Optimizing equine assisted reproductive technologies

Abstract

Application of assisted reproductive technologies (ARTs) is more common in the horse breeding industries, but there is still room for improvement. Embryo recovery rate after embryo flushing, embryo production rate after ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI), cryopreservation of equine embryos and pregnancy rates after transfer of these embryos can be improved. ARTs cause epigenetic changes in the early embryo, resulting in low birth weight in man and large offspring syndrome in cattle. It is unknown how this alter equine fetal development. Ultrasonographic evaluation of fetal development throughout gestation can help to monitor and detect abnormal development. Fetal growth curves of Dutch Warmblood mares are created as baseline for future monitoring of intrauterine development of foals produced by ARTs. Only aorta diameter and biparietal cross-sectional surface area increased linearly during late gestation. Mitochondria are one of the factors involved in success of fertilisation and early embryonic development. In vitro embryo production (IVEP) and maternal age affect mitochondria in oocytes and embryos by reducing their numbers and functionality. In this study it became evident that day (D) 7 embryos from older (>16 years) mares contain fewer mtDNA copy numbers than embryos from younger (<12 years) mares. D8 IVEP embryos had similar mtDNA copy numbers compared to D7 in vivo embryos. Maternal age and IVEP can also compromise onset of mtDNA replication, therefore the onset of mtDNA replication was determined in in vivo and IVEP embryos. The first significant post-fertilisation increase of mtDNA copy number took place between the early and expanded blastocyst stage. This onset of mitochondrial replication was preceded by an increase in TFAM [removed]gene involved in regulation of mitochondrial copy number) in IVEP embryos 96h after culture, 4 days prior to blastocyst formation. In D8 IVEP embryos, expression of genes involved in mitochondrial functioning GPX3 and mtPOLGB deviated from that observed in in vivo D7 embryos. mtPOLGB expression was doubled in IVEP embryos whereas GPX3 expression was halved. This reduced expression of GPX3 might lead to reduced protection against reactive oxygen species induced damage, while the overexpression of mtPOLGB may be part of an attempt to maintain mitochondrial number in the face of increasing rates of damage. The results of cryopreservation of equine embryos is still suboptimal and results demonstrated that exposure to cryoprotectants without freezing/cooling did not affect cellular structures like mitochondria, nucleus or cytoskeleton. Overall, small embryos cryopreserved by controlled-rate freezing suffered less damage than large controlled-rate frozen or vitrified embryos. Vitrification of large embryos changed mitochondrial distribution from a homogenous to a heterogenous pattern, that can be related to alteration in function. Mitochondrial activity is a difficult parameter to evaluate for damage caused by freezing/cooling. Healthy embryos have a ‘quieter’ metabolism than damaged embryos because they do not have to generate energy to repair damage. In this thesis it became evident that several ARTs are available in equine reproduction, but all are associated with (dis) advantages. Breeders should be well informed of the pro’s and con’s before application one of the techniques.