Intrauterine expression of LIF and its receptors in the cycling and early pregnant mare

Leukaemia Inhibitory Factor (LIF) plays a critical role in blastocyst development and implantation, as clearly demonstrated by the failure of wild-type mouse embryos to implant in the uterus of LIF-knockout female mice unless the latter receive LIF supplementation. Expression of LIF and its receptors (LIF-R and gp130) in the early pregnant horse have not been described. However, the horse may be an interesting animal in which to study such ‘implantation factors’ because various aspects of implantation occur both unusually late and in a temporally distinct fashion in this species. The aim of this study was to examine LIF, LIF-R and gp130 gene expression in the endometrium of cycling and early pregnant mares, and in the early conceptus. Endometrial biopsies were recovered from 4 mares at each of late oestrus, days 7 and 14 of dioestrus (cycling mares), and days 7, 14 and 21 of gestation. Conceptuses were recovered by uterine lavage (day 7) or using a video-endoscopically guided net (days 14 and 21). For day 14 and 21 conceptuses, only the bilaminar trophoblast was used in gene expression studies. Expression of mRNA for LIF, LIF-R and gp130 was analysed by rtPCR, with relative expression calculated with respect to the 3 most stable housekeeping genes using GeNorm analysis. A dramatic increase in LIF mRNA expression (p<0.01) was observed in both the endometrium and the trophoblast on day 21 of pregnancy. Expression of LIF-R and gp 130 increased significantly in the trophoblast on day 21 (p<0.01), but did not vary in the endometrium at the various stages examined. We propose that LIF plays a role in the adhesion between trophoblast and endometrium which, in the mare, cannot begin until the blastocyst capsule is dissolved somewhere between days 18 and 22 of gestation

Decision Making for Cryptorchid Castration; a Retrospective Analysis of 280 Cases

Abstract The location of an undescended testicle influences the choice of surgical technique for efficient cryptorchid castration. We review a standardized protocol for preoperative examination to dictate surgical approach to cryptorchidism. Cases are split into two periods: 2004–2006 and 2007–2014. In 2004–2006, conventional cryptorchidectomy and laparoscopic cryptorchid castration (standing) were both offered, but the choice of technique was based primarily on owners' preference for a recumbent or standing procedure. In 2007–2014, ultrasonography was used to locate the testes and dictate the preferred surgical approach; for abdominal testes, laparoscopic intraabdominal spermatic cord ligation without orchidectomy was preferred and for inguinal testes, conventional open orchidectomy. The numbers of animals requiring a second procedure to complete castration were compared between the two periods. In addition, failure rates for individual testes grouped by location were determined separately for the different techniques, and the value of preoperative ultrasonography to locate the retained testes was assessed. In 2004–2006, 15.3% (20/131) of the cryptorchids needed more than one surgery to complete castration, compared to 0.7% (1/144) in 2007–2014. Failure rates for laparoscopic castration were 0/168 (0%) for abdominal, 3/40 (7.5%) for inguinal, and 9/55 (16.4%) for scrotal testes; for conventional castration, failure was recorded for 3/12 (25%) abdominal and 0/92 (0%) inguinal testes. For 94% (156/166) of retained testes, ultrasound-based preoperative advice on surgical approach was correct. Using a standardized preoperative examination to determine choice of surgical technique significantly (P < .001) reduced the number of second surgeries needed to complete castration. Preoperative ultrasound is therefore a useful aid to determining the surgical approach to cryptorchid castration

Decision Making for Cryptorchid Castration; a Retrospective Analysis of 280 Cases

Abstract The location of an undescended testicle influences the choice of surgical technique for efficient cryptorchid castration. We review a standardized protocol for preoperative examination to dictate surgical approach to cryptorchidism. Cases are split into two periods: 2004–2006 and 2007–2014. In 2004–2006, conventional cryptorchidectomy and laparoscopic cryptorchid castration (standing) were both offered, but the choice of technique was based primarily on owners' preference for a recumbent or standing procedure. In 2007–2014, ultrasonography was used to locate the testes and dictate the preferred surgical approach; for abdominal testes, laparoscopic intraabdominal spermatic cord ligation without orchidectomy was preferred and for inguinal testes, conventional open orchidectomy. The numbers of animals requiring a second procedure to complete castration were compared between the two periods. In addition, failure rates for individual testes grouped by location were determined separately for the different techniques, and the value of preoperative ultrasonography to locate the retained testes was assessed. In 2004–2006, 15.3% (20/131) of the cryptorchids needed more than one surgery to complete castration, compared to 0.7% (1/144) in 2007–2014. Failure rates for laparoscopic castration were 0/168 (0%) for abdominal, 3/40 (7.5%) for inguinal, and 9/55 (16.4%) for scrotal testes; for conventional castration, failure was recorded for 3/12 (25%) abdominal and 0/92 (0%) inguinal testes. For 94% (156/166) of retained testes, ultrasound-based preoperative advice on surgical approach was correct. Using a standardized preoperative examination to determine choice of surgical technique significantly (P < .001) reduced the number of second surgeries needed to complete castration. Preoperative ultrasound is therefore a useful aid to determining the surgical approach to cryptorchid castration

Asynchronous Embryo Transfer Followed by Comparative Transcriptomic Analysis of Conceptus Membranes and Endometrium Identifies Processes Important to the Establishment of Equine Pregnancy

Preimplantation horse conceptuses require nutrients and signals from histotroph, the composition of which is regulated by luteal progesterone and conceptus-secreted factors. To distinguish progesterone and conceptus effects we shortened the period of endometrial progesterone-priming by asynchronous embryo transfer. Day 8 embryos were transferred to synchronous (day 8) or asynchronous (day 3) recipients, and RNA sequencing was performed on endometrium and conceptuses recovered 6 and 11 days later (embryo days 14 and 19). Asynchrony resulted in many more differentially expressed genes (DEGs) in conceptus membranes (3473) than endometrium (715). Gene ontology analysis identified upregulation in biological processes related to organogenesis and preventing apoptosis in synchronous conceptuses on day 14, and in cell adhesion and migration on day 19. Asynchrony also resulted in large numbers of DEGs related to &lsquo;extracellular exosome&rsquo;. In endometrium, genes involved in immunity, the inflammatory response, and apoptosis regulation were upregulated during synchronous pregnancy and, again, many genes related to extracellular exosome were differentially expressed. Interestingly, only 14 genes were differentially expressed in endometrium recovered 6 days after synchronous versus 11 days after asynchronous transfer (day 14 recipient in both). Among these, KNG1 and IGFBP3 were consistently upregulated in synchronous endometrium. Furthermore bradykinin, an active peptide cleaved from KNG1, stimulated prostaglandin release by cultured trophectoderm cells. The horse conceptus thus responds to a negatively asynchronous uterus by extensively adjusting its transcriptome, whereas the endometrial transcriptome is modified only subtly by a more advanced conceptus

Initiation of X Chromosome Inactivation during Bovine Embryo Development

X-chromosome inactivation (XCI) is a developmental process that aims to equalize the dosage of X-linked gene products between XY males and XX females in eutherian mammals. In female mouse embryos, paternal XCI is initiated at the 4-cell stage; however, the X chromosome is reactivated in the inner cell mass cells of blastocysts, and random XCI is subsequently initiated in epiblast cells. However, recent findings show that the patterns of XCI are not conserved among mammals. In this study, we used quantitative RT-PCR and RNA in situ hybridization combined with immunofluorescence to investigate the pattern of XCI during bovine embryo development. Expression of XIST (X-inactive specific transcript) RNA was significantly upregulated at the morula stage. For the first time, we demonstrate that XIST accumulation in bovine embryos starts in nuclei of female morulae, but its colocalization with histone H3 lysine 27 trimethylation was first detected in day 7 blastocysts. Both in the inner cell mass and in putative epiblast precursors, we observed a proportion of cells with XIST RNA and H3K27me3 colocalization. Surprisingly, the onset of XCI did not lead to a global downregulation of X-linked genes, even in day 9 blastocysts. Together, our findings confirm that diverse patterns of XCI initiation exist among developing mammalian embryos

Hindquarter Movement of Sporthorse Stallions During Semen Collection

Some stallions used for both breeding and show jumping perform less well in competitions during the breeding season. It has been suggested that the demands of semen collection on the back and pelvis may impair the range of motion (ROM) required for show jumping. To better understand hindquarter dynamics, this study assessed the ROM of the hindquarters during both the mounting phase (MP) and ejaculatory phase (EP) of semen collection and compared it to the ROM previously measured during show jumping. The kinematics of six warmblood stallions were studied during semen collection on a phantom. Skin markers were placed on the withers, tuber coxae, proximal femur and tibia, and distal tibia and metatarsus. During each phase, six angles were measured using recordings made with a home-video camera (60 Hz) positioned perpendicular to the phantom. The differences in joint angles between the two phases were compared statistically using commercially available software. The pelvis showed a significantly larger ROM during the MP than the EP (P <.05). The ROM of the hindquarters was significantly larger during both the MP and EP than reported during show jumping, and the pelvis was considerably more extended (P <.05). This relatively extreme flexion and extension of the pelvis during semen collection may impose different strains on musculoskeletal structures which, combined with asymmetrical lateral flexion and/or axial rotation on the phantom, may exacerbate subclinical hindquarter pain. Nevertheless, the outcome of this study would benefit from an additional, objective clinical locomotor examination combined with analysis of motion during show jumping using modern IMU sensor technology

The Role of Oviductal Cells in Activating Stallion Spermatozoa

Conventional in vitro fertilization is poorly successful with equine gametes. Although stallion spermatozoa bind to the zona pellucida in vitro, they fail to acrosome react and cannot therefore penetrate into the perivitelline space. Failed sperm penetration most likely relates to the absence in in vitro fertilization media of essential molecules required to fully support stallion sperm capacitation. In vivo, the oviductal lumen provides an environment that appropriately regulates interactions between the gametes and promotes fertilization. Identifying the oviductal “fertilization stimulating factors” would enormously benefit the development of equine in vitro fertilization media. This review focuses on the current understanding of equine sperm–oviduct interactions, which may hold essential clues to achieving successful in vitro fertilization with equine gametes