138 Heat-induced hyperthermia affects the periovulatory follicular fluid metabolome in lactating dairy cows

Hyperthermia affects the periovulatory follicle components after the LH surge to potentiate ovulation and corpus luteum formation and influence developmental competence of the maturing oocyte resident within. The aim of this study was to examine the follicular fluid metabolome when levels of cow hyperthermia varied. Lactating Holsteins were pharmacologically stimulated to have a dominant follicle. Cows were transported to a climate-controlled facility 35 h after PGF_2α was administered. After GnRH was injected (40 h after PGF_2α) to induce an LH surge, the temperature-humidity index (THI) was steadily increased for ~12 h to induce hyperthermia (final THI 83.2 ± 0.2). The average rectal temperature of hyperthermic cows ranged from 38.9 to 40.2°C (overall average, 39.5 ± 0.1°C). Thermoneutral cows were maintained at a THI of 65.8 ± 0.1; rectal temperatures ranged from 38.2 to 38.7°C and averaged 38.4 ± 0.1°C. After ~12 h, hyperthermic cows were returned to thermoneutral conditions. Fluid of the dominant periovulatory follicle was aspirated at 16 h (average, 16.4 h; range, 15.3–18.2 h) after GnRH (E2:P4 ratio, 1.43 ± 0.11; follicle diameter, 17.0 ± 0.5 mm). Follicular fluid samples from hyperthermic (n = 11) and thermoneutral (n = 11) cows were submitted for ultra-high performance liquid chromatography-high resolution mass spectrometry metabolomic analysis. Mixed model hierarchical regression was performed using metabolite abundance as the dependent variable. Mean rectal temperature of individual thermoneutral and heat-stressed cows was the independent variable. Best fit models were obtained using backward manual selection after ensuring lack of collinearity among regressors (e.g. follicular fluid and serum hormone levels, follicle sizes, days in milk, milk yield, and parity) and data normality. Of 68 unique metabolites identified in the periovulatory follicular fluid, 22 were affected by varying levels of hyperthermia (P < 0.05); most of these (n = 18) decreased with increasing rectal temperature. Metaboanalyst 6.0 was used for enrichment analysis. Homocysteine degradation (P = 0.0005, FDR = 0.0375) and methionine metabolism (P = 0.0008, FDR = 0.0375) were overrepresented. Homocysteine is a degradation product of methionine that is reported to play important roles in cellular growth and oxidative stress. Intrafollicular methionine has been related to fertility outcomes and is necessary for redox maintenance. Collectively, these and other findings provide important insights regarding the extent to which the periovulatory follicular fluid metabolome is affected by varying degrees of hyperthermia. Functional consequences on fertility-important periovulatory follicle components provide the basis of ongoing and future studies.

This project was supported by the state of Tennessee through UT Institute of Agriculture, AgResearch, the Department of Animal Science, and the USDA National Institute of Food and Agriculture, Multistate Project No. NE2227.