Search

The present study aimed to evaluate whether primed anoestrus mares are suitable recipients for embryos produced by intracytoplasmic sperm injection (ICSI). Anoestrus was confirmed in four mares and daily doses of oestradiol benzoate (6 mg in total) over 5 days were administered; after 3 days of rest, oral altrenogest was administered at 0.088 mg/kg and embryos (1 to 5 embryos per mare; 15 in total) were transferred 3.5 days after progesterone onset. Uterine lavage was conducted 48 h after transfer. The results revealed an 80% embryo recovery rate, and among the retrieved embryos, 67% showed evident intrauterine development. Hence, ICSI-derived embryos can be successfully transferred to primed anoestrus mares, but more studies are required to ensure further embryo development and foaling., (© 2024 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.)

Two methods for preimplantation genetic testing (PGT) have been described for equine embryos: trophoblast cell biopsy (TCB) or blastocoele fluid aspiration (BFA). While TCB is widely applied for both in vivo- and in vitro-produced embryos, BFA has been mostly utilized for in vivo-produced embryos. Alternative methods for PGT, including analysis of cell-free DNA (CFD) in the medium where in vitro-produced embryos are cultured, have been reported in humans but not for equine embryos. In Experiment 1, in vivo- (n = 10) and in vitro-produced (n = 13) equine embryos were subjected to BFA, cultured for 24 h, then subjected to TCB, and cultured for additional 24 h. No detrimental effect on embryonic diameter or re-expansion rates was observed for either embryo group (P > 0.05). In Experiment 2, the concordance (i.e., agreement on detecting the same embryonic sex using two techniques) among BFA, TCB, and the whole embryo (Whole) was studied by detecting the sex-determining region Y (SRY) or testis-specific y-encoded protein 1 (TSPY) (Y-chromosome), and androgen receptor (AR; X-chromosome) genes using PCR. Overall, a higher concordance for detecting embryonic sex was observed among techniques for in vivo-produced embryos (67-100 %; n = 14 embryos) than for in vitro-produced embryos (31-92 %; n = 13 embryos). The concordance between sample types increased when utilizing TSPY (77-100 %) instead of SRY (31-100 %) as target gene. In Experiment 3, CFD analysis was performed on in vitro-produced embryos to determine embryonic sex via PCR (SRY [Y-chromosome] and amelogenin - AMEL [X- and Y-chromosomes]). Overall, CFD was detected in all medium samples, and the concordance between CFD sample and the whole embryo was 60 % when utilizing SRY and AMEL genes. In conclusion, equine embryos can be subjected to two biopsy procedures (24 h apart) without apparent detrimental effects on embryonic size. For in vivo-, but not for in vitro-produced equine embryos, BFA can be considered a potential alternative to TCB for PGT. Finally, CFD can be further explored as a non-invasive method for PGT in in vitro produced equine embryos., (Published by Elsevier Inc.)

Transrectal and transabdominal ultrasonography is an established method to monitor pregnancy, fetal growth and wellbeing in different species. Growth charts with multiple bio-morphometric parameters to estimate days of gestation and days before parturition exist in small companion animals, sheep and goats, riding type horses and large ponies but not in small horse breeds like Shetland ponies. The aim of this study was to apply fetal biometric assessment and detailed description of physiologic fetal development to mid and late term pregnancies in Shetland mares and to generate reference data for clinical practice and for future research. Fetal parameters were collected starting on day 101 of pregnancy in five Shetland mares. The fetal biometric parameters determined consisted of aortic diameter, eye diameter, combined rib and intercostal distance (CRID), stomach length and width and different heart morphology parameters in sagittal and frontal plane. Additionally, fetal activity and organ development in terms of differentiation and changes in echogenicity were recorded. Considering reliably assessable parameters, fetal CRID was the best predictor for gestational age with ± 13.6 days and fetal aortic diameter the most accurate for prediction of days until parturition with ± 16.2 days., Competing Interests: Declaration of competing interest The authors do not report any conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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. Three groups of anestrous recipient mares treated with different duration of estradiol were used: long (LE), short (SE) and no treatment (NE). Day 8 embryos were transferred into recipient mares four days after long-acting progesterone administration and recovered 48h later to examine embryo growth and viability. The endometrial gene expression profile of selected genes was also investigated. The likelihood of recovering an embryo 48h after transfer was 46.1% (6/13), 62.5% (5/8) and 85.7% (6/7) for recipient mares from the NE, SE and LE groups, respectively (P = .09). Embryos recovered from the different groups of recipients did not, however, differ in size, morphology or the proportion of nuclei undergoing mitosis (P > .05). Abundance of mRNA for uterocalin (P19) and insulin-like growth factor 1 (IGF1) were increased in the LE compared to the NE group, while fibroblast growth factor 2 (FGF2), progesterone receptor (PGR) and insulin-like growth factor 1 receptor (IGF1R) transcript abundances were increased (P < 0.05) in the NE group compared to both SE and LE groups. In conclusion, a longer exposure of the endometrium to estradiol before progesterone tended to improve embryo survival within 48h of transfer. However, the grade, growth rate, and proportion of mitotic cells in surviving embryos did not differ among groups. If embryos are destined to fail in a suboptimal endometrial environment, they die and disappear quickly. Moreover, a more adequately estradiol-primed uterus, before the progesterone rise, seems to create a uterine environment, in terms of P19, IGF1, FGF2 and PGR gene expression, more conducive to embryo survival and further development., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Fetal age in Quarter Horses can be predicted within 2 weeks from 100- to 200- days of gestation using femur length, biparietal diameter (cranium diameter) and eye approximated volume. However, as pregnancy advances, the femur and cranium become too large to be imaged in their entirety using ultrasound and the corresponding biometric parameters can no longer be measured. In this longitudinal study, the proximal phalanx (P1) was evaluated as a novel biometric parameter for late gestation to predict fetal age and bone maturation. Transrectal ultrasound was performed in ten pregnant mares with known ovulation dates, every two weeks from 240- days of gestation until parturition. P1 was imaged in 69 % of the examinations. Inability to image P1 was due to obstructive positioning such as carpal or fetlock flexion, or posterior presentation of the fetus. Advancing fetal age did not affect visibility of P1. P1 length correlated significantly with days of gestation and a correlation equation was established: y = 0.3837x -69.55 where y is the predicted value of P1 length and x is the day of gestation (with day 0 being the day of ovulation). When P1 length was equal to or larger than the width of the ultrasound image (52.5 mm), 90 % of mares (9/10) were above 300- days of gestation. Ossification of the proximal and distal epiphysis of P1 typically appeared between 277- and 303 -days of gestation (mean: 288 days). The proximal epiphysis did not close before parturition whereas the distal one closed between 306- and 333-days of gestation (mean: 320 days). P1 epiphyseal appearance and closure occurred chronologically reflecting bone maturation. Radiographic findings at birth and prenatal ultrasound findings were in agreement, apart from timing of P1 distal epiphyseal closure. In conclusion, P1 length can be used as a new fetal biometric parameter to assess fetal age and growth after 240- days of gestation. The knowledge of P1 bone maturation process in utero as a marker for fetal bone development, may also be valuable in clinical decision-making when considering inducing parturition in the mare., (Copyright © 2024. Published by Elsevier Inc.)

The aims of this study were to determine the effect of the embryo flushing technique and the number of flushing attempts performed by operators of different experience on embryo recovery (ER). Ten non-lactating mares were inseminated with the same stallion in six cycles each (n = 60). Embryo flushing (EF) was performed 7-9 days after ovulation by three operators (OP; 20 EF cycles each): OP1 had performed >500 EF before the study, while OP2 and 3 had performed 0 EF. Each EF was performed with 2 flushing attempts (FA) using 1L of ringer's lactate "in-and-out" using two EF techniques: 1) uterine massage (UM): continuous ballottement and massage of the uterus per rectum during ringer lactate recovery, 2) gravity flow (GF): the ringer lactate was allowed to flow back without massaging the uterus. In both groups, 20 IU of oxytocin were administered at the second FA and the ringer lactate was allowed to remain in the uterus for 3 min before recovery. An extra FA was performed in each group using 0.5 L of ringer lactate and uterine massage. More embryos (P < 0.05) per ovulation were recovered in the UM (17/33, 0.51) than in the GF group (8/36, 0.22). For the UM group, 16/17 embryos (94.1 %) were recovered in the first FA, while only one embryo in the second FA (1/17, 5.9 %). In the GF group, 4 embryos were recovered in each FA. No embryo was found in the extra FA in the UM group, while seven additional embryos were found in the GF group (5/7 flushed by OP1; P < 0.05). The overall ER per cycle was 70, 40, and 45 % for OP1, 2 and 3, respectively. In conclusion, highest embryo recovery is achieved in EF performed with UM, with the majority of embryos being flushed in the first FA., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This retrospective study aimed at identifying factors that contribute to the success of equine in vitro embryo production by intracytoplasmic sperm injection (ICSI). A total of 7993 ovum pick-up (OPU) sessions were performed, totaling 2540 donor mares and semen from 396 stallions. Oocytes were aspirated at multiple sites in Brazil and were sent to the laboratory, within 6 h from OPU, in pre-maturation medium where they were in vitro matured (IVM) followed by ICSI and in vitro embryo culture for 7-8 days. The number of recovered oocytes, matured oocytes, cleaved embryos and blastocysts were used to explore the effect of age and breed of the donor mare, time of year in which the mare was aspirated and phase of the estrous cycle on the day of follicular aspiration. Mares between 6 and 15 years old were superior to other age groups in most parameters evaluated, including the average number of blastocysts per OPU. The impact of age was similar when evaluated within two breeds, American Quarter Horse (AQHA) and Warmblood mares. We observed that breed (AQHA, Warmblood, Crioulo, Lusitano and Mangalarga) had an important effect on most of the parameter evaluated, including number of oocytes recovered, blastocysts produced per OPU, and blastocyst rates. The overall impact of season was less pronounced than age and breed, with the only statistically significant difference being a higher rate of oocyte maturation during the summer season. Finally, most of the parameters evaluated were superior in follicular phase mares, with or without dominant follicle than luteal phase mares. In conclusion, this retrospective study revealed that breed, age, season and stage of estrous at the time of OPU are all important parameters for the success of equine embryo production by ICSI. This technology enables producing embryos all-year-round from mares of different breeds and ages from OPU-derived oocytes collected at multiple sites., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The present study was conducted to investigate the global proteome of 8-day-old equine blastocysts. Follicular dynamics of eight adult mares were monitored by ultrasonography and inseminated 24 h after the detection of a preovulatory follicle. Four expanded blastocysts were recovered, pooled, and subjected to protein extraction and mass spectrometry. Protein identification was conducted based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, and PepExplorer). Enrichment analysis was performed using g:Profiler, Panther, and String platforms. After the elimination of identification redundancies among search tools (at three levels, based on identifiers, peptides, and cross-database mapping), 1977 proteins were reliably identified in the samples of equine embryos. Proteomic analysis unveiled robust metabolic activity in the 8-day equine embryo, highlighted by an abundance of proteins engaged in key metabolic pathways like the TCA cycle, ATP biosynthesis, and glycolysis. The prevalence of chaperones among highly abundant proteins suggests that regulation of protein folding, and degradation is a key process during embryo development. These findings pave the way for developing new strategies to improve equine embryo media and optimize in vitro fertilization techniques., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Background: Equine embryos >300 μm require puncture before vitrification. Protocols that do not require pre-puncture would make vitrification easier and allow for its widespread use., Objectives: To design a successful vitrification protocol for embryos >300 μm without puncture as a pre-treatment., Study Design: Experimental in vivo study., Methods: Thirty-eight embryos were divided into 3 groups (G1: ≤300 μm, n = 11; G2: >300-500 μm, n = 20; G3: >500 μm, n = 7). Embryos were vitrified using a human vitrification kit. Following a 15 min exposure to equilibration solution (ES; 7.5% DMSO +7.5% ethylene glycol [EG] in a base medium [BM] of M199 HEPES-buffered medium [H199] + hydroxypropyl cellulose + gentamycin), embryos were exposed for ≤90 s to a vitrification solution (15% DMSO +15% EG + 0.5 M trelahose in BM), loaded onto a Cryolock and plunged into LN2. Warming was undertaken by plunging the Cryolock tip into 1 mL of H199 + 20% FBS + pen/strep +1 M sucrose at 42°C for 1 min. The embryos were then moved to a 0.5 M sucrose solution for 4 min, then placed in Vigro Hold for 4 min prior to transfer to a recipient., Results: Pregnancy rates were 81.8% (9/11) for G1, 80% (16/20) for G2, and 0% (0/7) for G3. The largest embryo to survive was 480 μm., Main Limitations: Limited numbers and only one pregnancy was followed to term., Conclusions: Equine embryos ≤480 μm can be successfully vitrified using a protocol with a longer exposure time to the ES. This does not appear to have a negative effect on early embryonic development., (© 2024 EVJ Ltd.)

Embryonic genome activation is a critical event in embryo development, in which the transcriptional program of the embryo is initiated. The timing and regulation of this process are species-specific. In vitro embryo production is becoming an important clinical and research tool in the horse; however, very little is known about genome activation in this species. The objective of this work was to identify the timing of genome activation, and the transcriptional networks involved, in in vitro-produced horse embryos. RNA-Seq was performed on oocytes and embryos at eight stages of development (MII, zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst; n = 6 per stage, 2 from each of 3 mares). Transcription of seven genes was initiated at the 2-cell stage. The first substantial increase in gene expression occurred at the 4-cell stage (minor activation), followed by massive gene upregulation and downregulation at the 8-cell stage (major activation). An increase in intronic nucleotides, indicative of transcription initiation, was also observed at the 4-cell stage. Co-expression network analyses identified groups of genes that appeared to be regulated by common mechanisms. Investigation of hub genes and binding motifs enriched in the promoters of co-expressed genes implicated several transcription factors. This work represents, to the best of our knowledge, the first genomic evaluation of embryonic genome activation in horse embryos., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Embryonic genome activation and dosage compensation are major genetic events in early development. Combined analysis of single embryo RNA-seq data and parental genome sequencing was used to evaluate parental contributions to early development and investigate X-chromosome dynamics. In addition, we evaluated dimorphism in gene expression between male and female embryos. Evaluation of parent-specific gene expression revealed a minor increase in paternal expression at the 4-cell stage that increased at the 8-cell stage. We also detected eight genes with allelic expression bias that may have an important role in early development, notably NANOGNB. The main actor in X-chromosome inactivation, XIST, was significantly upregulated at the 8-cell, morula, and blastocyst stages in female embryos, with high expression at the latter. Sexual dimorphism in gene expression was identified at all stages, with strong representation of the X-chromosome in females from the 16-cell to the blastocyst stage. Female embryos showed biparental X-chromosome expression at all stages after the 4-cell stage, demonstrating the absence of imprinted X-inactivation at the embryo level. The analysis of gene dosage showed incomplete dosage compensation (0.5 < X:A < 1) in MII oocytes and embryos up to the 4-cell stage, an increase of the X:A ratio at the 16-cell and morula stages after genome activation, and a decrease of the X:A ratio at the blastocyst stage, which might be associated with the beginning of X-chromosome inactivation. This study represents the first critical analysis of parent- and sex-specific gene expression in early equine embryos produced in vitro., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non-surgical transcervical procedure and divided into three groups based on their size (A ≤ 300 µm, 300 µm300 µm). However, more research is required to find the best method for embryos ≥700 µm., (© 2021 Wiley-VCH GmbH.)

We investigated the early effects of the equine embryo on maternal serum concentrations of insulin-like growth factor 1 (IGF1), leptin and adiponectin, uterine immune cells and genes and proteins related to embryo development and the maintenance of pregnancy. Ipsilateral endometrial expression was assessed on Days 7 and 13 after ovulation for the following transcripts: oestrogen receptor ERα (ESR1), progesterone receptor (PGR), progestin and adipoQ receptor family member 5 (PAQR5), oxytocin receptor (OXTR), prostaglandin-endoperoxide synthase 2 (PTGS2), raf-1 proto-oncogene serine/threonine kinase (RAF1), p21-activated kinase 6 (PAK6), fibroblast growth factor family member 9 (FGF9), IGF1 and its receptor (IGF1R), mucin 1 (MUC1), osteopontin (OPN), leptin receptor (LEPR) and adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2). Ipsilateral endometrial immunological cell infiltration and immunohistochemical protein localisation were evaluated on Days 7, 10 and 13 after ovulation for ERα, PGR, OXTR, PTGS2, IGF1, IGF1R, IGF2 and MUC1. Serum hormone concentrations were not affected by reproductive status. Pregnancy downregulated ESR1 and PGR mRNA levels, upregulated the expression of all other genes and affected the expression of all genes, except PGR, on Day 7 (compared with eight genes affected at Day 13). Proteins were affected by pregnancy or by its interaction with other variables (day of extraction and endometrial compartment). Pregnant mares had a higher lymphocyte count, which decreased towards Day 13. The effect of pregnancy on leucocytes and proteins was more evident in superficial endometrial compartments. The results of this study suggest that the equine embryo exerts prompt paracrine regulation of critical biological processes.

Timed artificial insemination (TAI) has boosted the use of conventional artificial insemination (CAI) by employing hormonal protocols to synchronize oestrus and ovulation. This study aimed to evaluate the efficiency of a hormonal protocol for TAI in mares, based on a combination of progesterone releasing intravaginal device (PRID), prostaglandin (PGF 2α ) and human chorionic gonadotropin (hCG); and compare financial costs between CAI and TAI. Twenty-one mares were divided into two groups: CAI group (CAIG; n = 6 mares; 17 oestrous cycles) and TAI group (TAIG; n = 15 mares; 15 oestrous cycles). The CAIG was subjected to CAI, involving follicular dynamics and uterine oedema monitoring with ultrasound examinations (US), and administration of hCG (1,600 IU) when the dominant follicle (DF) diameter's ≥35 mm + uterine oedema + cervix opening. The AI was performed with fresh semen (500 × 10 6 cells), and embryo was recovered on day 8 (D8) after ovulation. In TAI, mares received 1.9 g PRID on D0. On D10, PRID was removed and 6.71 mg dinoprost tromethamine was administered. Ovulation was induced on D14 (1,600 IU of hCG) regardless of the DF diameter's, and AI was performed with fresh semen (500 × 10 6 cells). On D30 after AI, pregnancy was confirmed by US. The pregnancy rate was 80.0% in TAIG and 82.3% in CAIG (p > .05). The TAI protocol resulted in 65% reduction in professional transport costs, and 40% reduction in material costs. The TAI was as efficient as CAI, provided reduction in costs and handlings, and is recommended in mares., (© 2020 Wiley-VCH GmbH.)

In horses, prostaglandin E 2 (PGE 2 ) is produced by embryos around Day 5 post-ovulation; PGE 2 functions directly at the oviduct promoting embryo transport into the uterus. Non-surgical collection of horse embryos for cryopreservation is recommended at Day 6.5-7 post-ovulation. It was proposed that misoprostol administered orally will hasten oviductal transport of horse embryos. In Experiment 1 (n = 15) there was comparison of time of embryo recovery (Day 6 and 6.5 post-ovulation) from mares administered misoprostol (Day 5 and 5.5) orally to that of untreated mares. On Day 6, embryo collections were attempted; if no embryo was collected, there was a second attempt on Day 6.5. In Experiment 2, (n = 16) misoprostol treatment was initiated on Day 4.5; there was the first embryo collection attempt on Day 5.5, followed by Day 6 and 6.5 if no embryo was collected. Blood samples were collected at 12 h intervals on Day 4.5 or 5, to Day 6.5. In Experiment 1, on days 6 and 6.5, respectively, there was collection of seven and one of a total of eight embryos detected at the time of collection per group (P = 1). In Experiment 2, 12 embryos were collected during 15 cycles with there being a total of three, two, and one collected from mares of both groups on Day 5.5, 6, and 6.5 post-ovulation, respectively (P = 1). Serum progesterone concentrations were not different (P ≥ 0.05). In conclusion, misoprostol, when administered orally, does not hasten oviductal transport of horse embryos., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The cardiovascular system is the first functional organ in the embryo, and its blood vessels form a widespread conductive network within the organism. Blood vessels develop de novo, by the differentiation of endothelial progenitor cells (vasculogenesis) or by angiogenesis, which is the formation of new blood vessels from existing ones. This review presents an overview of the current knowledge on physiological and pathological angiogenesis in the horse including studies on equine endothelial cells. Principal study fields in equine angiogenesis research were identified: equine endothelial progenitor cells; equine endothelial cells and angiogenesis (heterogeneity, markers and assessment); endothelial regulatory molecules in equine angiogenesis; angiogenesis research in equine reproduction (ovary, uterus, placenta and conceptus, testis); angiogenesis research in pathological conditions (tumours, ocular pathologies, equine wound healing, musculoskeletal system and laminitis). The review also includes a table that summarizes in vitro studies on equine endothelial cells, either describing the isolation procedure or using previously isolated endothelial cells. A particular challenge of the review was that results published are fragmentary and sometimes even contradictory, raising more questions than they answer. In conclusion, angiogenesis is a major factor in several diseases frequently occurring in horses, but relatively few studies focus on angiogenesis in the horse. The challenge for the future is therefore to continue exploring new therapeutic angiogenesis strategies for horses to fill in the missing pieces of the puzzle., (© 2019 The Authors. Anatomia, Histologia, Embryologia published by Wiley-VCH GmbH.)

Equine embryo vitrification is still not a well-established technique in equine practice. Notably, little work has been done on the effect of the warming system on viability of vitrified embryos. Our goal was to evaluate the effect of warming without cryoprotectants on in vitro - produced (IVP) embryo viability in culture, quality assessment parameters, and pregnancy after transfer. Equine IVP blastocysts were vitrified using commercial embryo vitrification media and a semi-closed vitrification device. In Exp. 1, we evaluated two warming temperatures (room temperature, RT, ∼22 °C; and 38 °C) for each of three warming systems: commercial warming solution (Kit); commercial embryo holding medium (EHM) with decreasing concentrations of sucrose (EHM + SS); or EHM alone without added sucrose. Embryos (n = 9 to 14 per treatment) were cultured in vitro for 24 h, stained with DAPI, TUNEL, and fluorophore-labelled phalloidin, and evaluated for nucleus number, mitotic rate, apoptotic rate, and actin filament distribution. In Exp. 2, to survey embryo viability in vivo, vitrified IVP blastocysts were shipped to an embryo transfer facility, then warmed immediately before transfer to recipient mares, using the warming treatments associated with the nominally best (Kit-RT, Kit-38, EHM-RT) and poorest (EHM + SS-38) assessed embryo quality in Exp. 1 (n = 7 to 8 per treatment). Subsequently, IVP blastocysts produced as part of our clinical program were vitrified and shipped, then warmed in embryo holding medium at an embryo transfer facility before transfer to recipient mares; fresh IVP embryos were shipped and transferred as controls. In Exp. 1, embryos increased significantly in diameter after culture (P < 0.01), with no difference among treatments. There was no difference (P > 0.05) in the number of viable nuclei, apoptotic rate, or microfilament distribution among treatments, or between vitrified-warmed and Control embryos. The mitotic rate was higher (P = 0.021) for Kit-RT (3.6%) when compared with the other treatment groups (1.5-2.0%). In Exp. 2, there was no difference (P > 0.05) in initial pregnancy (71.4-87.5%) or heartbeat (57.1%-85.7%) rates among warming treatments. In the clinical trial, there was no difference (P > 0.05) between vitrified-warmed and Control embryos in initial pregnancy (90.9% and 66.6%, respectively) or heartbeat (81.8% and 66.6%, respectively) rates. These results indicate that a semi-closed vitrification system using commercially-available media, and incorporating warming in the field in a single step using commercial embryo holding medium without cryoprotectants, can provide high pregnancy rates with IVP equine embryos., (Copyright © 2020. Published by Elsevier Inc.)

In order to determine whether differences in uterine blood flow between pregnant and non-pregnant mares can be used to predict the presence of the equine embryo prior to flushing in an embryo transfer program, power Doppler ultrasonography was used on a total of 52 mares on days 7 or 8 post-ovulation. Computer analysis of Doppler images was subsequently performed using ImageJ v1.48 software. Vascular perfusion of the endometrium was analyzed using spot meter techniques, measuring mean pixel intensity and area of blood flow. Mares with positive flushings presented a higher uterine blood flow area (one embryo: 54.01 ± 2.27 mm 2 or two embryos: 61.01 ± 6.73 mm 2 ) prior to embryo recovery compared to barren mares (21.77 ± 2.22 mm 2 ) (p≤0.05). However, significant differences in vascular perfusion were not detected between single or twin pregnancies. Blood flow area appears to be a good predictor for differentiation between pregnant and non-pregnant mares with an AUC: 0.869; p≤0.001 and an optimal cut-off value of 37.21 mm 2 . Both the mare's age and day of embryo recovery caused effects on uterine vascular perfusion. According to Youden's J statistics the uterine blood flow area of young pregnant mares was greater than 25.4 mm 2 on day 7 (with a sensitivity of 75% and a specificity of 87.5%) and greater than 21.02 mm 2 on day 8 post-ovulation (with a sensitivity of 93.8% and a specificity of 100%). The uterine blood flow area in adult pregnant mares was greater than 41.4 mm 2 on day 7 (with a sensitivity of 80% and a specificity of 85.5%) and greater than 35.55 mm 2 on day 8 after ovulation (with a sensitivity of 97.2% and a specificity of 85.7%). Evaluation on day 8 is therefore considered to be more reliable. Older and middle aged pregnant mares (5-18 years old) had increased uterine vascularization compared to young pregnant mares (2-5 years old) (p≤0.001). Conversely, older barren mares showed higher endometrial vascularity (35.06 ± 2.56 mm 2 ) than young (17.21 ± 1.26 mm 2 ) and middle aged non-pregnant mares (23.84 ± 1.50 mm 2 ) (p≤0.05). We hypothesized that the higher blood flow area seen in older barren mares may be a consequence of a subclinical endometritis due to repeated flushing for embryo recovery. The results of the present study indicate that power Doppler ultrasound combined with computer assisted analysis of images are reliable techniques to detect early pregnancy prior to embryo recovery., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The aim of this study was to compare the embryonic and early fetal development of horse embryos between recipient mules and mares from day 10-60 of pregnancy, in addition to hormonal (eCG and progesterone), ovarian, and uterine characteristics for approximately 4 months. Embryo donor mares (n = 5) and two groups of recipients (acyclic mules, n = 7; cyclic mares, n = 7) were used. Donor mares were monitored daily by transrectal ultrasonography and inseminated using fresh semen. Cyclic recipient mares were synchronized with the donor's ovulation using PGF2α and deslorelin acetate. Mules were prepared for the embryo transfers with estrogen and progestagen. Embryo collection and transfer were performed 8 days after ovulation of the donor mares. Pregnancy diagnosis with ultrasonography began 1 day after embryo transfer. After pregnancy confirmation, the recipient mules received long-acting progesterone once weekly for at least 120 days. The first day of detection (day 10) of an embryonic vesicle (EV) was similar between mules and mares. A period of extensive intrauterine mobility of the embryonic vesicle was observed similarly in mules and mares from days 10-17. The day of fixation of the EV in mules tended to be 1-day earlier than in mares; however, the diameter and growth rate of the EV did not differ between the two species. The embryo proper was first detected at day 20, and the crown-rump, width, and diameter were similar between the two recipient types. The heartbeat and allantoic sac tended to be detected 1 day later in mules than in mares, while the umbilical cord was first observed around day 40 in both species. Besides the expected differences found in ovarian aspects and eCG production, similar endometrial diameter, uterine tone and echotexture, and progesterone levels were seen between the two types of recipients. In conclusion, striking ultrasound similarities in equine embryo and fetal development, and uterine characteristics were seen between mules and mares used as recipients of horse embryos., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this work., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Previous work comparing the developmental mechanisms involved in digit reduction in horses with other mammals reported that horses have only a 'single digit', with two flanking metapodials identified as remnants of digit II and IV. Here we show that early Equus embryos go through a stage with five digit condensations, and that the flanking splint metapodials result from fusions of the two anterior digits I and II and the two posterior digits IV and V, in a striking parallel between ontogeny and phylogeny. Given that even this most extreme case of digit reduction exhibits primary pentadactyly, we re-examined the initial stages of digit condensation of all digit-reduced tetrapods where data are available and found that in all cases, five or four digits initiate (four with digit I missing). The persistent pentadactyl initiation in the horse and other digit-reduced modern taxa underscores a durable developmental stability at the initiation of digits. The digit evodevo model may help illuminate the biological circumstances under which organ systems become highly stabilized versus highly plastic.

The major histocompatibility complex (MHC) influences sexual selection in various vertebrates. Recently, MHC-linked social signaling was also shown to influence female fertility in horses (Equus caballus) diagnosed 17 days after fertilization. However, it remained unclear at which stage the pregnancy was terminated. Here we test if MHC-linked cryptic female choice in horses happens during the first days of pregnancy, i.e., until shortly after embryonic entrance into the uterus and before fixation in the endometrium. We exposed estrous mares to one of several unrelated stallions, instrumentally inseminated them with semen of another stallion, and flushed the uterus 8 days later to test for the presence of embryos. In total 68 embryos could be collected from 97 experimental trials. This success rate of 70.1% was significantly different from the mean pregnancy rate of 45.7% observed 17 days after fertilization using the same experimental protocol but without embryo flushing. Embryo recovery rate was not significantly dependent on whether the mares had been socially exposed to an MHC-dissimilar or an MHC-similar stallion. These observations suggest that MHC-linked maternal strategies affect embryo survival mainly (or only) during the time of fixation in the uterus.

Two cloned mares, produced from the same sample of skin fibroblasts, were bred during four breeding seasons from their second year of age, as embryo donors, in exactly the same conditions, using the same stallions for both cloned mares. The aim of this study was to test the embryo donor potential of cloned mares and to compare the results obtained from two cloned mares of the same mare with other embryo donor mares (n = 31-39 per breeding season) at the same stud. For both cloned mares, 19 embryos were recovered by 43 collection attempts (44%) (7/22 for one; 12/21 for the other), 16 (84%) pregnancies (5/7 for one, 11/12 for the other) were obtained at day 14 post-ovulation (D 14 ), and 12 (3/7 for one; 9/12 for the other) foals were born. One cloned mare was a less efficient donor mare than the other (p < .05), In control donor mares, 623 embryo collections were performed, with a recovery rate (80%-496/623) significantly higher than for cloned mares. The recovery rate in the subpopulation of 2-5-year-old control donor mares (same age of cloned mares) (89%-127/143) and The recovery rate in the subpopulation of 12 control mares bred with the seven same stallions as clones (55%-17/31), were both higher than for cloned mare (p < .05). The success rate of transfer was not different between embryos produced by cloned mares (84%-16/19) and those produced by control donor mares (79%-392/496). However, the foaling rate per embryo collection was significantly lower for cloned mares (28%-12/43) than for control donor mares (52% - 325/623) (p < .05)., (© 2019 Blackwell Verlag GmbH.)

Procaine directly triggers pH-dependent cytokinesis in equine oocytes and induces hypermotility in stallion spermatozoa, an important event during capacitation. However, procaine-induced hyperactivated motility is abolished when sperm is washed to remove the procaine prior to sperm-oocyte co-incubation. To understand how procaine exerts its effects, the external Ca2+ and Na+ and weak base activity dependency of procaine-induced hyperactivation in stallion spermatozoa was assessed using computer-assisted sperm analysis. Percoll-washed stallion spermatozoa exposed to Ca2+-depleted (+2 mM EGTA) procaine-supplemented capacitating medium (CM) still demonstrated hyperactivated motility, whereas CM without NaCl or Na+ did not. Both procaine and NH4Cl, another weak base, were shown to trigger a cytoplasmic pH increase (BCECF-acetoxymethyl (AM)), which is primarily induced by a pH rise in acidic cell organelles (Lysosensor green dnd-189), accompanied by hypermotility in stallion sperm. As for procaine, 25 mM NH4Cl also induced oocyte cytokinesis. Interestingly, hyperactivated motility was reliably induced by 2.5-10 mM procaine, whereas a significant cytoplasmic cAMP increase and tail-associated protein tyrosine phosphorylation were only observed at 10 mM. Moreover, 25 mM NH4Cl did not support the latter capacitation characteristics. Additionally, cAMP levels were more than 10× higher in boar than stallion sperm incubated under similar capacitating conditions. Finally, stallion sperm preincubated with 10 mM procaine did not fertilize equine oocytes. In conclusion, 10 mM procaine causes a cytoplasmic and acidic sperm cell organelle pH rise that simultaneously induces hyperactivated motility, increased levels of cAMP and tail-associated protein tyrosine phosphorylation in stallion spermatozoa. However, procaine-induced hypermotility is independent of the cAMP/protein tyrosine phosphorylation pathway., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Variable gestation length in the mare poses a challenge for determination of fetal readiness for birth. The objectives of this study were to describe sonographic characteristics of the fetal GI tract in the late gestation mare and identify changes that occur with progressing fetal maturity. Based on these characteristics, a grading scale modeled after the existing human and canine scoring system was developed. Weekly sonographic examination of the fetus by one observer beginning 2-3 weeks prior to a calculated due date of 330 days was performed. Fetal assessment included; presence of stomach rugae, bowel segment definition (uniform echogenic, some defined, clearly defined), bowel segment dilation (none, segmental dilation), intestinal contents (none, mixed echogenicity) and peristalsis (none, occasional, every 3 s, continuous). Based on these characteristics a phase was assigned to the GI tract (1-5). Phase 1 was defined as a uniform echogenic grey area caudal to the diaphragm. The differentiation of Phase 2-5 was based primarily on the frequency of peristalsis, with Phase 2 exhibiting no peristalsis, Phase 3 exhibiting occasional peristalsis, Phase 4 exhibiting peristalsis every 3 s and Phase 5 exhibiting continuous peristalsis. Only data from mares with a normal parturition and healthy foals were included in the statistics (N = 10). Associations amongst sonographic fetal GI characteristics and days prepartum were validated using a simulation-based bootstrap approach with 1000 replicates using Stata 14. Stomach rugae, peristalsis, intestinal contents, tail head relaxation and udder development were all highly correlated with days prepartum. Using a multiple linear regression model, tail head relaxation and peristalsis predicted days prepartum with a 95% CI ± 6 days. The same model predicted days prepartum with a 95% CI ± 4 days when day of gestation, tail head relaxation and peristalsis were included as variables. Based on these findings, characterization of the fetal GI tract could provide valuable information concerning the maturity of the equine fetus. Further studies are needed comparing high risk mares to normal mares before this information could be used with confidence in the clinical setting., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: In vitro embryo production (IVEP) is increasingly popular but data assessing the outcome of transferred embryos are scarce., Objectives: To determine the likelihood of pregnancy and embryonic loss after transfer of frozen-thawed IVP embryos and identify factors influencing success., Study Design: Retrospective clinical study., Methods: Blastocysts (n = 261) were produced from immature oocytes of Warmblood mares (n = 116) by Intracytoplasmic Sperm Injection (ICSI) and in vitro culture, and cryopreserved. Thawed IVP embryos were transferred into recipient mares on day 4, 5 or 6 after ovulation. The influence of donor mare (age, reproductive history), recipient mare (age, reproductive status, management; in-house vs. outpatient, day post-ovulation), embryo (interval from ICSI to blastocyst formation) and management factors (season when ovum pickup was performed, year and method of transfer) on likelihood of pregnancy and embryonic loss was examined, and the developmental stage of the IVP embryo at the time of transfer was estimated., Results: The percentage of mares pregnant 7-10, 23 and 37 days after transfer was 56% (147/261), 49% (129/261), and 48% (124/261), respectively. Development of IVP embryos after transfer equated to day 5 or 6 in vivo embryos. With the exception of year of transfer, none of the factors had an impact on the likelihood of pregnancy or embryonic loss. Nevertheless, the likelihood of pregnancy tended to be lower for IVP embryos from infertile mares or when embryos were transferred into recipient mares on day 6 after ovulation rather than on day 4 or 5. Finally, the diameter of the embryonic vesicle 7 days post transfer was lower for pregnancies that were lost compared to those that were maintained., Main Limitations: Small sample size in some of the donor and recipient mare categories., Conclusions: Cryopreserved IVP embryos should be transferred into recipient mares on day 4 or 5 after ovulation and a slower rate of post transfer vesicle expansion indicates a higher risk of subsequent embryonic loss The Summary is available in Portuguese - see Supporting Information., (© 2018 EVJ Ltd.)

Aluteal cycles were induced in the mare to evaluate the effects of progesterone deprivation on the gene expression of embryos and endometrium collected eight days after ovulation. We hypothesized that the transcript expression would be altered during induced aluteal (AL) cycles (low progesterone <1 ng/mL) when compared with control cycles during diestrus (high progesterone; > 4 ng/mL) for 1) the embryonic expression of progesterone-mediated transcripts and those related to normal embryo growth and development and 2) the endometrial expression of progesterone-mediated transcripts and those related to prostaglandin synthesis and normal pregnancy establishment. Seven cyclic mares with a median age of 6.5 years (range 3-16) were utilized in a crossover design. Mares in estrus were artificially inseminated to a fertile stallion and randomly assigned to control or AL groups. Mares received either saline solution (control mares) or PGF 2α (AL mares), twice daily on days 0, 1, and 2 and once daily on days 3 and 4. Serial blood samples were collected daily from day 0 (ovulation) until the day of embryo collection and endometrial biopsy on day 8. Mares were monitored until they returned to estrus, and artificially inseminated. Mares were switched to the opposite treatment group only after a successful embryo collection occurred during the previous cycle and only cycles that produced embryos were used for analyses. The study design resulted in paired samples from each mare for analyses. No significant rise in progesterone was observed in the AL group with mean concentrations of plasma progesterone remaining <1.0 ng/mL from ovulation until embryo collection on day 8. This is in sharp contrast to the control (luteal) cycle where a post-ovulatory rise in plasma progesterone was observed. Real-time RT-PCR was utilized to evaluate the expression of ESR1, PGR, CYP19A1, P19, SLC35A1, OCD, APOB, AQP3, NEU2 transcripts in the embryos and PTGS2, P19, ESR1, HK2, sPLA2, PGR, CTGF, IFNE, FGF9, SLC36A2 expression in the endometrium. Four transcripts showed increased expressed in embryos developed during AL cycles ESR1, P19, APOB and PGR (p < 0.05). Four transcripts showed increased expressed in endometrium developed during AL cycles sPLA2, PGR, ESR1, FGF9 (p < 0.05) and four transcripts showed decreased expression P19, CTGF, IFNE, HK2 (p < 0.05). Additionally, staining differences were present in endometrial staining for both ERα and PR receptor during AL cycles compared with control cycles. Embryos and endometrium developed in a progesterone-deprived environment during induced aluteal cycles demonstrated altered transcript expression. These results indicate that adequate progesterone levels may be a key mediator of the appropriate embryo-maternal environment during early preimplantation embryo development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: To determine the chondrogenic potential of cells derived from interzone tissue, the normal progenitor of articular cartilage during fetal development, compared to that of adult bone marrow-derived and adipose-derived mesenchymal cell isolates. The objective of this study was to compare the chondrogenic potential of fetal musculoskeletal progenitor cells to adult cell types, which are currently used therapeutically to facilitate joint cartilage repair in equine clinical practice. The hypothesis tested was that cells derived from interzone tissue have a chondrogenic potential that exceeds that of adult bone marrow-derived and adipose-derived mesenchymal cell isolates., Study Design: In vitro study., Animals: Six young adult horses (15-17 months of age) and 6 equine fetuses aged 45-46 days of gestation., Methods: Three-dimensional pellet cultures were established under chondrogenic conditions with fresh, primary cells isolated from adult (articular cartilage, bone marrow, adipose, dermis) and fetal (interzone, skeletal anlagen cartilage, dermis) tissues. Cellular morphology, pellet architecture, and proteoglycan synthesis were assessed in the pellet cultures. Steady state levels of ACAN (aggrecan core protein), COL2A1 (collagen type II), and COL1A1 (collagen type I) messenger RNA (mRNA) were compared among these cell types as pellet cultures and monolayer cultures., Results: Adult articular chondrocytes, fetal interzone cells, and fetal anlage cells generated the largest pellets under these chondrogenic culture conditions. Pellets derived from adult articular chondrocytes and fetal anlage cells had the highest scores on a neocartilage grading scale. Fetal anlage and adult articular chondrocyte pellets had low steady-state levels of COL1A mRNA but high COL2A1 expression. Anlage chondrocyte pellets also had the highest expression of ACAN., Conclusion: Adult articular chondrocytes, fetal interzone cells, and fetal anlage chondrocytes exhibited the highest chondrogenic potential. In this study, adult adipose-derived cells exhibited very limited chondrogenesis, and bone marrow-derived cells had limited and variable chondrogenic potential., Clinical Significance: Additional investigation of the high chondrogenic potential of fetal interzone cells and anlage chondrocytes to advance cell-based therapies in diarthrodial joints is warranted., (© 2019 The American College of Veterinary Surgeons.)

Equine embryos tolerate an unusually large degree of negative uterine asynchrony (recipient mare up to 5 days behind the donor mare). By contrast, positive asynchrony of more than 2 days results in a high incidence of early embryonic loss (EEL). Day 8 embryos range in diameter from approximately 130-1300 μm, with embryos smaller than 300 μm reported to suffer an increased incidence of EEL. However, it is not known whether this reduced viability is due to intrinsically poor embryo quality, or to inadvertent recipient uterine stage-embryo (positive) asynchrony. To examine whether small embryos survive better in Day 4-5 recipients than in recipients with a more advanced uterine stage, the likelihood of pregnancy (PR) and EEL for 62 small (<300 μm) and 215 larger Day 8 horse embryos were compared after transfer to recipients at different uterine stages (Days 4-5, 6-7 and 8-9) using logistic regression. Overall, EEL was higher (21.2%; P < 0.05) for small than larger embryos (7.1%). However, neither PR nor EEL were influenced by the recipient's uterine stage at the time of transfer (P > 0.1). The EEL for small embryos transferred into Day 4-5, 6-7 and 8-9 recipients was 20.8, 18.7 and 25.0%, respectively. We conclude that embryos recovered on Day 8 with a diameter <300 μm are at increased risk of EEL due to reasons other than inadvertent positive asynchrony with the recipient mare's uterus., (Copyright © 2018 Elsevier Inc. All rights reserved.)

There is a complex cascade involving proteins during early embryo development and maternal recognition, which is very important for maintenance of a conceptus. The aim of this study was to compare proteomic profile of uterine fluid after ovulation in pregnant and cyclic mares. In the first cycle, samples of uterine fluid of 30 cyclic mares were collected on days 7 (n = 10), 10 (n = 10) and 13 (n = 10) post ovulation and constituted the Cyclic group. In the second cycle, the same mares were bred to a fertile stallion. At days 7, 10 and 13 uterine fluid samples were collected. Immediately after sample collection, the mare's uteri were flushed, and those with an embryo recovered were assigned to the Pregnant group. Of the 30 mares flushed embryos were recovered from 6 mares on day 7, 6 on day 10 and 6 on day 13. Samples from the mares without embryo recovery were excluded from both groups. The uterine fluid samples were processed by two-dimensional electrophoresis technique followed by matrix assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry for the identification of relevant protein spots. From a total of 677 detected spots 19 were identified, 13 more abundant in Pregnant group and 6 in Cyclic group. In summary, pregnant and cyclic mares showed proteins with different abundance. Identified proteins were related to the transport of lipids through the embryo capsule, uterine motility, ATP generation, maternal immunological tolerance, cell proliferation, differentiation, metabolism and angiogenesis. Changes in the proteomic profile of uterine fluid during early embryo development in mares were related with the conceptus presence, suggesting that these alterations may be important for conceptus development and maternal recognition of pregnancy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Assisted reproduction techniques (ARTs) have become widespread in the equine breeding industry. In particular, the combination of oocyte recovery from live mares followed by IVM and intracytoplasmic sperm injection (ICSI) has increased markedly among the ARTs used with valuable or low-fertility animals. There is currently no consensus among research groups regarding the optimal oocyte maturation period to produce high-quality embryos. In this study, we report the maturation dynamics of equine oocytes at different time points, from 20 to 40h (Experiment 1). In addition, in Experiment 2, equine ICSI blastocysts were produced from oocytes that exhibited early (up to 24h) or late (28-30h) extrusion of the first polar body (PB). Blastocyst rates and diameter were recorded and embryo quality was assessed by analysing the number of apoptotic cells and Yes-associated protein 1 (YAP1) expression. By 20h of IVM, 42% of oocytes were mature, and the remaining oocytes matured within the next 17h of IVM. Although no differences were found in cell apoptosis or the number of YAP1-positive cells between groups exhibiting early and late PB extrusion, embryos from the early group (Group I) exhibited an improved total cell number and blastocyst rate compared to embryos from the late group (Group II) (18.60% vs 10.17% respectively).

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22-27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

The aim of this study was to evaluate ultrastructural and histological changes in the endometrium on days 7, 10 and 13 post-ovulation in pregnant and cyclic mares. Mares were routinely examined by transrectal palpation and ultrasonographic examination of the reproductive tract until estrus was detected. In the first cycle, endometrial biopsies from 30 cyclic mares (Cyclic group) were collected on days 7, 10 and 13 post-ovulation. In the second cycle, the same mares were bred by a fertile stallion. At days 7, 10 and 13 post-ovulation intrauterine biopsies were collected. Immediately after sample collection, the mare's uteri were flushed, and those mares with embryo recovery were assigned to the Pregnant group. From ovulation detection until day of uterine biopsy, blood samples to measure Progesterone concentrations were collected daily in cyclic and pregnant mares. A larger blood vessel caliber was observed in pregnant mares than in cyclic from day 7-13. On the 7th day of pregnancy a large loss of ciliated cells was evident in the group of pregnant mares in comparison with the Cyclic group and the superficial cells of the endometrium were more protruded, and a small amount of histotrophic material between the folds was observed. On the 10th day of pregnancy, the glandular histotrophic secretion and the secretion of luminal epithelium became more intense than the secretion of cyclic mares. On the 13th day of pregnancy, a very large amount of histotroph was observed within large glandular openings surrounded by ciliated cells. The concentrations of P4 were affected by day (P < 0.001), but were not affected by group. Changes occurred in the uterine environment thereupon the entry of the embryo into the uterus. In the stroma and in the lumen, these modifications may aid to provide the necessary nutrition for the initial development of the embryo and to promote changes at cellular structures that will interact in the embryonic signaling and future fixation, implantation and placentation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In this study we examined the timeline of mitotic events of invitro-produced equine embryos that progressed to blastocyst stage using non-invasive time-lapse microscopy (TLM). Intracytoplasmic sperm injection (ICSI) embryos were cultured using a self-contained imaging incubator system (Miri®TL; Esco Technologies) that captured brightfield images at 5-min intervals that were then generated into video for retrospective analysis. For all embryos that progressed to the blastocyst stage, the initial event of extrusion of acellular debris preceded all first cleavages and occurred at mean (±s.e.m.) time of 20.0±1.1h after ICSI, whereas 19 of 24 embryos that did not reach the blastocyst stage demonstrated debris extrusion that occurred at 23.8±1.1h, on average 4h longer for this initial premitotic event (P<0.05). Embryos that failed to reach the blastocyst stage demonstrated a 4-h delay compared with those that reached the blastocyst stage to reach the 2-cell stage (P<0.05). All embryos that reached the blastocyst stage expressed pulsation of the blastocyst with visible expansion and contraction at approximate 10-min intervals, or five to six times per hour. Using a logit probability method, we determined that 2- and 8-cell stage embryos could reasonably predict which embryos progressed to the blastocyst stage. Together, the results indicate that TLM for equine embryo development is a dynamic tool with promise for predicting successful embryo development.

Epigenetic perturbations during the reprogramming process have been described as the primary cause of the low efficiency of somatic cell nuclear transfer (SCNT). In this study, we tested three strategies targeting nuclear reprogramming to investigate effects on equine SCNT. First, we evaluated the effect of treating somatic cells with chetomin, a fungal secondary metabolite reported to inhibit the trimethylation on histone 3 lysine 9 (H3K9 me3). Second, caffeine was added to the culture medium during the enucleation of oocytes and before activation of reconstructed embryos as a protein phosphatase inhibitor to improve nuclear reprogramming. Third, we tested the effects of the histone deacetylase inhibitor trichostatin A (TSA) added during both activation and early embryo culture. Although none of these treatments significantly improved the developmental rates of the invitro aggregated cloned equine embryos, the first equine cloned foal born in Australia was produced with somatic cells treated with chetomin. The present study describes the use of chetomin, caffeine and TSA for the first time in horses, serving as a starting point for the establishment of future protocols to target epigenetic reprogramming for improving the efficiency of equine cloning. Cloning is an expensive and inefficient process, but has gained particular interest in the equine industry. In this study we explored different strategies to improve cloning efficiency and produced the first cloned foal born in Australia. Our data serve as a starting point for the establishment of future protocols for improving equine cloning efficiency.

Some basic parameters for equine invitro embryo production have not yet been established, including the optimum temperature for maturation and embryo culture, and the optimum CO2 concentration and pH during early embryo development. To explore this, we first performed cultures in incubators set at 37.2°C, 37.7°C or 38.2°C. At these temperatures, the corresponding maturation rates were 33%, 38% and 42%; cleavage rates were 84%, 86% and 88%; and blastocyst rates were 35%, 44% and 44% per injected oocyte. These rates did not differ significantly (P>0.2). We then evaluated three different CO2 concentrations (6%, 6.5% or 7% CO2) in 5% O2 for culture over Days 0-5 after intracytoplasmic sperm injection, using a commercial human embryo medium with added serum, at 38.2°C. The pH values of these media were 7.36, 7.33 and 7.29 respectively. In the presence of 6%, 6.5% or 7% CO2, cleavage rates were 68%, 80% and 70% respectively, and blastocyst rates per injected oocyte were 42%, 54% and 27% respectively. The blastocyst rate for the 7% CO2 treatment was significantly lower than that for the 6.5% CO2 treatment (P<0.05). We conclude that equine invitro embryo production is equally effective within the range of 37.2-38.2°C, but that equine early cleavage stage development is sensitive to small changes in CO2 atmosphere and/or medium pH.

Several studies report that a two-step culture where mammalian oocytes are first kept under meiosis-arresting conditions (prematuration) followed by IVM is beneficial to embryo development. The most promising results were obtained by stratifying the oocyte population using morphological criteria and allocating them to different culture conditions to best meet their metabolic needs. In this study, horse oocytes were characterised to identify subpopulations that may benefit from prematuration. We investigated gap-junction (GJ) coupling, large-scale chromatin configuration and meiotic competence in compact and expanded cumulus-oocyte complexes (COCs) according to follicle size (<1, 1-2, >2cm) and season. Then we tested the effect of cilostamide-based prematuration in compact COCs collected from follicles <1 and 1-2cm in diameter on embryo development. Meiotic competence was not affected by prematuration, whereas COCs from follicles 1-2cm in diameter yielded embryos with a higher number of cells per blastocyst than oocytes that underwent direct IVM (P<0.01, unpaired Mann-Whitney test), suggesting improved developmental competence. Oocytes collected from follicles <1cm in diameter were not affected by prematuration. This study represents an extensive characterisation of the functional properties of immature horse oocytes and is the first report of the effects of cilostamide-based prematuration in horse oocyte IVM on embryo development.

Embryo mobility occurs as a result of prostaglandin production by the embryo and endometrium, promoting uterine smooth muscle contractions, which propels the embryonic vesicle through the lumen. Non-steroidal anti-inflammatory drugs (NSAIDs), as flunixin meglumine, are routinely used in equine medicine and can alter the conceptus mobility if applied in early pregnancy, which may impair maternal recognition of pregnancy. The objective of this study was to evaluate and compare the effect of flunixin meglumine (FM; 1.1 mg/kg IV), firocoxib (FIRO; 0.2 mg/kg PO), and meloxicam (ML; 0.6 mg/kg, IV), on the embryo mobility. Thirty mares were divided into three groups (n = 10 per treatment). After the pregnancy diagnosis on day 12 after ovulation, the embryo mobility was evaluated by transrectal ultrasonography every 5 min for 1 h in order to visualize the location of the embryo. In all mares, three evaluations were performed: immediately before treatment (pre-treatment), after NSAID administration and 24 h after treatment. In group FM, embryo mobility decreased, from 5.8 ± 0.3 movements/hour (m/h) to 2.3 ± 0.5 m/h (p < 0.05) and, after 24 h the values were similar to the pre-treatment evaluation (5.9 ± 0.2 m/h). Likewise, ML treatment caused a decrease of embryo movements, from 5.9 ± 0.3 to 1.9 ± 0.3 m/h (p < 0.05), 24 h after treatment values were 5.7 ± 0.4 m/h. Treatment with FIRO did not interfere with embryo mobility (5.7 ± 0.4; 5.8 ± 0.3 and 5.6 ± 0.3 embryo movements in the first, second and third evaluation, respectively). In conclusion, FIRO was the only NSAID that did not alter the embryo mobility and may be the safest NSAID for use in early pregnant mares., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Oocyte activation is initiated when a fertilising spermatozoon delivers sperm-borne oocyte-activating factor(s) into the oocyte cytoplasm. Candidates for oocyte activation include two proteins, phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (PAWP; also known as WBP2 N-terminal like (WBP2NL)). We localised PLCZ1 and WBP2NL/PAWP in stallion spermatozoa and investigated the PLCZ1 content and sperm parameters as well as cleavage after intracytoplasmic sperm injection (ICSI). PLCZ1 was identified as 71-kDa protein in the acrosomal and postacrosomal regions, midpiece and principal piece of the tail. Anti-WBP2NL antibody identified two WBP2NL bands (~28 and ~32kDa) in the postacrosomal region, midpiece and principal piece of the tail. PLCZ1 and WBP2NL expression was positively correlated (P=0.04) in sperm heads. Flow cytometry evaluation of PLCZ1 revealed large variations in fluorescence intensity and the percentage of positively labelled spermatozoa among stallions. PLCZ1 expression was significantly higher in viable than non-viable spermatozoa, and DNA fragmentation was negatively correlated with PLCZ1 expression and the percentage of positively labelled spermatozoa (P<0.05). The use of equine sperm populations considered to have high versus low PLCZ1 content resulted in significantly higher cleavage rates after ICSI of bovine and equine oocytes, supporting the importance of PLCZ1 for oocyte activation.

The clinical use of intracytoplasmic sperm injection (ICSI) in horses usually involves the transfer of embryos into recipient mares, resulting in substantial cost increases. This is essential when subfertile mares are oocyte donors; but some donors are fertile, with ICSI compensating for limited or poor-quality spermatozoa. Fertile oocyte donors could carry pregnancies, eliminating the need for a recipient. We assessed the potential of using oocyte donors as recipients for their own ICSI-produced embryos during the same cycle. Donors in oestrus and with large dominant follicles were administered ovulation-inducing compounds to cause follicle and oocyte maturation. Maturing oocytes were collected, cultured and fertilised using ICSI. At 6 or 7 days after ICSI, developing blastocysts were transferred into respective donors' uteri, and pregnancy rates were determined. Twenty follicles were aspirated from nine mares and 12 oocytes were collected. After ICSI, 10 of the 12 oocytes (83%) cleaved, and eight (67% of injected oocytes) developed into blastocysts for transfer. Five pregnancies resulted from the eight transferred embryos (pregnancy rate 62% per embryo and 42% per sperm-injected oocyte). Following this synchronisation regime, ICSI-produced embryos can be transferred into oocyte donors' uteri during the same cycle, allowing donors to carry pregnancies after assisted fertilisation.

In recent years, the number of equine invitro-produced embryos (IVP) has increased markedly; as yet, there are few reports on what constitutes an 'ideal' recipient for an IVP embryo. This study retrospectively investigated the effects of recipient mare oestrous cycle characteristics on the likelihood of pregnancy after transfer of IVP (n=264) and invivo-derived embryos (n=262). IVP embryos tolerated only a narrow window of recipient mare 'synchrony', with transfer on Day 4 after ovulation resulting in a higher likelihood of ongoing pregnancy (69%) than transfer on Days 3, 5 or 6 (53.2%, 41.3% and 23.1% respectively; P=0.02). In contrast, Day 8 invivo-derived embryos tolerated a wide range of uterine (a)synchrony, with no difference in pregnancy or pregnancy loss for recipients that ovulated between Day 4 and Day 9 before transfer. However, transferring invivo-derived embryos to recipients that had a longer oestrus preceding transfer resulted in higher Day 12 and ongoing pregnancy rate (P<0.01). This effect was not significant in IVP embryos. In conclusion, Day 6-8 IVP blastocysts survive best after transfer to Day 4 recipient mares; Day 8 invivo-derived embryos survive equally well in Day 4-9 recipients, but do better in mares that have a long preceding oestrus.

The increased commercialisation of intracytoplasmic sperm injection (ICSI) in horses creates more opportunities to incorporate advanced reproductive technologies, such as sex-sorted, refrozen and lyophilised spermatozoa, into a breeding program. This paper reviews the status of these semen-handling technologies in light of their use in equine ICSI programs. Pregnancies have been achieved from each of these advanced technologies when combined with ICSI in horses, but refinements in the semen-handling processes underpinning these technologies are currently being explored to produce more reliable and practical improvements in the results from equine ICSI.

The timing of early mitotic events during preimplantation embryo development is important for subsequent embryogenesis in many mammalian species, including mouse and human, but, to date, no study has closely examined mitotic timing in equine embryos from oocytes obtained by ovum pick-up. Here, cumulus-oocyte complexes were collected by transvaginal follicular aspiration, matured invitro and fertilised via intracytoplasmic sperm injection. Each fertilised oocyte was cultured up to the blastocyst stage and monitored by time-lapse imaging for the measurement of cell cycle intervals and identification of morphological criteria indicative of developmental potential. Of the 56 fertilised oocytes, 35 initiated mitosis and 11 progressed to the blastocyst stage. Analysis of the first three mitotic divisions in embryos that formed blastocysts determined that typical blastocyst timing (median±IQR) is 30.0±17.5min, 8.8±1.7h and 0.6±1.4h respectively. Frequent cellular fragmentation, multipolar divisions and blastomere exclusion suggested that equine embryos likely contend with a high incidence of chromosomal missegregation. Indeed, chromosome-containing micronuclei and multinuclei with extensive DNA damage were observed throughout preimplantation embryogenesis. This indicates that time-lapse image analysis may be used as a non-invasive method to assess equine embryo quality in future studies.

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.

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. This study evaluated the frequency of micronucleus formation as a proxy for aneuploidy in invitro-produced (IVP) and invivo-derived horse blastocysts. Associations between IVP embryo morphology, frequency of nuclear abnormalities and the likelihood of pregnancy were investigated. IVP blastocysts exhibited a higher frequency of cells with micronuclei than invivo-derived embryos (10% vs 1% respectively; P=0.05). This indication of chromosomal instability may explain the higher incidence of pregnancy failure after transfer of IVP embryos. However, the frequency of micronuclei was not correlated with brightfield microscopic morphological characteristics. Nevertheless, IVP embryos reaching the blastocyst stage after Day 9 of invitro culture were less likely to yield a pregnancy than embryos that developed to blastocysts before Day 9 (27% vs 69%), and embryos that had expanded before transfer were more likely to undergo embryonic death than those that had not expanded (44% vs 10%). These findings indicate that current embryo culture conditions are suboptimal and that the speed of embryo development is correlated with pregnancy survival.

Intracytoplasmic sperm injection (ICSI) is used to produce equine embryos invitro. The speed of embryo development invitro is roughly equivalent to what has been described for embryos produced invivo. Morphological evaluations of ICSI-produced embryos are complicated by the presence of debris and the dark nature of equine embryo cytoplasm. Morulas and early blastocysts produced invitro appear similar to those produced invivo. However, with expansion of the blastocyst, distinct differences are observed compared with uterine embryos. In culture, embryos do not undergo full expansion and thinning of the zona pellucida (ZP) or capsule formation. Cells of the inner cell mass (ICM) are dispersed, in contrast with the differentiated trophoblast and ICM observed in embryos collected from uteri. As blastocysts expand invitro, embryo cells often escape the ZP as organised or disorganised extrusions of cells, probably through the hole incurred during ICSI. Quality assessment of invitro-produced early stage equine embryos is in its infancy, because limited information is available regarding the relationship between morphology and developmental competence. Early embryo development invivo is reviewed in this paper, with comparisons made to embryo development invitro and clinical assessments from a laboratory performing commercial ICSI for >15 years.

Embryo biopsy for fetal sexing has clinical application, but few reports are available of its use within an active embryo transfer program. We evaluated results on biopsy of 459 embryos over one breeding season. There were no significant differences in pregnancy rate between biopsied and non-biopsied embryos (72% vs 73%) or for biopsied embryos recovered at the centre (73%) compared with those shipped overnight (72%). However, the pregnancy rate decreased significantly in shipped embryos biopsied ≥20h after collection. Overall, 86% of biopsies provided a sex diagnosis. The likelihood of a positive genomic (g) DNA result was significantly higher for biopsies from large blastocysts (96%) than from smaller embryos (70-85%). In total, 38% of biopsies were positive for Y chromosome DNA (Y-DNA) and were diagnosed as male. Subsequently, 95% of Y-DNA-positive embryos were confirmed as male and 78% of Y-DNA-negative embryos were confirmed as female. The accuracy of prediction of female (Y-DNA negative) was significantly higher when the biopsy sample was probed for Y-DNA only compared with probing for both gDNA and Y-DNA. We estimate that by transferring only Y-DNA-negative embryos, 3% of potential female pregnancies may have been lost, and production of male pregnancies was reduced by 72%.

The increase in demand for in vitro produced horse embryos is fostering the development of commercial laboratories for this purpose. Nevertheless, blastocyst production after intracytoplasmic sperm injection (ICSI) is still not as great as desired in most of these laboratories. In relation to horse oocyte classification, both expanded and compact cumulus-oocyte-complexes (COCs) are used for in vitro embryo production. The aim of this study was to compare in vitro embryo developmental capacity of COCs from horses including those with only the corona radiata, frequently collected after aspiration procedures. Horse oocytes were collected by follicular aspiration of abattoir-derived ovaries. After classification as expanded, compact or corona radiata COCs, these were in vitro matured, fertilized by ICSI and in vitro cultured for 7.5 days. Maturation rate, cleavage rate and morula/blastocyst rates were recorded. No significant differences (P > 0.05) were detected among groups in maturation rate. Cleavage rate was less (P < 0.05) for embryos derived from oocytes with a corona radiata as compared to compact-derived embryos, but embryo development after 7.5 days of culture was similar among groups (P > 0.05). In conclusion, even if embryos derived from oocytes with corona radiata had a lesser cleavage rate after ICSI, the developmental capacity was similar to embryos derived from oocytes with a compact and expanded cumulus morphology, indicating these can be an useful source of embryos in horses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The development of techniques to produce equine embryos in vitro is reviewed with specific reference to intracytoplasmic sperm injection (ICSI). Unexplored 50 years ago, this technology has progressed rapidly in the last 20 years to become a commercial reality for the equine breeding industry. Improvements in our understanding of oocyte and embryo competence in the horse have been key factors in overcoming some of the initial problems associated with ICSI. It is now possible to obtain high nuclear maturation and cleavage rates in vitro and the most limiting factor, presently, is the low rate of development to the blastocyst stage. However, in spite of this, once obtained, these in vitro-derived blastocysts can result in pregnancy rates in excess of 60% following transfer., (© 2018 EVJ Ltd.)

Equine chorionic girdle trophoblast cells play important endocrine and immune functions critical in supporting pregnancy. Very little is known about the genes and pathways that regulate chorionic girdle trophoblast development. Our aim was to identify genes and signalling pathways active in vivo in equine chorionic girdle trophoblast within a critical 7-days window. We exploited the late implantation of the equine conceptus to obtain trophoblast tissue. An Agilent equine 44K microarray was performed using RNA extracted from chorionic girdle and chorion (control) from equine pregnancy days 27, 30, 31 and 34 (n = 5), corresponding to the initiation of chorionic girdle trophoblast proliferation, differentiation and migration. Data were analysed using R packages limma and maSigPro, Ingenuity Pathway Analysis and DAVID and verified using qRT-PCR, promoter analysis, western blotting and migration assays. Microarray analysis showed gene expression (absolute log FC >2, FDR-adjusted P < 0.05) was rapidly and specifically induced in the chorionic girdle between days 27 and 34 (compared to day 27, day 30 = 116, day 31 = 317, day 34 = 781 genes). Pathway analysis identified 35 pathways modulated during chorionic girdle development (e.g. FGF, integrin, Rho GTPases, MAPK) including pathways that have limited description in mammalian trophoblast (e.g. IL-9, CD40 and CD28 signalling). Rho A and ERK/MAPK activity was confirmed as was a role for transcription factor ELF5 in regulation of the CGB promoter. The purity and accessibility of chorionic girdle trophoblast proved to be a powerful resource to identify candidate genes and pathways involved in early equine placental development., (2018 The authors)

In recent years, the interest in equine foetal gender determination (FGD) during gestation increased remarkably. Ultrasonographic FGD can be performed in two different periods during gestation. The earliest examination can take place at a gestational age of 60-70 days, whereby the genital tubercle is used to differentiate between male and female foeti. The time window of the second approach is wider (120-210 days), and there are more characteristics to take into consideration. In this article, the feasibility and accuracy of ultrasonographic FGD in mid- to late gestation are evaluated. One hundred twenty-one mares from different breeds with a pregnancy stage between 120 and 270 days were examined once, using B-scale ultrasonography (Esaote MyLab™ClassC). None of the mares were sedated nor shaved, and the procedure was completed within 15 min. Diagnosis was firstly based on the gonads. The final judgement was made based on all visible foetal reproductive organs. In three cases with a pregnancy stage beyond 257 days, FGD was not possible. All of the examined mares in which a FGD could be performed gave birth to a healthy foal. In 98% of the examinations (116/118), the diagnosis was correctly made. In both cases of misdiagnosis, only one characteristic was seen during the procedure and wrongly interpreted. Beyond 210 days of pregnancy, the extremities can preclude a good visualization of the inguinal region. In conclusion, equine FGD in mid- to late gestation is an accessible and accurate technique, although a good ultrasound device is a prerequisite and experience and expertise is necessary., (© 2018 Blackwell Verlag GmbH.)