Donor Mares Research Articles

Doppler ultrasonography: an improvement in real-time recipient selection in equine ET programs

In this study, we test the hypothesis that a CL with greater blood perfusion has a more significant impact than luteal size and P4 concentrations on pregnancy per embryo transfer (P/ET). The study was carried out from November/2021 to May/2022 on a stud farm of the Mangalarga Marchador breed in Itaperuna-RJ, Brazil. Recipient mares (n=110), aged between 3-16 years, weighing 370-460 kg, with adequate BCS and on days 3 to 9 after spontaneous ovulation, received a single fresh grade I or II blastocyst, retrieved by uterine flushing of donor mares. Immediately before the ET, the CL characteristics were evaluated by B-mode and Color-Doppler ultrasonography (US) and a blood sample was collected for plasma P4 assay. Recipients were retrospectively classified according to CL area [small (≤6 cm2) or large (>6 cm2)], CL blood perfusion [low ≤55 % or high >55%] and plasma P4 concentration [low ≤9.98 ng/mL or high >9.98 ng/mL]. Pregnancy diagnosis was performed 30 days after ET and P/ET analysis was performed using the GLIMMIX of SAS procedure. The overall P/ET was 73% (83/110). The P/ET was not influenced by the luteal area, but effects of CL blood perfusion class (P=0.007) and the P4 plasma concentration class (P=0.02) on P/ET were observed. The P/ET was greater in the high luteal perfusion group [89.1%, 41/46 vs.65.6%, 42/64] and in the high P4 group [85.4%, 41/48 vs. 67.7%, 42/62]. When area, luteal blood perfusion and P4 concentration were compared with the probability of pregnancy through ROC curve analysis, luteal blood perfusion (AUC=0.66) was the most significant (P=0.01) predictor of pregnancy compared to area (AUC=0.53) and plasma P4 (AUC=0.60). Finally, recipients were grouped into four different groups by analyzing CL blood perfusion classes and P4 (high CLBP and high P4 vs. High CLBP and low P4 vs. low CLBP and high P4 vs. low CLBP and low P4) and compared with the P/ET using the Fisher test. P/ET differed between groups (P=0002), as indicated by the higher P/ET in the high CLBP and high P4 95% (19/20) and high CLBP and low P4 84.6% (22/26) groups, compared to animals with low CLBP and low P4 55.5% (20/36). In conclusion, although the P4 concentration is a potential indicator of pregnancy success, it cannot be used in real-time when selecting the most appropriate recipient in a commercial ET program. Also, luteal blood perfusion determined by Doppler-US is an innovative and easily accessible tool that can be used in real-time to select the recipients with the greatest chances of maintaining pregnancy in ET programs in horses.

Comparison of the efficacy of Lecirelin and hCG for ovulation induction in mares

Ovulating agents are very useful drugs in equine breeding, hCG being the first intention molecule, whereas GnRH agonists are especially interesting for any mare immunized against hCG, like repeat breeders and embryo donor mares. In Europe, no long acting GnRH agonist is available in veterinary practice. Saline solutions of buserelin or triptorelin, which is only available for human treatments in Europe are commonly used for equine ovulation induction (Dordas-Perpinya et al. Reprod. Dom. Anim. 2020; 55:374-383). An alternative, Lecirelin which is licensed in cattle and rabbits, has been evaluated in donkeys (Carluccio et al. Therio. 2007;68:914-919). The aim of our study was to compare the efficacy of lecirelin with hCG in horses. Forty mares underwent ovulation induction on four consecutive cycles, two cycles with hCG and two cycles with lecirelin, including one spontaneous estrus and one following chemical luteolysis. Mares were examined on a daily basis to check ovarian follicles diameter, uterine edema, and cervical softening. Once they presented a preovulatory follicle over 35 mm, and uterine edema (>=2) (Mc Kinnon. Equine diagnostic ultrasonography. 1998; 79-102) and a soft cervix, they were randomly injected with saline solution containing 200 µg of Lecirelin or 1500 IU of hCG intravenously. After induction, they were scanned every 12 hours until ovulation. Mares were inseminatedwith a mix of fresh semen of two fertile stallions 20 hours after induction on the spontaneous cycles. Pregnancy diagnosis was performed at day 12 and day 14. As each mare is her own control, statistical analysis was performed by χ², Fisher's test and Student's test for matched data. Follicle size at injection did not differ significantly between the two induction treatments (p>0,05), with or without distinction of spontaneous and induced cycles. Rates of ovulation in the 24-48 hours post-injection did not significantly differ between lecirelin (71%) and hCG (83%) on estrus following chemical luteolysis (p>0,05) or spontaneous estrus (43% for lecirelin and 68% for hCG) (p>0,05). We observed significantly more spontaneous ovulations occurring within the first 24 hours after induction with hCG (27% on spontaneous estrus, and 15% after chemical luteolysis) than with lecirelin (5% on spontaneous estrus, 7% after chemical luteolysis) (p < 0,05). Pregnancy rates were not significantly different following induction by lecirelin (67%) or hCG (57%) (p>0,05). These results highlight the usefulness of saline solution of lecirelin licensed in Europe for bovine and rabbit for induction and synchronization of ovulation for mares with control cycles. Lecirelin may be particularly suitable for embryo donor mares with a reduced hCG response.

Effect of cooled vs frozen stallion semen on the efficiency of equine in vitro embryo production

Since 2008, our practice has been optimizing the equine in vitro embryo production system. Oocyte collection, in vitro maturation and culture media, sperm processing techniques and the Piezo driven sperm injection have remained relatively unchanged since then. However, our population of patients is now younger donor mares (< 17 years old) and in some instances, the use of extended-cooled semen has been prioritized. The purpose of this clinical report is to provide evidence that utilization of cooled semen 24-36 h post collection for ICSI increases the overall embryo production efficiency. Mares (n=350, 90% <17 years old and 8 different breeds) were presented for transvaginal follicular aspiration (TVA) and ICSI. Antral follicle count was determined prior to TVA and oocyte retrievals (n=680) were performed between day 12-20 post ovulation, or 19-25 days after the last TVA. In vitro embryo production was performed as previously described (LF Campos-Chillon, CM Owen, JL Altermatt, J Equine Vet Sci. 2019; 73:51-55). Briefly, sperm injections were performed with a Piezo driven system with the client's stallion of choice (n=262), and in vitro embryo culture was done for up to 9 days. Data (frozen vs cooled semen) was analyzed by One-way ANOVA using Minitab. The average follicle count was 15 (2-35) with an oocyte recovery rate of 82 ± 8%. Oocyte maturation rate expressed as oocytes witha polar body was 70.6 ± 1.1%; for frozen (n=476) vs cooled (n=204) semen injections, cleavage (57.6 ± 1.8 and 70.1 ± 2.9%) and blastocyst (13.4 ± 1.2 and 25.8 ± 1.5%) rates were different (p<0.001) respectively. In addition, number of embryos produced per cycle were 1.16 ± 0.08 for frozen and 2.24 ± 0.13 for cooled semen (p<0.001). Pregnancy rates of fresh embryo transfers at day 35 were higher (p<0.004) for embryos obtained with cooled (n=90, 91.5 ± 0.04%) than with frozen (n=250, 72 ± 0.05%) semen. Furthermore, of the 23% of cycles (n=156) that did not produce an embryo in the first TVA attempt, 18 and 82 % were performed with cooled and frozen semen, respectively. Overall, when cooled semen was used, there was a one-fold increase in embryos obtained per TVA, 20 percentile points higher pregnancy rate, and only 4 % of cycles using cooled semen yielded no embryo vs 19% with frozen semen. These results suggest that cooled semen used for ICSI increases the overall efficiency of the in vitro embryo production system. However, further prospective studies controlling stallion and mare effect of cooled and frozen semen in the model are warranted. In addition, studies involving sperm motility/viability, decondensation parameters and PLCz content and its effect in semen samples as recently described by Gonzales-Castro (Repro Fert Dev. 2022, 35:150-150).

The use of prostaglandin applied in Bai Hui Acupoint on mare reproductive efficiency

SummaryProstaglandin F2α (PGF2α) is one of the most frequently used drugs for pharmacologic manipulation of the oestrous cycle in mares. Among its roles, PGF2α induces luteolysis, leading to an early oestrus return, which reduces the dioestrous phase and the inter‐ovulatory intervals. Interestingly, mares are more sensitive to PGF2α than other domestic species, hence lower doses are used; even so, the side effects are still adverse and inconvenient for animals and breeders. Recent studies of acupuncture, a traditional Chinese medicine technique, have been performed as an alternative to decrease the PGF2α dose for mares. The efficacy described of applying lower doses of PGF2α at specific acupoints is similar to that obtained with the regular dose. Therefore, the objective of this study was to analyse the reproductive performance and characteristics of oestrus in embryo recipient and donor mares using different doses at the Bai Hui—an acupoint located on the dorsal midline, also known as ‘the point of one hundred meetings’, as its region of effect encompasses many nerve trunks, nerve endings and vascular networks—a useful veterinary acupuncture treatment for ovarian disorders. For this study, 76 Quarter Horse mares were randomly assigned to three experimental groups: Control (CTR)—Intramuscular (IM) administration of 5 mg PGF2α; Bai Hui 5 mg (BH5)—application of 5 mg at the Bai Hui acupoint; and Bai Hui 2.5 mg (BH2.5)—application of 2.5 mg at the Bai Hui acupoint. In the results, there were no differences regarding uterine oedema, pregnancy rate or body condition score among the groups. However, oestrous expression was lower (p &lt; 0.001) in both BH groups compared with CTR. Lastly, the interval between application and ovulation was shorter in BH5 compared with CTR (p = 0.042). The results indicate BH5 and BH2.5 as viable alternatives to the IM administration.

Uterine Inflammatory Response After Prostaglandin E1 (Misoprostol) Infusion Prebreeding or Immediately After Embryo Flushing in Commercial Donor Mares

Misoprostol, a synthetic PGE1, is becoming a common therapy for mares with suspected uterine tube obstruction. Recently, there have been concerns that uterine administration of misoprostol induces exacerbated uterine inflammation; however, this has not been critically evaluated. This study aimed to assess the inflammatory response and potential systemic reactions after uterine administration of misoprostol, either during prebreeding or immediately after postembryo flushing. Privately owned embryo donor mares (n = 11) were randomly assigned in a crossover design to receive misoprostol (3 mL +200 µg) or sham (3 mL of lactate Ringer's solution) infusions, bilaterally deposited via deep-horn, at least 72 hours prebreeding (experiment 1) or immediately after embryo flushing (experiment 2). Each mare had one cycle for misoprostol and sham in both experiments and a breeding cycle (no sham or misoprostol) between experiments. Uterine edema, fluid accumulation, and the number of uterine PMN were assessed before each infusion and then daily for 72 hours. Uterine lavage was performed the day after each infusion across groups and experiments. Ovulation was hastened with a GnRH agonist and confirmed at 24 hour-intervals. Mares were bred with semen from one of six stallions per owner's choice. Embryo flushing was performed 8 to 9 days postovulation. In either experiment, misoprostol did not affect uterine edema or fluid accumulation (P > .05). However, both the sham and misoprostol infusions increased the number of PMN up to 48 hours postinfusion in both experiments. Embryo recoveries were similar between sham (45%, 5/11) and misoprostol cycles in experiments 1 (45%, 5/11; P > .05) and 2 (sham, 68%, 7/11; misoprostol, 45%, 5/11; P > .05). In conclusion, misoprostol did not induce exacerbated uterine inflammation in mares or systemic adverse reactions when infused prebreeding or immediately after embryo flushing.

Pentoxifylline reduces post-breeding uterine fluid accumulation in older embryo donor mares and may enhance embryo recovery

Most donor mares in embryo transfer programs have impaired uterine clearance due to uterine and cervical problems. In addition, repeated breeding cycles, gynecological procedures, and embryo flushing make mares prone to endometritis. Anecdotally, pentoxifylline, an anti-inflammatory, has been used in mares to enhance blood flow in the reproductive tract and reduce uterine inflammation. However, this has not been critically studied. This study aimed to assess the post-breeding inflammatory response and embryo recovery of subfertile donor mares treated with pentoxifylline. This study hypothesized that pentoxifylline supplementation mitigates the post-breeding inflammatory response in embryo donor mares. Forty-two estrous cycles of seven embryo donor mares (16±3.7years old; 13±3years donating embryos) were completed in the study. In the first two cycles, mares received pentoxifylline (17mg/kg/day) given in 50mL of Broodmare-Plus® (Botupharma), the two subsequent cycles served as control, and in the last two cycles, mares received 50mL of Broodmare®. Mares were monitored daily, and then once a preovulatory follicle was detected, ovulation was induced with deslorelin (1mg, i.m.). Immediately before (0h), 24, 48, and 72h post-breeding mares had transrectal ultrasonography performed to determine ovulation, quantify uterine edema and intrauterine fluid accumulation (IUF). At similar timepoints, uterine cytology was collected, stained with Diff-Quik®, and polymorphous nuclear cells were counted in ten hpf. Embryo collections were performed eight days after ovulation, and mares receivedan injection of 5mg of dinoprost (i.m., Lutalyse, Zoetis, USA) to return to estrus. Statistical analyses were performed with Graph Prisma (San Diego, CA). Neutrophil counts were analyzed with ANOVA repeated measures and Tukey's as post-hoc. Edema scores and intrauterine fluid accumulation were analyzed with Kruskal-Wallis and Dunn's post-hoc. Embryo recovery rates were evaluated with Fisher's test. There was an effect of time for neutrophil counts in the washout cycle (P=0.032) but not in the other two groups (P>0.05). Neutrophil counts didn't differ across groups (P>0.05). Edema scores had effects of time (P=0.046) but no effects of group (P>0.05). Similarly, there was an effect of time (P=0.038) for IUF, and mares receiving pentoxifylline had reduced post-breeding IUF in comparison with washout cycles (P=0.034) but not different than the group receiving just Broodmare® (P>0.05). Embryo recovery was not different across groups (P>0.05) but remarkably different as follows: washout cycle was 29% (4/14); Broodmare® was 36% (5/14), and pentoxifylline (Broodmare-Plus®) was 57% (8/14). Mares in the study were subfertile older mares used as embryo donors for multiple years, thus, justifying the low embryo recovery for the cycles with no pentoxifylline. In conclusion, supplementation with pentoxifylline may be an alternative to improve the post-breeding uterine clearance. It may enhance embryo recovery in mares serving as donors for multiple years; however, this remains to be confirmed with a larger group number of mares.

The mystery of capsular vesicles: Preliminary studies

An unexpected finding from a previous experiment was the finding of capsular vesicles in the uterine flush 48h after the transfer of a Day 8 embryo that had been punctured with an acupuncture needle. The authors of this study initially thought the vesicles originated from fragments of capsule, in the same way fragments of cultured trophoblast readily form vesicles. However, recovery of a capsule with the hole made by the acupuncture needle alongside the capsular vesicles showed that this was not the case. A preliminary study was undertaken to determine if trophoblast vesicles (TVs) could initiate capsule vesicle formation in recipient mares at different stages post ovulation when transferred for 24h. Day 11 embryos were recovered from donor mares and the capsule was removed before fragmenting the trophoblast and incubating the tissue in DMEMF12 (+ 5% FBS and pen/strep) for 24-48h. Between 20 and 50 TVs were transferred to the uteri of ten recipient mares. Recipient mares were divided into 3 groups; G1, Days 4 or 5 post ovulation (n = 2); G2, Days 6 to 9 (n = 5); and G3, Days 12 and 13 (n = 3). The uteri of the recipient mares were flushed 24h after transfer of the TVs and the recovered fluid searched. In all of the mares, TVs were recovered withvarying degrees of degeneration. In G1 no capsular vesicles were recovered, in G2 and G3 between 5 and 32 capsular vesicles were found in each flush. Although different numbers of vesicles were transferred to recipients the greatest number of capsular vesicles per transferred TV was in G2 compared to G3 (0.44 - 1.6 vs. 0.12 – 0.25 capsular vesicles/transferred TV, respectively). Proper formation of the capsule in the mare is believed to involve interaction of the embryo with the endometrium because in vitro produced embryos fail to produce a proper capsule until after transfer. In vivo, developmentally regulated changes in composition of the capsule have also been noted and the capsule increases in thickness up until at least Day 11. These preliminary results suggest that capsular vesicles can form in the uterus following transfer of TVs, providing that the uterus is at a stage when an embryo would normally be forming the capsular layer since transfers prior to Day 6 failed to produce capsular vesicles and reduced numbers were produced after Day 12. The mechanism of their formation remains a mystery as they are free in the uterus and did not envelop the trophoblastic vesicles.

Slow-freezing is more damaging than vitrification to post-thaw quality of large equine embryos collapsed by blastocoele aspiration

Embryo flushing in mares is best performed on day 7 or 8 after ovulation because the likelihood of recovering an embryo(s) is higher. However, embryos larger than 300 µm are less tolerant of cryopreservation unless the blastocoele is first collapsed. While vitrification of collapsed blastocysts has yielded good results, there are no reports of the relative success of slow-freezing punctured blastocysts. The aim of this study was to compare the effects on post-thaw embryo quality of vitrifying and slow-freezing day 7-8 equine blastocysts after blastocoele puncture. Embryos were collected from donor mares on day 7 or 8 after ovulation, graded (1 to 4), measured (µm), and divided into two size categories (330-550 µm, n= 11 and >550 µm, n= 16). Blastocoele fluid was aspirated using a micromanipulator, and the embryos then either slow-frozen (n= 14; 2 steps, 10% glycerol) or vitrified (n= 13; 2 steps,16.5% ethylene glycol/16.5% DMSO/0.5M sucrose). After thawing or warming, embryos were cultured for 24h before being graded and measured to determine degree of (re)expansion (%). Next, the embryos were stained with DAPI to identify dead cells, before being fixed and further labeled with Phalloidin (cytoskeleton), WGA (capsule), and TOPRO-3 (DNA) to assess live/dead cell proportions, cytoskeleton quality, and capsule integrity. Control embryos (>300-550 µm, n=3 or >550 µm, n=3)were collapsed and cultured for 24h without cryopreservation. Data were analyzed using one-way ANOVA, Kruskal-Wallis, or Fisher's exact tests. For 300-550 µm embryos, quality grade was negatively affected by cryopreservation but did not differ between slow-frozen (3±0) and vitrified (1.7±1) embryos. On the other hand, re-expansion was significantly reduced in slow-frozen (73±34%) compared to vitrified (148±26%) and control (179±10%) embryos. Live/dead cell proportion, cytoskeleton quality and capsule integrity were not influenced significantly by cryopreservation technique. For larger (>550 µm) embryos, re-expansion was also significantly reduced in slow-frozen (52±24%) compared to vitrified (115±7%) and control (129±12%) embryos. In addition, embryo quality was compromised more after slow-freezing (3±0.5) than vitrification (1.9±0.4) or 24h culture alone (control: 2±0). Furthermore, the proportion of dead cells and degree of cytoskeleton disruption was higher after slow-freezing than vitrification, although the incidence of capsule loss was not influenced by freezing method. In conclusion, slow-freezing of micromanipulator-collapsed equine blastocysts compromises embryo quality more than vitrification; therefore, after aspiration of blastocoel fluid, it is preferable to vitrify rather than slow-freeze equine embryos >300 µm.

Uterine inflammatory response after misoprostol during estrus and diestrus in commercial embryo donor mares

Uterine tube obstruction is the leading cause of unexplained subfertility in mares. Over the years, numerous techniques were developed to unblock uterine tubes with variable success. In recent years, the administration of misoprostol, a synthetic analog of prostaglandin E1, in the uterus, has regained popularity in equine practice. However, there have been some concerns regarding an exacerbated post-infusion uterine inflammatory response to misoprostol; nonetheless, this has yet to be critically studied. In addition, debate exists when mares should receive misoprostol. This study aimed to determine the uterine inflammatory response in mares after infusion with misoprostol during estrus or diestrus. We hypothesized that misoprostol infusion induces a greater inflammatory response than sham infusion; similarly, diestrus results in a greater inflammatory response than estrus. Forty-four estrous cycles of light breed mares (n=11) were sequentially randomly assigned to receive misoprostol or sham infusion during estrus and diestrus. The misoprostol and sham infusions were performed by directing a flexible pipette (Minitube, Germany) deep into each uterine horns, which received 200mcg of misoprostol diluted in 3 mL of Lactate Ringer's Solution (LRS), whereas the sham inoculation consisted of 3 mL of LRS. The cycles assigned for estrus treatment received it when preovulatory follicles ranged from 28-32 mm, and diestrus cycles were treated eight days post-ovulation. The day after each infusion, mares had uterine lavage performedwith LRS. Uterine edema and intrauterine fluid accumulation were performed by transrectal ultrasonography immediately before (0h), 24, 48, and 72 h after treatments. At similar time points, uterine cytology samples were harvested to count the number of neutrophils in a high-power field (400 ×). Ovulation was hastened with deslorelin (1mg, i.m.) during estrus or the subsequent estrus after diestrus infusion. Mares received 5 mg of dinoprost (i.m.) 24 h after infusion in estrus and diestrus. Statistical analyses were performed with Graph Prisma. Neutrophil counts were analyzed with ANOVA repeated measures and Tukey's as post-hoc. Edema scores and intrauterine fluid accumulation were analyzed with Kruskal-Wallis and Dunn's post-hoc. Embryo recovery rates were evaluated with Fisher's test. There were effects of time (P=0.04) but no effects of group (P=0.96), or stage of the estrous cycle (P=0.26), or interaction between group and time for edema scores (P=0.97). Similarly, there were effects of time (P=0.03) but no effects of group (P=0.32), stage of the estrous cycle (P=0.86), or interactions between group and time or stage of the estrous cycle for neutrophil counts (P=0.87). Uterine fluid scores did not change over time (P=0.39), groups (P=0.22), or stage of the estrous cycle (P=0.77). Embryo recovery was similar for estrus (Misoprostol 46% vs. sham 46%) vs. diestrus (Misoprostol 67% vs. sham 46%) (P>0.05). In conclusion, 200 mcg of misoprostol bilateral administration did not exacerbate uterine inflammation in mares.

Delayed embryonic development or long sperm survival in embryo donor mares

An embryo collection procedure is usually performed 7-8 days post-ovulation in mares. Embryos are subsequently transferred into a synchronized recipient mare that is expected to carry the pregnancy to term. Submission of a blood or hair root sample to an approved genetics laboratory for DNA parentage verification is a prerequisite for foal registration for most horse breed organizations. The goal of this report is to document two clinical cases in which the DNA parentage test of the foals produced by embryo transfer excluded the stallions whose semen was used on the estrous cycle yielding the embryo and identified as the genetic sire the stallion whose semen was utilized on the previous estrous cycle . Clinical Case # 1. A 21-year-old Arabian mare was inseminated with frozen-thawed semen from a deceased stallion (Stallion A) immediately after detection of a single ovulation. No embryo was recovered following uterine lavage 8 days later. Cooled-transported semen from a different stallion (Stallion B) was used on the subsequent cycle. An exceptionally large (2,466 µm in diameter) expanded blastocyst stage embryo was recovered 8 days post-ovulation and transferred into a recipient mare. The recipient mare carried the pregnancy to term. Genetic testing of the foal excluded Stallion B as the sire and confirmed Stallion A as the genetic sire of the foal. Clinical Case # 2 . A 11-year-old Quarter Horse marewas inseminated with cooled-transported semen from Stallion A and subsequently ovulated two follicles. Uterine lavage 6.5 days later yielded one embryo. Cooled-transported semen from a different stallion (Stallion B), housed at a different reproductive center, was used on the second cycle. The mare ovulated one follicle and uterine lavage 8 days post-ovulation yielded two embryos, one of which was substantially larger than expected. The recipient mare receiving the larger embryo carried the pregnancy to term. DNA parentage testing excluded Stallion B as the genetic sire and confirmed Stallion A as the genetic sire. The intervals from the dates of the original insemination with the genetic sire to the first cycle ovulation, second cycle ovulation and recovery of the embryos in question were 0 days (post-ovulation insemination), 18 days, and 26 days for the Arabian mare and 2 days, 18 days and 26 days for the Quarter Horse mare. The potential explanations for how an equine embryo could be recovered following an insemination 26 days previously include marked delayed fertilization or marked delayed embryonic development. Equine DNA parentage testing relies on the principle of exclusion, with the inheritance of a series of short tandem repeat (STR or microsatellite) markers evaluated in the foal, sire, and embryo donor mare. Parentage assignment based solely on breeding records would have been incorrect.

First report of mule-in-mule pregnancies with live births following embryo transfer

Many of the equid species can interbreed to produce viable but usually infertile offspring, with the mule (♂donkey [2n=62] × ♀mare [2n=64]) being the commonest of these hybrids. Despite its reduced ability to breed successfully, the mule has been shown capable of carrying and rearing horse and donkey foals using between-species embryo transfer (ET). These pregnancies have provided insight into the early development of the conceptus by highlighting the role of the uterine environment on endometrial cup development and lifespan (Allen et al., Reproduction, 1993); 98(1), 55-60). The aim of this preliminary study was to see if a mule (2n=63) was capable of carrying a mule pregnancy to term, which to the authors' knowledge has not previously been reported. Mule embryos were produced in vivo, by inseminating donor horse mares with fresh donkey semen. The uteri of donor mares were flushed on either Day 7.5 or 8 post-ovulation and three grade 1 expanded blastocysts and one grade 1 early blastocyst were recovered. In Argentina, two embryos were transferred using the Wilsher ET technique into each of two anestrous recipient muleson day 8 after initiating treatment with IM estradiol 17β for 3 days followed by long-acting progesterone every 7 days until day 100 of gestation. In Italy, two embryos were simultaneously transferred by a conventional ET technique into a cyclic recipient mule on day 5 after ovulation. The transfers resulted in two singleton pregnancies, in Argentina, and a twin pregnancy, which was manually reduced to a singleton, in Italy. Repeated ultrasound examinations showed normal conceptus and, subsequently, fetal development to term. Spontaneous delivery of three female mule foals occurred on Days 355, 342, and 335 of gestation, respectively and no placental abnormalities were recorded. No complications occurred during either parturition or peripartum, and the resulting mule fillies developed normally to weaning. To the best of the authors’ knowledge, this is the first report of mules foaling mules after embryo transfer. More studies are warranted to describe the hormonal profile of mule-in-mule pregnancy, especially equine Chorionic Gonadotrophin (eCG) which is known to be influenced by both maternal and fetal factors.

Transfer of Day 11 ruptured embryos can prevent luteolysis in recipient mares

Previous studies transferring Day 11 embryos to Day 11 or 12 recipient mares and then manually crushing the transferred embryo 24 hours later resulted in 83% (5/6) and 100% (6/6) mares, respectively, entering a period of luteostasis. Undertaking manual reduction 12 hours post transfer resulted in 100% (5/5) of Day 11 recipient mares becoming luteostatic. This work suggested that the mare could recognise the presence of an embryo within the uterus after only a short period of time. The present experiment aimed to shorten the window of the presence of an intact embryo still further by evaluating the ability of ruptured or damaged Day 11 embryos transferred to recipient mares to initiate a period of luteostasis. A total of sixteen Day 11 embryos were flushed from the uteri of donor mares. Embryos were moved to a petri dish containing 5mL of Vigro Hold Plus Solution where they were measured and graded. The intact embryos were ruptured using two 100µm diameter acupuncture needles, ensuring the blastocoele fluid was released into the medium. The ruptured embryo and the medium were then transferred to recipient mares at Day 11 (n = 8) or 12 (n = 8) post ovulation using a standard embryo transfer technique. Control Day 11 (n = 8) and 12 (n = 8) recipient mares received 5mL of Vigro Hold Plus. All recipient mares were monitored on alternate days post transfer using a combination of ultrasonography,including assessment of the corpus luteum using colour Doppler, and serial blood serum samples assayed for progesterone in peripheral blood to determine if, and when, they returned to oestrus. Mares with a functional corpus luteum with blood flow and progesterone levels >1ng/mL up to 25 post ovulation were deemed to have entered a period of luteostasis and at this time point luteolysis was achieved by administration of an i.m injection of prostaglandin. Transfer of the ruptured embryo and medium resulted in significantly more Day 12 recipients entering a period of luteostasis compared to Day 11 recipients (75% vs. 12.5%; 6/8 vs. 1/8; P= 0.04, respectively). Day 11 and 12 control recipient mares that received only medium had an incidence of luteostasis of 12.5% (1/8) in each group. Hence the presence of medium containing blastocoele fluid, a ruptured capsule and trophoblast within the uterus can prevent luteolysis around the time of maternal recognition of pregnancy (MRP) in the mare. If all or only some of the components of the ruptured Day 11 embryo are required to initiate luteostasis needs further study. If the mechanisms underlying luteostasis with this model mirror those of the MRP signal in pregnant mares this study also raises questions about the duration of the MRP signal and how long embryo migration within the uterus is needed to prevent luteolysis.

Success rate in a clinical equine in vitro embryo production program

In vitro embryo production (IVEP) via Ovum Pick-Up (OPU) and Intracytoplasmic Sperm Injection (ICSI) has become a popular breeding technique in Warmblood mares because of the high success rate and several practical advantages. IVEP offers a solution for a variety of reproductive issues including, but not limited to, sub-fertility in stallions or mares, poor quality or scarce frozen semen, difficulty in synchronizing donor and recipient mares, and inefficient use of recipient mares. In 515 OPU-ICSI sessions performed in our facility in 2021, a mean of 25.9 antral follicles were aspirated yielding an average 13.8 immature oocytes, which were shipped overnight to a specialized ICSI laboratory (Avantea). One or more blastocysts (range: 0–13 blastocysts) were produced from 78% of procedures with a mean of 2.12 blastocysts per session; the likelihood of pregnancy after transfer of a cryopreserved thawed IVP blastocysts in 2021 (n = 781) was 77.7%. Several donor mare, recipient mare, stallion and embryonic factors influence the likelihood of producing an in vitro blastocyst or achieving pregnancy. Approximately 60% of the transferred IVP blastocysts yield a foal; moreover, neither gestation length nor the health of foals is noticeably influenced by IVEP. On the other hand, a skewed sex ratio towards colts is apparent among IVEP foals resulting from day 7 but not day 8 embryos, suggesting that male embryos develop more rapidly in vitro. Although serious complications after OPU are uncommon, owners should be aware of their existence, because some complications can be life-threating.

57&#x2003;Influence of donor mare age on pre- and postimplanation embryo development within an equine ovum pick-up-intracytoplasmic sperm injection-embryo transfer (OPU-ICSI-ET) program over a three-year period.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Equine ovarian tissue xenografting: impacts of cooling, vitrification, and VEGF.

Ovarian tissue transplantation methods using cooled and cryopreserved samples have been attractive options for fertility preservation in animal models and humans. The aim of this study was to evaluate the impact of previous exposure to cooling, cryopreservation, and VEGF on the overall efficiency of equine ovarian tissue after heterotopic xenotransplantation in mice. The end points evaluated were follicular morphology and development, follicular and stromal cell densities, angiogenesis (i.e. the density of new and mature blood vessels), collagen types I and III fiber densities, and total fibrosis. Ovaries of adult mares were harvested after ovariectomy, and ovarian fragments were xenografted in the i.p. wall of BALB nude mice. Ten types of treatments involving different combinations of cooling, cryopreservation, xenografting procedures, and VEGF exposure were compared. The novel aspect of this study was the use of equine ovarian tissue xenotransplantation in mice, challenging the fragments with different combinations of treatments. The main findings were (i) cooling but not cryopreservation was effective in preserving the follicular morphology, (ii) a greater percentage of developing follicles but lower follicular and stromal cell densities were observed after ovarian tissue engraftment, (iii) exposure to VEGF increased new and mature vessels in cryopreserved-transplanted tissue, and (iv) an appropriate balance in the collagen types I and III fiber ratio in cooling-transplanted tissue was observed after exposure to VEGF. This study contributes to advancing knowledge in the preservation of ovarian tissue after cooling-cryopreservation and transplantation aiming to be applied to genetically superior/valuable horses, livestock, endangered animals, and, possibly, humans.Lay summaryDue to ethical limitations involving humans, the female horse (mare) has recently emerged as an alternative model for reproductive comparisons with women to optimize fertility restoration using ovarian tissue transplantation techniques. This study determined if ovarian tissue from donor mares (n = 3), exposed or not to vascular endothelial growth factor (VEGF) before transplantation, better survives for 7 days after transplantation into mouse hosts (n = 12). Tissues submitted to different combinations of cooling, freezing, and transplanting treatments, along with control groups, were evaluated using the parameters morphology, development, the density of immature eggs (follicles), the density of supportive (stromal) cells, collagen protein proportions, and density of blood vessels. Frozen-thawed treatments had lower percentages of normal follicles. Exposure to VEGF increased blood vessel densities in frozen tissue and favored adequate collagen levels in cooled-transplanted treatments. In conclusion, VEGF exposure seems to be beneficial for mare ovarian tissue transplantation and warrants further investigation.

Studies on Equine Embryo Transfer: Effect of Flushing Media and Environment in Maiden and Adult Arabian Mares

The advances in equine embryo transfer (ET) allowed its application in small facilities with promising results. ET has been carried in 27 donor mares where the procedures were applied in 2 facilities; 1) farm group (n=18) where the ET was carried out in stable confined group. 2) procedures were applied in the lab (lab group, n=9). Two solutions, phosphate buffered saline (PS) or ringer lactate (RL), were used as flushing media. ET was applied by directing the pipette with finger through the cervix. In farm group 39 flushes resulted in 17 embryos with 43.6% recovery rat with 63.2% pregnancy rate. In lab group 16 flushes resulted in 14 embryos with recovery rat 87.5% and pregnancy rate 71%. In conclusion, ET could be conducted in equine farms without affecting recovery or pregnancy rate, RL could be used as flushing media without decreasing pregnancy rate, and cervical penetration dose not decreasing pregnancy rate.

Horse Agribusiness in Rio de Janeiro, Brazil: trade and economic aspects of Mangalarga Marchador farm production

This study aimed to assess the economic and commercial performance of the Mangalarga Marchador horse breeding, and production costs in the Rio de Janeiro. Data was collected from the ABCCMM archives and from associated breeders, selected through stratified sampling by the mesoregions of the State of Rio de Janeiro. The main means of marketing the animals is selling on the farm. The average of horse’s sale per stud farm in the Rio de Janeiro is 14.92/year with the average of R$16,628.46/horse, generating an estimated sale at the farms of around R$192,026,783.88/year. The sale of mating (8.77 mating/year) and weaned foals (5.44 foals/year) contributed to the largest volume of sales at the stud farms. Donor mares (R$57,318,908.50) and mares (R$42,958,357.20) represented the highest in terms of market value throughout the Rio de Janeiro. The Coastal Lowlands mesoregion had the highest average of animals sold per stud farm/year (40.57 animals) and the Central Fluminense mesoregion had the lowest average (25.39 animals). The average commercialization per farm/year corresponds to R$385,667.90 and the average total value traded in the Rio de Janeiro is R$465,880,252.32/year. The average is 6.52 hired employees, which is equivalent to an average monthly labor gross cost of R$11,286.00. Rio de Janeiro employs around 5,584 people directly, the greatest numbers in the Metropolitan mesoregion (1,833 employees), and the lowest concentration in the Northern Fluminense mesoregion (530 employees). Together with the production costs, Mangalarga Marchador horse’s business turns over more than R$650 million per year in the Rio de Janeiro.

Relationships between blood and follicular fluid urea nitrogen concentrations and between blood urea nitrogen and embryo survival in mares

High blood urea nitrogen (BUN) concentration is linked to low fertility in cows and ewes; however, this relationship has not been reported in mares. The study characterized the relationship between BUN and follicular fluid urea nitrogen (FUN) during follicle growth (Experiment 1) and the impact of BUN from embryo donors on the pregnancy outcome of recipient mares (Experiment 2). In experiment one, follicular fluid and blood samples were collected from mares during diestrus with growing follicles and during estrus with pre-ovulatory follicles (n = 16 and 10 mares, respectively). In experiment two, BUN concentrations of embryo donors were related to pregnancy outcome after embryo transfer. In experiment one, there was a strong positive correlation between BUN and FUN (R = 0.83; P < 0.0001), with higher BUN in mares with growing follicles than with preovulatory follicles (P = 0.004) and higher FUN in growing follicles than in preovulatory follicles (P = 0.031). In experiment two, BUN was higher in donor mares that produced unsuccessful embryos compared to donor mares that produced embryos resulting in successful pregnancies at D14 (P < 0.03). Additionally, there was an effect of age (P = 0.01) and interaction between age and lactation (P = 0.009) in donor mares for embryo survival after embryo transfer. Donor mares with unsuccessful embryos were older than donor mares with successful embryos. Therefore, these experiments showed that BUN was related to follicular fluid environment as well as to the survival of Day 7–8 embryos after transfer to recipient mares.

Ovum pick up and in vitro embryo production in pregnant mares

The effectiveness of different treatments with recombinant equine FSH to stimulate follicular growth, multiple ovulations and embryo production in seasonally anovulatory mares was evaluated. During mid-winter season (July–August in Argentina, South America) forty light breed donor mares, presenting follicles <10 mm in diameter and no CL at ultrasound examination (deep-anestrus), were randomly assigned (n = 10/group) to one of the following treatments: Group 1: twice daily intramuscular (IM) injections of 0.65 mg reFSH (AspenBio Pharma, CO), Group 2: once daily IM injection of 1.3 mg reFSH, Group 3: twice daily IM injection of 0.32 mg reFSH, and Group 4: once daily IM injection of saline (control). Treatment was administered until a follicle of 35 mm was observed or for a total period of 10 days. When the largest follicle reached ≥35 mm in diameter, treatment was discontinued and 2500 IU hCG was injected intravenously (IV) 36 h later. Mares receiving hCG were inseminated with fresh semen every 48 h until ovulation(s) were detected or one dose of frozen semen (250 × 106 motile sperm) after the first ovulation was detected. Eight days after first ovulation, transcervical embryo recovery was performed. Recovered embryos were non-surgically transferred to anovulatory estrogen/progesterone treated recipients and pregnancy diagnosed by ultrasonography 7, 14 and 21 days later. All mares receiving reFSH, but none receiving saline control, responded to the treatment with follicular growth. On average, 6.5 days of reFSH treatment were required for mares to develop follicles of ovulatory size (>35 mm). Ovulations were detected in 80% of mares in Groups 1 and 2, 50% of mares in Group 3 and in none of Group 4 (Control). Among ovulating mares, no differences in number of ovulations, number of embryos recovered, or pregnancy rates were observed among reFSH treatments. Of treated mares, 6, 7, and 5 produced embryos in Groups 1, 2, and 3, respectively. The average embryo recovery rate per ovulated mare was 88%. The average embryo recovery rate per ovulation was 43%. Overall, a 59% pregnancy rate was achieved. These results indicate that treatment with reFSH during deep anestrus results in follicular development, ovulation of fertile oocytes, and production of embryos that established viable pregnancies after transfer. Also, a single daily administration of reFSH was as effective as two daily administrations, which allows for a simplified administration regimen.

Laboratory Production of Equine Embryos

Assisted reproduction technologies (ART) are well developed in humans and cattle and are gaining momentum also in the equine industry because of the fact that the mare does not respond to superovulation but can donate large numbers of oocytes through ovum pick up (OPU). After collection, the oocytes can be fertilized by intracytoplasmic sperm injection (ICSI) using a variety of stallion semen samples, even of poor quality, and the resulting embryos can establish high pregnancy rates after cryopreservation and transfer. The discoveries that equine oocytes can be held at room temperature without loss of viability and that an increase in vitro maturation time can double the number of embryos produced are fueling the uptake of the OPU technique by several clinics that are shipping oocytes of their client’s mares to specialized ICSI laboratories for embryo production and freezing. In this article, we present a retrospective analysis of 10 years of work at Avantea with a special focus on the last 3 years. Based on our data, an average production of 1.7 to 2 embryos per OPU-ICSI procedure can be obtained from warmblood donor mares with a pregnancy rate of 70% and a foaling rate in excess of 50%. OPU-ICSI offers the added value of freezing embryos that allows the development of embryo commercialization worldwide to the benefit of top horse breeders who are endorsing this technology as never before.