Establishment of Repertoire of Placentome-Associated MicroRNAs and Their Appearance in Blood Plasma Could Identify Early Establishment of Pregnancy in Buffalo (Bubalus bubalis)

Abstract

Precise early pregnancy diagnosis in dairy animals is of utmost importance for an efficient dairy production system. Not detecting a dairy animal pregnant sufficiently early after the breeding results to extending the unproductive time of their milk production cycle and causes substantial economic loss for a dairy producer. At present, the most conventional and authentic pregnancy confirmation practice in cows and buffaloes is rectal palpation of the reproductive organs at Days 35–40 after insemination, which sometime leads to considering an animal as false pregnant. Other alternative methods available for early pregnancy diagnosis lack either accuracy or reproducibility or require elaborate instrumentation and laboratory setup not feasible to practice at farmers’ doorstep. The present study was aimed at establishment of the microRNA (miRNA) repertoire of the placentome in buffaloes, which could capture the event of the cross talk between a growing embryo and a dam, through fetal cotyledons and maternal caruncles, and thus could hint at the early pregnancy establishment event in ruminants. Total RNA was isolated from buffalo placentome tissues during early stages of pregnancy (at Day < 25 and Days 30–35), and global small RNA analysis was performed by using Illumina single-end read chemistry and Bubalus bubalis genome. A total of 2,199 miRNAs comprising 1,620 conserved and 579 non-conserved miRNAs were identified. Stringent functional miRNA selection criteria could predict 20 miRNAs worth evaluating for their abundance in the plasma of pregnant, non-pregnant, cyclic non-bred, and non-cyclic prepubertal animals. Eight of them (viz., miR-195-5p, miR-708-3p, miR-379-5p, miR-XX1, miR-XX2, miR-130a-3p, miR-200a-3p, and miR-27) displayed typical abundance patterns in the plasma samples of the animals on Day 19 as well as Day 25 post-insemination, thus making them ambiguous candidates for early pregnancy detection. Similarly, higher abundance of miR-200a-3p and miR130a-3p in non-pregnant animals was indicative of their utility for detecting the animals as not pregnant. Most interestingly, miR-XX1 and miR-XX2 were very characteristically abundant only in pregnant animals. In silico target prediction analysis confirmed that these two miRNAs are important regulators of cyclooxygenase-2 (COX-2) and cell adhesion molecule-2 (CADM-2), both of which play a significant role in the implantation process during feto-maternal cross talk. We interpret that circulatory miR-XX1 and miR-XX2 in blood plasma could be the potential biomarkers for early pregnancy detection in buffaloes.

miR‐1246 is implicated as a possible candidate for endometrium remodelling facilitating implantation in buffalo (Bubalus bubalis)

“…Among the seven miRs selected for the expression dynamics in TE spent media, the miR‐1246 qualified as a novel candidate selected from our lab's NGS data (Sarwalia et al., 2021 , NCBI, Accession: PRJNA705293, ID: 705293) while the remaining six miRs were selected based on the previous literature (Inyawilert et al., 2015 , Chen et al., 2016 Krawczynski et al., 2015 ; Siristatidis et al., 2015 , Kaczmarek et al., 2020 ). The three miRs, namely, miR‐1246, miR‐let‐7a and miR‐let‐7b exhibited a very typical expression pattern on Day 21, the expected day of maternal recognition of pregnancy (MRP) (Figure 3 ) which corresponds to placentation in this experiment (Day 21+ 9 days of blastocyst formation).…”

“…The microRNA functional analysis was performed using a custom‐made mirVana™ miRNA mimic for the miR‐1246 (hsa‐miR‐1246, Assay ID: MC13182, miRBase version v 22.0). This micro‐RNA was selected based on the results of NGS data in our lab that indicated its highest abundance on Day 21 (Sarwalia et al., 2021 , NCBI, Accession: PRJNA705293, ID: 705293). Besides, it is a novel miR whose role in MRP has not been hitherto elucidated.…”

Circulatory extracellular vesicle derived miR-195-5p promotes cellular apoptosis and suppresses cell proliferation in the buffalo endometrial primary cell culture

“…In this study, we identified placental-origin EV-miRNAs in the blood of pregnant buffalo and investigated the functional impact of miR-195-5p on endometrial cells in vitro. Previous studies have reported differential expression of circulating extracellular miRNAs, including miR-195-5p, miR-200a-3p, miR-27, and miR-1246, in the blood plasma of pregnant cattle and buffaloes 31 , 35 , 36 . Therefore, we examined the presence of these miRNAs within EVs derived from blood plasma and found that all four miRNAs were indeed present within these vesicles.…”

Identification of circulatory microRNA based biomarkers for early pregnancy diagnosis in buffalo

“…Deciphering the precise miRNAs that can serve as biomarkers for early pregnancy detection in dairy animals is a current focus of numerous laboratories. Evidence of the existence of pregnancy-specific miRNAs and their role as potential markers in pregnancy diagnosis has recently been published in many journals ( Ioannidis and Donadeu, 2016 ; 2017 ; Sarwalia et al, 2021 ). The microRNA profiling in bovine at 8 weeks has been described for comparative studies ( Lim et al, 2021 ).…”

“…The cDNA library prepared by different protocols of library preparation ( Ioannidis and Donadeu, 2016 ; Ioannidis and Donadeu, 2017 ; Sarwalia et al, 2021 ) was analyzed using agarose gel electrophoresis and a fragment analyzer. A 147 bp band was visible on the agarose gel, indicating the presence of mature miRNA.…”