Expression of leukaemia inhibitory factor at the conceptus?maternal interface during preimplantation development and in the endometrium during the oestrous cycle in the mare.

Leukaemia inhibitory factor (LIF) plays a critical role in blastocyst development and implantation in several species. The present study investigated mRNA and protein expression for LIF, as well as the low-affinity LIF receptor (LIFR) and interleukin-6 signal transducer (IL6ST), in equine endometrium, trophoblast and histotroph during early pregnancy and in the endometrium during the oestrous cycle. Endometrial LIF mRNA expression was upregulated after Day 21 of pregnancy, whereas LIF immunoreactivity increased in the endometrium on Day 28. Expression of LIF mRNA in the yolk sac membrane increased from Day 21 of pregnancy, whereas LIF immunoreactivity increased from Day 28 in the trophoblast. LIFR and IL6ST mRNA was expressed in the endometrium during both the oestrous cycle and early pregnancy and, although LIFR and IL6ST protein were localised to the glandular epithelium during the cycle and first 14 days of pregnancy, from Day 21 they were located in the luminal epithelium. Trophoblast expression of LIFR and IL6ST increased as pregnancy proceeded. In conclusion, LIF expression increased at the conceptus-maternal interface during capsule attenuation. Because contemporaneous upregulation of both LIFR and IL6ST was also observed in the trophoblast, we propose that LIF plays an important role in the development of endometrial receptivity for trophoblast growth, apposition and adhesion in mares.

Effect of pregnancy on endometrial expression of luteolytic pathway components in the mare.

Endometrial oxytocin receptors (OXTR) and prostaglandin-endoperoxide synthase 2 (PTGS2) are central components of the luteolytic pathway in cyclic mares, and their suppression is thought to be critical to luteal maintenance during early pregnancy. We examined the effect of pregnancy on endometrial expression of potential regulators of prostaglandin (PG) F2α secretion in mares. Expression of the nuclear progesterone receptor and oestrogen receptor ERα was high during oestrus, and depressed when progesterone was elevated; the opposite applied to the membrane progesterone receptor. PTGS2 was upregulated on Day 14 of dioestrus, but not pregnancy. Although OXTR mRNA expression was not elevated on Day 14 of dioestrus, protein abundance was; this increase in OXTR protein was absent on Day 14 of pregnancy. Intriguingly, gene and protein expression for PTGS2 and OXTR increased markedly between Days 14 and 21 of pregnancy suggesting that, although initial avoidance of luteolysis during pregnancy involves their suppression, this is a transient measure that delays rather than abolishes luteolytic pathway generation. The only oxytocin-PGF2α feedback loop component downregulated on both Days 14 and 21 of pregnancy was the PGF2α receptor we propose that downregulation of the PGF2α receptor uncouples the oxytocin-PGF2α feedback loop, thereby preventing generation of the large PGF2α pulses required for luteolysis.

The Role of Conceptus-maternal Signalling in the Acquisition of Uterine Receptivity to Implantation in Mammals.

Implantation is a pivotal step in the establishment of mammalian pregnancy. Although implantation strategies vary between species, many aspects of conceptus-maternal signalling necessary to induce uterine receptivity to implantation are conserved. The temporal 'window' for the initiation of implantation is short and precisely controlled by endocrine, paracrine and autocrine factors. An invariable prerequisite for the development of uterine receptivity to implantation is continuous exposure of the endometrium to progesterone which, after a species-specific interval, downregulates progesterone receptor (PGR) expression in the epithelium and stimulates the production of progestamedins. Uterine receptivity involves temporal changes in the expression of genes, leading to modifications in surface, extracellular matrix and secretory characteristics that support growth, proliferation, migration and attachment of the conceptus. Moreover, a complex interplay between endometrial progestamedins and estramedins and conceptus-derived oestrogens, cytokines and interferons (INFs), prostaglandins (PGs) and cortisol is crucial to the preparation for implantation. Understanding the individual roles and combined actions of conceptus and endometrial autocrine and paracrine factors in the development of uterine receptivity to implantation is essential for translational research into strategies to reduce pregnancy loss in man and animals.

Vitrifying expanded equine embryos collapsed by blastocoel aspiration is less damaging than slow-freezing.

The cryotolerance of equine blastocysts larger than 300 µm can be improved by aspirating blastocoele fluid prior to vitrification; however, it is not known whether blastocoele aspiration also enables successful slow-freezing. The aim of this study was therefore to determine whether slow-freezing of expanded equine embryos following blastocoele collapse was more or less damaging than vitrification. Grade 1 blastocysts recovered on day 7 or 8 after ovulation were measured (>300-550 µm, n = 14 and > 550 µm, n = 19) and blastocoele fluid was aspirated prior to slow-freezing in 10% glycerol (n = 14), or vitrification (n = 13) in 16.5% ethylene glycol/16.5% DMSO/0.5 M sucrose. Immediately after thawing or warming, embryos were cultured for 24 h at 38 °C and then graded and measured to assess re-expansion. Control embryos (n = 6) were cultured for 24 h following aspiration of blastocoel fluid, without cryopreservation or exposure to cryoprotectants. Subsequently, embryos were stained to assess live/dead cell proportion (DAPI/TOPRO-3), cytoskeleton quality (Phalloidin) and capsule integrity (WGA). For 300-550 µm embryos, quality grade and re-expansion were impaired after slow-freezing but not affected by vitrification. Slow-freezing embryos >550 µm induced additional cell damage as indicated by a significant increase in dead cell proportion and disruption of the cytoskeleton; neither of these changes were observed in vitrified embryos. Capsule loss was not a significant consequence of either freezing method. In conclusion, slow-freezing of expanded equine blastocysts collapsed by blastocoel aspiration compromises post-thaw embryo quality more than vitrification.

Insulin-like growth factor system components expressed at the conceptus-maternal interface during the establishment of equine pregnancy.

In many species, the insulin-like growth factors (IGF1 and IGF2), their receptors and IGF binding proteins play important roles in preparing the endometrium for implantation, and regulating conceptus growth and development. To determine whether the IGF system may contribute to conceptus-maternal interaction during equine pre-implantation development, we evaluated mRNA expression for IGF system components in conceptuses, and endometrium recovered from pregnant and cycling mares, on days 7, 14, 21 and 28 after ovulation. We also investigated expression of IGF1, IGF2 and their receptors 6 and 11 days after transfer of day 8 embryos to synchronous (day 8) or asynchronous (day 3) recipient mares. Expression of IGF1 and IGF2, IGF1R, IGF2R, INSR and IGFBPs 1, 2, 4 and 5 was evident in endometrium and conceptus membranes during days 7-28. Endometrial IGF2, INSR, IGFBP1 and IGFBP2 expression increased between days 7 and 28 of pregnancy. In conceptus membranes, expression of all IGF system components increased with developmental stage. Immunohistochemistry revealed strong expression of IGF1, IGF2 and IGF1R in both endometrium and conceptus membranes, whereas INSR was highly expressed in endometrium but barely detectable in the conceptus. Finally, a negatively asynchronous uterine environment retarded IGF1, IGF2 and INSR expression in the conceptus, whereas in the endometrium only INSR expression was altered by asynchrony. The presence of IGFs, their receptors and IGFBPs in the endometrium and conceptus during early equine pregnancy, and down-regulation in the conceptus following asynchronous embryo transfer, suggest a role in conceptus-maternal communication during the preparation for implantation.

Advanced mare age impairs the ability of in vitro-matured oocytes to correctly align chromosomes on the metaphase plate.

BACKGROUND: Advanced mare age is associated with declining fertility and an increased risk of early pregnancy loss. Compromised oocyte quality is probably the primary reason for reduced fertility, but the defects predisposing to embryonic death are unknown. In women, advanced age predisposes to chromosome segregation errors during meiosis, which lead to embryonic aneuploidy and a heightened risk of miscarriage. OBJECTIVES: To evaluate the effect of advanced mare age on chromosome alignment and meiotic spindle morphology in in vitro-matured (IVM) oocytes. STUDY DESIGN: Morphometric and morphological analysis. METHODS: To investigate differences in spindle organisation and chromosome alignment between young and old mares, oocytes collected from slaughtered mares were divided into two groups depending on mare age (young, ≤14 years and old, ≥16 years), IVM and stained to visualise chromatin and alpha-tubulin. Spindle morphology, morphometry and chromosome (mis)alignment were evaluated by confocal microscopy and 3D image analysis. RESULTS: Oocytes from old mares showed a higher incidence of chromosome misalignment (47.4% vs. 4.5%; P<0.001) and a thicker metaphase plate (mean ± s.d.: 5.8 ± 1.0 µm vs. 4.9 ± 0.9 µm; P = 0.04) than oocytes from young mares. Although no differences in spindle morphometry were detected between old and young mares, an increased major spindle axis length was associated with chromosome misalignment (mean ± s.d.: 25.3 ± 6.1 µm vs. 20.8 ± 3.3 µm; P = 0.01) irrespective of age. MAIN LIMITATIONS: The oocytes were IVM and may not exactly reflect chromosome misalignment in vivo. CONCLUSIONS: Advanced mare age predisposes to chromosome misalignment on the metaphase II spindle of IVM oocytes. The compromised ability to correctly align chromosomes presumably predisposes to aneuploidy in resulting embryos and thereby contributes to the age-related decline in fertility and increased incidence of early pregnancy loss. The Summary is available in Portuguese - see Supporting Information.

Ultrasonographic measurements of localized fat accumulation in Shetland pony mares fed a normal v. a high energy diet for 2 years.

Health risks associated with obesity are more likely a factor of the localization of fat excess, rather than of elevated BW per se. The aim of this randomized controlled clinical trial was to determine the effect of a long-term high energy diet on BW, fat accumulation and localization. Eight Shetland pony mares, 3 to 7 years old, were randomly divided into a control and a high energy (HE) diet group fed either maintenance or double maintenance energy requirements (200% net energy (NE)) for two consecutive summers, with a low energy diet in the winter in between. Body condition score (BCS) did not differ between the groups at the onset of the study (control 5.6±0.75 v. HE 6.3±0.5). From 12 weeks after starting the diet, ultrasonography of five different locations (retroperitoneal, axillary, withers, intercostal and rump) for adipose deposition, BCS and BW were measured monthly during the period that ponies received different diets. Statistical analysis was performed using a linear mixed-effects model with post hoc Bonferroni testing. P values <0.05 were considered significant. At week 12 after the onset of the diet, fat thickness in the HE group was significantly greater than in the control group. During the monitoring period, the HE group showed a significant increase in mean (±SE) BW (+52%, 265±13.94kg) and BCS (+70%; to 9.0±0.4), while the control group was unchanged (BW 160±13.98 kg; BCS 3.8±0.4). At all locations, the fat depth in the HE group increased significantly, with the highest increase noted for retroperitoneal deposits. The conclusions were that a 200% NE diet induced subcutaneous and retroperitoneal fat accumulation, with the greatest increase in intra-abdominal deposits. The moderate increase of the subcutaneous fat depth followed by a plateau phase suggests the existence of a limit of adipose tissue expandability, as in man.

Relationship between colour flow Doppler sonographic assessment of corpus luteum activity and progesterone concentrations in mares after embryo transfer.

Colour-flow Doppler sonography has been described as a means of assessing corpus luteum (CL) function rapidly, because area of luteal blood vessels correlates well with circulating progesterone (P4) concentrations [P4] in oestrous cycling mares. The aim of this study was to assess the relationships between CL size and vascularity, and circulating [P4] during early pregnancy in mares, and to determine whether luteal blood flow was a useful aid for selecting an embryo transfer recipient. Equine embryos (n=48) were recovered 8 days after ovulation and were transferred to available recipient mares as part of a commercial program with the degree of synchrony in timing of recipient ovulation ranging from 1 day before to 4 days after the donor. Immediately prior to embryo transfer (ET), maximum CL cross-section and blood vessel areas were assessed sonographically, and jugular blood was collected to measure plasma [P4]. Sonographic measurements and jugular blood collection were repeated at day 4 after ET for all mares, and again at days 11, 18 and 25 after ET in mares that were pregnant. The number of grey-scale and colour pixels within the CL was subsequently quantified using ImageJ software. The CL blood flow correlated significantly but weakly with plasma [P4] on the day of transfer and on day 4 after ET in all mares, and on days 11 and 25 after ET in pregnant mares (r=0.30-0.36). The CL area and plasma [P4] were also correlated on each day until day 11 after ET (r=0.49-0.60). The CL colour pixel area decreased significantly after day 18, whereas CL area was already decreasing by day 4 after ET. The CL area, area of blood flow, or [P4] was predictive of pregnancy. Findings in the present study suggest that both CL area and blood flow are correlated with circulating [P4] at the time of transfer and in early pregnancy. Evaluation of the CL using B-mode or CF sonography, although practical, provides no improvement in the selection of recipients or prediction of pregnancy outcomes than methods employed currently.

Magnetic resonance microscopy atlas of equine embryonic development.

REASONS FOR PERFORMING STUDY: Equine embryogenesis post implantation is not well studied, and only two-dimensional illustrations are available. A thorough appreciation of the complex three-dimensional relationship between tissues and organs and their development is, however, crucial for understanding physiological and pathological mechanisms. OBJECTIVES: The goals were 2-fold: 1) to establish a freely accessible online atlas as a reference tool for the scientific and pedagogic communities; and 2) to create a framework for integration of data with known spatiotemporal distribution, such as gene expression or cell lineage. STUDY DESIGN: Descriptive anatomical study. METHODS: Magnetic resonance microscopy was performed on embryos of 28, 32, 35, 37, 39, 40, 42, 45, 50 and 65 days gestation using a 9.4 T magnet. Equine embryos were staged according to the Carnegie system. Acquired images were optimised using histogram optimisation and processed for easy online access. RESULTS: Magnetic resonance microscopy protocols for imaging of equine embryos and fetuses were developed. The wider spread of signal intensity values achieved by histogram equalisation increased visual contrast considerably. Despite their longer gestation, equine conceptuses appeared to reach the various Carnegie staging benchmarks earlier than human embryos. CONCLUSIONS: The equine atlas is designed to serve as an online reference tool for research and teaching. POTENTIAL RELEVANCE: The equine atlas may serve as a foundation and scaffold for improved anatomical labelling, spatial and temporal data integration and further understanding of physiological and pathophysiological processes involved in development and disease.

Efficacy of transvaginal ultrasound-guided twin reduction in the mare by embryonic or fetal stabbing compared with yolk sac or allantoic fluid aspiration.

Transvaginal ultrasound-guided pregnancy reduction (TUGR) is a procedure described for the management of twins post-fixation in the horse. Success rates are often disappointing but are reported to be more favorable for bilaterally situated twins and when intervention takes place before day 35 of gestation. This study aimed to determine whether stabbing the embryo/fetus rather than aspirating conceptus fluids improved the likelihood of success, measured as the birth of a normal live singleton foal. Data from 103 TUGR interventions were analyzed by logistic regression analysis; method of treatment, relative conceptus location (i.e., uni- vs. bilateral), and stage of gestation were included as interdependent factors that potentially influence the outcome. Overall, 34/103 (33%) TUGR interventions resulted in a single live foal. There was no significant difference (P = 0.14) in the outcome between TUGR based on fetal stabbing (12/28: 42.9%) versus fluid aspiration (22/75: 29.3%). There was also no significant influence (P = 0.11) of the conceptuses being located unilaterally (19/65: 29.2%) versus bilaterally (15/38: 39.5%). However, TUGR was numerically more successful (P = 0.05) when performed ≤ Day 35 of gestation (21/53: 39.6%), as opposed to > Day 35 (13/50: 26%). Day 45 may represent an even more critical time point because only 2 out of 15 TUGRs (13.3%) performed beyond this day resulted in the birth of a live foal, compared with 11/35 (31.4%) performed between Days 36 and 45. Although the numbers are low, this suggests that TUGR is not the method of choice for reducing > Day 45 twins. Four pregnancy losses were recorded 1 to 7 months post-TUGR (4/38: 10.5%), and although it is tempting to attribute the losses to TUGR, this rate of late gestation pregnancy loss is normal. We conclude that TUGR by fetal stabbing does not offer significant advantages over fluid aspiration. However, TUGR should be performed before Day 35 of gestation and is considered primarily a salvage procedure to be used when re-breeding is not a viable alternative.