Salivary miR-16, miR-191 and miR-223: intuitive indicators of dominant ovarian follicles in buffaloes
Estrus or sexual receptivity determination is utmost important for efficient breeding programs for female buffaloes. Prominent estrus behavioral symptoms are the result of several molecular and neuroendocrine events involving the ovary and the brain. Expression of estrus behavior is poor in buffaloes during the summer season. Hence, the discovery of biomarkers specific to the estrus stage or its related ovarian events, like the presence of dominant ovarian follicle, is helpful for developing an easy estrus determination method. MicroRNA are small non-coding RNA with a potential to be biomarkers. Therefore, the present study targeted to investigate the potential of estrogen responsive miRNAs (miR-24, miR-200c, miR-16, miR-191, miR-223 and miR-203) as estrus biomarkers in buffalo saliva, a non-invasive fluid representing animals’ pathophysiology. There was a significant (P < 0.05) increase in the salivary presence of the miR-16, miR-191 and miR-223 at 6th and 18th–19th days than the 0 day (estrus), 10th day and the following consecutive estrus day. These observations may indicate an association between the representative lower presence of these miRNA in saliva and the presence of dominant ovarian follicles. To test this association, pathway analysis, target gene identification, functional annotation and protein–protein interaction networks (PPI) were performed for miR-16, miR-191 and miR-223 by different bioinformatics tools. Interestingly, the top pathways (fatty acid biosynthesis and oocyte meiosis), target genes (FGF, BDNF and IGF1) and PPI hub genes (KRAS, BCL2 and IGF1) of these miRNAs were found essential for ovarian follicular dominance. In conclusion, the miR-16, miR-191 and miR-223 may not be the perfect estrus stage-specific biomarkers. However, their lower presence in saliva at estrus and 9th–10th day of estrous cycles, when the ovary usually has a dominant follicle in buffaloes, may intuitively indicate the follicular dominance. Further studies are needed to prove this association in a large population.
KeywordsSalivary microRNA qRT-PCR Estrus Dominant follicle Buffaloes
The authors are thankful to ICAR-NDRI and ICAR-NASF for their financial assistance to this work. We also thank Livestock Research Center, ICAR-NDRI, and farm personnel for the management and handling of buffaloes during the saliva sample collection.
Compliance with ethical standards
The present work was funded by ICAR-National Dairy Research Institute (ICAR-NDRI), Karnal, India (Project No. IXX10906), and ICAR-National Agriculture Science Foundation (ICAR-NASF), Indian Council of Agricultural Research, New Delhi, Government of India (Project No. OXX03374).
Conflict of interest
The authors have no financial and non-financial competing interests with other people and organizations influencing the paper content.
Author list discrepancy statement
We added an additional author Pankaj Singh as a third author in the revised manuscript. As the major revision needed the provision of an additional data to prove specificity of the amplified miRNA products, we have cloned and sequenced three amplified miRNA products. Pankaj Singh contributed the cloning and sequencing work. Hence, his name is included as a third author. All other co-authors agreed in this regard.
An institutional Animal Ethics Committee (IAEC) of the ICAR-National Dairy Research Institute, Karnal, India, a registered ethics committee of CPCSEA (Reg No. 1705/GO/ac/13/CPCSEA) approved animals for sample collection in this research work under the Project Nos. IXX10906 and OXX03374. Accordingly, “all applicable international, national, and/or institutional guidelines for the care and use of animals were followed”.
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