The current study used RNA sequencing to determine transcriptional profiles of equine endometrium collected 14, 22, and 28 days after ovulation from pregnant mares. In addition, the transcriptomes of endometrial samples obtained 20 days after ovulation from pregnant mares, and from non-pregnant mares which displayed and failed to display extended luteal function following the administration of oxytocin, were determined and compared in order to delineate genes whose expressions depend on the presence of the conceptus as opposed to elevated progesterone alone. A mere fifty-five transcripts were differentially expressed between samples collected from mares at Day 22 and Day 28 of pregnancy. This likely reflects the longer-term exposure to a relatively constant, progesterone-dominated environment with little change in factors secreted by the conceptus that would affect endometrial gene expression. The complement system was amongst the canonical pathways significantly enriched in transcripts differentially expressed between Day 14 and Day 22/28 of pregnancy. The expression of complement components 7 and 8 was confirmed using in situ hybridization. The expression of SERPING1, an inhibitor of the complement system, was confirmed by immunohistochemistry. In line with the resumed capacity of the endometrium to produce prostaglandin, prostaglandin G/H synthase 1 was expressed at higher levels at Days 22 and 28 than at Day 14 of pregnancy. Our data suggest that this up-regulation is enhanced by the presence of the conceptus; samples obtained from mares at Day 20 of pregnancy had significantly higher levels of prostaglandin G/H synthase 1 transcript than mares with extended luteal function.

Unlike traditional forage grasses, turfgrasses, which were developed to be tolerant of foot traffic and close mowing, may be suitable as alternative ground cover in areas of high hoof traffic such as dry lots. The objective of this study was to evaluate the potential of eight cool-season turfgrasses as alternative ground cover in heavy use areas. Cultivars were established via seeding in four replicated plots. To simulate horse traffic at a trot, a Baldree traffic simulator was driven over a section of the plot, either 0 (CON), 1 (LOW), or 2 (HIGH) times per week for 6 weeks followed by 4 weeks of rest in the spring, summer, and fall over a 2-year period. Variables assessed include compaction, biomass available for grazing, vegetative cover (persistence), and nutrient composition. Soil compaction increased with the application of traffic treatments (P < .0001). Traffic treatment reduced persistence following traffic by 19%–36% across all trials in both years (P = .0003). For most trials, biomass available for grazing was reduced after traffic treatment by 19% and 43% (P = .02). Overall, tall fescue cultivars were most traffic tolerant followed by hard fescue. Cultivars considered “low” in nonstructural carbohydrate (<15%) included creeping bentgrass in Year 1 and hard fescue and chewings fescue in Year 2. In Year 2, the Ca:P ratio declined beyond what is recommended for horses. Tall fescue, hard fescue, and creeping bentgrass cultivars are recommended for further evaluation as suitable ground cover in areas of heavy use on equine operations.

Sixteen cases of spontaneous pregnancy loss (11 of singletons and five of pairs of twins) are described. The losses occurred between gestation Days 13 and 25 in 12 mares being monitored almost daily by transrectal ultrasonography (for measurement of conceptus growth) and blood sampling (for determination of maternal plasma progesterone concentrations as evidence of luteolysis) in experimental studies of early pregnancy. In 10 of the 16 cases the uterus was flushed and eight conceptuses were recovered for morphological assessment. Five of the 11 losses of singletons occurred before Day 16 and, with one exception, were preceded or accompanied by luteolysis. The remaining six singleton pregnancies failed after Day 16, with two cases evidencing luteolysis beforehand. Thus, overall, 6/11 singleton losses were associated with luteolysis while 5/11 were not. The five cases of simultaneous loss or degeneration of twin conceptuses all occurred on Day 19 or 20, preceded by luteolysis in only one case. These observations suggest that while the causes of spontaneous early pregnancy failure are multifactorial, luteolysis might contribute to the problem more often than has been previously contended.

Steroid production and metabolism by early conceptuses are very important for the establishment and maintenance of pregnancy in horses. Our earlier work suggested the possible formation of 5alpha-reduced steroids in equine conceptuses. We have now demonstrated the formation of 5alpha-reduced metabolites of androstenedione, testosterone, and progesterone by the embryo and its membranes. A total of 44 conceptuses were collected from 26 mares between 20 and 31 days of pregnancy. Tissues from the embryo proper and from the separated components of the conceptus (bilaminar and trilaminar trophoblast, allantois) were incubated with tritium-labeled substrates. 5Alpha-reduced metabolites (5alpha-dihydro-and 3beta, 5alpha-tetrahydro-steroids) as radiolabeled products were identified from a series of chromatographic steps using four solvent systems for high-performance liquid chromatography. Use of a 5alpha-reductase inhibitor confirmed the metabolites were indeed 5alpha-reduced steroids. For the embryo, the only products from androstenedione were 5alpha-dihydroandrostenedione and 3beta, 5alpha-tetrahydroandrostenedione, with no evidence of more polar metabolites; there was some 3beta, 5alpha-tetrahydrotestosterone but no 5alpha-dihydrotestosterone from testosterone, and formation of androstenedione was followed by the production of 5alpha-dihydroandrostenedione and 3beta, 5alpha-tetrahydroandrostenedione. The major 5alpha-reduced product from progesterone was 3beta, 5alpha-tetrahydroprogesterone, with lesser amounts of 5alpha-dihydroprogesterone. For the membranes, reductions to tetrahydro, 5alpha-reduced steroids were prominent in most instances, but also present were considerable amounts of products more polar than the substrates. The well-recognized activity of some 5alpha-reduced steroids-for example, 5alpha-dihydrotestosterone in male sexual differentiation-provokes interest in their even earlier appearance, as seen in this study, and suggests a possible role for them in early embryonic development in horses and, more generally, in other species.

The main purpose of this autobiographical reminiscence of 40 years of embryo research is to provide young theriogenologists with a firsthand account of how career development can depend strongly on early influences that become modified by changing circumstances. With no intention of being didactic, I hope that my experience of coping with enormous changes in techniques and attitudes may be of use to some of those embarking on a further 40 years of change of at least equal enormity.

Twin ovulations are common in horses, but twin pregnancies are rarely carried to term. Theories of how one or both twins is/are naturally eliminated in early pregnancy, termed 'embryo reduction', have been based on ultrasonographic, not morphological, studies. Here we describe conceptuses recovered transcervically between Days 15 and 28 from 31 twin and two triplet pregnancies. Signs of contact between conceptuses were deduced from those seen in one pair that remained attached by their capsules on Day 18. Signs were found on capsules in two of 10 pairs before or during fixation (immobilisation) at Days 16-17 even though contact had not been seen by ultrasound. After fixation, the signs became stronger in seven of nine unilateral pregnancies, indicated adhesion between pairs and included effects on the vitelline circulation and/or degeneration of one twin. Conceptuses recovered from five of seven unilateral twin pregnancies after the time of capsule disruption (~Day 21) evidenced embryo reduction; in the two surviving pairs, attachment between twins was near the trilaminar/bilaminar yolk-sac wall border. The findings are consistent with the notions that: (1) the capsule plays a role in initiating adhesion between twins; and (2) twin survival depends on an unencumbered trilaminar yolk-sac wall and a functional vitelline circulation.

Tetrapod limb development has been studied extensively for decades, yet the strength and role of developmental constraints in this process remains unresolved. Mammals exhibit a particularly wide array of limb morphologies associated with various locomotion modes and behaviors, providing a useful system for identifying periods of developmental constraint and conserved developmental mechanisms or morphologies. In this study, landmark-based geometric morphometrics are used to investigate levels and patterns of morphological diversity (disparity) among the developing forelimbs of four mammals with diverse limb morphologies: mice, opossums, horses, and pigs. Results indicate that disparity among the forelimbs of these species slightly decreases or stays the same from the appearance of the limb ridge to the bud stage, and increases dramatically from the paddle through tissue regression stages. Heterochrony exhibited by the precocial opossum limb was not found to drive these patterns of morphological disparity, suggesting that the low disparity of the middle stages of limb development (e.g., paddle stage) is driven by processes operating within the limb and is likely not a result of embryo-wide constraint.

The early equine conceptus produces estrogens which are assumed to play a role in the interaction between the mare and the conceptus promoting the establishment and maintenance of pregnancy. It is well known that the extra-embryonic membranes such as the trophoblast synthesize estrogen by the presence of the enzyme aromatase (P450arom), which has also been detected in the hypoblast. Estrogen biosynthesis by the embryo proper has only recently been demonstrated in our investigations but the particular tissues involved remained unknown. To locate the site(s) of P450arom and estrogen receptors, immunohistochemical (IHC) staining was performed on paraffin embedded fixed sections of embryos proper at 24, 25, 26, and 27 (±1) days after conception. Trophoblast, endometrium and placenta were used as positive controls. P450arom was detected with mouse anti-human aromatase, specific for a single 55kDa band by immunoblot of horse trophoblast homogenate. Highest levels of P450arom were localized by IHC to trophoblast surface epithelium. Expression of P450arom in embryonic tissues was less pronounced but especially notable in the developing heart, mesonephros and the genital ridge. To locate embryonic tissues that might respond to estrogen, we surveyed the tissue distribution of estrogen receptors by IHC, using rabbit anti-mouse estrogen receptor (ER) alpha and mouse anti-human ER beta. With these antibodies, ER alpha and ER beta were detected in endometrial glands by IHC and as 55kDa and 59 kDA immunoreactive bands by immunoblots. Within the embryo proper both classes of estrogen receptors were located in many tissue types including developing heart, vascular endothelium, mesonephros, mesenchyme and genital ridge. In conclusion, this study reports the detection of P450arom at the earliest stage in development of an embryo proper for any mammalian species. It is suggested that the detection of aromatase and ERs may signify a direct role for estrogen biosynthesis in the equine embryo proper—perhaps related to differentiation and organogenesis. [Supported by NSERC, Equine Guelph, the Grayson-Jockey Club Research Foundation and OMAFRA.]

The embryo proper in early equine pregnancy has recently been shown to have a remarkable capacity for metabolism of oestrogens. High concentrations of oestrogens in yolk-sac fluid could provide substrate for local metabolism in tissues of the embryo proper and this activity could have significance for early development. Due to the high level of oestrogen metabolism in the embryo proper we examined the possibility that it could also biosynthesise oestrogens. Conceptuses were collected in the fourth week of pregnancy (n = 23) and the embryo was separated from extraembryonic tissues for incubation with [3H]androstenedione. Steroids were recovered from media by solid-phase extraction and eluted as unconjugated and conjugated fractions. Profiles of free and sulfoconjugated fractions, as well as the phenolic steroids extracted from them, were obtained by chromatography. Oestrone and oestradiol were seen clearly, indicating oestrogen biosynthesis, and the presence of more polar products, arising from metabolism of the primary oestrogens, gave further evidence that the embryo was capable of oestrogen biosynthesis. Aromatase activity was also demonstrated by detection of tritium loss, as 3H2O, from incubations (n = 3) with [1β-3H]androstenedione. It is suggested that its oestrogen biosynthesis may have significance for the remarkable development of the vasculature in the embryo proper at this stage.

To advance the understanding of early pregnancy and pregnancy failure in horses, this study determined how luteolysis induced by cloprostenol (an analogue of prostaglandin F2α) affects conceptus development. Mares were injected on Days 12, 14, 16 or 18 of pregnancy with either cloprostenol (treatment groups, total n=83 pregnancies) or saline (controls, n=81), and growth of the conceptuses was monitored and compared by daily ultrasonography until they were collected transcervically on Days 15-22, 1-4 days after the injections. The comparisons were extended in the recovered conceptuses by counting somites, measuring the volume and osmolality of yolk-sac fluid and its concentrations of proteins, estrone sulfate and progesterone, and by assessing the morphology of the capsule and vascular system. When luteolysis was initiated on or before Day 16, most pregnancies survived until the time of collection and the conceptuses in respective treated and control groups on Days 15-20 were very similar except for some effects of treatment on the capsule and vascular development. In contrast, after luteolysis was initiated on Day 18, abortion often ensued within 3 days and most conceptuses collected had degenerated, therein constituting a predictable system in which to study the pathogenesis of a particular cause of pregnancy failure.

Estrogen production by the trophoblast is considered important in early equine pregnancy and leads to high concentrations in yolk-sac (Y-S) fluid. The embryo proper is a potential site for their action. We examined estrogen metabolism in the embryo proper because some actions of estrogens are derived from locally formed metabolites. The embryo proper, as well as separated extraembryonic tissues, of conceptuses collected about day 25 of pregnancy, were incubated with (3)[H]-estrone (E(1)) and (3)[H]-estradiol (E(2)). Steroids were recovered from media by solid-phase extraction and eluted separately as unconjugated and conjugated fractions. Profiles of free and sulfo-conjugated fractions were obtained by HPLC. Some differences and similarities were noted for the embryo proper as compared to the extraembryonic tissues. No reduction of E(1) to E(2) was noted for the embryo proper and allantois, but some was seen with the bilaminar Y-S wall. Less conversion of E(2) to E(1) occurred in the embryo proper than in the extraembryonic tissues. Profiles for hydrolyzed sulfates from incubation of the embryo proper were very similar for both substrates, mainly with E(1) present. Thus, low levels of reductase and high levels of oxido- activities were apparent for the 17beta-hydroxysteroid dehydrogenase enzymes. Further evidence of an active role for the embryo proper was seen as minor, polar products, and an unknown compound eluting between E(2) and E(1). These findings show, for the first time, that the embryo proper can metabolize estrogens that are found in Y-S fluid - a function of potential significance at this stage in its development.

To investigate how equine conceptuses expand rapidly despite the hypo-osmolality of their yolk sac fluid, 18 conceptuses, aged 8-12 days and 0.8-10.0 mm in diameter, were examined by cryoscanning electron microscopy and energy dispersive X-ray microanalysis to determine the distribution of Na, Cl and K in their fluids. No osmotic gradient was found between central and peripheral yolk sac fluid. In conceptuses > or = 6 mm in diameter, the concentrations of both Na and K in the subtrophectodermal compartments were higher than those determined previously in uterine fluid, supporting the concept of osmotic intake of fluid from the uterine environment as far as the compartments. However, electrolyte concentrations in the compartments consistently exceeded those found in the yolk sac, making it likely that 'uphill' water transport, rather than a purely osmotic uptake, is involved in yolk sac fluid accumulation. We also speculate that capsule formation could actively contribute to conceptus expansion and thereby to fluid intake.

Carl Hartman's title of 47 years ago is invoked in tribute to his first recovery of a bovine embryo 30 years before that, and his legacy of an emphasis on the value of descriptive and comparative studies in reproductive biology. The horse's qualification as a farm animal has waned since those times but, in a conference understandably dominated by research in ruminants and pigs, there are lessons to be learned from some peculiarities of equine embryonic development. Morphological and physiological features of the conceptus and its interaction with its environment during the first month of pregnancy are described and discussed, with emphasis on conceptus expansion, experimental study of the capsule and its associated proteins, and steroid production and metabolism by the various tissues within the conceptus. It is also suggested that easy access to entire conceptuses at advanced stages of development in horses offers valuable opportunities for comparative investigation of early organogenesis and fetal membrane differentiation and, possibly, how they are affected by embryo manipulation in vitro.

During the second and third weeks of pregnancy, the equine conceptus expands rapidly while it is enclosed within a glycan capsule. Around day 16 of gestation, the conceptus loses its mobility in the uterus by a process termed 'fixation', coinciding with various changes in the capsule. Here, we compared the structure of the carbohydrate moieties expressed by the capsule during pre- and post-fixation periods. The glycan structures were studied by chemical analyses in combination with mass spectrometry. Capsule material from conceptuses collected before fixation (days 13-16) was observed to carry a sialylated core type 1 O-linked glycan, Neu5Ac-(2 -> 3)-Gal-(1 -> 3)-GalNAc-(1 -> Ser/Thr. By comparison, analysis of post-fixation capsules (days 17-19) revealed a desialylated core type 1, Gal-(1 -> 3)-GalNAc-(1 -> Ser/Thr. The equine embryonic capsule also furnished 4-substituted GlcNAc, 4-substituted Glc and 2,3,4,6-tetrasubstituted Glc residues, the concentrations of which did not change between pre- and post-fixation stages. The loss of sialic acid from the sialylated core type 1 in the capsule appears to be directly related to successful fixation of the conceptus, and thus critical to the continuance of pregnancy in horses. (c) 2007 Elsevier Ltd. All rights reserved.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in ANIREP 94 (2006) 6–7, doi:10.1016/j.anireprosci.2006.04.037 . The duplicate article has therefore been withdrawn.

Progress and changes in embryo technology in farm animals are briefly reviewed in terms of how well embryos can be made and used and what the subject has taught us about the maintenance of pregnancy and reproduction in general. Generalizations are made about the need to not accept dogma, how initially complex techniques always become simplified and thereby more applicable, and the need for the support of long-term and basic research. Personal views are offered on how best to prepare and motivate the next generation of scientists in the field, and the need for scientists to engage in the debate of how embryo technologies should be used responsibly in countering global inequalities.

Abstract Because estradiol (E2) production by the early equine conceptus is considered crucial to the establishment of pregnancy, the amounts of E2, estrone (E1), and their sulfates (E2S, E1S) were measured by RIA in yolk-sac fluid of 63 conceptuses collected by transcervical lavage over the period of 11–26 days after ovulation. Amounts increased significantly with age of conceptus, especially for E1S. Then, the metabolism of E2, which may be highly relevant for its action, was examined in the conceptus and endometrium over the period when the conceptus ceases to migrate within the uterus. Eleven conceptuses collected mainly on Days 12, 15, and 18, with endometrial biopsy samples taken immediately thereafter, were used for steroid metabolic studies. Trophoblastic and endometrial tissues were incubated with [3H]-labeled E2 or E1, and with [14C]-E1 in one experiment. Steroids were recovered from the media by solid-phase extraction (SPE) and eluted separately as unconjugated and conjugated fractions. Conjugation increased from Day 12 for the trophoblast (more so by bilaminar than trilaminar tissues on Day 18) and was much greater for endometrium, with almost all as sulfoconjugates. HPLC profiles of free and sulfate fractions were obtained from a gradient of acetonitrile/water. Interconversion (E2 ⇌ E1) by trophoblast varied with development; it favored E2 in older conceptuses, more in bilaminar than trilaminar tissues. Some more polar products were also noted, with loss of tritium seen as [3H]2O at SPE, and confirmed by HPLC in a second system with authentic reference steroids. Almost all radioactivity in the endometrium was present as E2 in both free and sulfate fractions. It was concluded that local metabolism of E2 is quantitatively significant and may play an important role in the actions of the large amounts of estradiol produced by the early equine conceptus.