Speed of in vitro embryo development affects the likelihood of foaling and the foal sex ratio.

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.

Effect of exogenous progesterone administration on luteal sensitivity to PGF during the early development of the corpus luteum in mares and cows.

The objective of this study was to determine the effect of exogenous progesterone administration at ovulation and during the early development of the CL, on its future sensitivity to a single administration of PGF2a in mares and cows. Horse Retrospective reproductive data from an equine clinic in the UK during three breeding seasons were used. Mares were divided into: control group, cycles with single ovulations; double ovulation group cycles with asynchronous double ovulations; and PRID group: cycles with single ovulations and treatment with intravaginal progesterone device (CIDR) immediately after the ovulation. All mares were treated with d-cloprostenol (PGF) at either: (i) 88 hr; (ii) 96 hr; (iii) 104 hr; or (iv) 112 hr after the last ovulation. Cattle A total of nine non-lactating Holstein cows were used. All cows were administered PGF14 d apart and allocated to one of two groups control group GnRH was administered 56 hr after the second PGF administration. CIDR group CIDR was inserted at the same time of GnRH administration. All cows were administered PGF at 120 hr post-ovulation. The complete luteolysis rate of mares with double ovulation (66.7%) and those treated with exogenous progesterone (68.4%) was significantly higher than the rate of mares with single ovulation (35.6%) at 104 hr. In the cow, however, the treatment with CIDR did not increase the luteolytic response in cows treated at 120 hr post-ovulation. In conclusion, the degree of complete luteolysis can be influenced by increasing the concentration of progesterone during the early luteal development in mares.

The mare as a model for luteinized unruptured follicle syndrome: intrafollicular endocrine milieu.

Luteinized unruptured follicle (LUF) syndrome is a recurrent anovulatory dysfunction that affects up to 23% of women with normal menstrual cycles and up to 73% with endometriosis. Mechanisms underlying the development of LUF syndrome in mares were studied to provide a potential model for human anovulation. The effect of extended increase in circulating LH achieved by administration of recombinant equine LH (reLH) or a short surge of LH and decrease in progesterone induced by prostaglandin F2α (PGF2α) on LUF formation (Experiment 1), identification of an optimal dose of COX-2 inhibitor (flunixin meglumine, FM; to block the effect of prostaglandins) for inducing LUFs (Experiment 2), and evaluation of intrafollicular endocrine milieu in LUFs (Experiment 3) were investigated. In Experiment 1, mares were treated with reLH from Day 7 to Day 15 (Day 0=ovulation), PGF2α on Day 7, or in combination. In Experiment 2, FM at doses of 2.0 or 3.0 mg/kg every 12 h and human chorionic gonadotropin (hCG) (1500 IU) were administered after a follicle ≥32 mm was detected. In Experiment 3, FM at a dose of 2.0 mg/kg every 12 h plus hCG was used to induce LUFs and investigate the intrafollicular endocrine milieu. No LUFs were induced by reLH or PGF2α treatment; however, LUFs were induced in 100% of mares using FM. Intrafollicular PGF2α metabolite, PGF2α, and PGE2 were lower and the ratio of PGE2:PGF2α was higher in the induced LUF group. Higher levels of intrafollicular E2 and total primary sex steroids were observed in the induced LUF group along with a tendency for higher levels of GH, cortisol, and T; however, LH, PRL, VEGF-A, and NO did not differ between groups. In conclusion, this study reveals part of the intrafollicular endocrine milieu and the association of prostaglandins in LUF formation, and indicates that the mare might be an appropriate model for studying the poorly understood LUF syndrome.

Follicle Diameter and Systemic Hormone Interrelationships during Induction of Follicle Collapse with Intrafollicular Prostaglandin E2 and F2α in Mares.

The objectives were to determine: (i) whether intrafollicular administration of PGE2 and PGF2α to mares would hasten follicle collapse and (ii) the differences in reproductive hormone characteristics in mares with spontaneous and prostaglandin-induced follicle collapses. Six mares were followed for two oestrous cycles each: when the mares reached a follicle diameter of 30-35 mm and showed mild-to-moderate endometrial oedema, mares were administered a single 0.5 ml dose containing 500 µg PGE2 and 125 µg PGF2α (treatment cycle) or a placebo (0.5 ml of water for injection; control cycle) into the preovulatory follicle (Hour 0). Blood samples were collected, and serial ultrasound examinations were performed until follicle collapse. Treated mares showed follicle collapse significantly earlier (20.0 ± 5.9 h) than the control mares (72.0 ± 10.7 h). The LH, progesterone, total oestrogens and oestradiol concentrations did not differ between groups; however, the progesterone concentration increased more between 48 and 72 h after follicle injection in the treatment compared to the control cycles (P < 0.05). In conclusion, intrafollicular treatment with PGE2 and PGF2α hastened follicle collapse in mares without the simultaneous use of an inductor of ovulation; despite the early induction of follicle collapse, the profiles of LH and oestradiol were not altered. This study provides information on the role of prostaglandins (PGs) in the process of follicle wall rupture and collapse and suggests that this may happen even before the beginning of the sharp rise in circulating LH at the final stage of the ovulatory surge.

Effect of intrafollicular administration of PGE2 or PGF2α in early estrus on ovulation, hemorrhagic anovulatory follicles formation, progesterone secretion and pregnancy outcome in the mare.

This experiment was performed to evaluate whether intrafollicular treatment of PGE2 or PGF2α administered in early estrus would induce normal ovulation, progesterone production (Experiment 1) and pregnancy (Experiment 2). In Experiment 1, mares in estrus after 2 days of endometrial edema were injected in all largest dominant follicles (28-35 mm in diameter) with 0.5 mL of sterile water containing 500 µg PGE2 (n = 6), 125 µg PGF2α (n = 6) or placebo (n = 7) (Hour 0). Ultrasound examinations were performed daily, until ovulation or anovulation was detected, and daily blood samples were taken for 8 days. In Experiment 2, mares with a dominant follicle ≥35 mm after at least three days of slight-to-moderate endometrial edema, were injected with 500 µg PGE2 diluted in 0.5 mL of sterile water for injection in the follicle (PGE2 group; n = 9 mares and 11 dominant follicles). No puncture was performed in the control group (n = 9 mares and 11 dominant follicles). Mares from both groups were inseminated. In Experiment 1, all mares (6/6) in the PGE2 group ovulated within 24 h of treatment. The mean interval from intrafollicular injection to ovulation was shorter (P < 0.001) in PGE2 mares (24 ± 0 h) than in control mares (77 ± 9 h). Mares from the PGF2α group developed hemorrhagic anovulatory follicles (HAF) more often (7/7) than control mares (2/7); P < 0.05). The progesterone concentration in mares from the PGF2α group was lower (P < 0.004) than control mares in the early post-ovulatory period. The first significant increase in post-ovulatory progesterone concentration occurred earlier (P < 0.05) in mares from the control group than in mares from the PGF2α and PGE2 groups. In Experiment 2, more mares from the control group (7/9, 78 %) became pregnant than from the PGE2 group (2/9, 22 %) (P = 0.015). In conclusion, PGE2 alone induced follicle collapse in all treated mares within 24 h of administrations, while PGF2α blocked ovulation and induced formation of HAFs. However, the post-ovulatory rise in progesterone production was delayed and the fertility reduced in mares with ovulation induced by PGE2 compared to control mares.

Use of a split or single prostaglandin F(2α) treatment in a 6-day synchronization protocol for nonlactating dairy cows.

The 6-d timed artificial insemination protocol has been designed to advance luteolysis after the first administration of GnRH so that the preovulatory follicular diameter at second GnRH is reduced and thereby pregnancy outcome may be improved. To achieve an earlier and complete luteolysis (5 to 6 d after the first GnRH treatment), an extra PGF(2α) treatment must be administered to cows 24 h after the initial PGF(2α) treatment. Although the use of 2 PGF(2α) treatments increases labor costs resulting from the increased handling of cows, no alternative and efficient protocol with a single PGF(2α) treatment has been found to date. The objective of this study was to compare the effect of a modified 6-d synchronization protocol on the luteolytic response and final preovulatory follicle diameter. The study followed a crossover design: 14 nonlactating dairy cows were included in 2 treatment doses. All cows received a presynchronization treatment consisting of 2 administrations of a PGF(2α) analog (PGF) 14 d apart followed by treatment with GnRH 11 d later. After the first GnRH administration, one treatment consisted of 150 µg of d-cloprostenol 5 and 6 d later (split dose) and the other treatment consisted of 375 µg of d-cloprostenol as a single dose 6 d after the first GnRH (single large dose). All cows were then treated with a second GnRH 8 d after the first. The luteolytic response to treatment was evaluated by blood progesterone concentration and CL area regression -1 to 3 d relative to the last PGF treatment obtained by ELISA and ultrasonography, respectively. Fewer cows of the split dose tended to have complete luteolysis 3 d after the last PGF treatment compared with the cows of the single large dose (35.7 and 64.3%, respectively). The final preovulatory diameter of the dominant follicle was similar in cows from the split dose and single large dose (13.7 ± 0.3 and 13.1 ± 0.5mm, respectively). Our results support the modification of the 6-d synchronization protocol by administering a single high dose of PGF 6 d after GnRH (with the subsequent reduction in labor resulting from reduced handling of animals) without detrimental effects on the luteolytic response of dairy cows and preovulatory diameter of the dominant follicle compared with the original protocol. However, this modification of the 6-d synchronization protocol should be tested in a large field study involving fertility data with lactating cows before its use can be recommended.

Ultrasound confirmation of ovulation in mares: a normal corpus luteum or a haemorrhagic anovulatory follicle?

The most common pathological anovulatory condition that occurs spontaneously during the breeding season in the mare is the haemorrhagic anovulatory follicle (HAF). A relatively high proportion of mares, soon after ovulation, develop a corpus haemorrhagicum (CH) with a central lacuna. This type of corpora lutea may resemble an HAF, which may complicate the accurate diagnosis of ovulation. The main objective of this study was to compare the ultrasound data of mares examined frequently with HAFs and CHs to elucidate whether it is possible to distinguish them from each other. A total of 135 ovulating mares were classified according to the morphology of the corpus luteum (CL) in mares with: a solid CL, a CH with small or with large central cavities. Ultrasound characteristics of the development of 11 HAF and 13 CHs with a large central cavity were compared. The pre-ovulatory follicular diameter of ovulatory mares was significantly correlated with the diameter of CH with large central cavities. The percentage of mares with post-ovulatory areas eligible to be mistaken with a CH was <25%. Although a predictive diagnosis of an HAF/CH can be made on the basis of several ultrasonographic endpoints, the only parameter that allows a definitive diagnosis is the thickness of the luteal border. This is <3 mm in HAFs in contrast to >5 mm in CHs. However, this only applies when the unidentified structure has non-organized contents.

The effect of dose and type of cloprostenol on the luteolytic response of dairy cattle during the Ovsynch protocol under different oestrous cycle and physiological characteristics.

The objective of this study was to characterize the effect of dose and type of cloprostenol (CLO) on the luteolytic response of dairy cattle during the Ovsynch protocol under different oestrus cycle and physiological characteristics. Twelve non-lactating dairy cows and 111 lactating dairy cows were used in three experiments. In Experiment I, cows were synchronized so that they had only a 5.5- to 6-day-old corpus luteum (CL) at the time of the prostaglandin F2α (PGF2α ) treatment of Ovsynch. In Experiment II, cows were synchronized so that they had at least a CL of approximately 14 days old at the time of PGF2α treatment and an accessory CL if they had responded to the first GnRH of Ovsynch. Furthermore, in each experiment, cows received either a standard or a double dose of d-CLO as the luteolytic treatment. In Experiment III, lactating cows were blocked by parity and assigned to one of three luteolytic treatments during Ovsynch: 500 µg d,l-CLO, 150 or 300 µg of d-CLO. In Experiment I, the dose of d-CLO had an effect (p = 0.08) on the percentage of cows with full luteolysis, but not in Experiment II (p > 0.1). More cows in Experiment II had full luteolysis than did cows of Experiment I (87% vs 58%, respectively; p = 0.007). In Experiment III, 87.1%, 84.4% and 86.2% lactating dairy cows had full luteolysis and 37.8%, 36.8% and 36.1% of cows became pregnant after treatment with 500 µg d,l-CLO, 150 or 300 µg of d-CLO, respectively (p > 0.05).

Effects of d-cloprostenol dose and corpus luteum age on ovulation, luteal function, and morphology in nonlactating dairy cows with early corpora lutea.

Luteolysis is a key event in cattle reproduction. A standard dose of exogenous PGF(2α) will induce full luteolysis in the majority of cows with a matured corpus luteum (CL). However, this will not occur in cows with a CL <5d old. To date, it is not known whether a larger dose will have a more potent luteolytic effect in cows during early diestrus. The objective of this study was to characterize the effect of 2 doses of d-cloprostenol (150 and 300 µg) on the progesterone concentration, luteal diameter, and ovulation rate in nonlactating dairy cattle 96 to 132 h postovulation. Twenty nonlactating dairy cows were included in the study. Each cow received 2 treatments of d-cloprostenol in 2 consecutive cycles: a standard dose of 150 µg and a double dose of 300 µg. The cows were allocated randomly to 1 of 4 groups (5 cows in each group) according to the age of the CL at the time of treatment: 96, 108, 120, and 132 h. The exact time of ovulation was known within 12h, because of twice per day ultrasound examination. The CL diameter and progesterone concentration were measured before treatment (d 0) and 2 and 4d after treatment. Within each CL age group, the effect of d-cloprostenol dose on luteolysis was determined. More cows treated with double dose tended to have full luteolysis compared with the standard dose (8/10 vs. 4/10, respectively). This effect was only apparent in cows with CL of 120 and 132 h but not in earlier CL. The interval from treatment to ovulation was shorter (3.3 ± 0.1d) in cows treated with a double dose than in cows treated with the standard dose (4.5 ± 0.4d).

The effect of systemic administration of cloprostenol on ovulation in mares treated with a prostaglandin synthetase inhibitor.

Prostaglandins (PGs) are essential to trigger the cascade of events that degrade the extracellular matrix of follicles leading to follicular rupture and ovulation. In mares, systemic administration of flunixin meglumine (FM), a PG synthetase inhibitor, blocks ovulation by inducing luteinized unruptured follicles (LUF). In the rat, the administration of PGF(2α) (PGF) and PGE restored ovulation in indomethacin treated animals. The mares were treated with FM 0, 12, 24 and 36 h after human chorionic gonadotrophin (hCG) administration to induce experimentally LUF (n = 15) or were left untreated (controls, n = 5). In addition, 250 µg of cloprostenol were administered intravenously to the mares 33, 35 and 36 h (CLO 33, n = 5) or 48, 49 and 50 h (CLO 48, n = 5) after hCG. One group was treated with FM but not with cloprostenol (FM-control, n = 5). The ovulation rate, follicular diameter and progesterone concentration were compared amongst groups. The ovulation rate at 48 h was higher (p < 0.05) in the controls (100%) than in the FM-control (0%), CLO 33 (0%) or CLO 48 (20%) mares. All but one FM treated mares developed LUF by 48 h after hCG administration. Two LUF collapsed between 48 and 60 h and 72 and 84 h in one mare from FM-control and from the CLO 33 group each, respectively. Progesterone concentration was significantly higher (p < 0.05) in the control mares than in any of the FM treated mares 5, 9 and 13 days after hCG. In conclusion, FM administered during the periovulatory period blocked ovulation in the mares. In contrast, the administration of cloprostenol, a PGF analogue, in the previously FM treated mares failed to restore ovulation.

Relationship between dose of cloprostenol and age of corpus luteum on the luteolytic response of early dioestrous mares: a field study.

The objective of this study was to establish and characterize the relationship between the dose of cloprostenol (37.5, 250, 500 and 750 µg) and the age of the early corpus luteum (CL) (80, 88, 96, 104 and 112 h) on the luteolytic response of mares. Behavioural oestrus and ultrasonographic signs of return to oestrus were considered as the occurrence of full luteolysis. A total of 298 mares were divided into groups according to dose of cloprostenol and CL age. There was an effect of dose of cloprostenol (p < 0.001) and age of the CL at the time of treatment (p < 0.001) on the percentage of mares with full luteolysis. The efficacy of 37.5 µg of d-cloprostenol was similar to that of 250 µg of d,l-cloprostenol (p > 0.05); and that of 500 similar to that of 750 µg (p > 0.05). The higher dose groups (500 and 750 µg) induced full luteolysis more frequently than the lower dose groups (37.5 and 250 µg) 96-104 h post-ovulation. There was no effect of CL age or cloprostenol dose on the interovulatory interval (p > 0.05). In conclusion, the effect of cloprostenol on the percentage of mares undergoing full luteolysis is dose-dependent. However, this effect is only evident in mares with CLs aged between 96 and 104 h. There is no advantage of administering more than 500 µg of d,l-cloprostenol (Estrumate(®)), to obtain a higher percentage of mares with full luteolysis in mares with CLs aged 80-112 h.

'Catching the spike and tracking the flow': Holter-temperature monitoring in patients admitted in a general internal medicine ward.

OBJECTIVE: To study if a 24-h continuous monitoring of temperature reveals information not accessible through conventional care. This included omitted fever peaks and circadian and complexity characteristics that may correlate with specific aetiologies. DESIGN: Ours was a prospective, observational study. A total of 62 patients, admitted to a general internal medicine ward, in whom a temperature > 38 °C had been observed the day before inclusion underwent a 24-h long continuous monitoring of both central and peripheral temperatures. The time series were recorded in a file, while they otherwise followed conventional care. Time series were analysed for standard statistics, chronobiological analysis (amplitude, mesor, acrophase, intra-daily variability) and complexity analysis (Approximate Entropy of both central and peripheral temperature, cross-ApEn). A month after discharge, the clinical reports were reviewed and a definitive diagnosis of the febrile syndrome was established. RESULTS: A total of 62 patients were initially included. In six cases, no time series could be obtained because of technical problems, leaving 56 patients accessible for analysis. In 10 cases, no definitive diagnosis was established. Continuous monitoring detected a mean of 0.7 (CI = 0.27-1.33) peaks of fever (central temperature > 38.0 °C) unobserved by conventional care per patient. A proportion of 16% (CI = 6-26) of patients considered afebrile by conventional care had at least one fever peak detected by continuous monitoring. Circadian rhythm persisted or was exacerbated in febrile patients. Circadian amplitude was increased in patients with tuberculosis. Complexity analysis did not differ among different diagnostic groups, although in subgroup analysis, viral infections had a higher complexity than other infectious diseases. CONCLUSIONS: Temperature Holter monitoring reveals fever peaks that pass otherwise unobserved. Furthermore, chronobiological and complexity analysis of the temperature profile may provide quick and easy 'hidden information', not available to conventional care.

The effect of time of insemination with fresh cooled transported semen and natural mating relative to ovulation on pregnancy and embryo loss rates in the mare.

One hundred and fifty-four mares were inseminated with fresh semen either during the pre- or post-ovulatory periods at different intervals relative to ovulation: 36-24 h (n = 17) and 24-0 h (n = 30) before ovulation; 0-8 h (n = 21), 8-16 h (n = 24), 16-24 h (n = 48) and 24-32 h (n = 14) h after ovulation. All mares received the same routine post-mating treatment consisting of an intrauterine infusion with 1 litre of saline and antibiotics followed 8 h later by an intravenous administration of oxytocin. Artificial inseminations (AI) from 36 h before ovulation up to 16 h post-ovulation were performed with transported cooled semen. While there was no data available for inseminations later than 16 h, data from natural mating after 16 h post-ovulation were included. Pregnancy rate (PR) of mares inseminated 36-24 h (29.4%) was significantly lower (p < 0.05) than mares inseminated 24-0 h before ovulation (60%), 0-8 h (66.7%) and 8-16 h (70.1%) post-ovulation. Embryo loss rate (ELR) was highest in mares mated 24-32 h after ovulation (75%). PR of mares mated 16-24 h post-ovulation (54.1%) did not differ significantly from any other group (p > 0.05); however, the ELR did increased markedly (34.6%) compared with inseminations before 16 h post-ovulation (<12%). At ≥ 30 days post-ovulation, PR of mares mated 16-24 h after ovulation (35.4%) was significantly lower than mares mated 0-16 h after ovulation (62%). Good PR with acceptable ELR can result from inseminations within 16 h of ovulation, at least with this specific post-mating routine treatment.

Risk factors for the development of haemorrhagic anovulatory follicles in the mare.

Haemorrhage into the dominant follicle during the reproductive season is a subtle but definitive cause of infertility in the mare population. This condition however can be of high relevance for an individual in which its incidence is abnormally high. Little is known about the nature and factors affecting the incidence of haemorrhagic anovulatory follicles (HAFs) in the mare. The objectives of the study were to define and characterize the ultrasonographic development and incidence of HAFs and to investigate possible risk factors influencing its occurrence. Detailed reproductive and ultrasound records of seven mares studied during their entire reproductive lives (>10 years and 612 oestrous cycles) were analysed retrospectively and computed into a statistical mixed model. Of all animal studied, two mares were found to have an unusually high incidence of HAFs of approximately 25%. Time of season and use of induction treatments (Cloprostenol) were found to influence its incidence. It appears that early-enhanced stimulatory effect of LH on an ovary with the presence of small and immature follicles might increase the risk of ovulatory failure of those follicles later in the cycle. Mares during the months of highest follicular activity (May to August) and after treatment with hormones to induce oestrus and ovulation are at greater risk to develop HAFs. The potential relevance of this study is two folds: clinical relevance for the practitioner to better understand this condition and so improve reproductive management of mares with abnormally high incidence; and to provide useful insights for researchers willing to further investigate the nature of this phenomenon.

Cloprostenol in equine reproductive practice: something more than a luteolytic drug.

Prostaglandin F(2α) and its analogues (PGF) are widely used in equine reproductive practice. The interval from PGF treatment to ovulation (ITO) varies greatly with a range from 2 to 16 days. Clinical observation suggests that mares mated and ovulated soon after PGF treatment may have poor fertility. Reproductive records of 329 cyclic Thoroughbred mares were analysed retrospectively. The following parameters were analysed: (i) use of cloprostenol; (ii) ITO and (iii) number of ovulations per cycle. According to these parameters, mares were classified into four groups. (i) mares with spontaneous ovulations, n = 57; (ii) mares induced with cloprostenol and ITO = 4-7 days, n = 77; (iii) ITO = 8-10 days, n = 89 and (iv) ITO = ≥ 11 days, n = 106. Differences in pregnancy (PR) and multiple ovulation (MO) rates among groups were tested using chi-squared test. PR rates for groups 1-4 were: 73.7%, 46.7%, 64% and 71.7% respectively (p < 0.05). Groups 1 and 2 had lower (p < 0.05) MO rate (24.6% and 20.8%) than groups 3 and 4 (40.4% and 44.3%). It appears that ovulation soon after PGF-induced luteolysis is detrimental to PR rates. It was found highly significant that in cloprostenol-treated mares, the MO rate was enhanced without subsequent increase in multiple pregnancies.

Monozygotic multiple pregnancies after transfer of single in vitro produced equine embryos.

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.

Effect of type of semen, time of insemination relative to ovulation and embryo transfer on early equine embryonic vesicle growth as determined by ultrasound.

Embryonic vesicle growth in the mare is easily monitored by ultrasound. Apart from pregnancy diagnosis, assessment of the embryonic vesicle in practice is also useful to evaluate its viability. Although subject to individual variation, embryo growth rate follows a constant pattern in the early stages of development in relation to embryonic age. Previous studies have shown a significant effect of some factors routinely used in practice, such as post-ovulation insemination and embryo transfer, on embryonic growth and the time in which the vesicle is first detected. This study attempts to confirm previous results in different settings and characterise the causes for this delay in growth. A total of 159 pregnancies from different mating protocols: (1) pre-ovulation natural mating, (2) pre-ovulation natural mating and transfer into recipient mares, (3) post-ovulation natural mating, and (4) post-ovulation AI with frozen/thaw spermatozoa were evaluated ultrasonographically from day 12 to 19 of pregnancy and vesicle diameters recorded. Regression analysis between embryonic vesicle diameters and embryonic ages was performed for each group and mean vesicle diameter at different age periods among groups were tested for statistical difference with a general linear model of variance. There was no significant difference between groups 1 and 2 (P=0.73) or between groups 3 and 4 (P=0.71). However both pre-ovulation groups (1 and 2) had larger vesicle diameters (P<0.000) at any embryonic age analysed than either of the post-ovulation groups (3 and 4). In conclusion, post-ovulation inseminations produced pregnancies with smaller vesicle diameters equivalent to approximately 1 day's growth.

Small day 8 equine embryos cannot be rescued by a less advanced recipient mare uterus.

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.

Effects of follicular ablation and induced luteolysis on LH and follicular fluid factors during the periovulatory period in mares.

Haemorrhagic anovulatory follicles (HAFs) are the most common pathological anovulatory condition in the mare. To enhance understanding of the physiopathology of HAFs, the aim of the present study was to determine the effects of an induced-follicular wave on LH concentrations and follicular fluid factors relevant to the ovulatory process. Mares were allocated to treatment or control groups (n = 7/group) in a crossed over design during 14 oestrous cycles with a period of one cycle occurring when there were no treatments between the times when treatments were administered. In the treatment group, all antral follicles ≥8 mm were ablated on Day 10 after ovulation followed by administration of a luteolytic dose of PGF2α. All mares of both groups were treated with 1500 IU of hCG when a follicle ≥32 mm was detected (Hour 0), and follicular fluid was aspirated 35 h later. Blood samples were collected every 48 h from Day 10 until Hour 0 from all mares. Follicular fluid was assayed for PGE2, estradiol and progesterone. Plasma was assayed for LH concentrations. A follicular wave followed follicle ablation in the treated mares. Concentrations of LH were greater (P = 0.05) in mares ot the treatment compared with control group. Concentrations of PGE2, estradiol and progesterone in follicular fluid did not differ between groups (P > 0.05). Treatment resulted in an earlier increase in circulating LH, however, there was no effect on concentrations of intra-follicular PGE2, estradiol or progesterone in hCG-stimulated preovulatory follicles.

Factors affecting the likelihood of pregnancy and embryonic loss after transfer of cryopreserved in vitro produced equine embryos.

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.