (<it>TFRC</it>) and ribosomal protein L32 (<it>RPL32</it>) were constantly classified as the less reliable controls.</p> <p>Conclusion</p> <p>This study underlines the importance of a careful selection of reference genes for qRT-PCR studies of exercise induced stress in horses. Our results, based on different algorithms and analytical procedures, clearly indicate <it>SDHA </it>and <it>HPRT </it>as the most stable reference genes of our pool.</p

Mitochondrial DNA diversity of five Italian autochtonous donkey breeds

AbstractTo investigate the mitochondrial DNA diversity of five Italian donkey breeds (Amiata, Martinafranca, Romagnolo, Asinara, and Ragusano), we sequenced the HVR I region (D-loop, 288 bp) and cytochrome b gene (274 bp) in 121 individuals. In the D-loop we found nineteen mutations corresponding to fourteen different haplotypes, while in cyt b coding gene only six mutations were found, originating five different haplotypes. In particular, three mutations out of six were non-synonymous, causing an aminoacidic substitution. About the D-loop region, the value of nucleotide diversity (π) observed within breeds was relatively low, but not far from values detected in other European breeds. Phylogenetic and network analyses disclosed the presence of two divergent maternal lineages within Italian donkeys. These haplogroups correspond to the well known lineages of ancestors (Equus asinus somaliensis and E. a. africanus), as donkeys were domesticated from distinct wild subspecies living in Eastern Africa regions. ..