Mare Age Research Articles

Multivariable analysis to determine risk factors associated with early pregnancy loss in thoroughbred broodmares

Early pregnancy loss (EPL) between days 15–65 after breeding has been shown to occur in 7.9% of equine pregnancies with substantial economical, welfare and safety implications. Whilst maternal age has been recognised as an important risk factor in relation to the incidence of EPL, few other risk factors have been conclusively identified. Further, multivariable data analysis of risk factors for EPL is sparse. A prospective cohort investigation of thoroughbred broodmares in the United Kingdom was conducted over the 2013 and 2014 breeding seasons. Information relating to 28 factors including mare, stallion, pregnancy and therapeutic interventions was collected using questionnaires and entered into a custom-designed Microsoft Access database. Mixed effects logistic regression was used to determine risk factors for EPL, including ‘mare’ as a random effect to account for repeat pregnancies in the same mare. Stallion, stud and veterinarian were also evaluated as random effects. Variables with a p-value of <0.25 in univariable analysis were taken forward for consideration in the multivariable model which was built using a forward stepwise approach. Data were collected on 2245 pregnancies in 1753 mares. Increasing mare age (OR = 1.11, 95% confidence interval (CI) = 1.04, 1.18, p = 0.001), having had one previous foal (OR = 3.52, 95% CI = 1.56, 7.95, p = 0.002) and presence of uterine cysts (OR = 1.76, 95% CI = 1.07, 2.91, p = 0.03) were all associated with increased odds of EPL following multivariable analysis. Increasing day 15/16 scan vesicle size (OR = 0.24, 95% CI = 0.16, 0.38, p < 0.001) and the use of ovulatory induction agents (OR = 0.31, 95% CI = 0.17, 0.55, p < 0.001) were negatively associated with EPL. Stallion, stud and veterinarian were not significantly associated with EPL. Analysis of a subpopulation of 344 multiple (twin and triplet) pregnancies found that the use of flunixin meglumine at the time of manual reduction of a multiple pregnancy resulted in reduced odds of EPL (OR = 0.34, 95% CI = 0.14, 0.84, p = 0.02). Results from this study can be used by stud farm personnel when assessing their broodmare population and by clinicians when deciding upon therapeutic strategies. Additional work can be focused around these risk factors to further our understanding of the pathophysiology of EPL.

In-vitro equine embryo production: an adventure in multi-unit collaboration

The accessibility of assisted reproductive techniques related to oocyte collection and in vitro fertilization in the mare is increasingly becoming more widespread for private equine practitioners and their clients. The necessity of hauling a valuable mare long distances to the closest facility for transvaginal aspiration (TVA) of oocytes and on site intracytoplasmic sperm injection (ICSI) over a period of potential weeks before obtaining a pregnancy no longer exists. Instead, a mare’s primary veterinarian can manage the patient’s cycle and briefly transport her to a nearby facility where TVA can be performed. With TVA services being offered with increasing frequency in densely equine populated areas it is increasingly common for TVA to be offered as an outpatient procedure even if ICSI laboratories are not present at that location. The majority of reports associated with TVA and ICSI are associated with benchtop research and typically do not continue beyond blastocyst development to report pregnancy rates. A cumulative pregnancy rate after TVA and ICSI were performed on immature oocytes, with shipment of blastocysts to an off-site recipient herd has been reported at 72% with a 52% foaling rate.1 In 2017, in collaboration with an exclusively equine reproduction veterinary practice, we worked together to provide TVA services for client-owned mares. Mares were referred from the veterinary practice to our university setting where outpatient TVA procedures were performed; 330 follicles were aspirated and 212 oocytes were recovered (mare age range from 4-26 years) and shipped overnight at room temperature in an Equitainer® to an ICSI laboratory via FedEx™. A total of 43 (26.1%) blastocysts resulting from 165 ICSI procedures were obtained; 29 embryos were vitrified for future use and 14 embryos were transported to the referring reproductive veterinary practice via courier for fresh transfer to recipient mares on day 7 or 8 after ICSI. A 14 day pregnancy rate of 71.4% (10/14) and a 50% (7/14) pregnancy rate at heartbeat check were recorded. Three recipient mares have already successfully foaled and four are due to foal later in the spring of 2018. In light of these results sending a mare to a local practice specializing in TVA, as opposed to a distant, self-contained facility that has both TVA and ICSI capabilities has benefits for referring equine practitioners, their clients and patients. Being a key element in the management of the mare’s cycle prior to TVA has allowed the primary veterinarian to remain an integral partner in providing these specialty services. Shorter duration stays at referral facilities translates into less expense for the mare owner. Finally, as collaborators in this working partnership we have found working as a team to offer such services has enhanced the working relationship between practitioners and specialists.

Advanced mare age impairs the ability of in vitro‐matured oocytes to correctly align chromosomes on the metaphase plate

SummaryBackgroundAdvanced mare age is associated with declining fertility and an increased risk of early pregnancy loss. Compromised oocyte quality is probably the primary reason for reduced fertility, but the defects predisposing to embryonic death are unknown. In women, advanced age predisposes to chromosome segregation errors during meiosis, which lead to embryonic aneuploidy and a heightened risk of miscarriage.ObjectivesTo evaluate the effect of advanced mare age on chromosome alignment and meiotic spindle morphology in in vitro‐matured (IVM) oocytes.Study designMorphometric and morphological analysis.MethodsTo investigate differences in spindle organisation and chromosome alignment between young and old mares, oocytes collected from slaughtered mares were divided into two groups depending on mare age (young, ≤14 years and old, ≥16 years), IVM and stained to visualise chromatin and alpha‐tubulin. Spindle morphology, morphometry and chromosome (mis)alignment were evaluated by confocal microscopy and 3D image analysis.ResultsOocytes from old mares showed a higher incidence of chromosome misalignment (47.4% vs. 4.5%; P<0.001) and a thicker metaphase plate (mean ± s.d.: 5.8 ± 1.0 μm vs. 4.9 ± 0.9 μm; P = 0.04) than oocytes from young mares. Although no differences in spindle morphometry were detected between old and young mares, an increased major spindle axis length was associated with chromosome misalignment (mean ± s.d.: 25.3 ± 6.1 μm vs. 20.8 ± 3.3 μm; P = 0.01) irrespective of age.Main limitationsThe oocytes were IVM and may not exactly reflect chromosome misalignment in vivo.ConclusionsAdvanced mare age predisposes to chromosome misalignment on the metaphase II spindle of IVM oocytes. The compromised ability to correctly align chromosomes presumably predisposes to aneuploidy in resulting embryos and thereby contributes to the age‐related decline in fertility and increased incidence of early pregnancy loss. The Summary is available in Portuguese – see Supporting Information

Ovarian function following immunocontraceptive vaccination of mares using native porcine and recombinant zona pellucida vaccines formulated with a non-Freund's adjuvant and anti-GnRH vaccines

An important determinant in the selection of any contraceptive agent is the impact on ovarian function, both in the short and longer term. In this study, ovarian activity was monitored in mares immunised with one of the following vaccine formulations; native porcine zona pellucida (pZP), recombinant zona pellucida proteins ZP3 and ZP4 (reZP), pZP and reZP combined or a commercially available anti-GnRH vaccine. The ZP antigens were prepared in an adjuvant formulation consisting of 6% polymeric adjuvant (Montanide™ PetGel A, Seppic, France) and 500 μg polyinosinic-polycytidylic acid - TLR3-agonist (Poly(I:C) HMW VacciGrade™, Invivogen, USA). A vehicle-only control group was administered the adjuvant formulation without antigen. Ovarian activity was monitored using clinical observations (transrectal palpation and ultrasonography of the reproductive tract) in addition to blood sampling for serum progesterone and anti-Müllerian hormone (AMH) concentrations while employing a low sampling frequency. Treatments and measurements were initiated in December (southern hemisphere summer) and subsequent data collection was performed in January, February, March and May. Both reZP and anti-GnRH vaccination were associated with clinically evident ovarian suppression in the short term. Ovarian activity in mares administered a reZP or anti-GnRH vaccine was significantly different to adjuvant control and pZP treated mares. Serum AMH concentrations were different between pZP and anti-GnRH treated mares 3.5 months after the final vaccination. Serum AMH concentrations were significantly correlated with mare age, serum progesterone and ovarian volume.

Influence of cycle stage, age and endometrial biopsy score on oxytocin receptor distribution and gene expression in the cervix and uterus of non-pregnant mares

Persistent breeding-induced endometritis (PBIE) or delayed uterine clearance (DUC) are major causes of mare subfertility. Oxytocin and its receptor are thought to play significant roles in the pathogenesis of DUC but the specific roles of oxytocin receptor (OR) distribution and gene expression remain undefined.In this study both OR distribution and gene expression in the endometrium, myometrium and cervix during both luteal and non-luteal phases in non-pregnant mares (n = 27) of differing age (young: 2–9 years, n = 17; old: > 10 years, n = 10) and endometrial biopsy score were described using immunohistochemistry (IHC) and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively.Immunohistochemistry showed a similar pattern of OR distribution in uterus and cervix, with the exception of the glandular epithelium, absent in the cervix. Uterine ORs were localized in endometrial luminal and glandular epithelia, transmural vascular endothelium, sub-epithelial and peri-glandular stromal cells and myometrial smooth muscle cells. The OR labeling intensity was consistently greatest in the vascular endothelium. Real-time qPCR showed a higher OR gene expression in myometrium compared to cervix (P = 0.001) and endometrium (P = 0.009). There was no difference in OR gene expression between cervix and endometrium (P = 1.0). Oxytocin receptor gene expression was significantly higher during the non-luteal phase in both combined uterine tissues (endometrium and myometrium) and myometrium. Oxytocin receptor distribution and gene expression were not influenced by a mare's age or endometrial biopsy score. As endometrial biopsy score and mare age were not predictors of OR gene expression, deficient OR gene expression is unlikely to be associated with DUC.

Association of equine sperm population parameters with outcome of intracytoplasmic sperm injections

Limited clinical information is available regarding sperm population parameters that are important for use with equine intracytoplasmic sperm injection (ICSI). Therefore, the appropriateness of a sample of sperm is typically not known before ICSI. The aim of our study was to determine which sperm population characteristics were predictive of ICSI outcome. Frozen-thawed sperm samples (n = 114) from 37 stallions in a clinical program were analyzed after ICSI for percentages of normal morphology (MORPH+), live as assessed by eosin/nigrosin stain (LIVE+), membrane intact as assessed by hypoosmotic swelling test (HOS+), and DNA fragmentation determined by sperm chromatin dispersion (DNA–). ICSI was performed on 147 oocytes, and cleavage (≥2 cells), embryo development (morula or blastocyst), and pregnancy status after embryo transfer were determined. Among the examined sperm parameters, LIVE + correlated positively with MORPH+ and HOS+, and MORPH + negatively with DNA–; no other significant correlations were observed. When used for ICSI, sperm population percentages for MORPH+ and DNA– were not predictive of ICSI outcome, including cleavage, embryo development, and establishment of a pregnancy. Sperm population percentages significantly affecting ICSI outcomes were LIVE+ and HOS + for oocyte cleavage, LIVE + for embryo development, and HOS + for establishment of a pregnancy. The probability of a pregnancy was significantly higher for sperm populations having HOS+ ≥40% than populations having HOS+ ≤20%. The mean age of the donor mare per sperm-injected oocyte did not differ for oocyte cleavage, embryo production, or establishment of pregnancy. In our study, the probability of sperm-injected oocytes to develop into an embryo (morula or blastocyst) improved when sperm were selected from a population with higher indicators of membrane integrity (LIVE+ and HOS+).

Insemination Dose and Site With Frozen Semen Affects the Sperm Transport and Inflammatory Response in Mares?

Persistent breeding-induced endometritis (PBIE) or delayed uterine clearance (DUC) are major causes of mare subfertility. Oxytocin and its receptor are thought to play significant roles in the pathogenesis of DUC but the specific roles of oxytocin receptor (OR) distribution and gene expression remain undefined.In this study both OR distribution and gene expression in the endometrium, myometrium and cervix during both luteal and non-luteal phases in non-pregnant mares (n = 27) of differing age (young: 2–9 years, n = 17; old: > 10 years, n = 10) and endometrial biopsy score were described using immunohistochemistry (IHC) and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively.Immunohistochemistry showed a similar pattern of OR distribution in uterus and cervix, with the exception of the glandular epithelium, absent in the cervix. Uterine ORs were localized in endometrial luminal and glandular epithelia, transmural vascular endothelium, sub-epithelial and peri-glandular stromal cells and myometrial smooth muscle cells. The OR labeling intensity was consistently greatest in the vascular endothelium. Real-time qPCR showed a higher OR gene expression in myometrium compared to cervix (P = 0.001) and endometrium (P = 0.009). There was no difference in OR gene expression between cervix and endometrium (P = 1.0). Oxytocin receptor gene expression was significantly higher during the non-luteal phase in both combined uterine tissues (endometrium and myometrium) and myometrium. Oxytocin receptor distribution and gene expression were not influenced by a mare's age or endometrial biopsy score. As endometrial biopsy score and mare age were not predictors of OR gene expression, deficient OR gene expression is unlikely to be associated with DUC.

Effects of mares' age and day of gestation on efficacy of transvaginal ultrasound-guided twin reduction.

Transvaginal ultrasound-guided aspiration (TUA) is a procedure which can be used for the reduction of twins post-fixation in the mare. The aim of this study was to evaluate the effect of the age of mares and the day of gestation on the outcome of TUA treatment. In 88 mares, diagnosed pregnant with twins, TUA of the yolk sac or allantoic fluid was performed between day 30 and 62 of gestation. Mares were aged 3-22 years. Ultrasonographic examination for a viable singleton pregnancy was performed by referring veterinarians 5-7 days and 4 weeks after TUA. Based on reported findings, effects of age and day of gestation on pregnancy rates were evaluated. Four weeks after TUA, 67% of the cases resulted in a viable singleton pregnancy. Five to 7 days after TUA treatment, the success rate was 74%. The gestational period did not affect the outcome, irrespective of the age of the mare. In contrast, success rates decreased with increasing age of the mares (84% ≤ 7 years vs. 67% 8-14 years vs. 57% ≥ 15 years). In mares aged 8-14 years, a decrease in singleton pregnancies was observed, if TUA was performed after day 35 of gestation. Success rates were slightly higher, if twin vesicles were localized within separate uterine horns (73%) as compared to the same horn (66%). Differences in singleton pregnancy rates were not statistically significant (p > 0.05). TUA was found to be an effective procedure for reduction of twin pregnancies performed at days 30-62 of gestation. Success rates for singleton pregnancies were high for young mares ≤ 7 years old (84%) and middle aged mares treated before day 36 of pregnancy (74%). Duration of pregnancy at the time of TUA did not have a major impact on the outcome. Nevertheless, the procedure should optimally be performed around days 32-35 of pregnancy to allow for the possibility of natural reduction before treatment and rebreeding in case of a total pregnancy loss after TUA.

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 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%).

Macroscopic characteristics of the umbilical cord in Standardbred, Thoroughbred and Warmblood horses

The umbilical cord (UC), the connection between mother and fetus via the umbilical vessels, carries nutrients and oxygenated blood to the fetus through the umbilical vein and removes deoxygenated blood and waste products via the umbilical arteries. It is designed to protect blood flow to the fetus during pregnancy. In equine medicine, only a few studies have described the UC, and most of these involved Thoroughbreds. The present study describes and compares the macroscopic features of the equine umbilical cord in three different breeds and in relation to the foal's gender. In addition, a possible correlation between UC features and maternal and perinatal factors is investigated. One hundred and twenty four healthy mares with normal pregnancies were enrolled in the study and were divided into three groups according to their breed: 70 Standardbreds (STB), 38 Thoroughbreds (THB) and 16 Warmbloods (WAB). The following data were recorded: mare's age and parity, gestation length, placental weight, presence of fetal membrane alterations, UC length and number of coils in the amniotic and allantoic portions, and the Umbilical Coiling Index (UCI), which is the ratio between total coils and total UC length. The UCI has not been investigated previously in veterinary medicine. Furthermore, immediately after foaling, APGAR score, foal's weight and sex were recorded. All the STB and WAB were housed in Italy and the THB were housed in New Zealand. Mares' mean age was higher in WAB than in THB and STB; the latter had a significantly shorter gestation length. The foal's weight was positively correlated with placental weight in all breeds; and in STB, foal weight was positively related to parity and gestation length. Mean total UC length was comparable to previous reports in THB, STB and WAB. The lengths of the two UC portions were statistically different between STB and THB, where the amniotic portion was longer than the allantoic one. In each breed, total UC length was correlated with total number of coils (THB and STB = 5 ± 1; WAB = 6 ± 1), the UC amniotic length was positively correlated with the number of amniotic coils and the allantoic length was positively correlated with the number of allantoic coils. The UCI values were 0.09 in STB and THB and 0.1 in WAB. This study provides reference values for UCI that could be included in the gross placental evaluation if its clinical importance were demonstrated.

Influence of composition of feed and lactation period on mineral composition of Mare's

Effects of lactation period and feed on essential minerals composition of mare's milk were studied. Average Ca, P, Na and Mg concentrations in feed DM were 0.66, 4.30, 0.13 and 2.21 g.kg-1 of DM (dry matter), respectively. In regard to milk all elements concentrations were not similar to each other due to the changes of the lactation day differences. Average Ca, P, Na and Mg concentrations (in DM) caused by breed differences and lactation days were 1.95, 1.08, 0.53 and 0.22 g.kg-1, respectively. During the milk period, a high-quality feed were maintaining the major mineral composition of mare's milk in 1.5 - 2 times higher amount than milk of mare fed with pasture and a low-quality feed. The colostrum stage of mare was much shorter than other animals, the mare's milk on the 2nd day of lactation showed properties of initial milk in which its composition, particularly in minerals, were two times higher when compare to that on milk periods that was started from 5th day after parturition. It seemed that some factors, except well physiological conditions, such as mare's age, number of foaling, sex of foal etc. did not affect milk nutrient composition.

DIAGNOSIS OF ENDOMETRITIS IN INFERTILE ARABIAN MARES USING ULTRASONOGRAPHY AND BACTERIOLOGICAL EXAMINATION

Endometritis is one of the biggest problems affecting horses after colic and lameness. The current study was carried out on 26 mares (4-19 years old) of Straight Egyptian Arabian mares located in different 4 private farms. Ultrasonography revealed that most cases of endometritis were showing intraluminal fluid accumulation, although there was certain cases did not show fluid accumulation yet they suffered from endometritis and subfertility. The degree and severity of fluid accumulation was increased with age of mares. Additionally, there was a relationship between the fluid accumulation and echogenicity and the type of isolated microorganism whereas isolation of Staph and Strept associated with fluid formation and observation of clinical endometritis while in contrast with E.coli, it produced little amount of fluid only high degree of uterine edema was evident. Concerning bacterial isolation there were many incriminated bacteria for example Staphylococcus spp (35.1%), E.coli (18.9%), Proteus vulgaris (13.5%), Streptococcus spp (10.8%), Klebsiella pneumoniae (5.5%), and Pseudomonas aeriogenosa (2.7%) and there was 13.5% bacteriologically negative cases.

65 Placental Function at Term is Altered in Broodmares Fed with Cereals from Mid Gestation

Recent data obtained in our laboratory suggest that feeding pregnant broodmares with cereal concentrates may affect both mare and foal metabolism in the short and long term. Here, we investigated feto-placental biometry and placental function at term in mares fed with cereals and forage or forage only. Twenty-two multiparous mares inseminated with the same stallion were allocated to 1 of 2 groups from 7 months of gestation: group F (n = 10) were fed forage only, whereas group B (n = 12) received forage and cracked barley until foaling. At 3 and 9 months of gestation, a glucose tolerance test (IVGTT) was performed to evaluate the insulin resistance of pregnant mares. At birth, placentas and foals were weighed and measured. Placental samples were collected above the umbilical cord insertion and snap frozen. An RNA sequencing (RNAseq) analysis was performed on 9 placentas of each group. After normalization, gene levels were analysed using the DESEqn 2 package of R software (https://www.r-project.org/). Enrichment of gene sets was analysed using the Gene Set Enrichment Analysis (GSEA) software using the Kyoto Encyclopaedia of Genes and Genomes (KEGG) and Gene Ontology [GO, biological processes (bp), molecular function (mf) and cellular components (cc)] databases. Gene analysis statistical results were considered significant for P-values &lt; 0.05 after false rate discovery (fdr) correction. The IVGTT results were analysed using a type 3 ANOVA on a mixed linear model with group as fixed effect and age of the mare as random effect. At 3 months of gestation, maternal glucose metabolism was not different between groups. At 9 months, B mares had a higher insulin area under the curve (AUC) after glucose injection than F mares (P &lt; 0.01), without any difference in glucose AUC, suggesting that B mares were more insulin resistant than F mares. At birth, no difference was observed for feto-placental biometry between groups. Gene-level analysis could not discern differences in gene expression between groups after fdr correction. The GSEA analysis, however, showed that 8 gene sets were down-regulated in C placentas (2 KEGG, 2 GObp, 3 GOmf, 1 GOcc) and 193 gene sets were up-regulated (15 KEGG, 144 GObp, 12 GOmf, 22 GOcc) in B placentas. The down-regulated gene sets were involved in neutral amino acids and anion transport, fatty acid oxidation, acetyl coA synthesis, cholesterol and folate degradation, and the up-regulated gene sets were involved in RNA expression, inflammation (activation and recruitment of immune cells, MAPK signalling, complement and coagulation cascades, pro-inflammatory cytokine production and signalling) and in vascularisation (vasculogenesis, angiogenesis and smooth muscle cells development). The results are consistent with the altered function observed in term placentas of women who suffer from gestational diabetes. In conclusion, feeding pregnant mares with cereal from mid gestation alters the placental function at term. The authors thank the GeT platform (Toulouse, France) for the sequencing of the samples.

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

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. 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). Retrospective clinical study. 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. 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). Retrospective design of the study. The involvement of several veterinary surgeons over the 3-year period could have affected data recording. 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.

Advanced age in mares affects endometrial secretion of arachidonic acid metabolites during equine subclinical endometritis

Even if mares continue to breed up to an advanced age, in aging mares reproductive failure is quite common. Subclinical endometritis, which occurs more often in aging mares than in younger counterparts, may cause prolongation or shortening of the inter-estrus period or the corpus luteum lifespan. We hypothesized that during subclinical endometritis the secretion of selected arachidonic acid metabolites may differ in aging mares compared to younger females. To verify this thesis, ex vivo organ cultures of endometrium were established with subsequent measurements of concentrations of prostaglandin E2 (PGE2), 6-keto-PGF1α and both leukotrienes (LTs), LTB4 and LTC4 in the culture supernatants. The endometrial biopsies were obtained from 82 mares of known breeding history. This study revealed that the concentrations of the selected arachidonic acid metabolites, which act both as immunological mediators and endocrine modulators in the reproductive organs, depends on the mares' ages. Spontaneous endometrial secretion of PGE2, 6-keto-PGF1α and LTC4 was increased in mares aged 16–23 years that suffered from subclinical endometritis, compared with control counterparts. Moreover, secretion of these metabolites was higher in endometritis-positive mares aged 16–23 years than in younger females. We conclude that advanced age in mares further disturbs the immuno-endocrine balance in endometritis-positive mares.

The influence of age, antral follicle count and diestrous ovulations on estrous cycle characteristics of mares

Reproductive aging must be well understood to optimize the reproductive management of older mares and to predict their reproductive life-span. The objectives of this study were to: 1) examine age-related differences in follicular dynamics, endocrine profiles, and primordial follicle counts, 2) evaluate the influence of antral follicle count (AFC) on age-related changes in follicular parameters, and 3) determine the influence of diestrous ovulations on the estrous cycle. Young (3–8yr; n = 10), middle-aged (9–18 yr; n = 16), and old (>18 yr; n = 19) light horse mares were examined with transrectal ultrasonography to monitor follicular growth over two consecutive estrous cycles. Jugular blood samples were taken and plasma progesterone and FSH concentrations were determined by an enzyme immunoassay and radioimmunoassay, respectively. Both interovulatory intervals and follicular phases were longer and the day of follicle deviation occurred later in aged mares. Furthermore, older mares had a tendency to ovulate smaller follicles. Neither follicular growth rate, the number of ovulations nor the length of luteal phase was influenced by mare age. Interestingly, as mare age increased, mares with low AFC had longer interovulatory intervals and follicular phases than mares with medium or high AFC. In addition, the number of primordial follicles declined with an increase in mare age but varied considerably between mares of the same age. Progesterone concentrations were positively influenced by age, whereas FSH concentrations were not, despite that FSH concentrations appeared higher in aged mares during the follicular phase. Estrous cycles with a diestrous ovulation had a longer interovulatory interval as well as a longer follicular and luteal phase while day of deviation occurred later. Progesterone concentrations were significantly higher on day 14 and 16 in estrous cycles with a diestrous ovulation than without a diestrous ovulation. In conclusion, aging in mares is associated with changes in follicular parameters which in turn are closely linked to differences in antral follicle count suggesting a relationship with ovarian reserve. Therefore, determination of antral follicle counts in aged mares can provide valuable information about the reproductive aging process. Finally, diestrous ovulations have a significant influence on different estrous cycle parameters.

Factors influencing reproductive efficiency and pregnancy in Pura Raza Española mares

The aim of this study was to analyse ovarian activity and pregnancy in 140 Pura Raza Espanola mares over a period of two consecutive years. The data were analysed using SPSS software to evaluate the effects of age, category of mares and season on pre-ovulatory follicle size, ovulation rate, interval from foaling to pregnancy, gestation length, incidence of twin pregnancies and pregnancy losses. The results showed that follicular diameter was significantly (P &amp;lt; 0.05) smaller in fillies (3-year-old) and maiden mares than in older and multiparous mares. Within the group of animals treated with gonadotropin hormone hCG, 87.23% ovulated 48 h following administration. Length of gestation varied from 305 to 356 days and all gestation pregnancies of less than 305 days resulted in a non-viable foal. In addition, foaling season affected (P &amp;lt; 0.05) the post-partum conception interval and the length of gestation, which were shorter and longer in the winter foaling, respectively. Regarding the other parameters, mares of this stud exhibited relatively very low rates of double ovulations, twin pregnancies and pregnancy losses. We conclude that age and category of mares and foaling season influence fertility and foaling rates by affecting pre-ovulatory follicle size, pregnancy loss and gestation length.

97 PRELIMINARY DESCRIPTIVE STUDY OF EQUINE PLACENTA GENERATED AFTER TRANSFER OF IN VIVO- AND IN VITRO-PRODUCED EMBRYOS

Placental changes associated with artificial reproductive technologies have been described in several species, but little information is available in horses. Joy et al. (2012) reported that human placentas from intracytoplasmic sperm injection derived embryos were heavier and thicker than those produced after natural conception. Despite the most growing interest and efficiency of artificial reproductive technologies in equine species, only recently, Pozor et al. (2016) described placental abnormalities in pregnancies generated by somatic cell NT, but there are no studies on equine placenta generated by intracytoplasmic sperm injection and traditional embryo transfer. In the present preliminary study, macroscopic differences of placentas generated after transfer of in vitro- or in vivo-produced embryos were registered. Twelve Standardbred recipient mares with pregnancy generated after transfer of in vivo-derived (Group 1) and in vitro-derived (Group 2) embryos were enrolled; 10 Standardbred mares with pregnancy derived by traditional AI were included as control (Group 3). All pregnancies were physiological, and newborn foals were healthy. Mare age, parity, length of pregnancy, gross evaluation and weight of placenta, total length of umbilical cord (UC), length of UC, number of UC coils, foal sex, and weight at birth were registered. Collected data are listed in Table 1 and are expressed as mean ± standard deviation. Differences between groups were evaluated by 1-way ANOVA, and the difference in proportion of overweight placentas was evaluated with the Fisher test. The gross evaluation of placenta revealed 8/12 placentas (2/4 Group 1; 6/8 Group 2) were heavier than 11% (Madigan, 1997) due to oedema of the chorioallantois. No overweight placentas were registered in Group 3. In Group 1, 1/4 placentas had villous hypoplasia, and in Group 2, 1/8 placentas had cystic pouches on the UC. There were no significant differences among groups. However, the proportion of overweight placentas between Group 2 (6/8) and Group 3 (0/10) approached significance (P = 0.06). Although preliminary, the results of the present study suggest that production of equine embryos in vitro may lead to alterations in placental development. Several studies in cattle and sheep have suggested that alterations in the placentas of pregnancies derived from in vitro-produced embryos are related to effects of culture on epigenetic regulation. Less is known in the horse about the effects of in vitro embryo production on placental development; thus, further research in this area is necessary. Table 1. Characteristics of full-term placentas derived from AI or embryo transfer with in vivo- and in vitro-produced embryos

Leading causes of pregnancy loss in the equine species: Review

The pregnancy loss are common problems in the creation of equine, being the cause of concern among professionals, the numerous economic losses to the agribusiness sector. Early miscarriages are more incidents late, being attributed to maternal, environmental and embryonic factors. The factors include maternal reproductive tract changes, the mare's age, progesterone deficiency, when the cover reproductive status, failure in maternal recognition of pregnancy covering the foal heat, among others. Environmental factors include maternal stress, nutrition and effect of semen. Late pregnancy losses are fewer incidents and were attributed mainly to infectious disorders related to fetal-placental interactions, as ascending placentitis of origin. Equine herpesvirus type I and leptospirosis are the most common infectious causes, making essential health control and vaccination of the squad. Due to the alarming figures, the aim of this study, a literature review describing the main causes of pregnancy loss in the equine species.

Dose-dependent inhibition of uterine contractility by nitric oxide: A potential mechanism underlying persistent breeding-induced endometritis in the mare

Nitric oxide (NO) may have a role in persistent breeding-induced endometritis in mares through an inhibitory effect on uterine contractility. The objectives of this study were to test the effect of NO on spontaneous uterine contractility in-vitro and to evaluate whether this effect varied between the longitudinal and circular muscle layers of the uterus. Reproductive tracts were collected from eight euthanized non-pregnant mares (age 4–19 years; body weight 405–530 kg). Transrectal examination of the reproductive tract was performed before euthanasia to evaluate stage of the estrous cycle and presence of any apparent abnormality. After euthanasia, one uterine tissue sample was collected for histological evaluation and four full-thickness uterine tissue strips (10–12 mm × 2 mm), two parallel to each muscle layer, were excised for in-vitro contractility evaluation. Strips were suspended in tissue chambers containing Krebs–Henseleit solution, with continuous aeration (95% O2–5% CO2; pH 7.4) at 37 °C. After equilibration, spontaneous contractility was recorded (pre-treatment) and strips excised in each direction were randomly allocated to each of two groups: 1) SNAP (S-nitroso-N-acetylpenicillamine, an NO donor); or 2) NAP (N-acetyl-d-penicillamine, vehicle and time-matched control). These were treated at 15 min intervals with increasing concentrations (10−7 M to 10−3 M) of SNAP and NAP, respectively. Contractility data was recorded throughout the experiment. An interaction effect of group-by-concentration was observed (P < 0.0001). The mean contractility after treatment with 10−4 M and 10−3 M SNAP were significantly lower than the pre-treatment contractility and the mean contractility after treatment with lower SNAP concentrations. In contrast, contractility did not change significantly in the NAP treated controls. The effect of treatment on uterine contractility was not influenced by age or weight of the mare, stage of estrous cycle, uterine histology grade, or muscle layer. Secondary findings included significant main effects of stage of estrous cycle (increased contractility in estrus compared to diestrus), uterine histology grade (decreased contractility in grade IIB compared to grade I) and age (decreased contractility in mares aged > 8 years compared to mares aged ≤ 8 years). In conclusion, results of this study indicate that NO has a dose-dependent inhibitory effect on spontaneous uterine contractility irrespective of the muscle layer in the mare.