PhD student Facultad de Veterinaria, Universidad de la República, Paysandu, Uruguay
Abstract Authors: Graciana R. Mendina1; Victoria de Brun2; Jorge Gil1; Maria Victoria Pons1; Rodrigo Vivian3; Maria de Lourdes Adrien1 ; Mario Binelli4; Ana Meikle2
1 Facultad de Veterinaria, Universidad de la República, Paysandú, Uruguay
2 Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay
3 Facultad de Agronomía, Universidad de la República, Paysandú, Uruguay
4 Department of Animal Sciences, University of Florida, Gainesville, Florida, USA
Abstract Text: We aimed to determine the effect of pregnancy on gene expression of Interferon-stimulated gene 15 (ISG15) and progesterone receptor (PGR) in the cervix of Holstein heifers. Sixteen heifers were synchronized with two injections of prostaglandin (Estrumate®, MSD, Argentina) separated by eleven days. Animals were sham-inseminated with semen extender only (open, n=6), or inseminated with frozen semen from one bull and were diagnosed pregnant by ultrasound at day thirty post insemination (pregnant, n=10). Blood by coccygeal venipuncture and cervical cells samples using a cytological brush were obtained at 14, 16, and 18 after insemination. Serum progesterone (P4) was determined by a solid-phase radioimmunoassay using a commercial kit. Total RNA was obtained with Trizol reagent (Life Technologies) and the concentration and purity of the RNA was determined using a spectrophotometer (nanodrop ND 1000). Total RNA was treated with DNase using a DNA-freeTM kit (Ambion, Austin, TX, USA). For each sample, copy DNA (cDNA) was synthesized by reverse transcription using a SuperScript III transcriptase (Invitrogen) with random primers and total RNA as a template. ISG15 and PGR gene expression was determined by real-time PCR (qPCR) using the Rotor-GeneTM 6000 kit (Corbett Life Sciences, Sydney, Australia). Gene expression was calculated by relative quantification to the exogenous control (β-actin) and normalized to the geometric mean of the endogenous control, taking into account the respective amplification efficiencies. Serum progesterone concentrations and the transcripts determined by real-time PCR were analyzed by a Glimmix procedure (Proc Glimmix; SAS Studio®) including group, day and their interaction as a fixed effect. Significance wasconsidered with alpha ≤ 0.05, and tendency between 0.05 and 0.10. Serum progesterone concentrations were higher in pregnant than open heifers (6.5 ± 0.48 vs 4.2 ± 0.57 ng/mL, P=0.0077). There was no effect of day or the interaction between group and day on progesterone concentrations. The cervical expression of ISG15mRNA was greater in pregnant than open heifers (1.76 ± 0.46 vs 0.13 ± 0.04, respectively, P< 0.0001), being different in all days (Fold-Change: 12.0, 21.5, and 9.4, on days 14, 16, and 18, respectively). There was an interaction between group and day (P=0.033), as pregnant heifers presented greater ISG15 mRNA expression on day 16 than on day 14 and 18 (P < 0.05), but no differences according to the day were found in open heifers. The PGR mRNA expression tended to be affected by the interaction between group and day (P=0.0779), while open heifers increased during the estrous cycle, pregnant heifers maintained PGR expression. We concluded that the cervical expression of ISG15 mRNA could be used as a pregnancy biomarker as early as day 14 post insemination.
