Comparative Proteome Profiling of Saliva Between Estrus and Non-Estrus Stages by Employing Label-Free Quantitation (LFQ) and Tandem Mass Tag (TMT)-LC-MS/MS Analysis: An Approach for Estrus Biomarker Identification in Bubalus bubalis.

Accurate determination of estrus is essentially required for efficient reproduction management of farm animals. Buffalo is a shy breeder and does not manifest overt signs of estrus that make estrus detection difficult resulting in a poor conception rate. Therefore, identifying estrus biomarkers in easily accessible biofluid such as saliva is of utmost interest. In the current study, we generated saliva proteome profiles during proestrus (PE), estrus (E), metestrus (ME), and diestrus (DE) stages of the buffalo estrous cycle using both label-free quantitation (LFQ) and labeled (TMT) quantitation and mass spectrometry analysis. A total of 520 proteins were identified as DEPs in LFQ; among these, 59 and four proteins were upregulated (FC ≥ 1.5) and downregulated (FC ≤ 0.5) during E vs. PE, ME, and DE comparisons, respectively. Similarly, TMT-LC-MS/MS analysis identified 369 DEPs; among these, 74 and 73 proteins were upregulated and downregulated during E vs. PE, ME, and DE stages, respectively. Functional annotations of GO terms showed enrichment of glycolysis, pyruvate metabolism, endopeptidase inhibitor activity, salivary secretion, innate immune response, calcium ion binding, oocyte meiosis, and estrogen signaling. Over-expression of SERPINB1, HSPA1A, VMO1, SDF4, LCN1, OBP, and ENO3 proteins during estrus was further confirmed by Western blotting. This is the first comprehensive report on differential proteome analysis of buffalo saliva between estrus and non-estrus stages. This study generated an important panel of candidate proteins that may be considered buffalo estrus biomarkers which can be applied in the development of a diagnostic kit for estrus detection in buffalo.

Ovarian Cysts in the Bitch: An Update.

Functional ovarian cysts occur as solitary or multiple fluid-filled structures of variable size that are unilateral or bilateral in the bitches of age 6-8 years. Though the pathogenesis is obscure, insufficient LH surge, intrafollicular changes in gonadotrophin receptors and growth factors are the possible reasons behind the occurrence of hormonally active ovarian cysts that predisposes the bitch to the development of cystic endometrial hyperplasia-pyometra complex and occasionally hyper estrogenism. In the presence of suggestive signs, ultrasonography is the practical imaging modality for the clinical diagnosis that can be confirmed by assay of ovarian steroids and histopathology. Medical management with gonadotrophin-releasing hormone analogues and human chorionic gonadotrophin is not preferred as they are not always successful. As uterine pathologies are highly likely by the time of diagnosis, ovariohysterectomy is the treatment of choice for the follicular and luteal cysts. Understanding the cellular and molecular changes in the hypothalamo-hypophyseal ovarian axis will improve our understanding on the canine ovarian cysts.

Kiss1 and its receptor: molecular characterization and immunolocalization in the hypothalamus and corpus luteum of the buffalo.

ABSTARCT The neuropeptide kisspeptin (Kp) through its receptor Kiss1r regulates the HPG axis by controlling GnRH release. Since buffalo is a seasonal breeder with problems of delayed puberty and postpartum anestrus, we characterized the Kiss1 and Kiss1r and investigated the immunolocalization in the hypothalamus and corpus luteum (CL). Kiss1 and Kiss1r genes were amplified from gDNA covering the coding region, cloned and sequenced with accession numbers MF168937 and MG820539, respectively. The Kiss1 DNA sequence had two exonic segment contained coding sequence (cds); 408 bp encoding a predicted protein of 136 aa with conservation of Kp-10 and shared 94.5-98.3% identity with ruminants. Kiss1r DNA sequence consisted of five exons with a cds of 1134 bp encoding a protein of 378 aa. Phylogenetic analysis of Kiss1 and Kiss1r revealed that it formed a monophyletic clade with cattle, which branched from sheep and goat. Immunofluorescence study revealed the presence of Kiss1 and Kiss1r in the neuronal soma and perinuclear area of preoptic and arcuate regions of the hypothalamus and luteal cells of the CL. This is the first report on molecular characterization of bubaline Kiss1 and Kiss1r genes that confirmed the presence of conserved Kp-10 like other ruminants and kisspeptinergic system is present in the hypothalamus and CL.

Endometrial transcripts of proinflammatory cytokine and enzymes in prostaglandin synthesis are upregulated in the bitches with atrophic pyometra.

Inflammatory markers of endometrial origin are valuable in order to differentiate the pyometra from cystic endometrial hyperplasia in the bitch. In the present study, we hypothesized that histological categorization would distinguish the differential regulation of the proinflammatory genes in the endometrium of bitches with pyometra. Ovariohysterectomy was done on bitches with confirmatory diagnosis of pyometra (n = 18). Using endometrium to myometrium ratio of 0.79 as threshold, the uteri (n = 8/group) were categorized into hyperplastic pyometra (HP) and atrophic pyometra (AP). Two samples were excluded as the diagnosis was inconclusive. In parallel, endometrial tissue was collected for total RNA extraction to study the differential expression of TLR4, IL-6, IL-8, COX-2 and PGFS through real time PCR. Diestrus uterus of non-pyometra bitches (n = 6) served as control. The mean fold change (2-ΔΔCt) for the target genes was determined using ß-actin as endogenous control and non-pyometra uterus as calibrator group. Except TLR4, other inflammatory genes were upregulated significantly by 1.82 to 3.74 times in the AP as compared to HP with maximum upregulation of COX-2 and PGFS. Further, correlation matrix with Spearman's rho revealed that IL-8 had strong positive correlation with COX-2 and PGFS in the AP group (P < 0.05). It is concluded that histological grading of pyometra into HP and AP revealed differential regulation of inflammatory cytokines and enzymes in the PG synthetic pathway in the canine endometrium that has diagnostic potential under clinical settings.