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Progesterone (P4), acting via its receptor, regulates uterine function and histotroph production, which are crucial to embryo growth. This study aimed to examine exogenous P4 effects on embryo size and differential endometrial gene expression at Day 19 of gestation using a 'dam size' sheep model of maternal constraint. Purebred Suffolk (S, genotypically large) embryos were transferred into recipient groups of Cheviot (C, genotypically small) or Suffolk ewes that had, or had not, been pre-treated with P4 from Days 0 to 6 of pregnancy. At Day 19S embryos were collected from four experimental groups: P4 pretreated S ewes (SP4; n=5), untreated S ewes (SnP4; n=15), P4 pretreated C ewes (CP4; n=7) and untreated C ewes (CnP4; n=21). Day-19 embryos from CP4 ewes were larger (P<0.05) than those from CnP4 ewes and similar in size (P>0.05) to embryos from SnP4 and SP4 ewes. Expression of mucin 1 (MUC1) and prostaglandin-endoperoxide synthase 2 (PTGS2) was upregulated in uterine horns ipsilateral to the corpus luteum from CP4 ewes. Prostaglandin receptor (PGR), MUC1 and PTGS2 expression was upregulated, whilst cathepsin L (CTSL) and radical S-adenosyl methionine domain-containing 2 (RSAD2) expression was downregulated in the ipsilateral horn of SP4 ewes. This suggests that pretreating ewes with exogenous P4 may alleviate early pregnancy maternal constraint via mechanisms that alter uterine function. However, further research is required to investigate the timing of P4 administration and its impact on conception rates.

Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphasearrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.

Progesterone (P4) administration in early pregnancy enhances embryo growth in sheep but is associated with decreased embryo survival. This study examined the effects of exogenous P4 administered during specific time periods between pregnancy Day 0 and Day 6 to determine the critical time point for advancement of embryo growth without pregnancy loss and to examine Day 6 and Day 19 endometrial gene expression. Suffolk (S) embryos were transferred into Cheviot (C) ewes that received exogenous P4 (CP4) on Days 0-3 (CP40-3), Days 0-6 (CP40-6), Days 2-4 (CP42-4) or Days 3-6 (CP43-6). Additionally, S embryos were transferred to C and S ewes that did not receive P4 (CnP4 and SnP4). Day 19 embryos from CP4 ewes were longer (P<0.05) than those from CnP4 ewes. CP42-4 ewes had embryos of similar size to those of CP40-3 and CP40-6 ewes but had higher pregnancy rates. There was altered expression of genes associated with embryo implantation and histotroph production: diacylglycerol-O-acyltransferase (DGAT2), hepatocyte growth factor (HGF) and prostaglandin endoperoxide synthase 2 (PTSG2) on Day 6 and endometrial galectin 15 (LGALS15) and mucin glycoprotein 1 (MUC1) on Day 19. This suggests that specific timing of P4 administration is critical to the enhanced embryo growth and survival observed. These findings provide a platform for further investigation aimed at advancing embryo development and survival.