Late gestation maternal overnutrition alters gene expression and histomorphology in neonatal foal testes

Reproductive health of male offspring following perturbations in maternal nutrition is not well-described in the horse; therefore, the objective of this study was to determine the effects of maternal overnutrition on neonatal foal testicular development. Sixteen Quarter Horse mares were used in a completely randomized design and stratified by expected foaling date, body weight, and body condition score into 1 of 2 dietary treatments beginning on gestational day 235: control (CON; n = 8) fed to meet nutrient requirements and overfed (HIGH; n = 8) fed 40% above CON. At 5 h postpartum, foals were euthanized, and testes from male foals were harvested, weighed, and stored for analyses. Samples were numerically coded in a blinded fashion and processed randomly to eliminate bias. Histomorphometric evaluations were performed following a hematoxylin & eosin stain and evaluated using ImageJ. All data were analyzed using the MIXED procedure in SAS v9.4. Horse within treatment served as a random effect, with a main effect of treatment. For RNA sequencing, differential gene expression analysis used DESeq2 1.20.0 R package to compare gene expression levels between CON and HIGH, and P-values were adjusted using the Benjamini-Hochberg correction. The threshold for significance was set at P ≤ 0.05 with trends toward significance at 0.05 < P ≤ 0.10, with sequencing data using adjusted P-values. Stringency was set to include differentially expressed genes with an unadjusted P-value < 0.05 for pathway enrichment analysis. Testes from HIGH foals (n = 4) were 61% heavier than testes from CON foals (n = 4; P < 0.01). There was no difference in individual seminiferous tubule area (P = 0.76) or Sertoli cell number within tubule (P = 0.57); however, testes from HIGH foals had 2.3 times fewer tubules per 10× magnification field than CON foals (P = 0.01). Further, tubules represented a lesser percentage of image area relative to interstitial tissue in testes from HIGH foals (P = 0.05). RNA sequencing revealed 56 differentially expressed genes between HIGH and CON testes (P-adjusted < 0.05). Differentially expressed genes represented biological processes related to steroidogenesis, cell regulation, and energy metabolism. Thus, late gestation maternal overnutrition alters fetal testicular development with potential long-term implications for reproductive function.