Summary
Background
Monozygotic multiple pregnancy is rare in horses, but may be more common after transfer of an in vitro produced (IVP) embryo.
Objectives
To determine the occurrence, incidence, ...characteristics and outcome of monozygotic siblings arising from in vivo and IVP equine embryos.
Study design
Retrospective case series.
Methods
A total of 496 fresh in vivo and 410 frozen‐thawed IVP blastocysts, produced by intracytoplasmic sperm injection (ICSI) of in vitro matured oocytes from Warmblood mares, were transferred into recipient mares. The likelihoods of pregnancy and multiple pregnancy were calculated, and the clinical features and outcome of any multiple pregnancy were recorded.
Results
The likelihood of pregnancy after transfer of a single IVP or in vivo embryo was 62% (254/410) and 83% (413/496) respectively. The incidence of multiple pregnancy was 1.6% (4/254) and 0% (0/413) for IVP and in vivo blastocysts, respectively. More specifically, three IVP blastocysts yielded twin embryo propers/fetuses, and one IVP conceptus developed three distinct embryonic bodies. Interestingly, only one embryonic vesicle was detected at all ultrasonographic examinations prior to embryo proper development. Multiple embryonic bodies only became apparent at later scans to check for an embryo proper and heartbeat, or when the recipient mare aborted. Two twin pregnancies aborted spontaneously at 3 and 9 months, respectively, while the heartbeat was lost from all three embryos in the triplet pregnancy before day 35 of gestation. Twin reduction by per rectum compression of one fetal thorax was attempted at day 50 of gestation in the fourth case; however, both fetuses were lost.
Main limitations
Small number of cases.
Conclusions
In vitro embryo production resulted in a higher incidence of multiple monozygotic pregnancy, which could only be diagnosed after development of the embryo proper and is likely to result in pregnancy loss later in gestation if left untreated.
Summary
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
Summary
Background
Previous surveys reported a positive association between the length of the follicular phase and subsequent fertility in embryo transfer donor and Thoroughbred mares. However, it is ...unclear whether a longer oestrus positively influences fertilisation and oviductal development (oocyte quality, oviductal environment), or uterine receptivity and survival of the embryo in the uterus.
Objectives
To determine the effect of length of oestrus (characterised by duration of endometrial oedema) on likelihood of pregnancy and early embryo loss (EEL) in recipient mares after embryo transfer (ET).
Study design
Retrospective clinical study.
Methods
A total of 350 embryos recovered from 161 donor mares were transferred into 231 recipient mares during three consecutive breeding seasons. The following variables were analysed via two binary logistic regression models to determine their effect on pregnancy and EEL: 1) year of transfer, 2) season of transfer, 3) age of the recipient mare, 4) age of the donor mare, 5) operator performing the transfer, 6) singleton or twin embryo, 7) embryo size, 8) number of transfers to a given recipient in any one season, the use of 9) d‐cloprostenol and 10) hCG in the recipient mare, 11) day of ovulation of the recipient mare at ET, 12) number of corpora lutea (CLs) at ET, and 13) duration of oestrus in the recipient mare.
Results
Age of the donor mare (P = 0.01), operator (P = 0.008), number of CLs at ET (P = 0.05) and the number of days of endometrial oedema during the oestrus preceding ET to the recipient mare (P = 0.004) influenced the likelihood of pregnancy. Early embryonic loss was influenced only by the year of transfer (P = 0.014).
Main limitations
Retrospective design of the study. The involvement of several veterinary surgeons over the 3‐year period could have affected data recording.
Conclusions
The likelihood of pregnancy in recipient mares is positively correlated with the duration of endometrial oedema during the oestrus preceding ET. This suggests a role for an adequate duration of oestrogenic priming during oestrus on uterine receptivity and embryo survival.
REASONS FOR PERFORMING STUDY: Haemorrhagic anovulatory follicle is the most common pathological anovulatory condition in the mare, but its cause remains unknown. An experimental model to induce ...luteinised unruptured follicles (LUF) with flunixin‐meglumine (FM) has been developed. Luteinised unruptured follicles share similar morphological and hormonal characteristics with haemorrhagic anovulatory follicles. OBJECTIVES: To test the effect of intrafollicular administration of prostaglandins PGE₂ and PGF₂α during the periovulatory period on ovulation and pregnancy in FM‐treated mares. STUDY DESIGN: In vivo experiment in a crossover design. METHODS: Five mares were followed during 2 oestrous cycles each. All mares were given FM at 1.7 mg/kg bwt i.v. every 12 h from Hour 0 (Hour 0 = human chorionic gonadotrophin treatment) to Hour 36. In treatment cycles (n = 5), at Hour 32 the preovulatory follicle was punctured and 0.5 ml of a solution containing 500 μg of PGE₂ and 125 μg of PGF₂α was deposited within the follicle. In control cycles, water for injection was administered into the follicle at the same time. In 3 control and 3 treatment cycles, mares were also inseminated at Hour 24. Diagnosis of ovulation/LUF formation and pregnancy was performed by ultrasound examination between Hours 36 and 72 and 14 days after ovulation/LUF formation, respectively. RESULTS: During the treatment cycles, all mares ovulated normally (100% ovulation rate) 36–48 h after human chorionic gonadotrophin, while in 4 of 5 control cycles the mares developed an LUF (80%, P<0.05). All 3 inseminated mares became pregnant in the treatment cycles, but not in the control cycles. CONCLUSIONS: Intrafollicular treatment with PGE₂ and PGF₂α overcame the anovulatory effect of FM. This sheds new insights into the knowledge on the possible therapeutic options for ovulatory failure in the mare.
Over several breeding seasons, eighteen inseminated mares were examined at 8(±1)h intervals from late oestrus until after ovulation. The time of ovulation was determined as 4h before the first ...detection of ovulation. Examinations for pregnancy began at 8h intervals between 236 to 260h (late Day 9 to 10) post ovulation. When present an embryo was reduced by compression with a total of 132 reductions on Days 10 (n=15), 11 (n=47), 12 (n=36), 13 (n=27), 14 (n=5) and 15 (n=2). Ten mares were used on multiple occasions, undergoing between 3 and 16 embryo reductions each. Mares were subsequently examined, first to determine non-pregnancy, and then to determine whether luteolysis or luteostasis occurred. Luteostatic mares maintained an ultrasonically identifiable corpus luteum, and between Days 22 and 25 were returned to oestrus by administration of PGF2α. Increasing numbers of mares became luteostatic following embryo reduction as the number of days post ovulation increased. The earliest initiation of luteostasis occurred when an embryo was reduced at 252h (10.5 days). Between the 8h examinations on Days 10 to 12 there was an increase in the proportion of mares undergoing luteostasis. When mares were used on multiple occasions, individual animals had aset time point within a ±8h window post ovulation when they responded to the MRP signal and after which, but not before, embryo reduction resulted in luteostasis. For the eight mares which had embryos reduced ≥6 occasions, the time when luteostasis occurred was consistent in an individual but varied between mares between Day 10 (244–252h) and Day 14 (348h). For example, one mare which did not recognise the presence of an embryo until Day 14 (348h), had 10 reductions undertaken between Days 11 to 13 (268–332h) and all resulted in luteolysis, but all 3 reductions at or after Day 14 (348h) resulted in luteostasis. Despite the late timing of when the MRP signal in this animal was recognised, no spontaneous embryo loss had been seen in three previous pregnancies. These results suggest that the timing of the luteolytic cascade is variable between mares but relatively constant in its timing in an individual animal. This would imply a consistent set of physiological processes that program the endometrium for the luteolytic cascade in any given animal. Further data is needed to investigate whether oestrous cycle length and variation in the oestrous and dioestrous phases of the mare play a role in initiating the mechanisms that enable the uterus to respond to the MRP signal.
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.
•Small embryos have an increased EEL than larger embryos.•The Day of ovulation of the recipient does not influence the EEL.•There is a strong correlation between the embryo diameter at ET and 4 days later.
The success of invitro embryo production (IVEP) in horses has increased considerably during recent years, but little is known about the effect of the speed of invitro embryo development. Blastocysts ...(n=390) were produced by intracytoplasmic sperm injection of IVM oocytes from warmblood mares, cryopreserved, thawed and transferred into recipient mares on Days 3, 4, 5 or 6 after ovulation. The time required for invitro-produced (IVP) embryos to reach the blastocyst stage was recorded (Day 7 vs Day 8). The likelihood of foaling was affected by the speed of invitro embryo development and recipient day after ovulation at transfer. The odds ratio for foaling was ~0.63 for transfer of Day 8 (46%) compared with Day 7 (56%) IVP blastocysts. The highest likelihood of pregnancy (72%) and foaling (60%) was observed when IVP blastocysts were transferred to recipient mares on Day 4 after ovulation. Finally, the sex (colt:filly) ratio was higher after transfer of Day 7 (71%:29%) than Day 8 (54%:46%) IVP blastocysts, suggesting that the speed of embryo development is sex dependent. In conclusion, the speed of invitro embryo development in our IVEP system affects the likelihood of foaling and the sex of the foal.
The diameter of embryos recovered from mares on Day 8 after ovulation varies greatly, from as little as 130 μm to as much as 2500 μm. Several factors have been proposed to affect embryo size at ...recovery, one of which is the type of semen (frozen vs fresh or cooled-transported) used to inseminate the mare. In addition, it has been shown that smaller embryos (<300 μm) recovered on Day 8 are less likely than larger embryos to result in successful pregnancy after transfer. However, whether the actual age of the embryo (interval from fertilization to flushing) in relation to its size also influences the post-transfer viability is unclear. The aims of this study were: a) To determine the effect of semen type (frozen-thawed vs cooled-transported) on embryo diameter after pre-ovulatory insemination; and b) To establish the relationship between embryonic age, embryo size and likelihood of pregnancy and pregnancy loss following transfer. A total of 179 embryos were recovered from mares inseminated with: frozen semen post-ovulation 8 days previously (G1; n = 35); cooled-transported semen pre-ovulation 8.5 days previously (G2; n = 95); frozen semen pre-ovulation 8.5 days previously (G3; n = 30); and frozen semen post-ovulation 9 days previously (G4; n = 19). The effect of embryonic age, type of semen, donor mare and its age, number of ovulations and embryos per flush on embryo diameter was tested using a general linear model of variance. In addition, the proportions and survivals of small embryos (<300 μm) in each group were compared with those of respective larger embryos by Fisher's exact test. Embryonic age (P < 0.001) and age of the donor mare (P = 0.07), but no other factor, influenced embryo diameter. The proportion of small embryos was 42.9, 10.5, 10.0 and 10.5% for Groups 1, 2, 3 and 4, respectively. The pregnancy status of recipient mares 35 days post-transfer for small embryos from Group 1 (12/15; 80.0%) was not different (P > 0.1) from that of recipients of small embryos from Groups 2 to 4 combined (8/15; 53.3%).
•Embryonic age but not the type of semen used for insemination influenced embryo diameter at flushing.•Young mares (3–5 years old) tended to produce larger embryos than older mares.•Approximately 10% of recovered embryos were small (<300 μm) even when flushing was delayed until Day 8.5–9.
The production of equine embryos by somatic cell nuclear transfer (SCNT) is limited by the availability of immature oocytes. The main source of oocytes for SCNT comes from ovaries obtained from ...slaughter houses. However, in some countries there is no horse abattoirs or their number is limited which precludes the use of SCNT for the production of equine cloned embryos. Ovum pick-up (OPU) is a reproductive assisted technique which is growing rapidly in the equine industry which allows obtaining oocytes from live mares. OPU is mainly used in combination to ICSI (intracytoplasmic sperm injection) for the production of in vitro embryos. Oocytes collected from live recipient mares by OPU could be an alternative for SCNT in countries in which there are limited number of equine slaughter houses. However, it is unknown the effect of the oocytes’ source on the development of equine cloned embryos. The aim of this study was to compare the development of equine SCNT embryos produced using oocytes either retrieved by OPU or recovered from abattoir-sourced ovaries. For the study, a total of 1128 oocytes were used, of which 663 were from abattoir-sourced ovaries and 495 were from live mares programmed for OPU. The harvested ovaries were transported to the lab within 1 h for processing. The methods used for in vitro maturation (IVM), SCNT andembryo culture in vitro were identical for both sources of oocytes. The rates of maturation, cell fusion, cleavage and blastocyst formation at Day 7 were evaluated. Transferrable grade blastocysts were transferred to recipient mares 4 to 5 days after ovulation and pregnancy was diagnosed at Days 14 and 42 by ultrasound. The maturation rate of OPU-derived oocytes was lower than that of abattoir-derived oocytes (50.3 ± 2.7% vs. 61.9 ± 3.4%; P < 0.05). However, the rates of cell fusion (90.7 ± 2.6% vs. 81.9 ± 5.2%), cleavage (68.8 ± 3.9% vs. 61.9 ± 5.0%) and blastocyst formation (34.3 ± 2.8% vs. 25.4 ± 2.1%) were greater (P < 0.05) for OPU-derived embryos compared with abattoir-derived embryos. While similar proportions of OPU- and abattoir-derived blastocysts initiated pregnancy at Day 14, a greater proportion of OPU-derived blastocysts developed to Day 42 of gestation (13 of 50 embryos) compared with abattoir-derived blastocysts (3 of 27 embryos; P < 0.05). The results show that abattoir-sourced oocytes subjected to IVM have the potential to support the development of SCNT embryos that can initiate pregnancy. However, given the observed difference in subsequent in vivo development, the use of OPU-derived oocytes for equine SCNT embryo production is recommended. Further research is needed to improve the IVM of abattoir-sourced oocytes
The interval from both spontaneous and prostaglandin (PGF)-induced luteolysis to ovulation is greatly variable in mares. Several reports have shown a positive association between the length of the ...interval from PGF treatment to ovulation (ITO) and the subsequent pregnancy rate (PR). However, it is not known whether this association also occurs in estrous cycles with spontaneous luteolysis. The main objective of this study was to determine the effect of the duration of estrus-like echotexture of the uterus during the follicular phase on the subsequent PR in both spontaneous and PGF-induced cycles. A total of 768 estrous cycles from 325 thoroughbred mares were analyzed (401 estruses were induced with exogenous PGF and 367 cycles were not treated with PGF). The following factors were taken into account to determine the effect on PR: age of the mare, stallion, year of breeding, month of season, reproductive status of the mare, use of PGF treatment, duration of follicular phase with estrus-like echotexture, interovulatory interval (IOI; in spontaneous cycles), and ITO (in PGF-induced cycles). The age of the mare (P = 0.017), mare status (P = 0.031), the ITO (P = 0.041), and the duration of the follicular phase with estrus-like echotexture (P < 0.001) influenced the PR. The PR increased with the duration of estrus and of endometrial edema in both PGF-induced and spontaneous cycles. The correlation between the duration of endometrial edema and the IOI and ITO was positive (r = 0.5) and significant (P < 0.05).
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