A. Claes

Serum anti-Müllerian hormone concentrations in stallions: Developmental changes, seasonal variation, and differences between intact stallions, cryptorchid stallions, and geldings

Anti-Müllerian hormone (AMH), a homodimeric glycoprotein, is secreted early in fetal life when it exerts a crucial function in sexual differentiation. The secretion of AMH in male humans persists after birth and is characterized by high prepubertal concentrations followed by a significant decrease at the onset of puberty. The expression of AMH in the normal and cryptorchid equine testis is well characterized but data regarding circulating AMH concentrations are lacking. The objectives of this study were to determine serum AMH concentrations in neonatal colts and fillies, prepubertal colts, and postpubertal stallions, and to evaluate variations in serum AMH related to season and gonadal status of stallions. In addition, we examined the presence and determined concentrations of AMH in seminal plasma of mature stallions. Serum AMH concentrations were significantly higher in neonatal colts than in neonatal fillies. Moreover, concentrations of AMH are high in prepubertal colts whereas significantly lower concentrations were detected after puberty. In intact mature stallions, season influenced AMH concentrations with significantly higher concentrations during spring and summer. Serum AMH concentrations were significantly higher in cryptorchid stallions compared with intact stallions or geldings. Finally, AMH was identified in seminal plasma of intact mature stallions, but there was no significant correlation between serum and seminal plasma AMH concentrations. In conclusion, serum AMH concentration varies with sex in the neonatal period, postnatal sexual development and season, and serum AMH concentration can be used as a biomarker for the presence of testicular tissue.

The interrelationship between anti-Müllerian hormone, ovarian follicular populations and age in mares.

REASONS FOR PERFORMING STUDY Anti-Müllerian hormone (AMH) is a granulosa-cell-derived glycoprotein, which plays an important inhibitory role during folliculogenesis. Concentrations of AMH are highly correlated with antral follicle counts (AFCs) in other species, which in turn are related to follicular reserve. Relatively little is known about AMH and AFC in the mare. OBJECTIVES To determine plasma AMH concentrations and AFCs in mares of different ages, to measure the repeatability of AMH concentrations and AFCs within and across oestrous cycles and to assess the relationship between plasma AMH concentrations and AFCs with regard to mare age and follicle size. STUDY DESIGN An observational study examining the relationship between AMH, AFC and age in 45 mares. METHODS Young (3-8 years), middle-aged (9-18 years) and old mares (19-27 years) were examined by transrectal ultrasonography over 2 or 3 oestrous cycles. Plasma AMH concentrations and AFCs were determined, and antral follicles were classified by size into different groups. RESULTS Plasma AMH concentrations varied widely between mares within similar age groups. Antral follicle counts were significantly lower in old mares than in young and middle-aged mares, and AMH concentrations were significantly lower in old than in middle-aged mares. A positive relationship was detected between AFC and AMH, and this relationship varied by mare age with a strong correlation in older mares (ρ = 0.86; P<0.0001), a moderate correlation in middle-aged mares (ρ = 0.60; P = 0.01) and no correlation in young mares (ρ = 0.40, P<0.4). The AMH concentrations were significantly related to the number of antral follicles between 6 and 20 mm in diameter, and the repeatability of AFCs and AMH concentrations was high within and between oestrous cycles. CONCLUSIONS Our findings indicate that the relationship between AMH and AFC varies across age groups, and concentrations of AMH might be a better reflection of reproductive age than calendar age.

Molecular changes in the equine follicle in relation to variations in antral follicle count and anti-Müllerian hormone concentrations.

REASONS FOR PERFORMING STUDY The wide variation in circulating anti-Müllerian hormone (AMH) concentrations between mares is attributed to differences in antral follicle count (AFC) which may reflect follicular function. There are few data regarding variations in AFC and associated regulatory factors for AMH in the equine follicle during follicular development. OBJECTIVES To examine molecular and hormonal differences in the equine follicle in relation to variations in AFC and circulating AMH concentrations during follicular development and to identify genes co-expressed with AMH in the equine follicle. STUDY DESIGN Observational study. METHODS Plasma AMH concentrations and AFC were determined in 30 cyclic mares. Granulosa cells, theca cells and follicular fluid were recovered from growing (n = 17) or dominant follicles (n = 13). The expression of several genes, known to be involved in folliculogenesis and steroidogenesis, was examined using real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. Intrafollicular oestradiol and AMH concentrations were determined by immunoassay. RESULTS Within growing follicles, the expression of AMH, AMHR2, ESR2 and INHA in granulosa cells was positively correlated with AFC and plasma AMH concentrations. In addition, the expression of ESR1 and FSHR was positively associated with plasma AMH concentrations. No significant associations were detected in dominant follicles. Furthermore, there was no association between AMH or oestradiol concentrations in follicular fluid and variations in AFC. Finally, the expression of AMH and genes co-expressed with AMH (AMHR2, ESR2 and FSHR) in granulosa cells as well as intrafollicular AMH concentrations decreased during follicular development while intrafollicular oestradiol concentrations increased and were inversely related to intrafollicular AMH concentrations. CONCLUSIONS This study indicates that variations in AFC and circulating AMH concentrations are associated with molecular changes in the growing equine follicle.

Biological Functions and Clinical Applications of Anti-Müllerian Hormone in Stallions and Mares

Anti-Müllerian hormone (AMH) plays a major role in sexual differentiation, Leydig cell differentiation, and folliculogenesis. In addition, AMH has clinical value in equine practice. In stallions, AMH can serve as an endocrine marker for equine cryptorchidism and as an immunohistochemical marker for Sertoli cell tumors. Considering that AMH is also an ovarian specific product, intact mares can be differentiated from ovariectomized mares. Peripheral AMH concentrations reflect the follicular population in mares, and therefore, are useful in the assessment of ovarian reserve and reproductive life-span of aged mares. Last, AMH is particularly suitable as a diagnostic marker for equine granulosa cell tumors.

Attempts to induce nocardioform placentitis (Crossiela equi) experimentally in mares

REASONS FOR PERFORMING STUDY Nocardioform placentitis in horses is poorly understood, and the development of an experimental model would be of help in understanding the pathogenesis of the disease. OBJECTIVES To investigate whether (1) intrauterine inoculation of Crossiela equi during the periovulatory period or (2) i.v., oral or intranasopharyngeal inoculation of C. equi during midgestation would result in nocardioform placentitis, and (3) before and after mating endometrial swabs present evidence of nocardioform placentitis-associated organisms (C. equi or Amycolatopsis spp.). METHODS In Study I, mares (n = 20) received an intrauterine inoculation of C. equi 24 h after artificial insemination. Endometrial swabs were obtained 24 h post inoculation for PCR analysis. In Study II, pregnant mares (at 180-240 days of gestation) were inoculated with C. equi by intranasopharyngeal (n = 5), oral (n = 4) or i.v. (n = 4) routes. Sixty contemporaneous pregnant mares maintained on the same farm served as control animals. In Study III, privately owned Thoroughbred mares (n = 200) had endometrial swabs collected before and within 24-48 h after mating for detection of nocardioform microorganisms. RESULTS In Study I, C.equi was identified by PCR in 3 of 20 mares following intrauterine inoculation. Pregnancy was established in 19 of 20 treated mares. There were 2 embryonic losses and one abortion at 177 days of gestation (undetermined cause). Sixteen mares delivered a normal foal and placenta. In Study II, one mare (oral inoculation) aborted at 200 days of gestation (unidentified cause). The remaining mares delivered a normal foal and placenta. In Study III, none of the mares yielded positive endometrial PCR for nocardioform microorganisms. CONCLUSIONS We were unable to induce nocardioform placentitis, and there was no evidence of nocardioform microorganisms in endometrial swabs of broodmares before or after mating. These findings suggest that nocardioform placentitis is not induced simply via the presence of nocardiform actinomycetes and that route, insufficient duration of exposure and dose may play a role in the development of disease. Additional predispositions may also be involved in the development of nocardioform placentitis.

Age and season affect serum testosterone concentrations in cryptorchid stallions

Cryptorchidism, characterised by inguinal and/or abdominal retention of one or both testes, is the most common developmental defect encountered in equine veterinary practice. Most cases present with stallion-like behaviour in males without palpable testes and questionable castration history. Evaluation of serum testosterone concentrations is commonly used to distinguish geldings from cryptorchid stallions without a scrotal testis, but results can be variable and difficult to interpret (Lu 2005, Raś and others 2010). Identifying factors that influence serum testosterone concentrations in cryptorchid stallions can be valuable in the interpretation of assay results. Both season and age are known to have a major effect on testosterone concentrations in intact stallions (Berndtson and others 1974, Raeside 1979, Stewart and Roser 1998), but little is known in cryptorchid stallions, with only one study reporting seasonal variation in serum testosterone concentrations (Cox and others 1988). Additionally, it is possible that serum testosterone concentrations in cryptorchid stallions are influenced by age as the cryptorchid testis is subjected to chronic elevated temperatures which could potentially adversely affect Leydig cell function. However, age-related changes in serum testosterone concentrations in cryptorchid stallions have not, to our knowledge, been described. Therefore, the objective of this study was to evaluate the effect of season and age on serum …

Likelihood of pregnancy after embryo transfer is reduced in recipient mares with a short preceding oestrus

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.

Effect of embryo transfer technique on the likelihood of pregnancy in the mare: a comparison of conventional and Wilsher’s forceps-assisted transfer

The aim of this study was to compare the success of embryo transfer (ET) performed by veterinarians with different degrees of experience using one of two ET techniques. Over three years, 179 embryos were transferred by three operators with moderate to high experience using a ‘conventional’ manual technique, and 170 embryos were transferred by four operators with little or no previous ET experience using a ‘Wilsher’ technique (Polansky speculum and Wilsher’s cervical forceps). The pregnancy status of recipient mares at the first pregnancy diagnosis and the embryo loss by the last examination were compared between groups and within group among operators. More recipients became pregnant (P<0.005) following ET with the Wilsher technique (157/170; 92.3 per cent) than with the conventional technique (127/179; 70.9 per cent), while the incidence of pregnancy loss did not differ between the groups (9.1 v 7.9 per cent, respectively). For the conventional technique, there was a significant operator effect (P<0.01) on the percentage of pregnant recipients at the first examination (50.9–79.7 per cent); no operator effect was apparent for the Wilsher technique (90.9–93.4 per cent). In conclusion, the Wilsher technique yielded high pregnancy rates (>90 per cent).

Horse embryo diameter is influenced by the embryonic age but not by the type of semen used to inseminate donor mares

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 Fishers 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%).

Effect of Embryo-Recipient Synchrony on Post-ET Survival of In Vivo and In Vitro-Produced Equine Embryos

Equine embryo transfer (ET) permits a relatively large degree of ovulation asynchrony between donor and recipient mare (+1 to –4 days). However, when the embryo is more than 4 days advanced than the uterus, its development is retarded after ET [1]; conversely, pregnancy rarely results in recipients that ovulate 2 days before the donor [2]. Embryos recovered from mares on day 8 after ovulation vary greatly in diameter (i.e. 130-2100 mm) [3], and small Day 7-8 embryos (<300 mm) have a reduced pregnancy rate (PR) and an increased risk of early embryonic loss (EEL) following ET [3]. However, it is unclear whether the reduced survival of small embryos is due to pre-existing poor quality (small for age) or an inadvertent increase in relative asynchrony with the recipients’ uterine stage. In vitro-produced (IVP) embryos usually reach the blastocyst stage 7 to 9 days after ICSI, at which point they are either transferred into a recipient mare or cryopreserved.When they reach the blastocyst stage, IVP embryos have a diameter of 128-160 mm. It is, however, difficult to estimate the ‘true’ developmental age of the embryo and, therefore, the optimal day after ovulation for a recipient. IVP embryos have been suggested to resemble Day 6 in vivo embryos and it is therefore advised that they be transferred into Day 4 to 6 recipients [4]. However, there is no information on the effect of recipient stage on post-transfer IVP embryo survival. The objectives of this study were to determine the effect of the recipients’ stage after ovulation on PR, embryonic vesicle growth and EEL for a) flushed embryos classified according to embryonic diameter; and b) IVP embryos frozen 7 to 8 days after ICSI.