Mule embryos share identical morphological features to horse embryos

Abstract

This study aimed to compare the morphometry of horse and mule embryos. The study's hypothesis was that the micronuclei and nuclear fragmentation indexes are higher in mule embryos than in horse embryos. Twenty-two mares were randomly assigned in a crossover design to receive semen from a horse and a donkey; thirteen horse and thirteen mule embryos were obtained. Embryos were recovered eight days post-ovulation and classified according to the stage of development and quality with a score from 1 (excellent) to 4 (degenerate). Embryos were stained with Hoechst33342, and images were acquired with a fluorescence microscope. Nuclei were categorized as compact, mitotic, or fragmented; the fragmented and mitotic indexes were calculated based on their proportion over the total amount of nuclei counted. Embryo size and nuclear morphometry were assessed through ImageJ. Data analyses were carried out with GraphPad using ANOVA and T-test; significance was set at P < 0.05. The number of positive flushes in cycles bred with donkey or stallion semen did not differ when compared per cycle or per ovulation (13 vs. 12) (P > 0.05). One set of twins was recovered from a mare bred to the stallion that had a double ovulation; a mule and horse embryos were both recovered from eight mares. There was no difference in size between mule and horse embryos (915.5 ± 288 μm vs. 575.8 ± 69.6 μm) (P > 0.05) size of the study. The mule embryos scored between grade 1 (n = 9) and grade 2 (n = 4); similarly, the horse embryos scored between grade 1 (n = 6) and grade 2 (n = 7). The evaluation of the nuclear morphometry revealed that horse and mule embryos have a similar number of compact nuclei per sector (148.7 ± 6.8 nuclei/sector in mule embryos vs. 156.5 ± 8.5 nuclei/sector in horse embryos) (P > 0.05); however, the number of mitotic nuclei tended to be higher in mule embryos (5.2 ± 0.82) than in horse embryos (3.3 ± 0.3) (P = 0.08). The fragmented nuclei index was similar between mule (0.25 ± 0.1%) and horse (0.22 ± 0.1%) embryos (P = 0.4); the mitotic nuclei index was higher in mule embryos (3.2 ± 0.4%) than in horse embryos (2.2 ± 0.2%) (P = 0.02). In conclusion, embryo morphology of mares bred to a donkey and a horse shares similar nuclear ultrastructure features, except that mule embryos have a higher mitotic index.