GSE128697
Persistent effects on bovine granulosa cell transcriptome after resolution of uterine disease
Metritis is associated with reduced fertility in dairy cows, but the mechanisms are unclear because the disease resolves several weeks before insemination. One hypothesis is that metritis causes persistent changes in granulosa cells during follicle development, which might be evident in the transcriptome of granulosa cells from dominant follicles weeks after parturition. To test this hypothesis we collected follicular fluid and granulosa cells from dominant follicles 63 days post partum from cows previously diagnosed with metritis, at least 6 weeks after resolution of the disease, and from cows not diagnosed with metritis (control cows). Bacterial lipopolysaccharide was detected in follicular fluid, and concentrations were associated with follicular fluid IL-8 and glucose concentrations. Transcriptome analysis using RNAseq revealed 177 differentially expressed genes in granulosa cells collected from cows that had metritis compared with control cows. The most upregulated genes were ITLN1, NCF2, CLRN3, FSIP2 and ANKRD17, and the most downregulated genes were ACSM1, NR4A2, GHITM, CBARP and NR1I3. Pathway analysis indicated that the differentially expressed genes were involved with immune function, cell-cell communication, cell cycle and cellular metabolism. Predicted upstream regulators of the differentially expressed genes included NF?B, IL-21 and lipopolysaccharide, which are associated with infection and immunity. Our data provide evidence for a persistent effect of metritis on the transcriptome of granulosa cells in ovarian follicles after the resolution of disease Overall design: It was a prospective cohort study conducted from June 2016 to February 2017 at the University of Florida Dairy Unit using a group of 45 lactating Holstein Bos taurus dairy cows fed a total mixed ration. Milk yield (AfiFlo milk meters, S.A.E. Afikim, Israel) and concentrations of fat, true protein, and lactose (AfiLab online real-time milk analyzer, S.A.E. Afikim) were recorded at each milking, and cows were weighed on a walk through scale (AfiWeigh, S.A.E. Afikim) immediately after each milking as they left the milking parlor. Data were collected for the first 280 days postpartum or until the cow died or was culled, whichever occurred first. Cows were evaluated every 48 h for the first 21 days after parturition for signs of metritis as defined by a fetid, red-brown watery vaginal discharge and rectal temperature > 39.5°C. Cows with metritis received antimicrobial treatment with 2.2 mg/kg body weight ceftiofur hydrochloride (Excenel, Zoetis, Parsippany, NJ, USA) for 5 consecutive days. The veterinarians and farm staff recorded other diseases diagnosed within 21 d post partum, including mastitis, ketosis, displaced abomasum and lameness. Cows were examined for endometritis, as determined by a purulent vaginal discharge after d 21 post partum, and for subclinical endometritis, as determined by > 5% neutrophils in endometrial cytology samples collected on the day of follicle aspiration, at 63 days postpartum. Cows started a an ovulation synchronization protocol with the double Ovsynch program (Souza et al. 2008) using 100 mg GnRH (gonadorelin diacetate tetrahydrate; Ovacyst, Bayer Animal Health, Whippany, NJ, USA) i.m. 53 ± 3 d post partum followed by 25 mg prostaglandin (PG) F2a (dinoprost tromethamine; Prostamate, Bayer) i.m. 60 ± 3 d post partum. The contents of the pre-ovulatory follicle were aspirated by transvaginal ultrasounography on d 63 ± 3 postpartum (see below for details), immediately before cows received a second GnRH injection 72 h after PGF2a. A second Ovsynch protocol was then started 7 d later, and the final GnRH injection was administered approximately 16 h before timed AI performed at 80 d postpartum. Pregnancy was diagnosed by transrectal ultrasonography 32 d after timed AI. Blood was collected from the coccygeal vessels into evacuated tubes containing sodium heparin (Vacutainer, Becton Dickson, Franklin Lakes, NJ, USA) on d 7, 21, 35, and 50 post partum.