Differences in endometrial mRNA expression of important genes related to fertility in repeat breeder and fertile cows.

P M JAUREGUIBERRY; LV MADOZ; S QUINTANA, ; M MARIN; M BURUCÚA,; M. TIZZANO; B RABAGLINO, ; R REARTE, ; ,MI PECORARO ; RL DE LA SOTA

Congreso; The 30th World Buiatrics Congress; 2018

Objectives: An altered uterine environment produced bychanges in the endometrial mRNA expression of key genesmay be responsible for reduced fertility in repeat breeder cows(RBC). The aim of the present study was to compare the endometrialgene expression of epidermal growth factor receptor(EGFR), nodal growth differentiation factor (NODAL), prostaglandin-endoperoxide synthase 2 (PTGS2), estrogen receptor1 (ESR1), and progesterone receptor (PGR) in RBC and fertilecows (FC) during diestrous by RT-qPCR.Materials and methods: This study was conducted in commercialdairy farms located in Castelli, Brandsen and Lobos,province of Buenos Aires, Argentina. A total of 82 grazing Holsteindairy cows (RBC and FC) were evaluated to select thecows that were included in the study. We considered a RBC asa cow without any clinical disease that had equal or greaterthan 3 unsuccessful artificial insemination (AI), and a FC as acow without any clinical disease and that became pregnant atfirst or second AI. Cows were evaluated by endometrial cytologyto exclude cows with subclinical endometritis (SCE) and ablood sample from the coccygeal vein was obtained to excludecows with less than 1 ng/mL of P4. Endometrial samples werecollected from each RBC and FC included in the study by cytobrushtechnique for RT-qPCR analysis. Samples were stored ina tube with RNA stabilizing solution at -20C. The RNA isolationwas performed using Trizol (TriReagent, Molecular ResearchCenter Inc., Cincinnati, OH, USA). Complementary DNA wassynthesized using a reaction mixture containing 1 μg of totalRNA, random hexamers (Promega, Madison, WI, USA) andMoloney murine leukemia virus reverse transcriptase (InvitrogenCorp., Carlsbad, CA, USA), following the procedures suggestedby the manufacturer. Reactions of qPCR for bovinegenes (ESR1, PGR, EGFR, NODAL and PTGS2) were carriedout using specific primers. The amplification conditions consistedof an incubation at 95C for 3 min and 40 cycling repeats of:denaturation at 95°C for 20 s, annealing for 20 s at indicatedannealing temperature and an elongation step at 72C for 20 s.A dissociation curve was generated at the end of the amplificationto confirm that a single product was amplified. To analyzethe relative level of expression of each mRNA, the 2-ΔΔCTmethod was used. Significance difference of endometrial geneexpression between RBC and FC was analyzed by general linearmodel procedure.Results: From a total of 31 RBC sampled, 6 were positive toSCE and 9 were not in diestrus (P4 less than 1 ng/mL); andwere not included in the study. Furthermore, from a total of 51FC sampled, 8 were positive to SCE, 18 were not in diestrus(P4 less than 1 ng/mL), 15 were not pregnant within the twofirst insemination and one was outlier; and were not included inthe study. Therefore, a total of 16 RBC and 9FC samples wereincluded in the study. Our results demonstrated that the mRNAexpression of EGFR (RP= -1.098; 95% IC= -1.66 ? -0.54; Pless than 0.01) and NODAL (RP= -3.227; 95% CI -4.15 ? -2.30;P less than 0.01) were higher in RBC than in FC, while themRNA expression of PTGS2 (RP= 0.439; 95% CI= 0.39 ? 0.49;P less than 0.01) was lower in RBC than in FC. On the otherhand, there were no significance differences in the mRNA endometrialexpression of ESR1 and PGR (RP= 0.229; 95% CI=-1.13 ? 1.59; P=0.73; RP= -0.517; 95% IC= -1.27 ? 0.23;P=0.17, respectively).Conclusions: In conclusion, there are significance differencesin the mRNA endometrial expression of EGFR, NODAL andPTGS2, but there are not in the mRNA endometrial expressionof ESR1 and PGR between RBC and FC during diestrous. Thisstudy provides important information that could be used as abase to future studies to elucidate the causes of reduced fertilityin RBC.