PSXI-17 In-vitro culture of bovine endometrial epithelial cells: A comparative analysis between virgin heifers and postpartum Holstein cows.

Abstract Postpartum uterine involution in cows restores the endometrium to its pre-pregnancy status to support subsequent pregnancy. Endometrial cells in the cow may differ from those in the heifer because cow endometrium arises from a regenerative process that occurs in the presence of microbial infection and tissue inflammation. The objective, therefore, was to determine if the quality and proliferative potential of bovine endometrial epithelial cells (bEECs) differed for heifers compared with postpartum cows. Postpartum Holstein cows (n=11) and virgin Holstein-Jersey heifers (n=5) were used. Sampling was performed for 5 consecutive weeks, either at 7, 14, 21, 28, and 35±3 days postpartum (cows) or starting at 365±7 days of age (heifers). Blood samples were collected for plasma progesterone (P4) and β-hydroxybutyrate (BHB) analyses. On day 35, the estrous cycles of all animals were synchronized using an injection of PGF2α. Seven days after injection, bEECs were collected in vivo by using a transcervical approach and Cytobrush Plus Cell collector (Cooper Surgical; Trumbull CT). Primary cell populations of bEECs were established according to the procedures of Rocha et al. (2022; JDS Communications 3:217-221) and grown to 80% confluency. Confluent cells were then dissociated with trypsin/EDTA, counted and analyzed for viability using a Countess II FL Automated Cell Counter (Thermo Fisher Scientific; Waltham, MA). The effects of status (cow versus heifer), cyclicity (P4), and metabolic activity (BHB) on the bEEC were evaluated by using PROC GLM in SAS. The results are presented as LSMEAN±SEM. Based on vaginal discharge score, none of the cows or heifers had uterine disease. The Cytobrush collection tended (P< 0.0904) to yield more cells from heifers compared with cows (2.16×107 and 9.22×106 fresh cells, respectively). Days to confluency after the initial plating (P0) was less (P< 0.0174) for heifers compared with cows (5.2±1.8 and 10.9±1.2 days, respectively). The total number (P< 0.0457; 5.15×105 and 1.47×105) and percentage of live cells (P< 0.0337; 50.5±7.5 and 29.3±5.0) was greater for heifers compared with cows (respectively) at the first passage (P1). There was no effect of cyclicity or subclinical ketosis (BHB >1.2 mmol/L) in cows on the number of cells collected or their growth in culture, but more cells were collected from cows with >5 ng/mL versus < 5ng/mL plasma P4 (2.6×107 versus 3.5×106 fresh cells, respectively; P< 0.0047). In conclusion, the bEEC isolated from cows differed from those isolated from heifers for their proliferative capacity (time to confluence) and viability following routine cell culture procedures (passaging). Cyclicity and metabolic disease in cows did not affect the development of cells in culture. Unique developmental properties of bEEC isolated from cows versus heifers may indicate that the regenerated uterine endometrium differs from the virgin endometrium. These differences may explain, in part, reduced fertility in postpartum cows compared with heifers.