Equine in vitro produced blastocysts: relationship of embryo morphology, stage and speed of development to foaling rate.

CONTEXT: Information on factors associated with developmental competence of equine in vitro -produced (IVP) blastocysts is lacking. AIMS: To determine the relationships of stage, grade, day of development, and specific morphological parameters of equine IVP blastocysts, to pregnancy and foaling rates. METHODS: Photomicrographs of 316 IVP embryos with known pregnancy outcomes were scrutinised individually by four observers. Inter-observer variation was assessed, and pregnancy outcome evaluated in relation to day of blastocyst development and assigned grade and stage. Individual component analysis was performed to determine the association of specific morphological parameters with foaling rate. KEY RESULTS: Overall pregnancy rate was 76.9% and foaling rate was 56.3%. The day of embryo development did not affect pregnancy rate but significantly affected foaling rate. Embryo stage did not affect foaling rate. Embryo grade affected foaling rate only for Day-9 embryos. Some morphological features in the bovine grading system did not predict outcome in equine IVP embryos. Significant individual parameters differed between Stage 5 and Stage 6 equine blastocysts. CONCLUSIONS: Day of blastocyst development is the major factor related to foaling rate for equine IVP embryos. Notably, there was no effect of embryo stage on foaling rate and no evidence that prolonging culture until embryos advance in stage increases foaling rate. The standard bovine grading system is not directly applicable to equine IVP embryos; equine-specific staging and grading systems are proposed. IMPLICATIONS: This information will allow laboratories to identify embryos with the highest developmental competence. Use of the proposed systems will increase consistency in embryo assessment among laboratories.

Successful in vitro fertilization in the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitation†.

Methods for standard in vitro fertilization have been difficult to establish in the horse. We evaluated whether prolonged sperm pre-incubation would support subsequent fertilization. Fresh sperm were pre-incubated with penicillamine, hypotaurine, and epinephrine (PHE) for 22 h. Co-incubation of cumulus-oocyte complexes (COCs) for 6 h yielded 43% fertilization; culture of presumptive embryos yielded 21% blastocysts. Sperm incubated similarly, but without PHE, did not fertilize oocytes. Use of extended semen in the system yielded 54% blastocysts and was applied in subsequent experiments. Transfer of three in vitro fertilization-produced blastocysts to recipient mares resulted in birth of three normal foals. When sperm were pre-incubated for 22 h, 47-79% of oocytes were fertilized after 1 h of co-incubation. Sperm pre-incubated for 15 min or 6 h before co-incubation yielded no fertilization at 1 h, suggesting that capacitation in this system requires between 6 and 22 h. Sperm assessed after 15 min, 6 h, or 22 h pre-incubation showed increasing protein tyrosine phosphorylation of the midpiece, equatorial band, and apical head; this pattern differed from that induced by high pH conditions and may denote functional equine sperm capacitation. Use of the final devised system, i.e., extended semen, with 22 h of sperm pre-incubation and 3 h of COC co-incubation, yielded 90% fertilization with a blastocyst rate of 74%. This is the first report of efficient and repeatable standard in vitro fertilization in the horse and the first report of in vitro production of blastocysts and resulting foals after in vitro fertilization.

Flow-cytometric analysis of membrane integrity of stallion sperm in the face of agglutination: the "zombie sperm" dilemma.

PURPOSE: To define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect. METHODS: Sperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 µm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions. RESULTS: Using PI-PSA, measured membrane integrity (MI; live sperm) was dramatically lower than was total motility (TMOT), indicating spurious results ("zombie sperm"). Sperm aggregates were largely of motile sperm. Loss of motility after A23187 treatment was associated with disaggregation and increased MI. On disaggregation using LD-PSA, MI rose, and MI then corresponded with TMOT. In equine sperm incubated after A23187 treatment, as the percentage of live acrosome-reacted sperm increased, TMOT decreased to near 0. CONCLUSION: Flow cytometry assesses only individualized sperm; thus, agglutination of viable sperm alters recorded membrane integrity. As viable sperm become immotile, they individualize; therefore, factors that decrease motility, such as A23187, result in increased measured MI. Disaggregation before assessment allows more accurate determination of sperm membrane status; in this case we documented a mismatch between motility and live acrosome-reacted equine sperm that may relate to the poor repeatability of A23187 treatment for equine IVF. These findings are of profound value to future studies on sperm capacitation.

Glucose concentration during equine in vitro maturation alters mitochondrial function.

The use of in vitro embryo production in the horse is increasing in clinical and research settings; however, protocols are yet to be optimised. Notably, the two most commonly used base media for in vitro maturation (IVM) supply glucose at markedly different concentrations: physiological (5.6 mM, M199) or supraphysiological (17 mM, DMEM/F-12). Exposure to high glucose has detrimental effects on oocytes and early embryos in many mammalian species, but the impact has not yet been examined in the horse. To address this, we compared the energy metabolism of equine COCs matured in M199-based maturation medium containing either 5.6 or 17 mM glucose, as well as expression of key genes in oocytes and cumulus cells. Oocytes were fertilised by ICSI and cultured. Analysis of spent medium revealed that COC glucose consumption and production of lactate and pyruvate were similar between treatments. However, the glycolytic index was decreased at 17 mM and analysis of mitochondrial function of COCs revealed that IVM in 17 mM glucose was associated with decreased ATP-coupled respiration and increased non-mitochondrial respiration compared to that for 5.6 mM glucose. We also found that the metabolic enzyme lactate dehydrogenase-A (LDHA) was downregulated in cumulus cells of oocytes that completed IVM in 17 mM glucose. There was no difference in maturation or blastocyst rates. These data indicate that COC mitochondrial function and gene expression are altered by high glucose concentration during IVM. Further work is needed to determine if these changes are associated with developmental changes in the resulting offspring.

Embryo development after vitrification of immature and in vitro-matured equine oocytes.

Effects of meiotic stage and cumulus status on development of equine oocytes after vitrification was evaluated. Immature oocytes with corona radiata (IMM); in vitro-matured oocytes with corona radiata (MAT CR+); and in vitro-matured oocytes denuded of cumulus (MAT CR-) were vitrified using the Cryotech® method. Warming medium was equilibrated either in 5% CO2 or Air. IMM oocytes underwent in vitro maturation after warming. Recovery, survival, and maturation rates, and cleavage and blastocyst rates after ICSI, were evaluated. Recovery was higher for oocytes warmed in CO2- than Air-equilibrated medium (86 ± 3 vs. 76.9 ± 4%, respectively). Maturation for all vitrified-warmed oocyte treatments (37 ± 6.5 to 45.9 ± 5.8%) was not different from control (50 ± 4.1%), except for MAT CR- CO2 (20.3 ± 4.6%). Cleavage for MAT CR- CO2 and Air groups was similar to control (67.7 ± 12.1, 71.4 ± 8.1, and 78 ± 5.3%, respectively). One blastocyst was produced (MAT CR + CO2), representing the first equine blastocyst reported after vitrification of an in vitro-matured oocyte.

Altered morphokinetics in equine embryos from oocytes exposed to DEHP during IVM.

Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine-disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time-lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidative status was assessed by confocal analysis. The possibility that verbascoside (VB), a bioactive polyphenol with antioxidant activity, could counteract DEHP-induced oocyte oxidative damage, was investigated. DEHP was detected in FF and in IVM media at concentrations up to 60 nM. Culture of oocytes in the presence of 500 nM DEHP delayed second polar body extrusion, reduced duration of the second cell cycle, and increased the percentage of embryos showing abrupt multiple cleavage, compared with controls. Mitochondrial activity and intracellular levels of reactive oxygen species were reduced in blastocysts from DEHP-exposed oocytes. VB addition during IVM limited DEHP-induced blastocyst damage. In conclusion, DEHP is detectable in equine FF and culture medium, and oocyte exposure to increased concentrations of DEHP during IVM affects preimplantation embryo development. Moreover, TLM, reported for the first time in the horse in this study, is an efficient tool for identifying altered morphokinetic parameters and cleavage abnormalities associated with exposure to toxic compounds.

Morphokinetics of early equine embryo development in vitro using time-lapse imaging, and use in selecting blastocysts for transfer.

The use of time-lapse imaging (TLI) in the evaluation of morphokinetics associated with invitro developmental competence is well described for human, cattle and pig embryos. It is generally accepted that embryos that complete early cleavage sooner are more likely to form blastocysts and that timing of later events, such as blastocyst formation and expansion, are predictive of implantation potential and euploid status. In the horse, morphokinetics as a predictor of developmental competence has received little attention. In this study we evaluated the morphokinetics of early equine embryo development invitro for 144 oocytes after intracytoplasmic sperm injection and report the timings of blastocyst development associated with ongoing pregnancy for the first time. There was a tendency for time of cytoplasmic extrusion and first cleavage to occur earlier in the embryos that went on to form blastocysts (n=19) compared with those that arrested, and for first cleavage to occur earlier in blastocysts that established pregnancies that were ongoing (n=4) compared with pregnancies that were lost (n=2). TLI was clinically useful in identifying blastocysts when evaluation of morphology on static imaging was equivocal.

Effect of intra-ovarian injection of mesenchymal stem cells in aged mares.

PURPOSE: This study aims to determine if intra-ovarian injection of bone marrow-derived mesenchymal stem cells (MSCs) improves or restores ovarian function in aged females. METHODS: Prospective randomized study of eight aged mares and six young mares receiving intra-ovarian injection of MSCs or vehicle. Main outcome measures were antral follicle count and serum anti-Müllerian hormone (AMH) (aged and young mares), and for aged mares, oocyte meiotic and developmental competence; gross and histological ovarian assessment; evaluation of presence of chimerism in recovered granulosa cells and in ovarian tissue samples; and gene expression in ovarian tissue as assessed by RNA sequencing. RESULTS: Injection of MSCs was not associated with significant changes in follicle number, oocyte recovery rate on follicle aspiration, oocyte maturation rate, or blastocyst rate after ICSI in aged mares, or in changes in follicle number in young mares. There were no significant changes in peripheral AMH concentrations, indicating a lack of effect on growing follicles. MSC donor DNA was not recovered in granulosa cells or in ovarian tissue, indicating lack of persistence of injected MSC. RNA sequencing revealed significant differences in gene expression between MSC- and vehicle-injected ovaries. CONCLUSIONS: Intra-ovarian injection of bone marrow-derived MSCs altered gene expression but did not improve ovarian function in aged mares.

Impact of equine assisted reproductive technologies (standard embryo transfer or intracytoplasmic sperm injection (ICSI) with in vitro culture and embryo transfer) on placenta and foal morphometry and placental gene expression.

Assisted reproductive technologies (ARTs) such as intracytoplasmic sperm injection (ICSI), in vitro embryo culture and embryo transfer (ET) may be associated with alterations in fetal and placental development. In horses, ET has been used for decades. More recently, in vitro embryo production by ICSI and in vitro culture, followed by embryo transfer (ICSI-C) has become an accepted method for clinical foal production. However, no information is available on the effects of ICSI-C or even of standard ET itself on placental and neonatal parameters in horses. We therefore evaluated placental and neonatal morphology and placental gene expression in reining- and cutting-type American Quarter Horse foals produced using different technologies. Thirty foals and placentas (naturally conceived (NC), ET and ICSI-C; 10 in each group) were examined morphometrically. The only parameter that differed significantly between groups was the length of the foal upper hindlimb, which was longer in ET and ICSI-C than in NC foals. Evaluation of placental mRNA expression for 17 genes related to growth and vascularisation showed no difference in gene expression between groups. These data indicate that within this population, use of ARTs was not associated with meaningful changes in foal or placental morphometry or in expression of the placental genes evaluated.

Vitrification of germinal-vesicle stage equine oocytes: Effect of cryoprotectant exposure time on in-vitro embryo production.

Previous studies have found low rates of blastocyst development (0-11%) after vitrification of germinal vesicle (GV)-stage equine oocytes. In this study, we systematically evaluated a short (non-equilibrating) system for GV-stage oocyte vitrification. In Exp. 1, we assessed oocyte volume in cumulus-oocyte complexes (COCs) exposed to components of a short protocol, using 2% each of ethylene glycol and propylene glycol in the first solution (VS1); 17.5% of each plus 0.3 M trehalose in the second solution (VS2); and fetal bovine serum as the base medium. Based on the time to oocyte minimum volume, we selected a 40-sec exposure to VS1. In Exp. 2, we evaluated exposure times to VS2 and, based on rates of subsequent maturation in vitro, we selected 65 s. In Exp. 3, we used the optimized vitrification system (40-VS1; 65-VS2) and evaluated three warming procedures. Blastocyst development after ICSI was equivalent (15%) for COCs warmed in either standard (trehalose stepwise dilution) or isotonic (base medium) solutions, but was reduced (0%) for COCs warmed in a highly hypertonic (1.5 M trehalose) solution. Exposure to the vitrification and warming solutions, without actual vitrification, was associated with reduced blastocyst development (0-5%; Exp. 4). We conclude that this optimized short protocol supports moderate blastocyst production after vitrification of GV-stage equine COCs. Oocytes can be warmed in isotonic medium, which simplifies the procedure. The systems used still showed a high level of toxicity and further work is needed on both vitrification and warming methods to increase the efficiency of this technique.

Nostopeptolide plays a governing role during cellular differentiation of the symbiotic cyanobacterium Nostoc punctiforme.

Nostoc punctiforme is a versatile cyanobacterium that can live either independently or in symbiosis with plants from distinct taxa. Chemical cues from plants and N. punctiforme were shown to stimulate or repress, respectively, the differentiation of infectious motile filaments known as hormogonia. We have used a polyketide synthase mutant that accumulates an elevated amount of hormogonia as a tool to understand the effect of secondary metabolites on cellular differentiation of N. punctiforme. Applying MALDI imaging to illustrate the reprogramming of the secondary metabolome, nostopeptolides were identified as the predominant difference in the pks2(-) mutant secretome. Subsequent differentiation assays and visualization of cell-type-specific expression of nostopeptolides via a transcriptional reporter strain provided evidence for a multifaceted role of nostopeptolides, either as an autogenic hormogonium-repressing factor or as a chemoattractant, depending on its extracellular concentration. Although nostopeptolide is constitutively expressed in the free-living state, secreted levels dynamically change before, during, and after the hormogonium differentiation phase. The metabolite was found to be strictly down-regulated in symbiosis with Gunnera manicata and Blasia pusilla, whereas other metabolites are up-regulated, as demonstrated via MALDI imaging, suggesting plants modulate the fine-balanced cross-talk network of secondary metabolites within N. punctiforme.

Lower blastocyst quality after conventional vs. Piezo ICSI in the horse reflects delayed sperm component remodeling and oocyte activation.

PURPOSE: The aim of this study was to evaluate the differential effects of conventional and Piezo-driven ICSI on blastocyst development, and on sperm component remodeling and oocyte activation, in an equine model. METHODS: In vitro-matured equine oocytes underwent conventional (Conv) or Piezo ICSI, the latter utilizing fluorocarbon ballast. Blastocyst development was compared between treatments to validate the model. Then, oocytes were fixed at 0, 6, or 18 h after injection, and stained for the sperm tail, acrosome, oocyte cortical granules, and chromatin. These parameters were compared between injection techniques and between sham-injected and sperm-injected oocytes among time periods. RESULTS: Blastocyst rates were 39 and 40%. The nucleus number was lower, and the nuclear fragmentation rate was higher, in blastocysts produced by Conv. Cortical granule loss started at 0H after both sperm and sham injection. The acrosome was present at 0H in both ICSI treatments, and persisted to 18H in significantly more Conv than Piezo oocytes (72 vs. 21%). Sperm head area was unchanged at 6H in Conv but significantly increased at this time in Piezo; correspondingly, at 6H significantly more Conv than Piezo oocytes remained at MII (80 vs. 9.5%). Sham injection did not induce significant meiotic resumption. CONCLUSIONS: These data show that Piezo ICSI is associated with more rapid sperm component remodeling and oocyte meiotic resumption after sperm injection than is conventional ICSI, and with higher embryo quality at the blastocyst stage. This suggests that there is value in exploring the Piezo technique, utilized with a non-toxic fluorocarbon ballast, for use in clinical human ICSI.

Assisted reproductive techniques in mares.

A wide variety of assisted reproductive techniques (ARTs) are available to aid in managing aspects of equine reproduction. Embryo recovery and transfer can be used to obtain more than one foal per mare per year, and to obtain foals from mares that cannot carry a foal to term. Oocyte recovery and either transfer to the oviduct of an inseminated recipient mare (oocyte transfer), or intracytoplasmic sperm injection (ICSI) and embryo culture can be used to obtain foals from mares with some types of subfertility, such as problems of the tubular tract. ICSI can be used to obtain foals when sperm number or quality is low. Because of its ease of use for the mare owner and efficiency, oocyte recovery and ICSI is being used in some cases for management of normally fertile mares and stallions. Oocytes can be recovered from live mares by the referring veterinarian, and shipped overnight to a laboratory for ICSI, without any decrease in oocyte or embryo viability. In case of unexpected death of a mare, ovaries or oocytes can be transported to the ICSI laboratory for production of embryos. Embryos produced both in vitro and in vivo can be biopsied to determine their genetic makeup before they are transferred. Equine embryos can be vitrified successfully; collapse of the blastocoele cavity allows efficient vitrification of expanded blastocysts. In contrast, cryopreservation of unfertilized equine oocytes still has low success. Genetics of valuable animals can be preserved via nuclear transfer (cloning) and several commercial companies offer this service clinically.

Influence of caudal epidural analgesia on cortisol concentrations and pain-related behavioral responses in mares during and after ovariectomy via colpotomy.

OBJECTIVE: To determine the influence of epidural detomidine and morphine on serum corticosteroid concentrations and pain-related behavioral responses in mares during and after ovariectomy via colpotomy. STUDY DESIGN: Blinded prospective study. ANIMALS: Nine university-owned mares. METHODS: Five of 9 horses received caudal epidural detomidine hydrochloride (0.01 mg/kg) and morphine sulfate (0.1 mg/kg) prior to surgery. All horses received local anesthetic around the ovarian pedicle, 0.02 mg/kg butorphanol IV at the start of the procedure and after first ovary removal, were sedated as required throughout the procedure, and were monitored for leg lifting, grunting, and abdominal tensing. Horses were monitored hourly for pain postoperatively. Heart rate was recorded every 4 hours, and photographs were taken to assess pain according to the horse grimace scale (HGS). Control group horses (n = 4) were treated with butorphanol (0.02 mg/kg IV) every 4 hours for 24 hours postoperatively. All horses received oral phenylbutazone 18 hours postoperatively. Serum cortisol was measured prior to the procedure, after first and second ovary removal, and 8 and 24 hours postoperatively. RESULTS: No differences were detected between horses receiving caudal epidural detomidine and morphine and those that received systemic opioids. A decrease in HGS score occurred after phenylbutazone administration. CONCLUSION: Administration of caudal epidural detomidine and morphine resulted in similar pain-related behavior and corticosteroid concentrations as did administration of systemic butorphanol every 4 hours for 24 hours postoperatively. CLINICAL SIGNIFICANCE: Caudal epidural detomidine and morphine may mitigate the requirement for frequent systemic opioid administration after a potentially painful procedure.

Blastocyst development after intracytoplasmic sperm injection of equine oocytes vitrified at the germinal-vesicle stage.

We evaluated the meiotic and developmental competence of GV-stage equine oocytes vitrified under different conditions. In a preliminary study, using dimethyl sulfoxide (D), ethylene glycol (EG) and sucrose (S) as cryoprotectants, the maturation rate was higher for cumulus-oocyte complexes (COCs) held overnight before vitrification (37%) than for those vitrified immediately (14%; P < 0.05). Thereafter, all COCs were held overnight before vitrification. In Experiment 1 we compared 1 min (1m) and 4 min (4m) exposure to vitrification and warming solutions; oocytes that subsequently matured were fertilized by ICSI. The maturation rate was similar between timing groups (29-36%), but was significantly lower than that for controls (73%). The 1m treatment yielded one blastocyst (11%), vs. 19% in controls. In Experiment 2, propylene glycol (PG) and trehalose (T) were also used. We compared two base solutions: M199 with 10% FBS (M199+), and 100% FBS; three cryoprotectant combinations: D-EG-S; PG-EG-S; and PG-EG-T; and two timings in vitrification solution: ∼30 s (30s) and 1 min (1m). The most effective treatment (FBS/PG-EG-T/30s) yielded 42% maturation, 80% cleavage and 1 blastocyst (10%), vs. 49%, 93% and 29%, respectively for controls (P > 0.1). In Experiment 3, we evaluated the toxicity of the M199/D-EG-S/1m and FBS/PG-EG-T/30s treatments, without actual vitrification. These treatments did not affect maturation but both significantly reduced blastocyst development (0% and 0%, vs. 21% for controls). This represents the second report of blastocyst development after vitrification of GV-stage equine oocytes, and presents the highest developmental competence yet achieved; however, more work is needed to increase the efficiency of this system.

Micromanipulation of equine blastocysts to allow vitrification.

Embryo cryopreservation presents an essential method for banking of valuable genetics. However, in equine species the cryopreservation of embryos is complicated by three interacting factors: (1) the late entry of the embryo into the uterus (~6 days after ovulation); (2) the rapid expansion of the blastocyst; and (3) the formation of the equine embryonic capsule, a glycoprotein membrane that forms between the embryo and zona. Efforts to freeze or vitrify equine expanded blastocysts were initially met with little success. In addition, it was thought that breaching the capsule led to loss of embryo viability. We found that micromanipulation with the Piezo drill to puncture the capsule and collapse the blastocyst before vitrification provided a means for successful cryopreservation of equine expanded blastocysts, and that this can be done successfully using a standard sperm injection pipette. Modification of cryoprotectants and methods for vitrification and warming resulted in a technique that allowed successful vitrification of expanded equine blastocysts up to 650 µm diameter, with pregnancy rates approaching those for fresh embryos. After blastocyst collapse, vitrification is performed with ethylene glycol and galactose as cryoprotectants, and the embryo is cooled in a low-volume micropipette tip. Vitrification of expanded equine blastocysts provides a valuable tool for use in exotic equids to preserve genetics.

Intracytoplasmic Sperm Injection, Embryo Culture, and Transfer of In Vitro-Produced Blastocysts.

Intracytoplasmic sperm injection is becoming a common clinical procedure in the horse, but little information is available on techniques for its performance. Each laboratory uses different procedures and different media for the steps involved with in vitro embryo production. This article outlines the procedures used in the Clinical Equine Intracytoplasmic Sperm Injection Program at Texas A&M University for in vitro blastocyst production during the past 3 years.

Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations.

Equine embryos develop in vitro in the presence of high glucose concentrations, but little is known about their requirements for development. We evaluated the effect of glucose concentrations in medium on blastocyst development after ICSI. In experiment 1, there were no significant differences in rates of blastocyst formation among embryos cultured in our standard medium (DMEM/F-12), which contained >16 mM glucose, and those cultured in a minimal-glucose embryo culture medium (<1 mM; Global medium, GB), with either 0 added glucose for the first 5 days, then 20 mM (0-20) or 20 mM for the entire culture period (20-20). In experiment 2, there were no significant differences in the rates of blastocyst development (31-46%) for embryos cultured in four glucose treatments in GB (0-10, 0-20, 5-10, or 5-20). Blastocysts were evaluated by immunofluorescence for lineage-specific markers. All cells stained positively for POU5F1. An inner cluster of cells was identified that included presumptive primitive endoderm cells (GATA6-positive) and presumptive epiblast (EPI) cells. The 5-20 treatment resulted in a significantly lower number of presumptive EPI-lineage cells than the 0-20 treatment did. GATA6-positive cells appeared to be allocated to the primitive endoderm independent of the formation of an inner cell mass, as was previously hypothesized for equine embryos. These data demonstrate that equine blastocyst development is not dependent on high glucose concentrations during early culture; rather, environmental glucose may affect cell allocation. They also present the first analysis of cell lineage allocation in in vitro-fertilized equine blastocysts. These findings expand our understanding of the factors that affect embryo development in the horse.

Regulation of axonemal motility in demembranated equine sperm.

Equine in vitro fertilization is not yet successful because equine sperm do not effectively capacitate in vitro. Results of previous studies suggest that this may be due to failure of induction of hyperactivated motility in equine sperm under standard capacitating conditions. To evaluate factors directly affecting axonemal motility in equine sperm, we developed a demembranated sperm model and analyzed motility parameters in this model under different conditions using computer-assisted sperm analysis. Treatment of ejaculated equine sperm with 0.02% Triton X-100 for 30 sec maximized both permeabilization and total motility after reactivation. The presence of ATP was required for motility of demembranated sperm after reactivation, but cAMP was not. The calculated intracellular pH of intact equine sperm was 7.14 ± 0.07. Demembranated sperm showed maximal total motility at pH 7. Neither increasing pH nor increasing calcium levels, nor any interaction of the two, induced hyperactivated motility in demembranated equine sperm. Motility of demembranated sperm was maintained at free calcium concentrations as low as 27 pM, and calcium arrested sperm motility at much lower concentrations than those reported in other species. Calcium arrest of sperm motility was not accompanied by flagellar curvature, suggesting a failure of calcium to induce the tonic bend seen in other species and thought to support hyperactivated motility. This indicated an absence, or difference in calcium sensitivity, of the related asymmetric doublet-sliding proteins. These studies show a difference in response to calcium of the equine sperm axoneme to that reported in other species that may be related to the failure of equine sperm to penetrate oocytes in vitro under standard capacitating conditions. Further work is needed to determine the factors that stimulate hyperactivated motility at the axonemal level in equine sperm.

Effect of holding equine oocytes in meiosis inhibitor-free medium before in vitro maturation and of holding temperature on meiotic suppression and mitochondrial energy/redox potential.

BACKGROUND: Evaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes. We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle's/Hank's' M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. We hypothesized that EH holding may affect mitochondrial function and that holding temperature may affect the efficiency of meiotic suppression. METHODS: Experiment 1 - Equine oocytes processed immediately or held in EH at uncontrolled temperature (22 to 27°C) were evaluated for initial chromatin configuration, in vitro maturation (IVM) rates and mitochondrial energy/redox potential. Experiment 2 - We then investigated the effect of holding temperature (25°C, 30°C, 38°C) on initial chromatin status of held oocytes, and subsequently repeated mitochondrial energy/redox assessment of oocytes held at 25°C vs. immediately-evaluated controls. RESULTS: EH holding at uncontrolled temperature was associated with advancement of germinal vesicle (GV) chromatin condensation and with meiotic resumption, as well as a lower maturation rate after IVM. Holding did not have a significant effect on mitochondrial distribution within chromatin configurations. Independent of treatment, oocytes having condensed chromatin had a significantly higher proportion of perinuclear/pericortical mitochondrial distribution than did other GV configurations. Holding did not detrimentally affect oocyte energy/redox parameters in viable GV-stage oocytes. There were no significant differences in chromatin configuration between oocytes held at 25°C and controls, whereas holding at higher temperature was associated with meiosis resumption and loss of oocytes having the condensed chromatin GV configuration. Holding at 25°C was not associated with progression of mitochondrial distribution pattern and there were no significant differences in oocyte energy/redox parameters between these oocytes and controls. CONCLUSIONS: Mitochondrial distribution in equine GV-stage oocytes is correlated with chromatin configuration within the GV. Progression of chromatin configuration and mitochondrial status during holding are dependent on temperature. EH holding at 25°C maintains meiotic arrest, viability and mitochondrial potential of equine oocytes. This is the first report on the effects of EH treatment on oocyte mitochondrial energy/redox potential.