2 ELEVATED NONESTERIFIED FATTY ACID CONCENTRATIONS HAMPER IN VITRO BOVINE OVIDUCTAL EPITHELIAL CELL PHYSIOLOGY

Abstract

Elevated nonesterified fatty acids (NEFA) have been recognised as an important link between lipolysis-based metabolic conditions and impaired fertility in high-yielding dairy cattle. However, NEFA effects on the oviductal micro-environment currently remain unknown. We hypothesise that elevated NEFAs may contribute to the complex pathology of subfertility and infertility by exerting a negative effect on bovine oviductal epithelial cell (BOEC) physiology. Therefore, the objectives of this study were to elucidate NEFA toxicity effects on BOEC, both qualitatively and morphologically, by assessing BOEC-sperm binding affinity, monolayer integrity, proliferation capacity and morphology. The BOEC of 4 bovine oviducts (4 replicates) at Day 3–5 of the oestrous cycle from a local slaughterhouse were mechanically isolated, pooled, and cultured in a polarized cell culture system (ThinCert, Greiner Bio-One, Frickenhausen, Germany) with DMEM/F12-based culture medium for 9 days until transepithelial electric resistance (TER) reached at least 700 Ω cm² to prevent leakage between the 2 compartments. At Day 10, monolayers were exposed to a 720 µM NEFA mixture of OA, SA, and PA, for 24 h in 4 treatment groups according to exposure side: control, basal (B), apical (A), and A+B. The BOEC were washed and monolayer quality was assessed by means of a sperm binding assay (30 min co-culture of BOEC monolayer and 10⁶ spermatozoa mL^–1), TER measurements (pre- and post-exposure) and a wound healing assay (8-h observation of BOEC proliferation capacity after over an artificial gap). Morphology of BOEC was assessed by scanning electron microscopy on cell polarity, presence of microvilli and cilia, and monolayer integrity. Data (mean ± s.d.) were analysed by mixed model ANOVA. In A+B, monolayers (31.28 ± 6.16 sp/0.05 mm²) showed a significantly reduced sperm binding affinity compared to the control (97.90 ± 10.76 sp/0.05 mm²; P < 0.05), and treatment A tended to be more affected (39.95 ± 19.30 sp/0.05 mm²) than treatment B (68.55 ± 15.38 sp/0.05 mm²; P = 0.051). The absolute TER increase post-NEFA exposure in the control (110 ± 11 Ω cm²) was significantly higher than in all the other treatments. Also, the TER increase differed significantly depending on the exposure side: in treatment A (3 ± 6 Ω cm²), the TER increase was lower than in treatment B (29 ± 8 Ω cm²), and monolayers in treatment A+B were even associated with a significant TER reduction (–15 ± 10 Ω cm²; P < 0.05). Cell proliferation capacity showed a significant closure of the gap in all treatments, but only the control group (41.64% closure) differed significantly (P < 0.05) from the other groups (B = 28.3%, A = 31.62%, A+B = 30.9% closure) irrespective of the exposure side; BOEC morphology was not affected. Depending on the exposure side, elevated NEFA exert a negative effect on BOEC physiology but not morphology. Ultimately, these physiological alterations in its micro-environment may result in suboptimal development of the pre-implantation embryo and a reduced reproductive outcome.