Recipient Mares Research Articles

A novel method for equine embryo transfer from contaminated recipient mares into second healthy recipients for surviving embryos

Embryo transfer (ET) is regarded as one of the reproductive methods for genetic improvement initiatives in the horse business. The current study aimed to increase the success rate of ET by re-flushing and re-transferring of embryos from contaminated recipient mares into second healthy recipients. The study involved 25 recipient mares that demonstrated embryonic vesicles inside intraluminal fluid (ILF) on ultrasound scanning 2 ± 0.5 days after ET. Seven recipient mares were left without re-flushing and re-transferring of embryos (control group) and 18 recipient mares (treated group) were subjected to re-flushing and re-transferring of the embryos to second healthy and prepared recipients. The retrieved fluid from treated mares was subjected to microbiological and cytological examinations. The results revealed mixed and single infections, as well as polymorphonuclear neutrophils (PMNs). The total bacterial count and cytology findings revealed that all first recipient mares had endometritis. All control mares showed embryonic death at 14 ± 1.1 days (13–15 days) of embryonic age. In treated group, microscopic evaluation of retrieved embryos revealed a well-developed thick capsule in 12/18 (66.7 %) of them and a normal capsule in 6/18 (33.3 %). The pregnancy rate in well-developed capsule embryos was greater (66.7 %) than in typical capsule embryos (33.3 %). This study illustrates the value of swabbing the uterus of the recipient mares during estrus prior to ET and their checking at 2 days after ET. In addition to boosting pregnancy rates in ET commercial programs, our novel method can protect the embryos from the harmful effects of the inflammatory process and help them survive.

PSII-14 Climate and conception: Investigating the resilience of equine embryo survival to ambient temperatures in southeast Texas

Abstract Amidst the unique challenges posed by the humid and hot climate of southeast Texas, this study aimed to assess the establishment of pregnancy from transferred equine embryos, hypothesizing that an increase in ambient temperature would lead to a decrease in pregnancy establishment. To achieve this, we utilized 17 embryo donors and 46 recipient mares (3 to 9 yr of age), all anatomically and clinically normal with no history of reproductive problems. Breeding records for 49 transferred d 8, grade-1 embryos from a single private horse farm in southeast Texas, along with the reported high and low ambient temperatures on the day of transfer, were analyzed using logistic regression via PROC GLIMMIX in SAS v9.4 with fixed effects for the reported high and low ambient temperatures on the day of transfer, with a random statement for donor and sire. The pregnancy rate for all embryo transfers was 79%. Surprisingly, our results revealed that neither the reported high ambient temperatures (P = 0.16) nor reported low ambient temperatures (P = 0.19) significantly influenced the likelihood of pregnancy establishment observed at d 14 post-ovulation of transferred equine embryos. Furthermore, no statistically significant influence (P > 0.05) on pregnancy establishment was observed when embryo transfers were conducted outside of the thermal neutral zone (5°C to 20°C). Within the context of our study, these results suggest that environmental temperatures in southeast Texas do not significantly impact equine pregnancy outcomes.

The influence of lactation on insulin and glucose metabolism, lipid profile, and cytokines in pregnant mares

Glucose metabolism adapts to gestation, resulting in progressive physiological insulin resistance and increased insulin secretion to maintain maternal euglycemia and glucose availability for the developing fetus. These changes can impact mare fertility and maternal and neonatal health. This is the first comparison of body condition, regional adiposity, insulin and glucose dynamics, lipid metabolism, and cytokine production between lactating and non-lactating mares before, during pregnancy, and early postpartum. Twelve pregnancies from 9 broodmares, five nonlactating (NL) and seven lactating (L), were used. Evaluations were performed on the day of ovulation, at 55, 110, 165, 220, 275, and 330 days of gestation (D55, D110, D165, D220, D275, D330) and 21 days postpartum (21pp). Mares in the L group had lower basal insulin and glucose at the beginning of pregnancy, smaller area under the curve of insulin and glucose, and greater insulin sensitivity and glucose tolerance. Resistin was higher in D110 and D165 than in D0, D275, 330 and 21pp, while leptin was higher in D55, and in D110, at D110 it was equal to D0, D220, and D275, but higher than at D330 and D21pp. As for the groups, L presented lower body condition score (BCS), crest neck score (CNS), rump fat thickness (RUM), basal insulin, glucose area under the curve (AUCg), MIRG and higher RISQI, adiponectin and tumor necrosis factor (TNFα). There was no effect over time in non-esterified fatty acids (NEFA) concentrations between the L mares; in the NL, D275 presented higher concentrations than those of D0, D55, and D110, which in turn were equal to the other time points; there were higher concentrations in NL mares than L in samples D165 and D275. In conclusion, a different metabolic profile during pregnancy was detected, and NL mares were closer to the metabolic threshold for the occurrence of metabolic syndrome during pregnancy. Understanding the impacts of these differences on mare's health and their offspring's future is fundamental as most of our recipient mares for embryo transfer are non-lactating. Therefore, we suggest that further studies be performed to evaluate lactation's influence on mares' metabolic parameters.

Intracytoplasmic sperm injection zone: insights and applications from a university-based assisted reproduction laboratory

Intracytoplasmic sperm injection (ICSI) has become one of the most important tools used for in vitro production of embryos (IVP) in equine reproduction management programs around the world. This procedure is often performed in the US for several breeds and is used primarily to optimize foal production for broodmares and performance mares but also for stallions with limited semen availability or poor semen quality. As such, there are a limited number of US laboratories capable of using this method, which requires advanced training of personnel and specialized equipment. Many veterinarians and breeding farms currently aspirate ovarian follicles in cycling mares and ship oocytes to ICSI-capable laboratories, where embryos can be produced and typically vitrified for ultralow temperature storage and transport for transfer to recipient mares and establishment of surrogate pregnancies. The Veterinary Assisted Reproduction Laboratory in the School of Veterinary Medicine at the University of California, Davis, maintains a 3-dimensional approach to equine assisted reproductive technology. We offer commercial solutions to breeders, educational advice and training to other laboratories, veterinarians, and visiting scholars. Moreover, as a research laboratory, our objective is to analyze and apply observations from IVP to elucidation of complex developmental problems such as embryonic and fetal loss. In 2018, we reported the birth of the first foal produced at UC Davis using ICSI, and we have since developed a commercial ICSI program for practitioners and horse breeders in the US. Today, our laboratory receives thousands of immature oocytes for ICSI sessions every year, with an average of 2 embryos per mare-session. Our research is focused on molecular, cellular and genetic aspects of gamete biology, perifertilization events, and early equine development using an array of tools including advanced microscopy, sequencing, and time-lapse imaging of developing embryos. In this review, we have highlighted our laboratory’s current methods for commercial equine IVP and research and clinical studies conducted to optimize IVP in horses.

Vaginal and Uterine Microbiota of Healthy Maiden Mares during Estrus.

This descriptive cross-sectional study compared the microbiota of the uterus, vagina, clitoral fossa (CF), and perineal skin in healthy maiden mares during estrus. Twelve synchronized, healthy maiden mares (3-4 years old) from one single recipient mare herd were included. Microbial communities were characterized by amplifying the V3-V4 region of the 16S rRNA gene using the Illumina MiSeq platform. The uterine and vaginal microbiota had significantly lower richness (Chao-1) than the skin (p < 0.05). The uterine and vagina bacterial composition was similar in presence and abundance and could be differentiated from that of the CF and perineal skin. The microbial composition (Jaccard and Bray-Curtis distances) significantly differed across body-site locations (p < 0.05), which explained approximately 14% and 19% of the variation in microbial composition for Jaccard and Bray-Curtis distances, respectively. Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes were the dominant taxa in the uterus and vagina, with higher proportions of Proteobacteria in the vaginal samples compared to the uterine samples. Streptococcaceae and Staphylococcaceae were present in high abundance in the uterine and vaginal samples, while Lactobacillaceae were not (<10%). We demonstrate that the uterine and vaginal microbiota of healthy maiden mares during estrus is similar but both distinct from that of the CF and perineal skin.

Development of the first equine blastocyst produced by conventional IVF and in vitro culture in Europe resulting in the birth of a foal

In this case report, the production of the first equine blastocyst using conventional in vitro fertilization (IVF) and in vitro embryo culture methods in Europe is described. A healthy foal was born after transfer of a blastocyst using a complete in vitro production process, including in vitro oocyte maturation, in vitro sperm capacitation, fertilization in vitro and culture to the blastocyst stage in vitro. Oocytes were recovered from ovaries of slaughtered mares. After in vitro maturation, the oocytes were fertilized by conventional IVF and cultured for nine days. One embryo reached the blastocyst stage and was vitrified. After the selection of a suitable recipient mare that had ovulated four days earlier, the blastocyst was thawed and transferred. Five days after embryo transfer, a single embryonic vesicle was detected by transrectal ultrasonography. After a normal pregnancy of 323 days, a healthy colt was born. Parentage testing via microsatellite genotyping confirmed that the recipient was excluded as the foal’s dam and that the stallion whose semen was used, qualified as the sire.

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

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

Factors affecting the efficiency of equine embryo transfer (EET) in polo mares under subtropical conditions of Pakistan.

Equine embryo transfer (EET) is a prominent technology in the equine breeding industry, and its efficacy is affected by a number of factors. The current study aimed to determine the effects of the breed of donor/recipient mares, estrus/ovulation induction treatment, cooled transportation of embryos, and synchrony between donor and recipient mares on the efficiency of the EET under subtropical conditions of Pakistan. A total of eighty-four (n = 84) Polo-playing donor mares (Argentino-polo = 41 and Anglo-Arab = 43) and seventy (n = 70) recipient mares (light breed = 26 and heavy breed = 44) were used for EET. The donor mares exhibiting natural estrus (n = 28) were detected by teaser a stallion, and corpus luteum (CL) having mares (n = 56) were treated with prostaglandin (150 μg of Cloprostenol) for estrus induction. The mares' follicular growth was monitored through ultrasonography until the dominant follicle's size reached 35 mm or more with a moderate to obvious uterine edema score. Afterward, the mares were treated either with GnRH, i.e., 50 μg of Lecirelin acetate (n = 41) or Ovusyn, i.e., 1500 IU hCG (n = 43). Insemination with chilled semen was performed 24 hours later. The embryos were collected non-surgically, 7 or 8 days after ovulation, from the donor mares. The collected embryos were transferred into the well-synchronized recipient mares as fresh (n = 44) or chilled (n = 26) embryos. The pregnancy after ET was checked through ultrasonography. Statistical analysis revealed that the embryo recovery rate (ERR) remained significantly higher (P<0.05) for the Prostaglandin (PG) treated group of donors as compared to the natural heat group of donors. The breed of donor mares, type of ovulatory treatment given, and day of embryo collection did not significantly (P>0.05) affect the ERR. There was no significant effect of the type (fresh vs chilled), classification, and stage of development of embryo on pregnancy outcomes (P>0.05). ET pregnancy rate was significantly affected by the breed of recipient mares and ovulation synchrony between donor and recipient mares (P<0.05). In conclusion, under the subtropical conditions of Pakistan, PG-based estrus induction of donor mares, breed of recipient mares, and ovulation synchrony between the donor and recipient mares had a substantial effect on the efficiency of EET.

Embryo transfer into anestrous recipient mares prepared using clomiphene citrate and short-acting progesterone

Embryo transfer (ET) is an important technology for genetic improvement programs in the equine industry. Many protocols have been used for preparation of recipients. This study evaluates a new protocol for preparation of acyclic embryo recipient mares using clomiphene citrate (CC) and short-acting oral progesterone (Altrenogest). Seventy-two native breed recipient mares were divided into 2 groups. Group 1(G1) included 60 non-cyclic mares with follicular structures ≤ 15 mm in diameter that received CC and Altrenogest. Group 2 (G2) included 12 cyclic recipient mares that served as a control group. G1 mares were treated with oral CC at a dose of 250 mg for 4 days followed by oral administration of Altrenogest for 6 days. Ultrasonography was carried out for evaluation of uterine echotexture response to hormonal treatment, and pregnancy diagnosis post ET. Serum estradiol and progesterone concentrations were also assessed. All data were statistically analyzed. The results revealed that the serum progesterone concentrations in G1 were higher than G2 (P < 0.001). There was no difference in the estradiol concentrations between both groups during diestrus phase (P > 0.05). The pregnancy rate was higher in G1 (83.3 %) than G 2 (66.6 %). In conclusion, using oral CC and Altrenogest, as a new protocol, was effective for preparation of acyclic recipient mares in this study.

Effect of hCG and prostaglandin on ovarian, luteal development, and hormonal changes in embryo donor mares during the hot summer months in subtropics.

Commercial embryo flushing of horses has required hormonal management of both the donor and recipient mares throughout the breeding season. This study aimed to find out the effect of using human chorionic gonadotropin (hCG) and prostaglandin F2α (PG) on the ovarian and uterine dynamics and hemodynamics, estradiol (E2), progesterone, oxidants-antioxidants, and blood biochemicals in embryo donor mares during the hottest months of the year in a subtropical climate. Three Control estrous cycles of native mares (10-20 years; N = 10) followed by two treated cycles with hCG and PGF2α were examined daily from May to August using Doppler ultrasound with blood sampling. Circulating, progesterone (P4), total cholesterol, total proteins, albumin, haptoglobin, nitric oxide (NO), catalase, alkaline phosphatase, lactate dehydrogenase (LDH), and myeloperoxidase were measured in blood serum. Days during the control estrous cycle impacted the dominant follicle (DF) diameter ( p < 0.0001), antrum diameter ( p < 0.0001), area ( p < 0.0001), antral area ( p < 0.0001), and color area % (p > 0.05), and corpus luteum (CL) diameter ( p < 0.0001). PG tended to impact DF diameter (p > 0.05) but influenced its antrum diameter (p < 0.05), color area (p < 0.05), CL diameter (p < 0.01), and area (p = 0.013). Days after hCG tended to impact DF antrum diameter (p > 0.05) and the antrum area (p > 0.05), but influenced CL diameter ( p < 0.0001). PGF2α and hCG increased uterine horn area (p = 0.016) and color area (p = 0.023), total cholesterol ( p < 0.0001), and NO ( p < 0.0001) levels but hCG increased the levels of myeloperoxidase (p < 0.005), total proteins (p < 0.001), and albumin ( p < 0.0001). Globulins achieved the highest level (p = 0.054) but the Albumin/globulin ratio reached a minimum value on Day 0 of the control mares ( p < 0.0001). PGF2α increased LDH ( p < 0.0001) and sharply declined (p = 0.028) progesterone. In conclusion, the treatment protocols of hCG and PGF2α showed minimal effects on the produced ovulating follicles and can be used during the summer season to manage embryo donor mares.

Current state of horse embryo transfer technology in Russia and factors influencing its effectiveness

The article presents data on activity in the field of embryo transfer in the Russian horse breeding system and analyzes of the factors influencing the success of the use of this technology. The results of Russian reproductive specialists work over the past 15 years have been collected (144 procedures of embryo recovery in 52 mares). The influence of age and degree of synchronization of ovulations between donors and recipients on the level of embryo recovery and survival rates was analyzed. It has been established that the highest embryo recovery rate is observed in mares of 2- 10 years old (yo) - 84.6%. In mares of 11-16 yo, this figure decreased to 64.3% (p˂ 0.05), and for 17-24 yo mares - to 42.8% (p˂ 0.001). The survival rate of embryos in the uterus of recipient mares of 3-14 yo remains at 70- 71%, in mares of 15-20 yo it sharply decreased to 42.8%. It has been shown that embryo transfer to a recipient whose ovulation occurred 1 day earlier or within a period of up to 5 days after the donor's ovulation provides a survival rate of 79% until the 35th day of pregnancy. When the recipient ovulation occurs 2-4 days earlier than the donor ovulation, the result of embryo transfer was dramatically reduced (7 ,7±7,4%, p˂0.001).

A simplified grading system for in vivo and in vitro derived vitrified equine embryos

The ability to predict the viability of embryos before vitrification and thawing has important commercial applications in any breeding program. The aim of this study was to develop and evaluate a simplified embryo grading system for both in vivo- and in vitro-derived vitrified day 8 embryos. The in vivo derived (n=109) and in vitro – intracytoplasmic sperm injection derived (n=145) embryos were produced in a commercial embryo program. The embryos were classified as Grade 1, 2 or 3 based on the amount of extruded material between the trophoblast and the zona pellucida observed during the vitrification process. The embryos were vitrified at day 8 of development in a two-step system with increasing concentrations of dimethylsulfoxide and ethylene glycol and 0.5 M sucrose in the final solution. Each embryo was thawed in 0.3 M and then 0.15 M sucrose before transfer into holding medium for non-surgical transfer into a recipient mare. Analysis of the relationship between the embryo grading system and pregnancy rates after vitrification, thawing and transfer of in vivo and in vitro derived embryos confirmed that there was a significant effect of origin (in vivo vs in vitro; P ≤ 0.05), and embryo grade (P ≤ 0.001) on embryo survival after transfer. In conclusion, this simplified grading system is predictive of embryo survival for both in vivo- and in vitro- derived vitrified equine embryos.

Age and treatment on the day of embryo transfer in recipient mares affect likelihood of pregnancy.

This retrospective evaluation of data from a large commercial embryo transfer facility aimed to determine the extent to which age and treatment on the day of embryo transfer in recipient mares influence the likelihood of pregnancy. Embryo recovery was carried out on days 8-10 post-ovulation using transcervical uterine flushing. Recipient mares grouped according to their age were treated once on the day of embryo transfer (Day 3-8 post ovulation) and were assigned randomly to 1 of 3 groups: Mares in Group A (n=101) received antispasmodic, antimicrobial, and anti-inflammatory drugs. Mares in Group B (n=100) received gentamicin and flunixin meglumine. Group C (control) (n=103) did not receive any treatment. Detomidine (0.008 mg/kg bwt i.v.) was administered to all recipients before transfer of the embryo. The influence of treatment and recipient´s age was calculated using binary logistic regression. Day 16 post-transfer pregnancy rates were highest in Group A (74/101, 73.3a%), when compared to Group B (60/100, 60%), and Group C (57/103, 55.3b%) (a vs b, p<0.05). Pregnancy loss rates at D45 were not different between groups, A (8/74, 10.8%), B (5/60, 8.3%), and C (6/57, 10.5%), respectively (p>0.05). Pregnancy losses were increased in recipient mares 17-22 years (33.3a%) compared to younger recipient mares (2-6 years 7b%, 7-11 years 10%, 12-16 years 8b%) (a:b p<0.05). The regression model showed that the predicted probability for pregnancy after embryo transfer decreased as the age of the recipient mare increased for treated recipients in Group A (p=0.012), there was no effect of treatment and recipient´s age in Group B, and a decreased likelihood of pregnancy in recipients of advanced age (≥12 years of age) in untreated recipients (group C). Likelihood of pregnancy increased following single administration of antispasmodic, antimicrobial, and anti-inflammatory drugs at the time of embryo transfer in recipients 2-12 years of age. Likelihood of pregnancy in recipients decreased in recipients≥12 years of age. These results, obtained under the conditions of a large commercial embryo transfer program, offer an opportunity to improve pregnancy rates in recipient mares≤12 years of age.

Seasonal absence of supplementary corpora lutea in pregnant mares and the relationship with pregnancy loss

Two experiments were conducted in the Northern (UK) and Southern (Brazil) hemispheres to determine the effect of season (month of conception) on the development of supplementary CL (SCL) and the relationship with pregnancy loss. In experiment 1, 199 pregnancies were followed between Day 14 and term, to determine the number of SCL and pregnancy viability (Northern Hemisphere). From the 199 pregnancies, 178 were obtained from inseminations during the breeding season (March–September), while the rest, 21 pregnancies resulted from conceptions in the non-breeding season (October to February). Pregnancies conceived in the breeding season were more likely (P < 0.01) to have at least 1 SCL (75.8 %, 135/178) than pregnancies from the non-breeding season (33 %, 7/21). However, the pregnancy loss between Days 35 and 120 of pregnancy in mares with no SCL was similar (3.5 %, 2/57; P >0.1) than from mares with SCL (1.4 %, 2/142). In Experiment 2 (Southern Hemisphere), three groups of recipients were used based on their ovarian activity at the time of embryo transfer: Anestrus (n = 8), transitional (n = 7) and cyclic (n = 7) recipient mares. While all transitional and cyclic mares developed at least 1 SCL, only 50 % of anestrous recipients (4/8) developed SCL by 120 of gestation. In conclusion, the development of SCL in pregnant mares is influenced by the time of season of conception, therefore it appears to be regulated by the photoperiod and the endogenous seasonal variation in gonadotropin concentrations. Mares with no SCL were not at increased risk of pregnancy loss.

A touch of glass: cryopreservation of equine embryos by vitrification

The cryopreservation of equine embryos larger than 300 µm in diameter has proved problematic for many decades. However, there now exist protocols to undertake vitrification of such embryos with good pregnancy rates following their subsequent warming and transfer to recipient mares. This article discusses the reasons why equine embryos have proved so difficult to cryopreserve, and looks at cryopreservation methods before focusing on embryo vitrification. It also provides a historical perspective on the development of vitrification and details the latest techniques and emerging methodologies aimed at making embryo cryopreservation more accessible for practitioners.

Use of Doppler ultrasonography for selection of recipients in embryo transfer programs in horses

We aimed to evaluate the impact of corpus luteum (CL) and uterine characteristics accessed by B-mode and Color-Doppler ultrasonography in recipient mares at the time of embryo transfer (ET) on pregnancy outcomes. Recipient mares (n = 110), between days 3–9 after spontaneous ovulation, received a fresh embryo. Immediately before ET, the reproductive system was assessed by transrectal palpation for the following parameters: uterine tone (0–3), CL echogenicity (0–6), CL type (homogeneous, trabecular or anechoic center), luteal area (cm2), uterine echogenicity (0–3), uterine edema (0–3), luteal blood perfusion (0–100%) and uterine blood perfusion (1–4). Additionally, a blood sample was collected by puncture of the jugular vein for plasma P4 dosage. Retrospectively, recipients were classified according to the luteal area (small [≤ 6 cm2] or large [> 6 cm2]), luteal blood perfusion (low [≤ 55%] or high [> 55%]), and plasma concentration of P4 (low ≤ 9.98 ng/mL or high > 9.98 ng/mL). Pregnancy diagnosis was performed at 12 and 30 days of gestation. Luteal blood perfusion was significantly higher (P = 0.04) in pregnant recipients (n = 83) than in non-pregnant recipients (n = 27). Overall P/ET was higher (P ≤ 0.02) in mares with high luteal blood perfusion and high P4. Luteal blood perfusion was the most adequate significant (P = 0.01) predictor of pregnancy compared with the luteal area and plasma P4 concentration. Only luteal blood perfusion showed a linear (P = 0.03) and cubic (P = 0.004) effect on P/ET. In conclusion, CL blood perfusion determined by color-Doppler can be used in real-time to select recipients with the greatest chance of maintaining pregnancy in equine ET programs.

Single closed-tube quantitative real-time PCR assay with dual-labelled probes for improved sex determination of equine embryos

In addition to fulfilling many breeders’ curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro-produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro-produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications.

Estradiol treatment prior to progesterone favors embryo survival in anestrous recipient mares

Previous studies have shown a positive correlation between the length of estrus prior to ovulation and fertility in cyclic donors and embryo recipient mares. Furthermore, higher endometrial expression of Uterocalin (P19) was found in anestrous mares treated with a 5 day estradiol protocol, compared to recipient mares with a short or no estradiol priming. However, it is not known how this extended estradiol exposure influences embryo survival. The objective of this study was to determine the effect of different lengths of estradiol treatment on embryo survival, growth and proportion of mitotic cells. Three experimental groups of anestrous recipient mares were used: (1) mares treated with long-acting progesterone (LA P4) only (NE); (2) mares treated with a single administration of 2.5mg of estradiol benzoate (SE) 2 days before LA P4; and (3) mares treated with 8mg of estradiol split into increasing doses for 5 consecutive days (LE) ending 3 days before LA P4 treatment. Embryos were recovered from donor mares 8 days after ovulation, and embryo diameter and grade were recorded. Only grade I and II expanded blastocysts were used for this study; they were transferred into recipient mares using a modified Wilsher's technique 4 days after LA P4. Embryo recovery was performed 2 days after transfer by uterine lavage to examine embryo survival and growth rate inthe three experimental groups. After a positive recovery, embryos were evaluated for grade, size and fixed in 1% paraformaldehyde for subsequent staining with Hoechst 33342 for nuclear evaluation by confocal microscopy. The proportion of mitotic cells was assessed for 4 areas (10,000µm2) per embryo. The likelihood of recovering an embryo 2 days 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). The embryos recovered from the three different groups of recipients 48h after transfer did not, however, show any differences in size, morphology or in the proportion of nuclei undergoing mitosis (p>0.05). In conclusion, the duration of estradiol treatment before progesterone administration tended to influence embryo survival in anestrous recipients within 2 days of transfer. However, the grade and growth rate of surviving embryos did not differ among groups. This may indicate that if the endometrial environment is not optimal to support embryo development following transfer, the embryo dies and disappears relatively quickly, i.e. within 48 h, whereas surviving embryos continue to develop normally.

Influence of lactational status of the surrogate mare during early pregnancy on inflammatory markers in subsequently born foals

Nutrition during pregnancy has a significant impact on the offspring and affects health, growth and metabolism in several species, including equines. Studies have associated orthopedic problems in foals, such as osteochondritis dissecans (OCD), with diet of the broodmare during pregnancy resulting in high glucose concentrations, causing suppression of the central regulation of growth hormone (GH) secretion and subsequent disruption of the GH/IGF-1 (insulin-like growth factor-1) axis. It is very common to use non-lactating mares as embryo recipients, however little is known about the effect of lactational status on pregnant mare metabolism and the effect on the resulting embryo, fetus and the offspring's metabolism and development. Our study aimed to compare inflammatory cytokines, osteocalcin and IGF-1 in foals born from lactating recipient mares (LM; n=7) and non-lactating recipient mares (NLM; n=5) from days 1-28 after birth. Twelve healthy Mangalarga Paulista mares aged 5 to 15 years of age were used. Twelve foals, eight males and four females, born by eutocia were evaluated. Recipient LMs were lactating from the day of ovulation, until a maximum of 170 days of pregnancy (124 to 170). Foal evaluations were performed at birth (D1) and at 7, 14, 21 and 28 days of age (D7, D14, D21 and D28). Leptin (ng/mL), adiponectin (µg/mL), resistin (ng/mL) analyses were performed using ELISA, and interleukin6 (IL6; pg/mL), interleukin 10 (IL10; pg/mL), tumor necrosis factor (TNFα; pg/mL), osteocalcin (ng/mL) and IGF-1 (ng/mL) were evaluated using immunoassay kits. We used a factorial arrangement between group (LM x NLM) and time (D1, D7, D14, D21 and D28). The results are the mean and standard error of the mean. There was no significant interaction in any of the variables. Concentrations of IGF-1, IL6, IL10 and TNFα were higher in the NLM group (IGF1: 29.48±3.82 vs 176.53±65.8, p=0.0002; IL6:9.89±0.74 vs 13.57±1.33, p=0.01; IL10: 75.37±3.89 vs 100.75±8.84, p=0.0006 and TNFα: 8.08±1.08 vs 12.89±1.75, p<0.001). Only in IL10 concentration, there was a difference over time (D1: 114.11±1.04; D7: 72.08±8.13; D14: 12±6.96; D21: 72.98±5.25 and D28:100.44±9.25; p<0.001). Osteocalcin correlated positively with leptin (p<0.05; r=0.27). In this study, the recipient mares’ lactational status during the first trimester of pregnancy influenced concentrations of inflammatory markers in the subsequently born foal up to 28 days of life, with higher concentrations of IGF-1, IL6, IL10 and TNFα than in foals born from non-lactating recipient mares. This raises an important discussion on the impact of lactational status of the recipient dam on the future of her offspring. Acknowledgements: FAPESP (2017/05425-0 and 2020/10260-3) and CAPES

The mystery of the capsular vesicles: preliminary studies

An unexpected experimental finding was the presence of capsular vesicles (CVs) in the uterine flushings of mares 48h after transfer of a punctured Day 8 embryo. These CVs were thought to originate from fragments of capsule, in the same way that trophoblast fragments readily form vesicles in vitro. However, recovery of the capsule with the puncture hole alongside numerous CVs showed this was not the case. A preliminary study was undertaken to see if CVs formed in recipient mares following transfer of trophoblast vesicles (TVs) for 24h and if CV formation was uterine stage specific. Day 11 embryos were recovered from donor mares and the capsule removed before fragmenting the trophoblast. Small fragments of trophoblast were incubated in DMEMF12 (+ 5% FBS and pen/strep) for 48h in a Tri-gas incubator at 38°C. After 48h any degenerate TVs were discarded, and the remainder were washed in H199 (+ 5% FBS + pen/strep). Twenty to 50 TVs were transferred to the uteri of 25 recipient mares divided into 4 groups; G1, Days 3–5 post ovulation (n=6); G2, Days 6–8 (n=7); G3, Days 9–11 (n=6); Days 12–14 (n=6). The recipient mares’ uteri were flushed 24h after TV transfer and the recovered fluid searched. In all groups TVs were recovered with varying degrees of degeneration. Data were analyzed using aOne Way ANOVA on ranks with a post-hoc Dunn's test. The median number of CVs varied between G1–4 (0 vs. 68 vs. 17.5 vs. 6.5, respectively), with G1 versus G2 varying significantly (P<0.05). When adjusted for CVs per transferred TV, median values for G1–4 were (0 vs. 1.6 vs. 0.51 vs. 0.225), with G1 versus G2 remaining significantly different (P<0.05). The highest number of CVs recovered was 148 (2.96 per TV) from a Day 8 recipient, with a trend for a reduction in CV production after Day 8. These results suggest that CVs can form in the uterus following TV transfer providing the uterus is at a stage when an embryo would normally be forming a capsule, since transfers before Day 6 failed to produce CVs. Immunohistochemistry needs to be undertaken to confirm the CVs are identical in composition to in vivo produced capsules. Capsule formation in the mare is believed to involve interaction of the embryo with the endometrium, as in vitro produced embryos fail to produce a proper capsule. In vivo, developmentally regulated changes in composition of the capsule occur as its thickness increases up until at least Day 11. The mechanism of the formation of capsular vesicles remains a mystery as they are free in the uterus and do not appear to form around the trophoblastic vesicles.