2010: Early embryonic death in mares Early embryonic death in mares Dale L. Paccamontia and Elaine Carnevaleb aDepartment of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA; bEquine Reproduction Laboratory, Colorado State University, Fort Collins, CO Introduction Early embryonic death (EED) is defined as loss of the conceptus before organogenesis is complete, generally regarded as before 40 days of gestation in the mare. Using ultrasonography, estimates of EED range from 5-23% between 12 to 50 days of gestation.1-4 Whether critical periods exist during which the incidence of EED is highest has not been firmly established.1 Overall, pregnancy losses are greatest early in gestation, before 35 days or even earlier before the embryo enters the uterus.1,4,5 Estimates of the incidence of EED rely on the method used to diagnose pregnancy and the ability to detect EED. Keywords: Early embryonic death, mare, infertility Diagnosis Although continued low progesterone is not compatible with pregnancy maintenance, a single low progesterone value does not necessarily indicate impending EED. Low progesterone concentration at more than one sampling is required before a diagnosis of EED or non-pregnant can be made.6-8 Estrone sulfate is a product of a viable conceptus and, therefore, can be used as an indicator of pregnancy.9 However, estrone sulfate is not a reliable indicator of fetal viability until at least 44 days of gestation,10 and may not be useful as a valid indicator until after 80 to 90 days of gestation. Early pregnancy factor, based on the rosette inhibition test, has been proposed in a number of species as a means to diagnose pregnancy and therefore detect EED at a very early stage. Although shown to be unreliable for detection of pregnancy and non-pregnancy in cattle11,12 and horses,12 it has been suggested to be useful for monitoring the viability of equine embryos.13 When EED occurred between days 11-15 after ovulation, no ultrasonographic indication of impending EED was noted.14 Later in gestation, however, indications of impending EED include an abnormal appearance of the conceptus, continued mobility of the vesicle, fluid surrounding the vesicle, absence of a heartbeat, decreased volume of conceptual fluids, disruption of conceptual membranes or edema of the endometrial folds.14-16 (Figures 1 and 2) Between days 20 and 45 of gestation, EED was first characterized by a speckled or granular appearance of the conceptus.17 The conceptus then decreased in size and lost clear definition before disappearing completely within four to seven days of the onset of the granular appearance.17 Doppler ultrasonography has been suggested as a tool to assess embryo viability or impending death.16,18 In a normal pregnancy, blood flow is greater in the uterine arteries of the gravid horn. A relative decrease in flow in the arteries of the gravid horn compared to the nongravid horn was observed after the administration of exogenous prostaglandin F2a (PGF).18 Embryos that are smaller than normal may be lost at an increased rate compared to normal sized embryos, while oversized vesicles are not at risk.19 Abnormal embryo development has been associated with the formation of trophoblastic vesicles that can be observed after day 22 of gestation that lack an embryo proper.20 Potentially, these structures are formed after embryonic insult and death of the inner cell mass, the cells within the embryo that will form the embryo proper. (Figure 3) The trophoblast layer of the embryo continues to grow, resulting in a vesicle without development of the embryo proper. The vesicle may be small compared to a normal vesicle. Although the characteristic bulge associated with pregnancy may not be palpable, uterine and cervical tone are usually typical of a pregnant mare. Diagnosis of EED by palpation and ultrasound is facilitated when the vesicle collapses and the contents spread out into the uterus. Although pregnancy loss can usually be detected by a loss of uterine tone and the palpable bulge, the bulge in the uterus may remain palpable for up to a week after conceptus death.9 Cervical patency at the time of embryonic loss indicates that embryos are probably lost by passage through the cervix rather than by resorption.21 If the mare is on supplemental progesterone, the progression of embryo loss may be changed, as the cervix will remain closed. 99 Figure 1. Embryonic vesicle in the left uterine horn (left image), with fluid within the lumen of the right uterine horn (right image). The fluid within the uterine lumen moved to surround the embryonic vesicle. In most cases, embryonic death will occur. Figure 2. Aborting fetuses with disruption of membranes and granular debris. Heartbeats were not imaged in the thorax, and fetal features are less defined. After being lavaged from the uterus of a mare, the fetus and membranes were intact, but degenerative changes were occurring. 100 Figure 3. A 7-day embryo collected from the uterus of a mare. The embryo has a layer of trophoblast cells that will form the placental membranes. The thicker area of cells represents the inner cell mass, the cells responsible for formation of the embryo proper Causes Fertilization rates are apparently very high in horses,22,23 although differences may exist between normal and subfertile mares.22 One problem in the assessment of fertilization and EED is that the equine embryo remains in the oviduct for approximately 5.5 days before entry into the uterus,24 and ovulatory or oviductal problems may affect early pregnancy rates. When recently ovulated oocytes or early embryos were recovered from the oviducts of mares, significantly fewer recently ovulated oocytes or embryos were recovered from old mares (≥ 20 years) than young mares (two to ten years), suggesting failure of ovulation or oviductal pickup of the oocyte. Therefore, fewer oocytes had the potential for fertilization and embryo development in the older mares, although fertilization rates were not different.25 Genetic abnormalities play a significant role in pregnancy loss in other species,26 and similar findings can be expected in horses. Chromosomally abnormal cells have been identified in morphologically normal equine embryos27 and an increased incidence of EED has been associated with certain family lines28 or stallions.29 The majority of chromosomal abnormalities leading to EED may not be inherited but arise during formation and ageing of gametes. These abnormalities may result in an inability of the resulting zygote to develop into a viable embryo.30 Early embryonic death due to aged gametes may be increased when mating is not closely synchronized with ovulation.30-32 Although insemination after ovulation can result in pregnancy, the incidence of EED is higher than when insemination occurs before ovulation33 and increases as the interval from ovulation to insemination lengthens.32 Observable or unobservable defects in the embryo soon after its formation may alter or affect its metabolic function. Differential transport of unfertilized ova and embryos by the oviduct is apparently a function of the embryo rather than the oviduct.23,34 Prostaglandin E2 (PGE), secreted by the equine embryo, is apparently responsible for instigating embryo transport.35,36 Administration of exogenous PGE has reportedly hastened embryo transport.34 Some degree of synchrony between the embryo and the uterus is critical for successful establishment of pregnancy. Therefore, pregnancy might not be established if an embryo was unable to produce sufficient quantities of PGE to induce 101 oviductal transport at the required time. Similarly, conditions resulting in increased concentrations of PGE may hasten transport resulting in early arrival of the embryo. Substances produced by the embryo may be important for its continued survival after arrival in the uterus. Equine embryos produce estrogen which may be important in maternal recognition and maintenance of pregnancy.37 Estrogen plays a role in the increased tone seen in the early pregnant mare which is influential in fixation of the embryo38 and has a synergistic effect with progesterone to stimulate uterine specific proteins39 which contribute to histotrophe. In a retrospective study, embryo morphology was graded before transfer as excellent, good (minor morphological imperfections) or fair/poor (moderate or severe changes in morphology).40 After transfer into recipient mares, pregnancy rates at 16 days were significantly higher for excellent and good embryos than for fair/poor embryos. However, although pregnancies were established at 16 days, the embryo loss rate from days 16 to 50 was higher for good than excellent embryos, and the embryo loss rate was similar for good and fair/poor embryos.40 Results of the study demonstrate that relatively minor morphological defects in embryos may indicate reduced potential for normal embryo and fetal development. Unrestricted conceptus mobility is essential for pregnancy maintenance and prevention of luteolysis.41 If uterine contractions, the main propulsive force stimulating embryo movement,14 are not sufficient to move the embryo throughout the uterus and inhibit luteolysis, EED may result. Uterine contractility is reduced in old versus young mares.42 Removal of the endogenous progesterone source resulted in a reduction in uterine contractions and conceptus mobility and a failure of embryonic fixation.21 The embryo was subsequently found more often in the uterine body,21 a condition associated with increased EED.43 It appears that prostaglandins also play a key role in embryonic mobility.44 Inhibition of prostaglandins with flunixin meglumine results in a marked decrease in embryonic migration.44 Experimental restriction of conceptus mobility has resulted in luteolysis followed by conceptus loss; however, administration of a progestogen was able to rescue the pregnancy.41 Pathology of the uterus such as transluminal adhesions or endometrial cysts could restrict conceptus mobility sufficiently to mimic the experimental model resulting in EED. A critical period in the establishment of pregnancy occurs at 14 to 16 days of gestation when the presence of the conceptus is needed to inhibit luteolysis.41 Factors of conceptus origin inhibit the ability of the endometrium to release PGF between days 12 to 16 after ovulation and are essential for maintenance of luteal function.45 If function of the corpus luteum is not maintained because the conceptus fails to block PGF release, either from inadequate migration or from retarded development of the conceptus, pregnancy loss will result.41 (Figure 4) A twin pregnancy rarely results in the birth of two healthy viable foals. If twins survive until late gestation, abortion, stillbirth or dystocia are likely outcomes. Fortunately, a natural reduction mechanism exists, provided both conceptuses are fixed in the same uterine horn, whereby one conceptus of a pair undergoes EED. The mechanism for embryonic reduction is hypothesized to be the result of deprivation of nutrients due to one conceptus preventing contact of the trilaminar omphalopleure of the other conceptus with the endometrium.46 Reproductive status of the mare may reflect maternal-embryonic interactions. Not surprisingly, pregnancy rates are lower and the incidence of EED higher for infertile mares than for normal mares.47,48 Embryos were recovered at a reduced rate from subfertile mares,49-52 although in one study, a majority of subfertile mares contributed a transferable embryo on at least one occasion after repeated embryo collection attempts.53 Similar pregnancy rates have been found between subfertile and normal mares at two days after ovulation; however, the pregnancy rate at day 14 was reported to be greatly reduced in subfertile mares.54 In general, subfertile mares tend to be older than fertile mares; therefore, some aspects of subfertility may be directly associated with aging. In the past, fertility was considered to be primarily dependent on uterine environment. Pregnancy rates were similar for embryos from normal and subfertile mares when transferred into normal mares.49 Moreover, initial pregnancy rates were similar after transfer of embryos to normal and subfertile recipient mares; however, subsequent EED was greater for subfertile recipients.51 Recent evidence indicates other factors are involved. Transfer of embryos from normal mares to normal and subfertile recipients resulted in similar pregnancy rates at day 12 or 28, indicating that the uterus of a subfertile mare is capable of supporting an embryo from a normal mare.55 Embryos collected from the oviducts of normal and subfertile donors four days after ovulation and transferred 102 Figure 4. An embryonic vesicle at 11 days (left image) with a regressing CL on the right ovary. Unless exogenous progesterone is administered, the mare will return to estrus, and EED will occur. to normal recipients resulted in lower pregnancy rates at day 14 for mares receiving embryos from subfertile donors than for mares receiving embryos from normal donors.22 Other researchers have also found a lower pregnancy rate and an increased incidence of EED after transfer of embryos from subfertile mares when compared to those obtained from maiden mares.52 In addition, abnormal embryos have been collected more often from subfertile mares than from maiden mares.56 Maternal aging has been shown to affect fertility and embryo loss in the mare. Extremes of age, either young or old, negatively affect fertility.57,58 The high rate of EED in yearling mares has been attributed to immaturity, inadequate nutrition or physical stress.57 Older mares were found to have a lower pregnancy rate and a higher loss rate compared with younger mares,22,48,58 and a decrease in live foaling rate.2,58-60 In a study of nearly 1400 mares in Newmarket, mares three to eight years of age experienced a 5% pregnancy loss between 15 and 35 days of gestation, while mares nine to 13 years of age had a 14% loss and mares 14 years of age or older showed a marked increase to approximately 22% EED.3 In a controlled experiment, using semen from the same stallion and similar breeding management, the embryo loss rate between 12 and 39 days was 11% for young mares (five to seven years) and 62.5% for older mares (≥ 15 years),42 suggesting differences in the ability of mares of different ages to establish and maintain a pregnancy. The effect of advanced age, however, is difficult to separate from acquired subfertility61 and is confounded by the effect of parity since the two are generally correlated.52 In one study, the incidence of embryonic resorption increased with parity.2 The effect of age on fertility, at least in part, originates in the oocyte. When embryos were collected from the oviducts of young mares (two to nine years) and old mares (≥ 20 years) significantly fewer cells and poorer morphology were noted for embryos from old mares.25 In a subsequent study, the researchers used oocyte transfer to compare fertility of oocytes collected from the follicles of young mares (six to ten years) or old mares (20 to 26 years).62 Mature oocytes were transferred into the oviducts of young, inseminated recipients, and embryo development rates were determined. More oocytes from young than old mares (11/12, 92% and 8/26, 31%) resulted in embryonic vesicles. Results of the study indicate that intrinsic defects occur in the oocytes of old mares, and these defects may affect fertility and early embryo loss. The repeatability of EED may depend on the inciting cause. Some authors indicate that mares which experience EED are more likely to again experience loss,63 while others have failed to find 103 such an association.48 Oviductal pathology may result in EED or fertilization failure. During post mortem examinations, salpingitis, which might affect embryo transport, oviductal secretions, and embryo support, was not uncommon.64 Almost all oviducts were patent, although adhesions involving infundibula were common.64 Uterine pathology may affect histotrophe production, which the early embryo relies on, and result in retarded embryonic development. Higher pregnancy rates have been reported when recipient mares had normal endometrium compared with mares that had moderate uterine pathology.50 Endometrial cysts are common in older mares. Most mares over 17 years of age have some cysts, and mares over 11 years of age are four times as likely to have cysts as younger mares. The effect of cysts on fertility is unclear, because studies are often confounded by other factors such as age and parity. A study of nearly 300 Thoroughbred mares found that cysts did not affect the ability to establish or maintain pregnancy; however, the time of initial pregnancy examination was not controlled and embryonic losses occurring before pregnancy examinations were not detected.65 The odds ratio of establishing pregnancy was suggestive of a negative effect.65 Other studies have corroborated the quantitative effect of cysts on fertility. If endometrial cysts are present in sufficient number or size, a decrease in fertility results; however, the effect on fertility is less than that caused by delayed uterine clearance. Although endometrial cysts may interfere with maternal recognition of pregnancy, once pregnancy is established they do not increase the likelihood of pregnancy loss.65 Progesterone, normally supplied by the corpus luteum, is essential for the maintenance of pregnancy.63,66,67 Evidence pointing to primary luteal insufficiency as a major cause of EED is lacking.5,68 Secondary luteal insufficiency due to premature luteal demise as a result of uterine pathology is a more likely cause of EED.8 Pregnancy loss after ovariectomy or PGF administration can be prevented by progestogen supplementation.6,7,63,66 Progesterone dependent endometrial proteins have been demonstrated in the mare, and the lack of these proteins following luteolysis may be associated with a decrease in the quality of histotrophe, contributing to EED.69 Supplemental progesterone, however, has not improved pregnancy rates in normal mares receiving embryos.70 Although PGF release from the uterus is normally responsible for lysis of the corpus luteum, other potential sources of PGF exist which can cause either complete luteolysis or decreased luteal function resulting in EED.71 Endotoxemia or other febrile conditions can stimulate PGF release sufficient to cause decreased luteal activity precipitating EED.71 Mares in early gestation, before endometrial cup formation, are at more risk of pregnancy loss. Although it is commonly believed that the developing corpus luteum is unaffected by PGF until five days after ovulation, studies have shown that some mares will respond as early as three days after ovulation.72 Furthermore, Irvine, et al.73 have shown that repeated low doses of PGF are very effective in inducing luteolysis. These studies would seem to indicate that inflammatory conditions, even soon after ovulation, warrant attention. Prostaglandin inhibitors such as flunixin meglumine are effective in blocking PGF release, but must be administered soon after the onset of endotoxemia.71,74 Delaying flunixin until two hours after endotoxin administration failed to prevent pregnancy loss.74 Exogenous progestogens, however, can maintain pregnancy even after luteal activity is compromised,71,74 but should be continued until placental progestins are capable of maintaining pregnancy.75 Human chorionic gonadotropin, although generally regarded as luteotrophic, has been shown to induce luteolysis, resulting in EED when given between 24 and 39 days of gestation.76 Endometritis is an important cause of EED in mares48 and usually causes loss before maternal recognition of pregnancy. Once pregnancy is established, endometritis is a less frequent cause of EED,4 provided anatomical defects are corrected. Embryonic death may result either indirectly as a result of inflammatory mediated prostaglandin release affecting luteal function or embryo migration or from a direct embryotoxic effect of the inciting agent or inflammatory process. For example, altered intrauterine concentrations of PGE, which play an important part in oviductal transport of embryos,34 has been associated with endometritis in mares.77 Direct embryotoxic effects of pathogenic organisms have been reported,56 as well as luteolysis secondary to induced endometritis.15 An inflammatory reaction to sperm cells in the uterus is part of the normal physiologic response to mating. In normal mares, fluid and debris are easily evacuated from the uterus and the inflammatory response subsides relatively quickly. A delay in uterine clearance results in persistent mating-induced endometritis (PMIE) which will result in EED, either directly or through luteolysis.78 Treatment of PMIE often includes administration of drugs such as oxytocin or cloprostenol, a PGF analog, to 104 augment uterine contractility and improve clearance. Oxytocin is generally regarded as safe when administered during the periovulatory period. Cloprostenol, on the other hand, has been shown to depress progesterone concentrations during mid-diestrus when given in the immediate post-ovulatory period.79-82 Although the literature on the effect of post-ovulatory cloprostenol on pregnancy rates is equivocal,79,81,82 it would seem advisable to err on the side of caution and restrict cloprostenol use to the pre-ovulatory period and use only oxytocin after ovulation. Contagious equine metritis, a transmissible venereal disease caused by Taylorella equigenitalis, commonly results in EED after initial infection.83 Clinical signs are confined to the mare and vary from acute endometritis to an inapparent carrier state.83 The stallion can also maintain the organism as a carrier.83 Control has been achieved by identifying carriers, improved hygiene during breeding, use of artificial insemination and surgical ablation of the clitoral sinuses.83 Mare reproductive loss syndrome is associated with both EED and abortion. Although the exact etiology is still unclear, reproductive losses have been linked to ingestion of eastern tent caterpillars, specifically the setae of the caterpillars.84 Administration of West Nile virus vaccine to pregnant mares has been shown to be safe and is not associated with EED.85 Early embryonic death has been associated with conception occurring too soon after foaling.59,86 Other studies have not substantiated these claims and have found that although pregnancy rates may be lower, no greater losses occur after foal heat breeding than after later breedings.48 Examining factors associated with an increase in EED, McKinnon et al. reported that pregnancy rates are lower in mares bred on foal heat when intrauterine fluid was observed ultrasonographically.87 A similar relationship was found between the presence of intrauterine fluid at foal heat and a greater risk of EED.88 In a study examining recovery rate and embryo quality in mares bred on foal heat compared with mares bred at a later estrus, the authors concluded that EED was not the reason for lower pregnancy rates in mares bred on foal heat, and hypothesized that sperm transport or oviductal conditions were more likely to blame.89 The effect of lactation on the incidence EED is likewise unclear. Lactating mares have been reported to have a lower,59 a higher,86 or the same60 incidence of EED as nonlactating mares. An increased incidence of pregnancy loss has been associated with nutritional stress.90,91 Mares suffering from malnutrition experienced an increase in EED, which could be averted by supplemental feeding.91 In a later study, the authors reported an association of poor quality protein in the ration with increased pregnancy losses, which they attributed to inadequate production of progesterone by the corpus luteum.92 Restriction of dietary intake was reportedly successful in reducing twin pregnancies to singletons in 60% of treated mares.93 Although reportedly beneficial in cows, nutritional supplementation with chelated or inorganic minerals failed to show an effect on conception, pregnancy rate, or EED in mares.94 No adverse effects on reproduction have been observed in obese mares.90,95 When mares at 16 to 22 or 32 to 38 days of gestation were hauled for nine hours without food or water, progesterone and adrenocorticotropic hormone increased, indicating acute stress; however, pregnancy losses were found to be no different than in non-transported mares.96,97 Stress due to pain, infectious disease or emotional disturbances such as weaning have been associated with decreased progesterone.98 Administration of corticosteroid similarly has resulted in a drastic yet transient decrease in progestogen which is apparently not a result of luteolysis mediated by PGF.98 Repeated palpation or ultrasound examinations have not been associated with an increase in EED.99,100 Long-term anabolic steroid treatment has untoward effects on subsequent reproductive performance. A trend towards higher EED was observed in mares treated during the previous year with anabolic steroids.47 Occasionally, there may be a need to induce EED. Choice of the best method depends on the stage of gestation.101 Within five days of ovulation, no consistently effective method exists because the developing corpus hemorrhagicum is relatively unresponsive to the effects of PGF and because the embryo, still within the oviduct, is protected from the uterine environment. From five days after ovulation until the formation of the endometrial cups at approximately 35 to 38 days, administration of PGF is the preferred method to terminate pregnancy. Other methods such as manual crushing of the vesicle and uterine infusions, though effective, carry the possibility of untoward side effects.101 After the formation of the endometrial cups, repeated injections of PGF are usually needed to induce EED. In the case of twins, manual crushing of one vesicle is the technique usually employed to 105 reduce the pregnancy to a singleton. Most other reported methods do not consistently achieve the same success.101 Prevention or treatment Effective methods to reduce contamination at breeding and treat endometritis should be employed to allow establishment of pregnancy. Procedures such as Caslick’s surgery are necessary if anatomic defects such as poor perineal conformation are present. Maintenance of good general health and nutrition will likely improve pregnancy rates and reduce EED. Failure to maintain pregnancy due to premature release of PGF attributable to inflammatory processes has been successfully treated with flunixin meglumine.102 In the same report, repeated EED attributed to a failure of the normal process of maternal recognition of pregnancy to maintain luteal function was successfully prevented with altrenogest despite regression of the primary corpus luteum and low endogenous progesterone.102 Although nonsteroidal anti-inflammatory drugs can inhibit PGF release resulting from endotoxin infusion, administration must occur within a very short time of endotoxin release, limiting their usefulness under natural conditions.71,74 Conceptus death may not occur until as long as 48 hours after luteolysis,74 and embryos can apparently continue to grow for some time after loss of endogenous progesterone.21 Therefore, sufficient time exists to institute progestogen therapy to rescue the pregnancy.21,71,74 Pregnancy has been maintained in mares lacking sufficient endogenous progesterone with the daily administration of progesterone in oil or altrenogest, but was subsequently lost if therapy was stopped before day 50 of gestation.66 Results of ovariectomy after day 50 are variable with some mares able to maintain pregnancy without exogenous progestogen. To ensure pregnancy maintenance, exogenous progestogen therapy should be continued through the first four months of gestation, until the placenta can provide adequate progestogens for pregnancy support. Some workers suggest that if the uterine environment is normal, progesterone therapy is not needed.8 The difficulty lies in determining if the uterine environment is normal from the viewpoint of the conceptus. Administration of altrenogest on days six to 20 to supplement endogenous progesterone or buserelin on day ten to bolster endogenous progesterone production did not significantly affect progesterone concentrations or conceptus size.103 If exogenous progestogen therapy is to be instituted, sufficient dosages should be used. Minimum dosages to maintain adequate circulating concentrations in plasma include daily administration of 200 to 300 mg progesterone in oil or 22 mg altrenogest.7,104 Lower doses or less frequent administration of these products were insufficient to maintain pregnancy.6,7 Aqueous forms of progesterone are cleared too rapidly to be of use for pregnancy maintenance.7 A long-acting injectable formulation of progesterone administered on a weekly basis has been shown to be effective in maintaining pregnancy.105 Pregnancy was not maintained in ovariectomized mares using a number of synthetic progestogens, including medroxyprogesterone, norgestomet and megestrol acetate; however, altrenogest at 0.044 mg/kg was able to maintain pregnancy.106 Although 22 mg altrenogest is reportedly sufficient for maintaining pregnancy,67 other workers suggest that higher doses are needed to provide optimal uterine and cervical tone and are preferred for pregnancy maintenance.67,107 McKinnon, et al.67 propose that if the progesterone concentration at 24 hours after exogenous progestin therapy is 2.5 ng/ml or more, exogenous therapy may be unnecessary. Viability of a pregnancy should be reaffirmed when long-term progestogen therapy is practiced. Retention of a conceptus after fetal death may result from continued treatment.108 Intensive management alone may accomplish the objective of getting a mare to carry a foal to term. In a report of six mares which had lost their pregnancies two to five times in the preceding six years, five had normal term pregnancies without any special treatment other than good management and being mated to a highly fertile stallion.109 Acknowledgement. Figures courtesy of Dr. Elaine Carnevale. References 1. Ball BA: Embryonic loss in mares. Vet Clin North Am Equine Pract 1988;4:263-290. 2. Chevalier-Clement F: Pregnancy loss in the mare. Anim Reprod Sci 1989;20:231-244. 106 3. Morris LHA, Allen WR: Reproductive efficiency of intensively managed Thoroughbred mares in Newmarket. Equine Vet J 2002; 34:51-60. 4. Ricketts S W: Early pregnancy failure as seen during the course of equine stud farm practice in Newmarket, 1996- 2003. Pferdeheilkunde 2003;19:633-638. 5. Forde D, Keenan L, Wade J, et al: Reproductive wastage in the mare and its relationship to progesterone in early pregnancy. J Reprod Fertil Suppl 1987;35:493-495. 6. Shideler RK, Squires EL, Voss JL, et al: Progestagen therapy of ovariectomized pregnant mares. J Reprod Fertil Suppl 1982;32:459-464. 7. Squires E, McKinnon AO: Hormone therapy for control of reproduction in mares and stallions. Vet Clin North Am Large Anim Pract 1987;3:81-99. 8. Stabenfeldt GH, Hughes JP 1987: Clinical aspects of reproductive endocrinology in the horse. Compend Cont Educ Pract Vet 1987;9:678-684. 9. Iuliano MF, Squires EL: Effect of exogenous progesterone on pregnancy rates after surgical embryo transfer in mares. Theriogenology 1986;26:291-298. 10. Kasman LH, Hughes JP, Stabenfeldt GH, et al: Estrone sulfate concentrations as an indicator of fetal demise in horses. Am J Vet Res 1988;49:184-187. 11. Gandy B, Tucker W, Ryan P, et al: Evaluation of the early conception factor (ECFTM) test for the detection of nonpregnancy in dairy cattle. Theriogenology 2001;56:637-647. 12. Metcalf ES, McCue PM, Jasko DJ, et al: Evaluation of a test for equine early conception factor. Proc Annu Conv Am Assoc Equine Pract 2004. p. 518-520. 13. Takagi M, Nishimura K, Oguri N, et al: Measurement of early-pregnancy factor activity for monitoring the viability of the equine embryo. Theriogenology 1998;50:255-262. 14. Ginther OJ: Dynamic physical interactions between the equine embryo and uterus. Equine Vet J Suppl 1985;3:41- 47 15. Ball BA, Shin SJ, Patten VH, et al: Embryonic loss in pony mares induced by intrauterine infusion of Candida parapsilosis. Theriogenology 1988;29:835-847. 16. Stabenfeldt GH, Hughes JP: Clinical aspects of reproductive endocrinology in the horse. Compend Cont Educ Pract Vet 1987; 9:678-684. 17. Simpson DJ, Greenwood RES, Ricketts SW, et al: Use of ultrasound echography for early pregnancy diagnosis of single and twin pregnancy in the mare. J Reprod Fertil Suppl 1982;32:431-439. 18. Chen YH, Stolla R: Using "uterine index" to diagnose embryonic death in mares. J Equine Vet Sci 2006;26:219- 224. 19. Ginther OJ, Bergfelt DR, Leith GS, et al: Embryonic loss in mares: incidence and ultrasonic morphology. Theriogenology 1985;24:73-86. 20. Vanderwall DK, Squires EL, Brinsko SP, et al: Diagnosis and management of abnormal embryonic development characterized by formation of an embryonic vesicle without an embryo in mares. J Am Vet Med Assoc 2000;217:58-63. 21. Kastelic JP, Adams GP, Ginther OJ: Role of progesterone in mobility, fixation, orientation, and survival of the equine embryonic vesicle. Theriogenology 1987;27:655-663. 22. Ball BA, Little TV, Weber JA, et al: Survival of day-4 embryos from young, normal mares and aged, subfertile mares after transfer to normal recipient mares. J Reprod Fertil 1989; 85:187-194. 23. Peyrot LM, Little TV, Lowe JE, et al: Autotransfer of day 4 embryos from oviduct to oviduct versus oviduct to uterus in the mare. Theriogenology 1987;28:699-708. 24. Freeman DA, Weber JA, Geary RT, et al: Time of embryo transport through the mare oviduct. Theriogenology 1991;36:823-830. 25. Carnevale EM, Griffin PG, Ginther OJ: Age-associated subfertility before entry of embryos into the uterus in mares. Equine Vet J Suppl 1993;15:31-35. 26. Berepubo NA, Long SE: A study of the relationship between chromosome anomalies and reproductive wastage in domestic animals. Theriogenology 1983;20:177-190. 27. Rambags BPB, Krijtenburg PJ, VanDrie HF, et al: Numerical chromosomal abnormalities in equine embryos produced in vivo and in vitro. Mol Reprod Dev 2005;72:77-87. 28. Moberg R: Untersuchungen uber das Auftreten und die Atiologie von embryonalem Fruhtod bei der Stute. Proc 7th Int Cong Anim Reprod Artificial Insemin; 1972. Vol 1. p. 779-784. 29. Moberg R: The occurrence of early embryonic death in the mare in relation to natural service and artificial insemination with fresh or deep-frozen semen. J Reprod Fertil Suppl 1975;23:537-539. 30. Bishop MWH: Paternal contribution to embryonic death. J Reprod Fertil 1964;7:383-396. 31. Hunter RHF: Gamete lifespans in the mare's genital tract. Equine Vet J 1990;22:378-379. 32. Koskinen E, Lindeberg H, Kuntsi H, et al: Fertility of mares after postovulatory insemination. Zentralbl Veterinarmed A 1990;37:77-80. 33. Woods J, Bergfelt DR, Ginther OJ: Effects of time of insemination relative to ovulation on pregnancy rate and embryonic-loss rate in mares. Equine Vet J 1990;22:410-415. 34. Weber JA, Freeman DA, Vanderwall DK, et al: Selective oviductal transport mechanism of the mare. Proc Annu Meet Soc Therio 1991. p. 178-182. 35. Weber JA, Freeman DA, Vanderwall DK, et al: Prostaglandin E2 hastens oviductal transport of equine embryos. Biol Reprod 1991;45:544-546. 36. Weber JA, Freeman DA, Vanderwall DK, et al: Prostaglandin E2 secretion by oviductal transport-stage equine embryos. Biol Reprod 1991; 45:540-543. 107 37. Heap RB, Hamon M, Allen WR: Studies on oestrogen synthesis by the preimplantation equine conceptus. J Reprod Fertil Suppl 1982;32:343-352. 38. Bessent C, Cross DT, Ginther OJ: Effect of exogenous estradiol on mobility and fixation of the early equine conceptus. Anim Reprod Sci 1988;16:159-167. 39. McDowell KJ, Sharp DC, Grubaugh W: Comparison of progesterone and progesterone + oestrogen on total and specific uterine proteins in pony mares. J Reprod Fertil Suppl 1987;35:335-342. 40. Carnevale EM, Ramirez RJ, Squires EL, et al: Factors affecting pregnancy rates and early embryonic death after equine embryo transfer. Theriogenology 2000;54:965-979. 41. Sharp DC, McDowell KJ, Weithenauer J, et al: The continuum of events leading to maternal recognition of pregnancy in mares. J Reprod Fertil Suppl 1989;37:101-107. 42. Carnevale EM, Ginther OJ: Relationships of age to uterine function and reproductive efficiency in mares. Theriogenology 1992;37:1101-1115. 43. Ginther OJ, Garcia MC, Bergfelt DR, et al: Embryonic loss in mares: pregnancy rate, length of interovulatory intervals, and progesterone concentrations associated with loss during days 11 to 15. Theriogenology 1985;24:409- 417. 44. Stout TAE, Allen WR: Role of prostaglandins in intrauterine migration of the equine conceptus. Reproduction 2001;121:771-775. 45. Stout TAE, Allen WR: Prostaglandin E-2 and F-2 alpha production by equine conceptuses and concentrations in conceptus fluids and uterine flushings recovered from early pregnant and dioestrous mares. Reproduction 2002;123:261-268. 46. Ginther OJ: The nature of embryo reduction in mares with twin conceptuses: deprivation hypothesis. Am J Vet Res 1989;50:45-53. 47. Villahoz MD, Squires EL, Voss JL, et al: Some observations on early embryonic death in mares. Theriogenology 1985;23:915-924. 48. Woods GL, Baker CB, Baldwin JL, et al: Early pregnancy loss in brood mares. J Reprod Fertil Suppl 1987;35:455- 459. 49. Douglas RH: Some aspects of equine embryo transfer. J Reprod Fertil Suppl 1982;32:405-408. 50. Pascoe DR, Liu IKM, Spensley MS, et al: Effect of endometrial pathology on the success of non-surgical embryo transfer. Equine Vet J Suppl 1985;3:108-110. 51. Squires EL, Imel KJ, Iuliano MF, et al: Factors affecting reproductive efficiency in an equine embryo transfer programme. J Reprod Fertil Suppl 1982;32:409-414. 52. Vogelsang SG, Vogelsang MM: Influence of donor parity and age on the success of commercial equine embryo transfer. Equine Vet J Suppl 1989;8:71-72. 53. Douglas RH, Burns PJ, Hershman L: Physiological and commercial parameters for producing progeny from subfertile mares by embryo transfer. Equine Vet J Suppl 1985;3:111-114. 54. Ball BA, Little TV, Hillman RB, et al: Pregnancy rates at days 2 and 14 and estimated embryonic loss rates prior to day 14 in normal and subfertile mares. Theriogenology 1986;26:611-619. 55. Ball BA, Hillman RB, Woods GL: Survival of equine embryos transferred to normal and subfertile mares. Theriogenology 1987;28:167-174. 56. Schlafer DH, Dougherty EP, Woods GL: Light and ultrastructural studies of morphological alterations in embryos collected from maiden and barren mares. J Reprod Fertil Suppl 1987;35:695. 57. Mitchell D, Allen WR: Observations on reproductive performance in the yearling mare. J Reprod Fertil Suppl 1975;23:531-536. 58. Vanderwall DK, Woods GL: Age-related subfertility in the mare. Proc Annu Conv Am Assoc Equine Pract 1990. p. 85-89. 59. Badi AM, O'Byrne TM, Cunningham EP: An analysis of reproductive performance in Thoroughbred mares. Ir Vet J 1981;35:1-12. 60. Bain AM: Foetal losses during pregnancy in the Thoroughbred mare: a record of 2,562 pregnancies. N Z Vet J 1969;17:155-158. 61. Ricketts SW, Alonso S: The effect of age and parity on the development of equine chronic endometrial disease. Equine Vet J 1991;23:189-192. 62. Carnevale EM, Ginther OJ: Defective oocytes as a cause of subfertility in old mares. Biol Reprod Mono 1991. p. 209-214. 63. Ginther OJ: Embryonic loss in mares: nature of loss after experimental induction by ovariectomy or prostaglandin F2a. Theriogenology 1985;24:87-98. 64. Vandeplassche M, Henry M: Salpingitis in the mare. Proc Annu Conv Am Assoc Equine Pract 1977. p. 123-131. 65. Eilts BE, Scholl DT, Paccamonti DL, et al: Prevalence of endometrial cysts and their effect on fertility. Biol Reprod Mono 1 1995. p. 527-532. 66. Holtan DW, Squires EL, Lapin DR, et al: Effect of ovariectomy on pregnancy in mares. J Reprod Fertil Suppl 1979;27:457-463. 67. McKinnon AO, Squires EL, Carnevale EM, et al: Ovariectomized steroid-treated mares as embryo transfer recipients and as a model to study the role of progestins in pregnancy maintenance. Theriogenology 1988;29:1055- 1063. 68. Darenius K, Kindahl H, Madej A: Clinical and endocrine aspects of early fetal death in the mare. J Reprod Fertil Suppl 1987;35:497-498. 69. Sharp DC, Zavy MT, Vernon MW, et al: The role of prostaglandins in the maternal recognition of pregnancy in mares. Anim Reprod Sci 1984;7:269-282. 108 70. Iuliano MF, Squires EL: Effect of exogenous progesterone on pregnancy rates after surgical embryo transfer in mares. Theriogenology 1986;26:291-298. 71. Daels PF, Stabenfeldt GH, Kindahl H, et al: Prostaglandin release and luteolysis associated with physiological and pathological conditions of the reproductive cycle of the mare: a review. Equine Vet J Suppl 1989;8:29-34. 72. Oxender WD, Noden PA, Bolenbaugh DL, et al: Control of estrus with prostaglandin F2a in mares: minimal effective dose and stage of estrous cycle. Am J Vet Res 1975;36:1145-1147. 73. Irvine CHG, McKeough VL, Turner JE, et al: Effectiveness of a two-dose regimen of prostaglandin administration in inducing luteolysis without adverse side effects in mares. Equine Vet J 2002;34:191-194. 74. Daels PF, Stabenfeldt GH, Hughes JP, et al: Effects of flunixin meglumine on endotoxin-induced prostaglandin F2a secretion during early pregnancy in mares. Am J Vet Res 1991;52:276-281. 75. Daels PF, Stabenfeldt GH, Hughes JP, et al: Evaluation of progesterone deficiency as a cause of fetal death in mares with experimentally induced endotoxemia. Am J Vet Res1991;52:282-288. 76. Allen WE: The effect of human chorionic gonadotrophin and exogenous progesterone on luteal function during early pregnancy in pony mares. Anim Reprod Sci 1983;16:223-228. 77. Watson ED, Stokes CR, David JSE, et al: Concentrations of uterine luminal prostaglandins in mares with acute and persistent endometritis. Equine Vet J 1987;19:31-37. 78. Troedsson MHT: Uterine clearance and resistance to persistent endometritis in the mare. Theriogenology 1999;52;461-471. 79. Brendemuehl JP: Effect of oxytocin and PGF2a on luteal formation, function, and pregnancy rates in mares. Proc Annu Conv Am Assoc Equine Pract 2001. p. 239. 80. Mocklin CM, Paccamonti DL, Eilts BE, et al: Effect of post-ovulatory PGF2a or cloprostenol on plasma progesterone concentration in mares. Anim Reprod Sci 2006;94:220-222. 81. Nie GJ, Johnson KE, Wenzel JGW, et al: Effect of administering oxytocin or cloprostenol in the periovulatory period on pregnancy outcome and luteal function in mares. Theriogenology 2003;60:1111-1118. 82. Troedsson MHT, Ababneh MM, Ohlgren AF, et al: Effect of periovulatory prostaglandin F2a on pregnancy rates and luteal function in the mare. Theriogenology 2001;55:1891-1899. 83. Timoney PJ, Powell DG: Contagious equine metritis - epidemiology and control. J Equine Vet Sci 1988;8:42-46. 84. Bernard WV, LeBlanc MM, Webb BA, et al: Evaluation of early fetal loss induced by gavage with eastern tent caterpillars in pregnant mares. J Am Vet Med Assoc 2005;225:717-721. 85. Vest DJ, Cohen ND, Berezowski CJ, et al: Evaluation of administration of West Nile virus vaccine to pregnant broodmares. J Am Vet Med Assoc 2004;225:1894-1897. 86. Merkt H, Gunzel AR: A survey of early pregnancy losses in West German Thoroughbred mares. Equine Vet J 1979;11:256-258. 87. McKinnon AO, Squires EL, Harrison LA, et al: Ultrasonographic studies on the reproductive tract of mares after parturition: effect of involution and uterine fluid on pregnancy rates in mares with normal and delayed first post partum ovulatory cycles. J Am Vet Med Assoc 1988;192:350-353. 88. Malschitzky E, Schilela A, Mattos ALG, et al: Intrauterine fluid accumulation during foal heat increases embryonic death. Pferdeheilkunde 2003;19:646-649. 89. Huhtinen M, Reilas T, Katila T: Recovery rate and quality of embryos from mares inseminated at the first postpartum estrus. Acta Vet Scand 1996;37:343-350. 90. Henneke DR, Potter GD, Kreider JL: Body condition during pregnancy and lactation and reproductive efficiency of mares. Theriogenology 1984;21:897-909. 91. van Niekerk CH: Early embryonic resorption in mares. J S Afr Vet Med Assoc 1965;36:61-69. 92. van Niekerk FE, van Niekerk CH: The effect of dietary protein on reproduction in the mare. VII. Embryonic development, early embryonic death, foetal losses and their relationship with serum progestagen. J S Afr Vet Med Assoc 1998;69:150-155. 93. Merkt H, Jungnickel S, Klug E: Reduction of early twin pregnancy to single pregnancy in the mare by dietetic means. J Reprod Fertil Suppl 1982;32:451-452. 94. Ley WB, Thatcher CD, Swecker WS, et al: Chelated mineral supplementation in the barren mare: a preliminary trial. J Equine Vet Sci1990;10:176-181. 95. Kubiak JR, Evans JW, Potter GD, et al: Postpartum reproductive performance in the multiparous mare fed to obesity. Theriogenology 1989;32:27-36. 96. Baucus KL, Squires EL, Ralston SL, et al: The effect of transportation stress on early embryonic death in mares. Proc 10th Equine Nutr Physiol Symp 1987. p. 657-662. 97. Baucus KL, Ralston SL, Nockels CF, et al: Effects of transportation on early embryonic death in mares. J Anim Sci 1990; 68:345-351. 98. van Niekerk CH, Morgenthal JC: Fetal loss and the effect of stress on plasma progestagen levels in pregnant Thoroughbred mares. J Reprod Fertil Suppl 1982;32:453-457. 99. Irwin CFP: Early pregnancy testing and its relationship to abortion. J Reprod Fertil Suppl 1975;23:485-488. 100. Villahoz MD, Squires EL, Voss JL, et al: Some observations on early embryonic death in mares. Theriogenology 1985;23:915-924. 101. Paccamonti DL: Elective termination of pregnancy in mares. J Am Vet Med Assoc 1991;198:683-688. 102. Darenius K, Fredriksson G, Kindahl H: Allyl trenbolone and flunixine meglumine treatment of mares with repeated embryonic loss. Equine Vet J Suppl 1989; 8:35-39. 103. Stout TAE, Tremoleda JL, Knaap J, et al: Effects of treatments to prevent early pregnancy loss in the mare. Pferdeheilkunde 2003;19:711. 109 104. Allen WR: Is your progesterone therapy really necessary? Equine Vet J 1984;16:496-498. 105. Vanderwall DK, Williams JL, Woods GL: Use of a compounded proprietary long-acting progesterone formulation for maintenance of pregnancy in mares. Proc Annu Meet Soc Therio 2003. p. 8. 106. McKinnon AO, Lescun TB, Walker JH, et al: The inability of some synthetic progestagens to maintain pregnancy in the mare. Equine Vet J 2000;32:83-85. 107. Hinrichs K, Sertich PL, Kenney RM: Use of altrenogest to prepare ovariectomized mares as embryo transfer recipients. Theriogenology 1986;26:455-460. 108. Meyers PJ, Varner DD: Abortion of a mummified fetus associated with short uterine body in a mare. J Am Vet Med Assoc 1991;198:1768-1770. 109. Darenius K, Kindahl H, Madej A: Clinical and endocrine studies in mares with known history of repeated conceptus losses. Theriogenology 1988;29:1215-1232. 110