Uterine inflammatory response, embryo recovery and morphometry in interspecies breeding

Problem mares presumably have better fertility when bred with donkeys; this could be due to differential post-breeding uterine inflammatory response modulated by seminal plasma (SP) or embryonic ultrastructural features; however, these hypotheses have not been studied. This study aimed to assess the: (i) uterine inflammatory response; (ii) embryo recovery and morphometry; (iii) the potential crosstalk between the proteomic and metabolomic of SP and uterus of mares bred with donkey or horse semen or infused with the SP of either species. Twenty-two mares were randomly assigned in a crossover design, with a washout cycle in-between, to receive semen and SP from both species and saline in five different estruses. The uterine inflammatory response was assessed via fluid accumulation measured by ultrasound, neutrophils on uterine cytology, and cytokines on uterine fluid (IL-1a, IL-1b, IL-4, IL-6, CXCL-8, IL-10) at 0-, 6, 24, 48-h-post-AI/infusion. Embryos were recovered 8d post-ovulation, classified, graded, measured, and the nuclei morphometrics were assessed with ImageJ on images acquired with a fluorescent microscope. Proteomics and metabolomics of SP were identified with LC-MS/MS and analyzed with R-studio, PANTHER, and Perseus. A mixed linear model and Kruskal-Wallis rank-sum test was used for normally and not-normally distributed variables. Uterine fluid increased at 6-h post-infusion/AI (P<0.05) but did not vary across groups (P>0.05). There was an effect of time (P=0.001) and of time*groupinteraction (P<0.001) but no effect of group (P=0.86) on neutrophil counts. At 6h post-infusion, the neutrophil count in the mares infused with donkey-SP tended to be the lowest (P=0.07). Cytokine concentrations peaked at 6h (P<0.05); horse semen triggered higher IL-6 and lower IL-1b concentrations than donkey semen (P<0.05). Donkey- and horse-SP shared 16 proteins; each species had 99 and 64 unique proteins, respectively. The metabolomic fingerprinting revealed a higher number of anti-inflammatory metabolites, such as choline, epinephrine, palmitoleic acid, and palmitic acid, in donkey-SP compared to horse-SP, suggesting these molecules can play a role in the uterine inflammatory response. Embryo recovery was similar between donkey-bred (59%) and horse-bred (55%) (P<0.05). Horse and mule embryos had similar sizes, grades, number of compact nuclei per sector, fragmented nuclei index mule, and micronuclei index (P>0.05); however, the mitotic nuclei index was higher in mule embryos (P=0.03). In conclusion, this is the first study to compare the uterine inflammatory response, embryo recovery, and morphology of mares bred with donkeys and horses. Horse semen triggered a greater uterine inflammatory response than donkey semen, and donkey-SP tended to have the lowest. The SP composition may explain the post-breeding uterine inflammatory response. Equid embryos share similar nuclear ultrastructure features, except that mules have a higher mitotic index.