Culture and analysis of endometrial cells isolated from dairy heifers compared with postpartum cows
No Thumbnail Available
Authors
Meeting name
Sponsors
Date
Journal Title
Format
Thesis
Subject
Abstract
Reproductive efficiency is fundamental to the sustainability and profitability of dairy production systems, where uterine health plays a pivotal role in determining fertility outcomes. The postpartum period represents a critical window for reproductive recovery, characterized by tissue remodeling and microbial exposure that can alter endometrial function. In contrast, the endometrium of virgin heifers provides a physiological baseline free from postpartum inflammation and metabolic stress. Understanding how parity and physiological status influence endometrial cell behavior is key to improving reproductive performance and mitigating uterine disease in dairy cows. This thesis investigated the cellular and molecular differences between endometrial cells derived from virgin heifers and postpartum Holstein cows, emphasizing inflammatory responses, metabolism, and microbial associations by using two complementary in-vitro models. Chapter 3 focused on bovine endometrial stromal cells (bEStCs). Under the established culture conditions, only stromal cells proliferated successfully, highlighting a methodological limitation in epithelial cell recovery, likely due to their lower abundance or greater fragility during enzymatic digestion and seeding. The bEStCs were challenged with increasing doses of lipopolysaccharide (LPS; 1, 10, and 100 ng/μL) to evaluate dose-dependent inflammatory sensitivity. Both heifer- and cow-derived stromal cells exhibited marked responsiveness to LPS, with upregulation of serum amyloid A3 (SAA3) and interleukin-8 (IL8) even at the lowest concentrations tested, confirming the high sensitivity of bovine endometrial tissue to gram-negative bacterial components. No significant differences between parities were detected, although the small sample size (heifers = 3; cows = 2) limited statistical power. The statistical model used log-transformed fold changes (log₀) for GAPDH, IL8, and SAA3 expression to reduce variance. GAPDH expression remained stable across type, dose, and interaction effects (P > 0.13), validating its use as a reference gene. SAA3 expression was significantly affected by dose (P = 0.0179) but not by animal type (P = 0.54) or their interaction (P = 0.66), indicating similar responses between cows and heifers. Likewise, IL8 expression showed a significant dose effect (P = 0.0041) but no effect of type (P = 0.23) or interaction (P = 0.49), suggesting a primarily dose-dependent pro-inflammatory response independent of parity. Chapter 4 expanded the analysis to both stromal and epithelial compartments. Endometrial cells collected in vivo were cultured and compared between heifers and postpartum cows to assess differences in proliferation, viability, metabolic profiles, vaginal microbiota, and transcriptomic responses to LPS. Cytobrush sampling tended (P = 0.09) to yield more fresh cells from heifers than cows (2.16 × 10⁷ vs. 9.22 × 10⁶), and heifer-derived epithelial cells reached confluence more rapidly (5.2 ± 1.8 vs. 10.9 ± 1.2 days; P = 0.02) and exhibited greater viability (50.5 ± 7.5 % vs. 29.3 ± 5.0 %; P = 0.03). Cows with plasma progesterone > 5 ng/mL yielded significantly more cells (2.6 × 10⁷ vs. 3.5 × 10⁶; P = 0.005), suggesting an endocrine influence on endometrial regeneration. Microbiome profiling revealed parity-dependent differences, with postpartum cows showing transient increases in Peptoniphilus indolicus, Helcococcus ovis, Trueperella pyogenes, and Clostridium perfringens. Principal component analysis (PCA) of RNA-seq data confirmed that cell identity was the dominant source of variation: samples from five heifers clustered distinctly by donor, with PC1 explaining 87 % of the variance and separating stromal (DD1) from epithelial (DD0) populations. Transcriptomic analyses identified extensive parity- and status-specific gene expression changes after LPS stimulation, with GO enrichment indicating strong regulation of inflammatory, immune, and tissue-remodeling pathways. Repeated LPS exposure revealed transcriptional evidence of “inflammatory memory,” characterized by progressive downregulation of proliferative pathways in epithelial cells and expanded antiviral and reparative gene networks in stromal cells. Collectively, these findings demonstrate that the regenerated postpartum endometrium differs functionally from the virgin endometrium at cellular, microbial, and molecular levels. Postpartum cows face compounded challenges of negative energy balance, microbial exposure, and tissue regeneration, which may impair uterine recovery and contribute to reduced fertility. In contrast, virgin heifers maintain metabolic stability and robust endometrial function. Understanding these mechanisms provides a foundation for developing targeted interventions to enhance uterine resilience, reproductive efficiency, and the long-term sustainability of modern dairy herds.
Table of Contents
PubMed ID
Degree
M.S.