BACKGROUND:Exercise is a physiological challenge inducing intensity-dependent hypothalamic-pituitary-adrenal (HPA) axis responses. The hypothesis was that training and racing sessions induce different HPA axis activation. AIMS/OBJECTIVE:To assess whether different exercise sessions affect equine adrenal responsiveness according to age and sex. METHODS:Ten clinically healthy Thoroughbred racehorses (6 females and 4 males) were recruited. Four were 2 years old and six were 3 years old. Horses were subjected to two exercise conditions: training and competitive racing sessions. Blood samples were collected at rest and at 5- and 30-min post-exercise to assess circulating adrenocorticotropin (ACTH) and cortisol changes. To determine the effects of exercise, age, and sex on hormonal responses over baseline values, one-way analysis of variance (ANOVA) was applied. RESULTS:Compared to baseline concentrations, ACTH and cortisol concentrations increased at 5 and 30 min (P < .001) after both training and racing sessions. Compared with training, racing horses had greater ACTH increases at baseline (Δ% 64) and 5 min (Δ% 110), and 30 min (Δ% 283) post-exercise. Cortisol concentrations increased more after racing than training at 5 min (Δ% 89) and 30 min (Δ% 128). CONCLUSION:Results showed that training and competitive racing differentially affected the HPA axis response, with a potential preparatory or anticipatory reaction to exercise before racing.
BACKGROUND:Transrectal ultrasonography during stallion andrological examinations often requires sedation, yet the effects of alpha2-agonist protocols and their pharmacological reversal on ejaculate characteristics remain unknown. OBJECTIVES:This study aimed to evaluate whether sedation with xylazine and detomidine followed by yohimbine reversal alters macroscopic, kinetic, or flow-cytometric seminal parameters in stallions. It was hypothesized that the sedation-reversal protocol would not impair seminal quality. METHODS:Five healthy stallions were assessed in a three-period crossover design, totaling 15 ejaculates per treatment. Subjects received three treatments: CONTROL (saline/saline), GROUP 1 (xylazine + detomidine/yohimbine), and GROUP 2 (saline/yohimbine). Yohimbine alone increased gel-inclusive and gel-free ejaculate volumes (P < 0.05) and reduced sperm concentration per milliliter (P < 0.05), while total sperm count remained unchanged. Sperm kinetics did not differ among treatments. RESULTS:Flow cytometric analyses revealed reductions in plasma membrane stability and mitochondrial membrane potential in GROUP 2 (P < 0.05), whereas mitochondrial superoxide anion generation was unaffected. Post-hoc power analysis indicated power ≥ 80% for flow cytometric variables, but limited power for sperm concentration parameters. Sedation with xylazine and detomidine followed by yohimbine reversal did not alter seminal quality under these conditions. CONCLUSION:These findings support that sedation with xylazine and detomidine followed by yohimbine reversal did not alter seminal quality under the conditions of this exploratory study, although larger trials are required to evaluate fertility outcomes.
The global spillover of clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus into mammalian hosts has become a concern for public health as well as for the equine industry. However, previous reports on H5N1 natural infection and the presence of specific antibodies in horses remain limited. Therefore, we conducted a serosurveillance study to assess the prevalence of H5N1 antibodies in serum samples collected from 400 horses in Japan. Samples were screened using competitive ELISA (cELISA) and hemagglutination inhibition (HI) tests against H5-recombinant protein and H5N1 viral antigen, respectively. Of the 400 samples, 399 (99.75%) were negative by cELISA, while one sample (0.25%) yielded a doubtful result. However, all samples showed HI titers of <8, confirming the absence of H5N1 antibodies. These findings indicate that the current risk of H5N1 infection among horses in Japan remains limited. Nonetheless, continuous global surveillance is warranted to monitor the potential risk of H5N1 spillover into equine populations.
BACKGROUND:Harness racing performance in horses is a complex polygenic trait influenced by genetic background, breeding history, training, and environment. Understanding genomic patterns shaped by long-term artificial selection provides insights into performance-related traits and breed-specific genomic variation. AIMS/OBJECTIVES:This study aimed to characterize genomic differentiation, population structure, and candidate genomic regions potentially influenced by historical selection in Standardbred horses using comparative whole-genome sequencing involving diverse horse breeds. METHODS:Whole-genome sequencing data from 86 horses representing 16 breeds were retrieved from public repositories and processed using a standardized bioinformatics pipeline. Population structure was investigated using principal component analysis (PCA), ADMIXTURE, Neighbor-Joining phylogeny, and ChromoPainter-based haplotype sharing. Genomic diversity was evaluated using runs of homozygosity (ROH) and nucleotide diversity (π), whereas genetic differentiation was assessed using fixation index (FST) analyses with functional annotation. RESULTS:After quality control, 18,384,176 autosomal SNPs were retained. Standardbred horses consistently formed a distinct genomic group, with principal components 1 and 2 explaining 5.00% and 3.66% of genomic variation, respectively. ADMIXTURE supported K = 2 as the best-supported clustering solution (cross-validation error = 0.523). ROH analyses revealed variation in genome-wide homozygosity among breeds, while FST and nucleotide diversity analyses identified differentiated genomic regions containing candidate genes associated with growth, skeletal development, muscle function, signaling, metabolism, and nervous system function, including LCORL, NCAPG, PDE1A, CDH13, and HTR1A. CONCLUSION:This comparative whole-genome analysis identified candidate genomic regions potentially shaped by historical selection in Standardbred horses and contributes to understanding genomic differentiation associated with breed history and functional specialization.
Background the protocol for mobilization of the fetlock region prior to acquiring a contrast tenogram radiograph is not clearly defined. Objective to determine if walking exercise improves identification of DFTS pathology on contrast tenography. Methods Prospective cohort study. Nineteen horses that underwent contrast tenography and subsequent surgical tenoscopy were included. A tenogram protocol was developed to enable comparison of diagnostic accuracy after increasing mobilization of the affected limb post-injection. Following contrast injection three tenogram radiographs were taken, (1) immediately post-injection, (2) after manual fetlock flexion, (3) after walking. Tenoscopic findings were compared with radiological diagnosis. Results The detection rate for both MF and DDFT tears improved with increasing limb mobilization (static<post-flexion<post-walking), increasing from 37% to 47% to 68% for detecting MF tears for each condition, and 26% to 47% to 79% for DDFT tear detection. Sensitivity and specificity had an overall upward trend for detecting MF and DDFT tears. For detection of DDFT tears sensitivity increased with increasing limb mobilization from 22%, to 50% post flexion; and to 71% post walking with a consistent 100% specificity throughout. For detection of MF tears sensitivity and specificity went from 67% and 50%; to 50% and 58% post-flexion; increasing to 60% and 71% post-walking. In five horses (5/19, 26%) radiographic diagnosis changed from incorrect prior to walking, to correct following walking exercise. Conclusions Walking exercise prior to contrast tenogram acquisition improves diagnostic accuracy for detecting MF and DDFT tears. Walking exercise should be undertaken as standard for contrast tenograms for accurate diagnosis.
BACKGROUND:During transvaginal oocyte aspiration (TVA), follicle selection is based primarily on ultrasonographic diameter, although follicles of similar size may differ in their physiological and endocrine status. AIMS:This study aimed to characterize the intrafollicular endocrine environment of equine follicles at aspiration and evaluate associations with oocyte developmental outcomes. METHODS:Follicular fluid was collected from antral follicles ranging from 5 to 40 mm in diameter prior to routine oocyte aspiration, and concentrations of luteinizing hormone (LH), anti-Müllerian hormone (AMH), and inhibins were quantified. Recovered cumulus-oocyte complexes were subjected to in vitro maturation, intracytoplasmic sperm injection, and embryo culture to assess maturation and blastocyst development. RESULTS:AMH concentrations were highest in 15 mm follicles and declined in larger follicles. Inhibin B was positively correlated with AMH, indicating coordinated granulosa cell endocrine activity. Inhibin A concentrations were significantly higher in follicles from which oocytes were not recovered. No consistent associations were detected between follicular fluid hormone concentrations and oocyte maturation, and blastocyst formation could not be robustly evaluated because of the limited number of embryos produced. Substantial variation in hormone concentrations was observed, particularly within the 10-25 mm range, which is commonly aspirated during TVA. CONCLUSION:These findings indicate that intrafollicular AMH and inhibins primarily reflect granulosa cell endocrine status, and their role as predictors of oocyte competence remains to be determined in future studies. The marked endocrine variability among follicles of similar diameter suggests that follicle size alone does not reliably represent functional follicular stage in the mare.
Developmental abnormalities of the Müllerian ducts, such as uterus unicornis, are rare in the horse. Uterus unicornis refers to a form of segmental aplasia in which one of the Müllerian ducts does not develop completely (Hughes, JP, Equine Reprod. 1993; 408–414). In cattle, this has been associated with a hereditary recessive trait; a genetic basis has not been identified in the horse. An 8-year-old maiden Quarter Horse mare was presented for breeding with cooled-transported semen. Transrectal palpation identified 2 ovaries with palpable ovulation fossae and a normal right uterine horn, uterine body, and cervix. The left uterine horn, however, seemed on palpation to be a thin band extending from the cranial uterine body toward the left ovary. Transrectal ultrasound confirmed a right uterine horn and body with moderate edema, a vestigial remnant of the left horn, and 2 active ovaries. Hysteroscopy further supported the diagnosis, revealing a normal cervix, uterine body, and right horn, with absence of a uterine bifurcation and left horn. Given the limited uterine capacity and concern for placental insufficiency leading to pregnancy failure or compromised fetal development, embryo transfer was selected. The left ovary was removed via standing flank laparoscopy in efforts to prevent the possibility of the mare ovulating from that ovary and improve reproductive efficiency. The urinary tract was not specifically evaluated in this case; however, no obvious absence of the left kidney was appreciated during surgery. Due to the shared embryologic origin of the urinary and reproductive tracts, evaluation of the urinary system should be considered in similar cases. One month postoperatively, the mare returned to estrus with a dominant follicle on the remaining right ovary. She was inseminated with cooled-transported semen, and an embryo was successfully recovered by uterine flush 8 d later. Transfer of the embryo into the uterus of a recipient mare resulted in a healthy foal after 341 d of gestation. The mare returned the following year and underwent a similar protocol, again producing a viable embryo, successful embryo transfer into a recipient mare, and a healthy foal after 349 d. This case report demonstrates that embryo recovery and transfer can be an effective and repeatable breeding strategy in a mare with uterus unicornis.
Current information on estrous cycle–associated variations in the equine reproductive tract microbiota is scarce. Our aim was to investigate changes in the uterine bacterial load of estrous mares after insemination when semen was processed with or without antibiotics (AB). Adult healthy mares (n = 9) were monitored over 4 consecutive estrous cycles. Double-guarded endometrial swabs were collected at 4 time points: (1) beginning of estrus (progesterone < 2 ng/mL, low–moderate endometrial edema, follicle(s) = 28 mm), (2) peak estrus (strong endometrial edema, preovulatory follicle = 35 mm), (3) end of estrus (36 h after injection of 1,500 IU hCG), (4) 10 d after ovulation when an embryo collection was done. Mares were left uninseminated in the control cycle. They were inseminated in 2 cycles separated by one resting cycle. Insemination was performed 24 h after hCG with fresh semen diluted with Equiplus (Minitube) extender with AB (penicillin plus amikacin; cycle AB) or without AB (cycle NAB) in a cross over design. Endometrial samples were cultured on (i) Columbia Blood agar (incubated under aerobic and anaerobic conditions) (ii) MacConkey II agar, and (iii) Columbia CNA Blood agar. Colony-forming units (cfu) were counted 48 h after incubation at 37°C. Single colonies were identified using MALDI-TOF mass spectrometry (Bruker). Out of 416 plates, 291 (70%) yielded no growth. Corynebacterium spp. and Streptococcus spp. were the most frequently isolated microorganisms (19/108 samples each). The cfu/mL among cycles and different time points were analyzed using a general linear model for repeated measures. There was a reduction in cfu count from time 2 to 4 with Columbia Blood agar (anaerobic) and Columbia CNA (both P < 0.05) irrespective of the cycle. There were no differences in cfu count among cycles or over time. Embryo collection rate did not differ among insemination cycles (AB cycle: 6/9, NAB cycle 7/9). These findings suggest that AB in the semen extender do not result in considerable changes of the endometrial bacterial load in healthy mares.
The global rise of in vitro-produced (IVP) embryo use has driven the development of simplified thawing methods that maintain embryo quality and pregnancy rates while reducing time and technical demands. These advances underscore the need for a fast-thawing method that allows safe IVP embryo transfer without specialized facilities or advanced expertise, improving usability in field settings. The aim of this study was to evaluate whether a novel single-step thawing method for equine IVP embryos produced by somatic cell nuclear transfer (SCNT) yields pregnancy outcomes comparable to the commercial 3-step KitazatoⓇ thawing protocol. A total of 77 cloned equine embryos were included, with 45 thawed using the 3-step protocol and 32 using the novel single-step method. These embryos were derived from 31 different donor cell lines across both groups. Only d 6 to 7 grade I and II (IETS) blastocysts vitrified by a single embryologist using the KitazatoⓇ vitrification media and CryotopⓇ were selected. Embryo transfer was performed conventionally into d 4 synchronized mares within 5 min of thawing. For the novel single-step thawing method, the CryotopⓇ containing the vitrified embryo was transferred directly from liquid nitrogen into 0.1 mL of pre-warmed media at 38.5°C (containing H-SOF + 10% FBS and 0.2 M sucrose) within a sterile 0.25-mL straw. Thawing was conducted by operators with varying levels of experience, including an embryologist, reproductive veterinarians, and an inexperienced technician. Stereomicroscopic assessment confirmed correct embryo placement within the transfer straw in 100% of procedures. Pregnancy rates at 14 d were significantly higher for embryos thawed using the single-step compared with the 3-step method (68.8% vs. 35.6%, P < 0.05). However, pregnancy loss before 25 d (36.4% vs. 25%), beyond 25 d (35.7% vs. 16.7%), and live foal rates (28.1% vs. 22.2%) did not differ significantly (P = 0.05). Overall, the single-step thawing technique represents a practical, safe, and simplified alternative to conventional laboratory-based thawing, eliminating the need for specialized equipment or training and reducing handling time without compromising overall pregnancy outcomes.
Climate change has intensified extreme weather events. Brazil recorded 9 heatwaves in 2023, one of the hottest years on record, followed by 8 additional events in 2024. Although it is widely recognized that heat stress compromises the reproductive efficiency of cattle through complex hemodynamic and endocrine alterations, data quantifying these detrimental effects in mares under rigorous field conditions remains scarce. Given the widespread use of embryo transfer in tropical regions, the aim of this retrospective study was to quantify the impact of a real-world heatwave exposure on embryo recovery rates and morphological quality. Data from 110 embryo flushing procedures involving 12 donor mares were analyzed during the years 2023 and 2024. Information regarding ambient temperature was obtained from the national meteorological institute. Cycles in which mares presented with endometritis or that were performed during seasonal transition periods were excluded. Cycles were assigned to groups based on local meteorological data: Control (n = 47; no excessive heat exposure) or Heat Wave (n = 63; defined as exposure to temperatures = 5°C above the historical average for = 3 consecutive days, from ovulation induction [largest follicle = 35 mm] to the day of uterine flushing). The embryo recovery rate was analyzed using logistic regression, and embryo (n = 87) gross development (morula, initial blastocyst, blastocyst), and quality (excellent, good, poor, degenerate, unfertilized; grades 1 to 5) were evaluated using generalized linear models, with all parameters adjusted for intra-individual correlation via mare clustering. The results revealed a significant impact of the presence of a heat wave on reproductive efficiency, with recovery rates of 87.2% (41/47) and 73.0% (46/63) for Control and Heat Wave groups, respectively. Exposure to heat waves was associated with a 16% reduction in embryo recovery (OR: 0.4; P = 0.042). However, among the successfully recovered embryos, Heat Waves did not affect embryo gross development and quality (blastocyst grade 1; P = 0.142) or morphological quality grade (P = 0.161). In conclusion, these findings suggest that heat waves may have minor but significant effects on equine fertility.
This study evaluated serum estradiol (E2) concentrations in anestrous mares treated with oral clomiphene citrate (CC) as an alternative protocol for preparing recipient mares. Fifteen anestrous mares were allocated into 3 groups (n = 5/group): control, oral clomiphene citrate, and injectable 17β-estradiol (17β-estradiol; Botupharma). Blood samples were collected at d 0 and d 3, with mares in the treated groups receiving their respective medications daily. Serum progesterone was measured at d 0 to confirm anestrus (<1.0 ng/mL). Transrectal ultrasonography was performed daily during the experimental period to evaluate uterine edema using a 0 to 4 grading scale. Statistical analysis was performed using repeated-measures ANOVA followed by Tukey’s post hoc test, with significance set at P < 0.05. In the CC group, E2 concentrations increased from d 0 (15.01 ± 3.33 pg/mL) to d 3 (20.62 ± 4.05 pg/mL; P < 0.01). A similar increase was observed in control mares (12.96 ± 1.54 to 18.83 ± 2.52 pg/mL; P < 0.01). In contrast, 17β-estradiol treatment induced a marked rise in E2 concentrations (20.54 ± 6.21 to 89.30 ± 8.44 pg/mL; P < 0.001). At d 3, E2 concentrations did not differ between CC and control groups (P > 0.05) but were significantly higher in 17β-estradiol-treated mares (P < 0.001). Uterine edema developed rapidly in treated mares. All mares (5/5) treated with either clomiphene citrate or 17β-estradiol developed grade 4 uterine edema within 24 h of treatment, whereas control mares maintained grade 0 edema throughout the observation period. These findings provide the first description of serum estradiol profiles in anestrous mares treated with clomiphene citrate and demonstrate that CC promotes pronounced uterine edema while maintaining physiological circulating estradiol concentrations. This protocol may represent a practical alternative to injectable estrogen treatments for preparing recipient mares, consistent with protocols previously described by Derbala et al. (J. Equine Vet. Sci. 2024; 133:105006).
This study compared the effects of estradiol cypionate (ECP) and altrenogest (ALT) treatments on placental efficiency in mares with experimentally induced ascending placentitis. Criollo mares were assigned to groups: healthy controls (n = 5), ECP treatment (n = 5), and ALT treatment (n = 5). Placentitis was induced in ECP and ALT mares by inoculation of 107 cfu of S. equi zooepidemicus, at 300 d of gestation. Treatment began 48 h after experimental induction of placentitis and mares received trimethoprim–sulfamethoxazole and flunixin meglumine with their respective hormonal therapy for 10 d. Chorioallantoic samples from 4 regions were collected and analyzed using digital pathology to quantify microcotyledonary and capillary areas. Gestation length (days) differed among groups (P = 0.026), with ECP-treated mares (345 ± 11) showing longer gestation than ALT mares (322 ± 10), whereas controls showed intermediate values (336 ± 13). Histomorphometric analysis showed significant treatment effects on placental vascularization (P < 0.001), with ECP group showing greater capillary area than ALT group. Additionally, the interaction between anatomical location and experimental group (P < 0.001) showed hormonal treatment effects on microcotyledonary area varied by placental region. The ECP group showed superior preservation of microcotyledonary architecture in metabolically active regions—gravid horn (5.04 × 106 µm2) and non-gravid horn (4.99 × 106 µm2)—maintaining values comparable to healthy controls. In contrast, the ALT group showed pronounced reduction, especially in the non-gravid horn region (4.01 × 106 µm2). This pattern may reflect differences in gestational duration among groups, as preterm delivery is commonly associated with reduced microcotyledonary development in the uterine horns. Neonatal outcomes varied significantly, with foal birth weight (kg) substantially reduced in the ALT group (29 ± 5) compared with controls (39 ± 6) and ECP mares (36 ± 2). The placental-to-foal weight ratio was significantly elevated in the ALT group (20 ± 3), indicating compromised placental efficiency compared with controls (11 ± 1) and ECP group (13 ± 2). These findings show that ECP maintains gestation length, preserves placental structure and vascularization, and improves neonatal viability in mares with ascending placentitis, outperforming altrenogest therapy in maintaining feto-placental homeostasis.
Overnight holding of equine oocytes, either for transportation or scheduling of fertilization, has become standard practice over the last several years. Glass vials have been favored due to a potential for toxicity arising from plastic tubes, but plastic tubes are more readily available and somewhat easier to use. The purpose of this study was to compare blastocyst production rates in oocytes stored overnight at 22°C in glass vials (Thermo-Scientific) or polystyrene culture tubes (Corning Life Sciences) that had been screened by lot with mouse embryo assay. Oocytes were collected by transvaginal ultrasound-guided follicular aspiration from Quarter-type mares, pooled in holding medium and divided between glass vials and plastic culture tubes. The oocytes were stored at 22°C overnight. The following morning the oocytes were placed in the laboratory’s standard maturation medium under mineral oil in an atmosphere of 4.7% CO2, 5% O2, 90.3% N2 at 38.2°C. The oocytes were subjected to ICSI 28–30 h later using semen from a single frozen straw. Spermatozoa were selected by centrifugation and swim-out. The presumptive zygotes were cultured in the laboratory’s standard culture medium in the same triple gas atmosphere and temperature and evaluated for cleavage on d 2 following ICSI and blastocyst formation on d 5 through 9. Seven replicates were performed involving a total of 704 oocytes. Data were analyzed by chi-squared. There was no difference in maturation 204/354 (57.6%) versus 205/350 (58.6%), cleavage 169/204 (82.8%) versus 159/205 (77.5%), and blastocyst 70/204 (34.4%) versus 73/205 (35.6%) rates for plastic versus glass, respectively. This study shows that appropriately screened plastic tubes can be used for equine oocyte holding and transport.
Artificial insemination using cooled semen is widely applied in horses but remains poorly developed in donkeys. Strategies such as cholesterol supplementation, egg-yolk freezing extenders, and seminal plasma (SP) removal improve sperm preservation and fertility in mares. However, cryoprotectant exposure and absence of SP have been linked to reduced conception in jennies inseminated with frozen-thawed semen, raising uncertainty regarding their suitability for cooled semen. Therefore, this study compared processing protocols for cooled donkey semen and evaluated fertility in jennies. Four ejaculates from 7 jacks (n = 28) were diluted in a milk-based extender containing cholesterol (BotuSemen GoldⓇ; BG) or an egg-yolk extender with (BotuCrioⓇ; BC) or without permeable cryoprotectants (BCnC). A secondary experiment compared BG with centrifuged semen resuspended without SP (BG-SP). Semen was stored at 5°C for 72 h and evaluated for total (TM) and progressive motility (PM), rapid sperm (RAP) by CASA, and plasma membrane integrity (PMI), mitochondrial membrane potential (HMMP), and reactive oxygen species (ROS) by flow cytometry. In a crossover trial, 16 jennies (61 cycles) were inseminated with semen from 2 jacks cooled for 24 h (1 or 2 × 109 sperm) in BG, BC, BCnC, or BG-SP. Ovulation was induced with histrelin acetate (250 µg), and uterine cytology performed before and 6, 24, and 48 h post-AI. The TM, PM, RAP, and PMI were higher in BC and BCnC than BG at 24–72 h (P < 0.05). The HMMP did not differ (P > 0.05), whereas ROS was lower in BC and BCnC (P < 0.05). BG-SP improved TM and PM at all times and PMI and RAP at 72 h (P < 0.05). Fertility was unaffected by extender (BG 60% [9/15]; BC 60% [9/15]; BCnC 43.8% [7/16]; BG-SP 53.3% [8/15]) or in vitro sperm quality, and post-breeding inflammation was similar across groups (P > 0.05). A higher insemination dose tended to increase pregnancy probability (OR, 3.14; P = 0.06). In conclusion, neither cryoprotectant exposure nor SP removal influenced conception rates in jennies AI with cooled semen. Although BC, BCnC, and BG-SP improved in vitro sperm parameters, these differences did not translate into higher fertility, suggesting that conventional sperm quality metrics may not reliably predict reproductive outcomes in donkey AI. However, higher insemination doses may be beneficial for this species.
Ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) represents a breakthrough technology in equine production. A major advantage of OPU is that multiple early blastocysts can be produced in a single session. However, research into equine OPU and ICSI is limited in Japan. The objectives of this study were (1) to evaluate the feasibility of OPU-ICSI in Hokkaido native horses and crossbreds, and (2) to assist conservation of this valuable native breed through the application of OPU-ICSI. Ten Hokkaido native or crossbred mares were used as oocyte donors. OPU was performed on mares without a dominant follicle and with a high number of follicles = 10 mm in diameter. All follicles = 2 mm in diameter were aspirated using a 12 G double-lumen needle. After puncture and aspiration of follicular fluid, each follicle was flushed 6 times with oocyte recovery medium while gently scraping the follicular wall. Aspirated fluid was filtered, and cumulus–oocyte complexes (COC) were identified under a stereomicroscope. Recovered COC were matured in vitro for 30 h. Following in vitro maturation (IVM), oocytes that reached the metaphase II (MII) stage were subjected to ICSI. Injected oocytes were cultured in the incubation until d 7–8 post-ICSI. Embryos that developed to the early blastocyst stage were vitrified and stored in liquid nitrogen. A total of 41 OPU-ICSI sessions were performed in this study. A total of 509 oocytes were recovered from 1,216 aspirated follicles (overall recovery rate of 41.9%). Of the 451 COC selected for IVM, 304 matured to the MII stage, yielding a maturation rate of 67.4%. Following ICSI, 304 embryos were produced, of which 151 developed to at least the 2-cell stage (i.e., a cleavage rate of 49.8%). Ultimately, 12 reached the early blastocyst stage, and 2 developed to the blastocyst stage; all were cryopreserved. Subsequently, 8 early blastocysts were thawed and transferred to recipient mares and one healthy foal was born. This study reports the first successful birth of a foal produced by OPU-ICSI in Japan. These findings demonstrate that OPU-ICSI may be a feasible assisted reproductive technology in Hokkaido native horses. With optimization, this technology is expected to contribute to the conservation of rare equine breeds and to the genetic improvement of horses.
Equine, human, and bovine cumulus cell (CC) transcriptomes were compared with identify a cross-species conserved signature of oocyte developmental competence, defined by blastocyst attainment (Bl+, reached blastocyst stage; Bl−, did not). An equine CC RNA-seq data set was generated from oocytes recovered by transvaginal aspiration from 8 healthy live mares. CCs were collected from individual oocytes after in vitro maturation, paired MII oocytes were subjected to intracytoplasmic sperm injection (ICSI) using sperm from an ICSI-proven stallion. Each CC sample was retrospectively labeled by outcome (Bl−, n = 11; Bl+, n = 15). A total of 75 oocytes were received; 44.0% matured to MII and were injected, yielding 84.8% cleavage and 45.5% blastocyst/ICSI. Public CC transcriptome data sets were obtained for human (Bl−/ Bl+, n = 10 each) and bovine (n = 5 each). Within each species, Bl− versus Bl+ log2 expression-changes were estimated, and conserved candidates were defined as 1:1 orthologs showing the same direction of change across all 3 species. Conserved candidate genes were grouped into UP (higher in Bl+) and DOWN (higher in Bl−) sets. For each sample, gene-set scores were computed by standardizing expression for each gene across samples (z-score) and averaging within UP-only, DOWN-only, or a combined (UP–DOWN) module score. Discrimination of Bl− versus Bl+ was quantified by ROC/AUC with bootstrap 95% confidence intervals. External validation of candidates was performed in an independent public human CC microarray data set of Bl− and Bl+ cohorts (n = 40 and n = 41, respectively). In exploratory evaluation of predefined filtering strategies, a minimal UP module (15 genes; average normalized expression ≥ 20th percentile; minimum absolute fold-change ≥ 0.20 across species) showed robust discrimination (AUC 0.784; bootstrap 95% CI 0.691–0.879), with higher scores in the Bl+ group. Identified genes are involved in ovarian endocrine regulation and cumulus microenvironment homeostasis, highlighting the importance of these pathways in oocyte competence. This cross-species CC module provides a practical readout for competence-associated CC states and may support objective assessment of how in vitro culture or maturation media influence CC transcriptomes, thereby informing evaluation and optimization of culture conditions.
This study investigated whether nuclear donor cell origin (mesenchymal stem cells vs. fibroblast cells) influences placental features and neonatal outcomes in cloned foals. Clinical and placental data were collected from 28 cloned foalings (13 fibroblast-derived [FB]; 15 mesenchymal stem cell-derived [MSC]). Mares were closely monitored during parturition, and foals were followed clinically until 7 d of life. Fetal membranes underwent systematic gross examination, and samples of placental tissue were submitted for histopathology. The gross area of the placenta was measured as described elsewhere; Allen et al. (Reproduction 2002; 123:445–453). The placenta:foal weight ratio was calculated as placental weight/foal weight × 100. Continuous variables were analyzed using t-test, and categorical by Fisher’s exact test. Data are expressed as mean ± SD. Significance was set at P < 0.05. Gestation length and foal birth weight did not differ between groups. Placental weight was higher in the FB group than MSC (7.35 ± 2.50 vs. 5.23 ± 1.60 kg; P = 0.03). The placenta:foal weight ratio was also higher in FB placentas (17.31% ± 4.04 vs. 14.16% ± 1.82 for MSC; P = 0.048), exceeding the normal equine reference range (11–14%). Histologically, FB placentas showed a markedly higher frequency of inflammatory infiltrates (100% vs. 36.4%), and placental edema was observed exclusively in FB cases (27.3% vs. 0%). Total placental area and morphometric measurements did not differ between groups. Despite increased placental weight and volume in FB clones, gross area of placenta remained unchanged, indicating that the weight increase resulted primarily from edema and inflammation rather than a larger functional exchange surface. Early neonatal mortality showed a trend toward being higher in FB foals (38.5% [n = 5/13] vs. 6.7% [n = 1/15]; P = 0.069). Although serum creatinine concentrations did not differ significantly between groups, FB foals (3.46 ± 2.16 mg/dL) showed elevated values above the laboratory reference range (1.6–2.55 mg/dL), whereas MSC foals (2.55 ± 1.37 mg/dL) remained at the upper limit of normal). Use of fibroblasts as nuclear donor cells was associated with placental compromise characterized by placental edema, increase in placental weight disproportionate to fetal weight, inflammation, and a tendency toward a higher mortality rate.
The mechanisms of pregnancy recognition (MRP) in mares remain unclear, partly due to a lack of in vitro models. In vitro studies of conceptus derived trophoblastic vesicles (TRV) and extracellular vesicles (EV), which are key factors in embryo-maternal communication, may fill this niche. The objectives of this study were to compare histologic, ultrastructural features, estradiol (E2) secretion and EV production of TRV derived from d 10 and d 14 equine conceptuses. Post-ovulation d 10 (n = 6) and d 14 (n = 6) equine conceptuses were collected transcervically, washed, separated from the capsule, cut into 1–2 mm pieces, and cultured in 300 or 500 µL, respectively, in DMEM/F12 supplemented with 10% exosome depleted fetal bovine serum in a humidified incubator with 5% CO2, 6% O2, and 89% N2 at 37.5°C. Culture medium (CM) was collected every 12 h for 4 d, pooled, filtered, and stored at −80°C with TRV number determined at each time point. The TRV were processed for toluidine blue and Oil Red O staining, transmission (TEM) and scanning electron microscopy. The EV were isolated from CM and characterized by TEM, immunoblot analysis for EV markers (Hsp70, Hsp90, Tsg101), and protein content using Bradford assays. Estradiol in CM was measured by ELISA. Concentrations were log-transformed and analyzed using linear mixed-effects models, with time, conceptus collection day (group), number of TRV and time-group interaction as fixed effects, and conceptus ID as a random effect, at P < 0.05. The TRV were spherical 1–2 cell layer structures with microvilli, supranuclear vesicles and mitochondria, tight junctions, multivesicular bodies, rough endoplasmic reticulum, and multiple lipid droplets. Most d 14 TRV showed morphological degeneration by 84–96 h. The E2 was dependent on TRV number (P < 0.001) and there was an interaction between day and time (P < 0.001); at 96 h mean ± SD CM E2 was d 10 8.8 ± 5.9 and d 14 0.6 ± 0.4 ng/mL. Both d 10 and d 14 TRV released small EV (30–50 nm). In conclusion, TRV exhibit E2 secretion and have morphological features similar to an equine conceptus. These properties highlight the potential of TRV for exploring the molecular processes involved in equine MRP in vitro.
The ability to re-expand and resume rhythmic contractions (pulses) after cryopreservation and warming has been associated with post-thaw survival and implantation potential of embryos in other species. In equine practice, vitrification is widely used to preserve in vitro-produced embryos, but various warming procedures are used with little comparative information on their influence on post-warming embryo physiology. This study aimed to characterize pulsing and extrusion dynamics of equine intracytoplasmic sperm injection (ICSI) blastocysts following vitrification and warming using protocols with 1, 2, or 4 steps. Embryos were produced by a commercial ICSI laboratory using frozen-thawed sperm from a single proven stallion and oocytes collected from middle-aged Quarter Horse mares, matured in vitro. Grade A or B (A–D scale) blastocysts formed on d 7–8 post-ICSI were vitrified on a Vitrific device (WTA) following a 2-step protocol (vitrification medium 1 [2 M ethylene glycol]/5 min, vitrification medium 2 [8 M ethylene glycol + 0.8 M galactose]/40 s) and later assigned to a warming protocol: 1-step (ABT holding medium/5 min), 2-step (warming solution 1 [0.4 M sucrose]/1 min, GMOPS+/4 min), or 4-step (warming solution 1/1 min, warming solution 2 [0.2 M sucrose]/3 min, warming solution 3 [0.1 M sucrose]/3 min, GMOPS+/1 min) (n = 10/group). Post warming, embryos were individually cultured in a time-lapse incubator (Miri, Esco) for 48 h (38.2°C, 6.5% CO2, 5% O2), images captured every 5 min. Embryos were evaluated for number of pulses, time to first expansion, time to initial extrusion (protrusion of cells through the zona pellucida), and grade of extrusion (1–4). Data were tested for normality and analyzed using one-way ANOVA or Kruskal–Wallis tests. No differences (P = 0.3) were detected for number of pulses (12 ± 1.2, 12 ± 1.2, 15 ± 2.6), time to first expansion (3.8 ± 0.7, 3.6 ± 0.7, 3.3 ± 0.6 h), time to initial extrusion (23 ± 3.3, 23 ± 2.4, 24 ± 3.4 h), and grade of extrusion (2.2 ± 0.3, 2.4 ± 0.4, 2.4 ± 0.3). All embryos exhibited at least 3 pulsing events, and extrusion behavior was observed in 93% of embryos across all treatments. In conclusion, equine ICSI blastocysts maintained comparable morphokinetics following 1-, 2-, or 4-step warming protocols. Further studies are required to clarify the clinical relevance of these embryo behaviors.
Equine in vitro maturation (IVM) remains inefficient, with < 10% of retrieved oocytes resulting in a viable pregnancy. Biphasic IVM (temporary meiotic arrest before IVM) has been described in several species. A recent experimental study using slaughterhouse-derived equine oocytes and semen from a single stallion reported improved equine embryo development after biphasic IVM (Muhammad Fakhar et al. Int. J. Mol. Sci. 2025; 26:5495). The objective of this clinical case series was to evaluate whether temporary meiotic arrest before IVM could improve in vitro embryo production (IVP) after oocyte retrieval. Mares (n = 35) yielding at least 12 oocytes were included. From each mare, 3 sibling oocytes (n = 105) were randomly assigned to pre-IVM for 6, 12, or 24 h in CNP-supplemented medium after a 4–6 h holding period, whereas the remaining oocytes (n = 315) followed the routine clinical holding and IVM protocol. After maturation, all oocytes with a polar body underwent ICSI using semen from the same client-selected stallion within each case (15 stallions overall) and were cultured for 9 d in a time-lapse incubator. Outcomes assessed were maturation, cleavage, blastocyst development, and cleavage normality. Pre-IVM for 24 h was detrimental, reducing maturation to 40% and cleavage to 50%, and yielding no blastocysts (0%; P < 0.05). In contrast, pre-IVM for 6 and 12 h resulted in maturation (67.4–71.4%), cleavage (75.9–76.7%), and blastocyst (17.2–26.7%) rates similar to those observed with the routine clinical protocol (67.9%, 70.1%, and 23.4%, respectively; P > 0.05). Cleavage normality did not differ among groups (P = 0.1327). Under clinical conditions, short pre-IVM periods (6–12 h) did not improve IVP outcomes, whereas prolonged meiotic arrest (24 h) impaired development. Unlike the earlier single-stallion experimental report, this clinical case series did not demonstrate a benefit of biphasic IVM under routine clinical conditions.
