Effect of duration of estradiol exposure on embryo survival and endometrial gene expression in anestrous embryo recipient mares.

Previous studies indicate a positive correlation between the duration of estrus prior to ovulation and likelihood of pregnancy in embryo recipient mares. However, the mechanisms by which the duration of estrus before may affect fertility remains unclear. This study aimed to determine the effect of different durations of estradiol exposure, prior to progesterone administration, on embryo viability in anestrous recipient mares, and endometrial expression of genes thought to influence embryo survival.

In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos.

In vitro production (IVP) of equine embryos is increasingly popular in clinical practice but suffers from higher incidences of early embryonic loss and monozygotic twin development than transfer of in vivo derived (IVD) embryos. Early embryo development is classically characterized by two cell fate decisions: (1) first, trophectoderm (TE) cells differentiate from inner cell mass (ICM); (2) second, the ICM segregates into epiblast (EPI) and primitive endoderm (PE). This study examined the influence of embryo type (IVD versus IVP), developmental stage or speed, and culture environment (in vitro versus in vivo) on the expression of the cell lineage markers, CDX-2 (TE), SOX-2 (EPI) and GATA-6 (PE).

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.

Microtubule nucleation from the fibrous corona by LIC1-pericentrin promotes chromosome congression.

Error-free chromosome segregation in mitosis and meiosis relies on the assembly of a microtubule-based spindle that interacts with kinetochores to guide chromosomes to the cell equator before segregation in anaphase. Microtubules sprout from nucleation sites such as centrosomes, but kinetochores can also promote microtubule formation. It is unclear, however, how kinetochore-derived microtubules are generated and what their role is in chromosome segregation.

Parental genomes segregate into distinct blastomeres during multipolar zygotic divisions leading to mixoploid and chimeric blastocysts.

Background: During normal zygotic division, two haploid parental genomes replicate, unite and segregate into two biparental diploid blastomeres.

Results: Contrary to this fundamental biological tenet, we demonstrate here that parental genomes can segregate to distinct blastomeres during the zygotic division resulting in haploid or uniparental diploid and polyploid cells, a phenomenon coined heterogoneic division. By mapping the genomic landscape of 82 blastomeres from 25 bovine zygotes, we show that multipolar zygotic division is a tell-tale of whole-genome segregation errors.

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.

Effect of Overfeeding Shetland Pony Mares on Embryonic Glucose and Lipid Accumulation, and Expression of Imprinted Genes.

Maternal overfeeding is associated with disturbances in early embryonic epigenetic reprogramming, leading to altered expression of imprinted genes and nutrient transporters, which can affect both fetal and placental development and have lasting effects on the health of resulting offspring. To examine how maternal overfeeding affects the equine embryo, Shetland pony mares were fed either a high-energy (HE: 200% of net energy requirements) or maintenance (control) diet. Mares from both groups were inseminated, and day-seven embryos were recovered and transferred to recipients from the same or the alternate group.

Dual spindles assemble in bovine zygotes despite the presence of paternal centrosomes.

The first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded, and instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In nonrodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active.

Effect of a long-term high-energy diet on cardiovascular parameters in Shetland pony mares.

Background: Changes in cardiovascular parameters, including blood pressure (BP) and cardiac anatomical dimensions, are an inconsistent feature of the equine metabolic syndrome. The order in which these changes arise is unknown.

Objectives: Determine the order in which EMS-associated changes in cardiovascular parameters arise.

Compromised MPS1 Activity Induces Multipolar Spindle Formation in Oocytes From Aged Mares: Establishing the Horse as a Natural Animal Model to Study Age-Induced Oocyte Meiotic Spindle Instability.

Aneuploidy originating during meiosis in oocytes is the major cause of reduced fertility, implantation failure and miscarriage in women beyond their mid-thirties. Loss of chromosome cohesion, and defective microtubule dynamics and spindle assembly are, in turn, the major contributors to the error-prone nature of chromosome segregation in the oocytes of older women. However, the underlying molecular defects are not well understood.

Overfeeding Extends the Period of Annual Cyclicity but Increases the Risk of Early Embryonic Death in Shetland Pony Mares.

Obesity has been associated with altered reproductive activity in mares, and may negatively affect fertility. To examine the influence of long-term high-energy (HE) feeding on fertility, Shetland pony mares were fed a diet containing 200% of net energy (NE) requirements during a three-year study. The incidence of hemorrhagic anovulatory follicles (HAF) and annual duration of cyclicity were compared to those in control mares receiving a maintenance diet.

The horse as a natural model to study reproductive aging-induced aneuploidy and weakened centromeric cohesion in oocytes.

Aneuploidy of meiotic origin is a major contributor to age-related subfertility and an increased risk of miscarriage in women. Although age-related aneuploidy has been studied in rodents, the mare may be a more appropriate animal model to study reproductive aging. Similar to women, aged mares show reduced fertility and an increased incidence of early pregnancy loss; however, it is not known whether aging predisposes to aneuploidy in equine oocytes.

Effect of long-term overfeeding of a high-energy diet on glucose tolerance in Shetland pony mares.

Background: Overfeeding is associated with obesity and insulin dysregulation (ID), which are both risk factors for equine metabolic syndrome. How chronic overfeeding affects development of these factors is poorly understood.

Objectives: To examine the influence of long-term high-energy diet provision on body condition and ID.

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).

In vitro production of horse embryos predisposes to micronucleus formation, whereas time to blastocyst formation affects likelihood of pregnancy.

Invitro embryo production is an increasingly popular means of breeding horses. However, success is limited by a high incidence of early embryo loss. Although there are various possible causes of pregnancy failure, chromosomal abnormalities, including aneuploidy, are important potential contributors.

Vitrifying immature equine oocytes impairs their ability to correctly align the chromosomes on the MII spindle.

Vitrified-warmed immature equine oocytes are able to complete the first meiotic division, but their subsequent developmental competence is compromised. Therefore, the present study investigated the effects of vitrifying immature horse oocytes on the chromosome and spindle configuration after IVM. Cumulus-oocytes complexes (COCs) were collected and divided into two groups based on mare age (young ≤14 years; old ≥16 years).

Effect of the duration of estradiol priming prior to progesterone administration on endometrial gene expression in anestrous mares.

Field data indicate that a longer period of estrus prior to ovulation correlates positively with fertility. To test the hypothesis that the duration of exposure to estrogens prior to progesterone dominance influences endometrial function, we used anestrous mares to simulate varying durations of estrus (3 groups of 5 mares): long (LE), short (SE), and no estrus (NE), as determined by the duration of estradiol priming prior to progesterone treatment: 7, 2 and 0 days for the LE, SE and NE, respectively. Endometrial biopsies were recovered 4 days after progesterone administration in all groups for real time quantitative reverse transcription PCR (RT-qPCR) and immunohistochemical analyses.

Expression of glucose transporters in the endometrium and early conceptus membranes of the horse.

Introduction: Glucose is the primary energy substrate for early conceptus development and, for the first 40 days of gestation, the equine conceptus depends solely on glucose available in the histotroph; thereafter, histotrophic glucose provision continues to support transport across the definitive placenta.

Methods: To investigate glucose provision routes during early equine pregnancy we examined expression of glucose transporters in conceptus membranes and endometrium recovered on days 7, 14, 21 and 28 after ovulation. To further differentiate the contributions of maternal progesterone priming and conceptus-endometrium crosstalk in regulating glucose transporter expression, day 8 embryos were transferred to recipient mares on day 8 (synchronous) or day 3 (asynchronous) after ovulation; conceptuses and endometrium were recovered 6 or 11 days later.

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.

Amino acid transporter expression in the endometrium and conceptus membranes during early equine pregnancy.

Maternally derived amino acids (AA) are essential for early conceptus development, and specific transporters enhance histotrophic AA content during early ruminant pregnancy. In the present study we investigated AA transporter expression in early equine conceptuses and endometrium, during normal pregnancy and after induction of embryo-uterus asynchrony. 'Normal' conceptuses and endometrium were recovered on Days 7, 14, 21 and 28 after ovulation.

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.

Negative uterine asynchrony retards early equine conceptus development and upregulation of placental imprinted genes.

Introduction: Placental imprinted genes appear to be sensitive indicators of an inappropriate pre-implantation environment. This study examined the effects of negative uterine asynchrony after embryo transfer (ET) on early horse embryo development, and yolk-sac membrane expression of DNA methyltransferases (DNMTs) and equine specific placental imprinted genes.

Methods: Day 8 embryos were transferred to recipient mares on day 8 (synchronous) or day 3 (asynchronous) after ovulation, and conceptuses were recovered 6 or 11 days later (day 14 or 19 of development).

Likelihood of pregnancy after embryo transfer is reduced in recipient mares with a short preceding oestrus.

Background: Previous surveys reported a positive association between the length of the follicular phase and subsequent fertility in embryo transfer donor and Thoroughbred mares. However, it is unclear whether a longer oestrus positively influences fertilisation and oviductal development (oocyte quality, oviductal environment), or uterine receptivity and survival of the embryo in the uterus.

Objectives: To determine the effect of length of oestrus (characterised by duration of endometrial oedema) on likelihood of pregnancy and early embryo loss (EEL) in recipient mares after embryo transfer (ET).

From Peptide Masses to Pregnancy Maintenance: A Comprehensive Proteomic Analysis of The Early Equine Embryo Secretome, Blastocoel Fluid, and Capsule.

Early pregnancy in the mare is a poorly understood, high risk period during which the embryo communicates its presence to the maternal endometrium. Remarkably, the maternal recognition of pregnancy signal is unknown in the horse. This study aimed to profile the proteins secreted by equine blastocysts into their immediate environment, along with proteins contained in the blastocoel and within the acellular embryo capsule.

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.