115 EXPRESSION OF RADICAL S-ADENOSYL METHIONINE DOMAIN CONTAINING-2 (RSAD2) AND TRANSFORMING GROWTH FACTOR β (TGF-β) IN BOVINE PERIPHERAL BLOOD MONONUCLEAR CELLS DURING PREGNANCY: IDENTIFICATION OF POTENTIAL BIOMARKERS TO DETERMINE PREGNANCY STATUS

Abstract

During the bovine peri-implantation period, immune cells are exposed to endocrine and paracrine signals, which support feto-placental development. Recent studies in cattle have shown that the antiviral factor Mx2, known to be up-regulated by conceptus-derived interferon-τ in pregnancy, is detected in peripheral blood mononuclear cells (PBMC) and can be used for pregnancy detection on Day 18. The objectives of the current study were to correlate mRNA expression levels of pregnancy associated factors and immune modulators RSAD2 (radical S-adenosyl methionine domain containing-2) and TGF-β (transforming growth factor β) in circulating PBMCs, with pregnancy status in dairy cows and heifers. Holstein-Friesian heifers (n = 12) and lactating dairy cows (n = 17) were synchronised to estrus and artificially inseminated (bred) or not (non bred, n = 4). Blood samples were collected on Day 0 (day of AI or estrus), 5, 7, 13, 16, 20, and 25 for PBMC isolation. Pregnancy was diagnosed retrospectively by transrectal ultrasonography at Days 30 and 60 after breeding. The relative quantification of RSAD2 and TGF-β transcript abundance, analysed by qPCR, was normalised against PPIA and calculated according to the relative standard curve method. Data from duplicates were pooled, and a mixed model was run on the pooled Cq values for each stage. Genes were considered as differentially expressed if the mixed model P value was <0.05. RSAD2 mRNA abundance was 2-fold higher in pregnant heifers compared with non-bred heifers on Day 20 (P < 0.01) and Day 25 (P < 0.03) post AI. Likewise, RSAD2 mRNA expression was greater (2.25 fold) in pregnant heifers compared with heifers that were inseminated but not pregnant (P < 0.007). In contrast, RSAD2 expression was not significantly different in pregnant cows compared to non-bred or bred but non-pregnant cows. TGF-β mRNA abundance was significantly lower in both pregnant cows (3.5-fold, P < 0.04) and pregnant heifers (4.5-fold, P < 0.01) compared to non-bred animals as early as Day 13. This suppression of expression was maintained until Day 20 in heifers (3.9-fold, P < 0.02). In addition, PBMC TGF-β mRNA expression was lower in pregnant cows compared with bred non-pregnant cows (1.65-fold, P < 0.06). In conclusion, Day 20 PBMC RSAD2 expression may be a useful predictor of pregnancy maintenance in dairy heifers. However, decreased TGF-β expression as early as Day 13 appears to be a more powerful indicator of the presence of a viable embryo in both dairy heifers and cows and may play a role in the establishment and maintenance of pregnancy through the restriction of monocyte recruitment or activation during the time of maternal recognition of pregnancy.