Effect of oral urea supplementation on the endometrial transcriptome of mares

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Highlights

Abstract

An intravenous large dose of protein led to an increased blood urea nitrogen (BUN), resulting in a lesser uterine pH and altered uterine gene expression in mares. The objective of the present study was to evaluate effects of a more physiological methodology to increase BUN on the endometrium of mares. Mares were fed hay and a treatment or control diet (n = 11 mares/treatment) in a crossover design starting at time of ovulation detection (D0) and continuing until D7. Mares of the treated group were fed urea (0.4 g/kg BW) with sweet feed and molasses, and those of the control group were fed sweet feed and molasses. Blood samples were collected daily, 1 hour after feeding, for BUN determination. Uterine and vaginal pH were determined after the last feeding on D7, and endometrial biopsies were performed. The RNA sequencing of the endometrium of a subset of mares (n = 6/treatment) was conducted. Differentially expressed genes (DEGs) between treatments were calculated (FDR-adjusted P-value<0.1). Urea-treated mares had greater BUN (P < 0.05), with no differences in uterine and vaginal pH compared to control mares. A total of 60 DEGs were characterized, those with largest fold change were SIK1, ATF3, SPINK7, NR4A1 and EGR3. Processes related to necrosis and cellular movement were predicted with the DEGs. Dietary administration of urea resulted in transcriptomic changes in the endometrium of mares related to necrosis, tissue remodeling and concentration of lipids. The observed changes in gene expression after an increased BUN might result in a disruption to the endometrium.

Introduction

Equine embryos are transported into the uterus approximately 6 days after ovulation (Freeman et al., 1991; Battut et al., 1997) and are dependent on histotroph (uterine milk) to grow and develop (Allen and Wilsher, 2009). An optimal intrauterine environment is important for a pregnancy outcome. Consequently, physiological changes to the endometrium occur during diestrus to ensure that the uterine milieu is optimal for a developing embryo (Sharp, 2000; Klein et al., 2010; Klein et al., 2013). Furthermore, nutritional factors in the dam such as large amounts of protein in diets have detrimental effects on fertility of cows and ewes (Butler et al., 1996; McEvoy et al., 1997; Butler, 2000; Rhoads et al., 2006). There have been no studies addressing the association between protein concentration in maternal diets and fertility in mares.

Diets with a large amount of protein or urea supplementation can lead to increased BUN concentrations in horses (Martin et al., 1996; Connysson et al., 2006), cows (Elrod et al., 1993; Rhoads et al., 2006), and ewes (McEvoy et al., 1997; Fahey et al., 2001). In a recent study, there was a short-term (6 hours) intravenous infusion of urea as a model for when there are large amounts of dietary protein fed to mares. The results of this study indicated there were urea affects endometrial transcripts related to cell pH, solute carriers, and ion homeostasis (Boakari et al., 2019). To more precisely mimic the effects of a high protein diet on the endometrial transcriptome, however, development of a dietary approach where mares were fed the compound being evaluated for a longer period of time would be preferred.

Currently, there have been no studies conducted to elucidate the effects of a diet containing large amounts of protein on the uterine transcriptome of mares. Because urea affects the endometrial transcriptome in mares and also pregnancy rates in cows and ewes as previously described, the hypothesis for the present study was that ingestion of urea included in a feed source would lead to increases in blood urea nitrogen (BUN) concentrations and alter the endometrial transcriptome of mares. The aim of the current study, therefore, was to develop an approach to mimic the feeding of a diet containing large amounts of protein to evaluate the effects of greater than optimal concentrations of BUN on the uterine and vaginal pH and endometrial transcriptome at D7 of diestrus.

Section snippets

Animals

All animal procedures were completed following the Institutional Animal Care and Use Committee of the University of Kentucky (Protocol #2011-0876). Clinically healthy mares of different breeds, ranging from 5 to 15 years of age were used in this study. All mares underwent a reproductive examination and transrectal ultrasonography for reproductive tract evaluation.

To induce ovulation, mares were administered 2,500 IU of human chorionic gonadotropin (hCG) (Chorulon; Intervet, Millsboro, DE)

Blood urea nitrogen

There was no effect of treatment (n = 11 mares/treatment, P =  0.57); or day (P =  0.10), and there was an interaction between treatment and day (P < 0.0001). On the first day of urea supplementation to the diet (day of ovulation, D0), there was no difference in BUN concentrations between the two treatments (P >  0.05). There was an increase in BUN concentration on D1 through D7 in treated mares. On the last day of treatment, D7, the mean BUN concentration was of 12.75 ± 0.99 and 28.57 ± 2.25

Discussion

Results of this study provide the first evidence that dietary supplementation with urea alters the expression of genes associated with necrosis, invasion of cells and concentration of lipids in the endometrium of mares during diestrus. The DEGs identified after urea treatment in this dataset serve to formulate possible molecular effects of this metabolite in the endometrium of animals that might be related to fertility.

The dietary supplementation of urea in the present study was an effective

Conclusion

In conclusion, most of the DEGs in the dataset accrued in the present study are associated with necrosis, cell movement, tissue remodeling and lipid concentration (Fig. 6). These initial results allow for elucidation of novel mechanisms through which the relatively greater systemic BUN concentrations, might alter the endometrial transcriptome in mares. Additional studies need to be conducted to evaluate how the changes in endometrial transcriptome that were detected in the present study will

Funding

This work was supported by Science without Borders-CNPq-GDE-USA [grant number 208518/2014-2]; and USDA-ARS National Program 215, Grass, Forage and Rangelands Agroecosystems (grant number 5042-21000-003-00D).

Declaration of Competing Interest

None

CRediT authorship contribution statement

Yatta Linhares Boakari: Conceptualization, Methodology, Data curation, Data curation. Hossam El-Sheikh Ali: Conceptualization, Methodology, Data curation, Data curation, Visualization. Pouya Dini: Methodology, Data curation, Data curation, Visualization. Shavahn Loux: Data curation, Data curation, Visualization. Claudia Barbosa Fernandes: Methodology, Data curation. Alejandro Esteller-Vico: Funding acquisition, Conceptualization, Methodology, Data curation. Kirsten Scoggin: Methodology, Data

Acknowledgments

The authors would like to thank Dr. Yamê Fabres Robaina Sancler-Silva for her assistance with sample collection.

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