2 CIRCULATING microRNA SIGNATURES ASSOCIATED WITH EARLY BOVINE PREGNANCY

Abstract

High-producing dairy cattle are characterised by low fertility, which extends calving intervals and reduces annual milk production. Early pregnancy diagnosis before Day 21 of gestation can shorten calving intervals, improve milk production, and increase overall profits from modern dairy herds. At present, accurate diagnosis is only possible after the third week of pregnancy. Circulating microRNA (miRNA) have been proposed as diagnostic biomarkers of numerous human conditions such as cancer and diabetes. Moreover, distinct circulating miRNA profiles have been associated with mid to late stages of human pregnancy. The aim of this study was to determine whether specific circulating miRNA signatures that occur during early pregnancy in cattle could be used for diagnostic purposes. To do this, we have compared miRNA levels in plasma samples from cycling, nonpregnant (n = 24) and cycling, early pregnant (D16, n = 11; D24, n = 11) Holstein heifers using next-generation sequencing and PCR arrays. Using small-RNA sequencing on 46 individual samples, we generated 2.8 million miRNA reads, and up to 328 miRNAs were detected in each sample. There were no significant differences (false discovery rate <0.1) in miRNA levels between nonpregnant and pregnant (D16, D24) heifers. In addition, 12 pooled samples (n = 9 for nonpregnant, n = 3 for D24) were analysed for the expression of 176 miRNA using PCR arrays; 16 miRNA had differentially expressed levels (2- to 3-fold; false discovery rate <0.1) in pregnant heifers. The QPCR validation of miRNA candidates obtained from the high-throughput analyses identified 6 miRNA that were differentially expressed in pregnant heifers. These included bta-miR-26a, bta-miR-29c, bta-miR-138, and bta-miR-204, which were differentially expressed at D16, bta-miR-1249, which was differentially expressed at D24, and hsa-miR-4532, which was differentially expressed at both D16 and D24 of pregnancy (P < 0.05, fold-change ≤2.4). The miRNA miR-26a and miR-204 have been previously associated with early (D20) and late pregnancy in pig and human respectively. Furthermore, miR-1249 has been associated with the regulation of ovarian function, and miR-26a is involved in immune regulation, suggesting a potential role for this miRNA as an immuno-suppressor during early pregnancy. In summary, we identified a miRNA signature associated with early pregnancy in cattle. We are currently using next-generation sequencing to identify differentially expressed miRNA at other stages of pregnancy, including Day-60 heifers, and this data will also be presented.

This study was funded by Zoetis and BBSRC.