Early Embryonic Loss in the Mare

REFEREED

ORIGINAL RESEARCH

Early Embryonic Loss in the Mare Dirk K. Vanderwall, DVM, PhD, Diplomate ACT

From the Northwest Equine Reproduction Laboratory, Department of Animal and Veterinary Science and Center for Reproductive Biology, University of Idaho, Moscow, ID. Reprint requests: Dirk K. Vanderwall, Northwest Equine Reproduction Laboratory, Department of Animal and Veterinary Science and Center for Reproductive Biology, University of Idaho, Moscow, ID 83844. 0737-0806/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2008.10.001

a weighted-mean across studies of 8.6% (Table 1). The highest embryonic loss rates (20% to 30% or higher) have been detected in mares greater than 18 years of age.1-6 The lack of a practical method of diagnosing pregnancy prior to day 10 makes detection of embryonic loss between fertilization and day 10 difficult. In addition, pregnancy loss prior to day 10 must be differentiated from fertilization failure. Data compiled from three studies7-9 that used the recovery of cleaved ova on day 2 post-ovulation as an estimate of the fertilization rate found that fertilization rates were 91% in young mares and 85% in aged mares inseminated with fresh, fertile semen under experimental conditions. In contrast, Carnevale et al10 reported the estimated fertilization rate on day 1.5 was significantly higher in young versus aged mares (88% vs 45%, respectively); however, that difference may have been due to delayed embryonic development in the aged mares, because the cleavage rate on day 3 was not different for young and aged mares (100% for both groups), and the embryos from aged mares were about one cleavage division delayed in their development compared with the young mares. Therefore, it seems that under optimal conditions (ie, fresh, fertile semen), fertilization rates are very high (>90%) in young mares and may be slightly lower (85%) in aged mares. Although fertilization rates seem to be high, embryo recovery rates on days 6 to 9 post-ovulation are markedly lower for aged versus young mares,11,12 which implies a high rate of embryonic loss during the first week of gestation in the latter mares. Similarly, a more recent report13 demonstrated that the detected day-14 pregnancy rate per ovulation was lower in aged mares compared with young mares (ie, fewer detected embryonic vesicles compared with the number of ovulated follicles in the aged mares), which like the embryo recovery data, is consistent with a higher incidence of embryonic loss in the aged mares. Collectively, these data are supported by experimental evidence that the estimated embryonic loss rate between fertilization and day 14 was <10% for young mares compared with 60% to 70% for aged mares.7,14 Based on the data described above, the cumulative incidence of early embryonic loss between fertilization and approximately day 40 may be as low as 10% to 20% in young mares to more than 70% in aged mares. Under field conditions, the detected incidence of embryonic loss between days 12 and 40 is on the order of 10% to 15% for young mares, and 20% to 30% for aged mares.1 At those levels, early embryonic failure represents a considerable economic loss

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Keywords: Equine; Embryonic loss; Pregnancy

INTRODUCTION Early embryonic loss in the mare is generally defined as pregnancy failure that occurs between fertilization and day 40 to 60 of gestation. The diagnosis of early embryonic loss and recognition of factors contributing to its occurrence have been dramatically improved by the routine use of transrectal ultrasonography for early pregnancy diagnosis. Under field conditions, transrectal ultrasonography is typically used for an initial pregnancy diagnosis as early as day 12 to 14 post-ovulation, whereas under experimental conditions it may be used as early as day 10 or 11; therefore, ultrasonography allows direct (and repeatable) assessment of the conceptus during approximately three quarters of the interval when early embryonic loss occurs. Prior to day 10, the conceptus is too small to be visualized with standard ultrasonographic equipment, therefore other techniques have been used to study embryonic loss during that interval. Specifically, clinical procedures such as embryo transfer and oocyte transfer and experimental techniques such as in vitro embryo culture and light/electron microscopy of oocytes/embryos have been used to study early embryonic loss prior to day 10.

INCIDENCE Because of the practicality and accuracy of using transrectal ultrasonography for the diagnosis of pregnancy as early as day 10, a considerable amount of data has been generated on the incidence of embryonic loss after that time. Although it can be difficult to compare data across experiments because of variables such as differences in the interval studied, and whether experiments were conducted under controlled conditions or under field conditions, based on serial examination with ultrasonography, the reported incidence of embryonic loss prior to day 60 of gestation has ranged from 2.6% to 24.0%, with

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Table 1. A review of studiesa on early embryonic loss in mares detected with transrectal ultrasonography Reference Chevalier and Palmer, 1982 Simpson et al., 1982 90 Ginther et al., 1985b 82

Interval Studied 81

Villahoz et al., 1985c 91 Woods et al., 1985d 46 Woods et al., 1987d 1 Forde et al., 1987 92 Chevalier-Clement, 1989d 31 Vogelsang et al., 1989 74 Villahoz, 1989 93 Irvine et al., 1990 94 Baucus et al., 1990b 59 Woods et al., 1990e 52 Lowis and Hyland, 1991 95 Meyers et al., 1991 50 Bruck et al., 1993 96 Tannus and Thun, 1995 32 Pycock and Newcombe, 1996f 36 Newcombe, 1997d 97 Papa et al., 1998 84 Barbacini et al., 1999 54 Carnevale et al., 2000g 98 Morris and Allen, 2002 51 Hemberg et al., 2004 4 Blanchard et al., 2004h 48 Carnevale et al., 2005i 5 Newcombe and Wilson, 2005 47 Allen et al., 2007h 6

23  6 to 43  10 14 to 63 11 to 50 15 to 50 14 to 48 14 to 48 18 to 42 22  5 to 44  12 15 to 35 10 to 45 17 to 42 12 to 50 11 to 40 18 45 12 to 40 24 to end of season 14 to 40 14 to 30 11 to 39 10 to 30 14 to 50 12 to 50 15 to 35 14 to foaling 14 to 41 16 to 50 14 to 45 15 to 42 Weighted-mean

# Losses/ # Pregnancies 69/1,295 13/326 5/27 37/154 61/354 42/404 60/559 13/437 165/2,989 85/1,085 23/135 17/179 7/54 20/85 10/132 34/509 103/1,379 20/229 17/168 8/313 17/128 31/349 65/419 119/1,144 49/391 18/395 42/201 131/547 238/3,194 1,519/17,581

% 5.3 4.0 18.5 24.0 17.2 10.4 10.7 3.0 5.5 7.8 17.0 9.5 13.0 23.5 7.6 6.7 7.5 8.7 10.1 2.6 13.3 8.9 15.5 10.4 12.5 4.6 20.9 24.0 7.5 8.6

a

Only studies with at least 25 mares were included. Experimental mares. c Embryo recipient and experimental mares. d Singleton pregnancies only. e Experimental mares inseminated pre- or post-ovulation. f Only includes untreated control mares. g Embryo transfer recipient mares. h Calculated empirically from reported data. i Oocyte transfer recipient mares. b

to the equine industry in the form of increased costs associated with additional breeding of mares and/or decreased foal production. This article reviews current information on the etiology, diagnosis, and management of early embryonic loss in the mare. Although a considerable body of information about early embryonic loss was generated before the advent of transrectal ultrasonography, due to the limitations of such studies (accuracy, interval studied, etc), this review will focus upon data obtained using transrectal ultrasonography.

ETIOLOGY Factors that may contribute to the occurrence of embryonic loss in the mare have been classified as intrinsic, extrinsic, and embryonic.15,16 Intrinsic factors include endometrial disease, progesterone insufficiency, maternal age, lactation, foal-heat breeding, time of insemination relative to ovulation, site of intrauterine fixation of the embryonic vesicle, and maternal chromosomal abnormalities. Extrinsic factors include stress; nutrition; season/climate; transrectal palpation/ultrasonography; sire and/or semen

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processing/handling; and gamete handling/manipulation for assisted reproductive techniques. Embryonic factors include chromosomal anomalies or other inherent characteristics of the embryo; currently, it appears that embryonic factors are linked to intrinsic (eg, maternal age) and/or extrinsic (eg, oocyte handling/manipulation) factors.

INTRINSIC FACTORS Endometrial Disease Endometrial disease is further classified as inflammatory or noninflammatory. Inflammatory forms of endometrial disease include acute and chronic endometritis, while noninflammatory forms include periglandular fibrosis and endometrial cysts. Acute endometritis is characterized by an influx of neutrophils into the stroma of the endometrium and the uterine lumen; the two primary pathological forms of acute endometritis are persistent mating-induced endometritis or infectious endometritis. Intrauterine fluid that accumulates during persistent mating-induced endometritis and infectious endometritis can adversely affect fertility by: (1) impairing spermatozoal motility and/or viability if breeding/insemination is performed while the uterus is inflamed17,18; or (2) by inducing embryonic loss if the endometritis persists beyond day 5 post-ovulation when the embryo enters the uterine lumen from the oviduct19 and the corpus luteum (CL) becomes sensitive to prostaglandin F2a (PGF2a).20 It has been demonstrated that acute inflammation associated with intrauterine fluid collections during diestrus increases the incidence of embryonic loss.3,21,22 Similarly, mares with a history of endometritis had significantly higher early embryonic loss rates compared with mares with no history of endometritis.1 Therefore, routine therapy for persistent mating-induced and infectious endometritis is directed at removing the fluid that has accumulated in the uterine lumen and, in the case of infectious endometritis, eliminating the inciting microbial agent(s). In contrast to acute endometritis, chronic endometritis is characterized by an influx of lymphocytes into focalized areas of the endometrial stroma. Chronic inflammation seems to develop in response to any disturbance within the uterus (both normal [eg, pregnancy] and abnormal [eg, infectious endometritis]) and is commonly identified on uterine biopsy specimens. Chronic inflammation does not seem to impair fertility, because aged mares with chronic inflammation can support and maintain early embryonic development,23 and the degree of chronic inflammatory infiltrations in the endometrium was not different for aged mares (R15 years) that maintained pregnancies compared with those that lost their pregnancies between days 12 and 39 of gestation.3 Clinically, noninfectious abnormalities of the endometrium such as periglandular fibrosis have been considered

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an important factor in the occurrence of both early embryonic loss and fetal death,24 and because this type of pathological change in the endometrium is generally more severe in aged mares,25,26 it seemed to be a foregone conclusion that periglandular fibrosis caused higher embryonic loss rates in aged mares. However, when Ball et al23 tested that hypothesis by transferring morphologically normal, day-7 or -8 blastocysts into the uterus of young (minimal pathology) and aged (extensive pathology) recipient mares, embryo survival rates (55% and 45% at day 12) and embryonic loss rates between days 12 and 28 (9 and 11%) were not significantly different for the young and aged recipient mares, respectively. Their results indicated the uterine pathology (periglandular fibrosis and chronic inflammation) present in the aged mare’s uteri did not result in a higher incidence of early embryonic loss in those mares; therefore, it seems other factors are primarily responsible for the higher early embryonic loss rates observed in aged mares. Another common form of noninflammatory endometrial pathology is cystic dilatation, which can be either glandular or lymphatic (for review, see Stanton et al27). Essentially, all cysts that are grossly or ultrasonographically detectable are lymphatic in origin28 and range in size from a few millimeters to several centimeters in diameter. The incidence of endometrial cysts increases with mare age,27 which is a potentially confounding factor when assessing the effect of cysts on fertility. There are two plausible mechanisms by which cysts could adversely affect fertility. First, large cysts (>3 cm) might impair intrauterine mobility of the conceptus, which could lead to failure of maternal recognition of pregnancy caused by an inability of the conceptus to adequately block endometrial PGF2a secretion with subsequent regression of the corpus luteum.29 Second, if a conceptus were to become fixed in direct contact with a cyst(s), the conceptus might be deprived of adequate nutrient exchange in a manner similar to adjacent twin conceptuses through the deprivation hypothesis of spontaneous twin reduction proposed by Ginther.30 Several reports have described an adverse effect of endometrial cysts on fertility (ie, decreased pregnancy rates and/or increased early embryonic loss rates);21,31,32 however, when mare age was accounted for when analyzing the data, there was no evidence of an overall effect of endometrial cysts on embryonic loss rates in mares.33 Despite the lack of conclusive evidence that endometrial cysts have a detrimental effect on fertility, treatment may be pursued in mares that have large and/or numerous cysts in conjunction with a poor reproductive history.27 Specific treatments that have been used to remove/eliminate cysts include manual rupture/ablation, drainage/aspiration, mechanical removal by ensnaring the cysts with obstetrical wire, endoscopic electrocoagulation, and laser therapy27). Anecdotally, cyst removal may help some mares, because in a group of 39 aged mares (for which complete follow-up

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data was available) that had been barren for 1 year, 62% of the mares became pregnant after cyst removal using laser photoablation.34 Progesterone insufficiency Despite a paucity of scientific evidence supporting its efficacy, prophylactic administration of exogenous progesterone to pregnant mares in an effort to enhance maintenance of pregnancy continues to be a widespread practice. Although low progesterone levels caused by primary CL insufficiency has been proposed as a cause of early embryonic loss in mares that could warrant administration of exogenous progesterone, its occurrence has not been clearly documented.35 Without a specific indication (or contraindication) for its use, progesterone supplementation is often empirically performed in mares that have a history of repeated pregnancy failure when no specific factor causing pregnancy loss is identified. Despite the fact that there is no evidence that routine use of exogenous progesterone will decrease the incidence of early embryonic loss, two studies using the gonadotropin-releasing hormone (GnRH) agonist buserelin in mares (40 mg administered on days 10 or 11 of pregnancy) have shown a reduction in embryonic loss rate before day 30,36,37 which may be due to a beneficial effect of increased levels of endogenous progesterone; however, further work is needed to confirm the beneficial effect (if any) of this GnRH agonist and its mechanism of action. Although primary luteal insufficiency has not been documented, it has been shown that some pregnant mares will undergo luteolysis on days 14 to 16 despite the presence of an embryo in the uterus.38-41 This condition is characterized by prominent edema of the endometrial folds and can be confirmed retrospectively by demonstrating a plasma progesterone concentration of less than 1 ng/ mL. Prompt treatment with exogenous progesterone may prevent impending pregnancy loss, but treatment must be continued until the mare forms an accessory CL and/or fetoplacental production of progestins begins. Maternal age Because the uterine environment of aged mares seemed suitable for the maintenance of pregnancy (previously discussed), Ball et al14 then performed an experiment designed to compare the viability of embryos collected from young and aged mares following their transfer to the uterus of young recipient mares. Day-4 embryos were collected from the oviducts of young and aged mares and transferred to young recipient mares; the survival rate of embryos from young mares was significantly higher than those from aged mares (84 vs 25%, respectively). Because the embryos from the aged mares had lower survival rates after transfer to a normal uterine environment, and the embryos had never been exposed to the uterine environment of the aged

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mares, it suggested that adverse effects of the oviductal environment and/or inherent embryonic defects were responsible for the lower survival rate (ie, higher embryonic loss) of the embryos from the aged mares. In a subsequent experiment, Carnevale and Ginther42 used transvaginal ultrasound-guided follicle aspiration (TVA) and oocyte transfer to compare the viability of oocytes/embryos from young (6 to 10 years) and aged (20 to 26 years) mares. Oocytes were collected from the donor mares using TVA and then surgically transferred to the oviducts of young recipient mares that were inseminated preand post-transfer. The use of oocyte transfer allowed fertilization and embryonic development to occur in the recipient mare, avoiding potentially deleterious effects of the oviductal environment on the oocytes (ie, in the aged mares). When pregnancy was diagnosed on day 12 with transrectal ultrasonography, significantly more oocytes from young versus aged mares resulted in embryonic vesicles (92% vs 31%, respectively). These results implicated inherent defects within the oocytes of aged mares as an important cause for their reduced viability, because the oocytes were never exposed to the tubular genitalia of the donor mares. To further investigate oocyte quality, Carnevale et al43 used light and transmission electron microscopy to compare quantitative and qualitative differences in oocytes from young and aged mares. When imaged with light microscopy, significantly more oocytes from aged versus young mares contained large intracellular vesicles. In addition, individual oocytes from aged mares had morphological anomalies not imaged in oocytes from young mares, including large vesicles in the ooplasm or associated with the nucleus; oblong or irregular shapes; areas of ooplasm without organelles; and sections of oolemma with sparse microvilli. More recently, Rambags et al44 identified that after in vitro maturation, the number of mitochondria was significantly less in oocytes from aged (R12 years) versus young (%11 years) mares; in addition, transmission electron microscopy demonstrated that the mitochondria in the oocytes from the aged mares were often swollen and exhibited extensively damaged cristae. Collectively, these studies have begun to characterize the types of degenerative and/or inherent morphological defects present in oocytes from aged mares; however, it remains to be determined how and/or when specific oocyte abnormalities affect embryonic development. Recent data from a clinical oocyte transfer program involving subfertile mares has shed further light on agerelated effects on oocyte quality.5 Although day-16 pregnancy rates were not different in recipient mares that received oocytes from mares <20 years compared to oocytes from mares R20 years (39 and 40%, respectively), the pregnancy loss rates between days 16 and 50 was 16% for the oocytes from mares <20 years compared with

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26% for oocytes from the mares R20 years of age. An encouraging aspect of this recent clinical work with oocyte transfer, is that it is enabling many aged, subfertile mares to produce offspring. Lactation It is plausible the energy demands of lactation and/or hormonal changes associated with lactation could affect the incidence of early embryonic loss. For example, progesterone levels were found to be significantly lower in lactating mares compared with nonlactating mares;45 however, 2 large field studies1,46 did not detect any difference in embryonic loss rates between lactating and nonlactating mares. In contrast, a more recent report47 that retrospectively examined records for 12 breeding seasons (over 3,000 mareyears) determined that embryonic loss rates were 31.4% in lactating mares compared with 15.4% in non-lactating barren mares. Clearly, further work is needed to more fully characterize whether lactation directly impacts early embryonic development/loss. Foal-Heat Breeding There is conflicting data on the effect of foal-heat breeding on the incidence of early embryonic loss. Two studies1,48 found no difference in embryonic loss rates between mares bred on foal-heat versus those bred on subsequent cycles. In contrast, other studies6,49-51 reported higher embryonic loss rates in mares bred at foal-heat compared with those bred at subsequent heats. It seems likely this discrepancy may be due to other factors such as management decisions, which could include such factors as determining which (if any) mares are bred on foal-heat. For example, mares bred at foal-heat that were treated for intrauterine fluid accumulation had significantly higher embryonic loss rates compared with foal-heat mares that did not require treatment.48 This is certainly another area that warrants further study. Time of Insemination Relative to Ovulation Insemination of mares after ovulation has been associated with an increased incidence of early embryonic loss. Woods et al52 found that embryonic loss rates between days 15 and 40 of gestation were significantly higher in mares inseminated within 24 hours post-ovulation compared with mares inseminated before ovulation with fresh semen (34 vs 14%, respectively). Similarly, Koskinen et al53 reported that 5 of 13 mares (38.5%) inseminated post-ovulation lost their pregnancies between days 16 and 25 of gestation compared with no pregnancy losses in 15 mares inseminated before ovulation. The underlying cause of the observed higher incidence of embryonic loss in mares inseminated after ovulation is not known. One possibility is that oocyte quality is altered such that fertilization is not adversely affected, but that embryonic viability is

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affected. Another possibility is that the delay in fertilization causes a proportional delay in embryonic development that could compromise the ability of the conceptus to block luteolysis. The latter possibility is supported by the finding that embryos were significantly smaller in mares inseminated post-ovulation compared with mares inseminated before ovulation,52 and that 4 of the 5 pregnancy losses that occurred in mares inseminated post-ovulation failed between days 16 and 21,53 which corresponds with the time that progesterone levels would have decreased if the conceptus had not been able to block luteolysis. In contrast to the previous data, a more recent study54 did not detect any difference in embryonic loss rates in mares inseminated within 6 hours post-ovulation and those inseminated before ovulation with frozen semen. Collectively, these results are not incompatible, in that it may simply reflect that the adverse effects of insemination after ovulation are not manifested unless insemination occurs more than 6 hours after ovulation. Site of Intrauterine Fixation of the Embryonic Vesicle The embryonic vesicle undergoes extensive intrauterine mobility from the time it first becomes evident with transrectal ultrasonography on day 10 or 11 through day 16, at which time the vesicle becomes ‘‘fixed’’ in the base of one of the uterine horns.55 Fixation is thought to occur because of an interaction of increasing uterine tone, increasing size of the embryonic vesicle, and a physical impediment to further movement caused by the sharp curvature or flexure of the uterine horns at this point.55 Although it is an uncommon event, the embryonic vesicle can undergo fixation in an aberrant location in the uterus, particularly the uterine body. In a recent retrospective analysis of clinical breeding records, the outcome of 30 ‘‘body pregnancies’’ was determined.56 Nine of the 30 body pregnancies were in the cranial uterine body and the remaining 21 pregnancies were in the caudal 6 cm of the uterine body near the cervix. Of the 9 cranial body pregnancies, 7 were carried to term, one mare lost her pregnancy between days 35 and 42, and one mare aborted at 9 months of gestation. Of the 21 pregnancies located in the caudal body, 3 were terminated by administration of PGF2a between days 23 and 46, leaving 18 pregnancies for which a spontaneous outcome was determined. Of those 18 caudal body pregnancies, only 3 were successfully carried to term; 13 (72%) of the caudal body pregnancies were spontaneously lost by day 43, while 2 were lost at 4 and 9 months of gestation. Although fixation in the cranial uterine body did not seem to alter early embryonic development (only 1 of 9 [11%] conceptuses lost during early gestation), fixation in the caudal uterine body was associated with an extremely high rate of early embryonic loss as noted above; however, the reason for the adverse effect of the caudal body location on maintenance of pregnancy is not known.

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Maternal Chromosomal Abnormalities Although maternal chromosomal abnormalities such as X monosomy (ie, XO, Turner’s Syndrome) are generally associated with profound primary infertility due to an absence of cyclical reproductive activity, more subtle forms of chromosomal abnormalities can be associated with repeated early embryonic loss. For example, it was recently reported that 3 mares with repeated pregnancy loss before day 65 of gestation each had a chromosomal translocation, which as the name suggests, involves the breakage of a section of a chromosome and its attachment to another chromosome.57 All 3 mares had regular estrous cycles, and one mare had produced 2 foals before experiencing repeated pregnancy loss; therefore, a history of producing offspring does not preclude the possibility of a chromosomal abnormality. In mares, particularly young mares, with seemingly idiopathic cases of repeated embryonic loss, karyotyping may be warranted to investigate the possibility of subtle chromosomal abnormalities.

EXTRINSIC FACTORS Stress It is plausible that maternal stress could contribute to the occurrence of early embryonic loss because it has been demonstrated that stress associated with severe pain (ie, colic), infectious disease, and weaning resulted in a 30% to 50% decrease in circulating progesterone levels in pregnant mares.58 The adverse effect of stress on progesterone levels is apparently mediated through adrenal corticosteroids because administration of 150 mg of prednisolone to 2 pregnant mares resulted in a sharp, although transient, decrease (approximately 30% to 40%) in progesterone level.58 A subsequent study59 demonstrated that both cortisol levels and progesterone levels increased significantly when mares were transported for 9 hours during early gestation compared with nontransported control mares; however, there was no difference in early embryonic loss rates between the transported and nontransported mares. Besides the potential for adverse effects of stressful conditions to be mediated through increased glucocorticoid levels, the deleterious effects of certain metabolic (eg, enteritis) and/or infectious (eg, metritis) diseases may be mediated through the systemic effects of bacterial endotoxin. Intravenous administration of bacterial endotoxin to 9 pregnant mares between days 23 and 55 of gestation resulted in pregnancy loss in 7 of the mares within 5 days of treatment; in contrast, infusion of endotoxin to 10 mares between 56 and 318 days of gestation did not induce any pregnancy losses.60 Luteal activity was compromised in all mares by 9 hours after treatment with endotoxin, and progesterone concentrations were consistently lower in mares that lost their pregnancies (1 to 2 ng/mL) than

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those that did not lose their pregnancies. The adverse effect of the endotoxin on luteal function occurs due to release of endogenous PGF2a, and although the release of PGF2a can be abrogated by the administration of flunixin meglumine, in order to be efficacious it must be administered at a very early stage of the endotoxemia, when clinical signs are often not yet apparent.61 Another therapeutic option to prevent endotoxin-induced pregnancy loss is to administer exogenous progesterone, because daily administration of altrenogest prevented pregnancy loss associated with endotoxemia.62 Nutrition A seminal study on the effect of nutrition on the fertility of mares was reported by Henneke et al63 in 1984. They randomly assigned 32 foaling Quarter Horse mares to 1 of 4 dietary treatments designed to reach the following nutritional endpoints: (1) attain high body condition from 90 days prepartum to foaling and then maintain high body condition through 90 days post-foaling; (2) attain high body condition from 90 days prepartum to foaling then lose body condition to 90 days post-foaling; (3) lose body condition from 90 days prepartum to foaling then maintain low body condition until 90 days post-foaling; and (4) lose body condition from 90 days prepartum to foaling then gain weight after foaling to attain high body condition by 90 days post-foaling. At the start of the study, the average body condition score for all 4 groups of mares was between 6.0 and 6.7. At foaling, average body condition scores of groups 1 and 2 were 7.7 and 7.5, respectively, while the condition scores for groups 3 and 4 were 3.4 and 3.8, respectively. At 90 days post-foaling, the condition scores for groups 1 to 4 were 7.1, 4.7, 3.7, and 6.8, respectively. Mares were not bred at foal heat, but were inseminated beginning with the second postpartum estrus; if mares did not become pregnant they were bred a maximum of 3 cycles. The results demonstrated a profound effect of nutrition on fertility in group 3, the mares fed to lose condition prepartum, and then maintain poor body condition through 90 days post-foaling. The overall per-cycle pregnancy rate was significantly lower for group 3 compared with the other groups, and the pregnancy loss rate between days 30 and 90 was significantly higher for group 3 (75%) compared with the other groups (0 to 12%). In addition, when compared across treatment groups, mares in the study that foaled with a body condition score less than 5.0 had significantly lower pregnancy rates, and none of the mares that were maintained with a condition score of less than 5.0 after foaling were pregnant at 90 days of gestation. In agreement with the data of Henneke et al63 is a retrospective study by Newcombe and Wilson47 that determined 71% of mares that maintained their pregnancy

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gained weight during the breeding season, while 58% of mares that lost their pregnancy between days 14 and 45 lost weight during the breeding season. In another report,64 van Niekerk and van Niekerk examined the effect of total protein intake and protein quality on the incidence of early embryonic loss in lactating and nonlactating mares. They found that 5 of 14 mares (35.7%) receiving low quality protein in their diet underwent pregnancy loss between days 14 and 90 of gestation compared with only 3 of 41 mares (7.3%) that received higher quality protein in their diets. Collectively, these reports highlight the importance of nutrition for optimum fertility in the mare. All indications are that to maximize reproductive efficiency and minimize early embryonic loss, mares should receive good quality feedstuffs in sufficient quantity to maintain optimum body condition. In addition to nutritional effects of energy intake/ body condition and/or nutrient content/quality on embryonic loss, ingestion of environmental toxicants may adversely affect embryonic development. For example, ingestion of endophyte infected fescue has been implicated as a possible cause of early embryonic loss, because 3 of 6 mares consuming endophyte-infected fescue underwent early embryonic loss between days 16 and 35 of gestation compared with 0/8 and 0/7 mares consuming endophyte-free fescue or nontoxic endophyte fescue, respectively,65 and there was a tendency (P ¼ .2) for higher embryonic loss rates between days 14 and 21 in mares consuming endophyte infected fescue compared with mares consuming endophyte-free fescue.66 However, other studies have not found a relationship between consumption of endophyte-infected fescue and early embryonic loss.67,68 Another example of an apparent environmental effect on early embryonic loss is the association between ingestion of Eastern Tent caterpillars and early embryonic loss as part of Mare Reproductive Loss Syndrome (MRLS).69 Season/Climate In cattle, it has been demonstrated that seasonal and/or climatic conditions such as heat stress can adversely affect early embryonic development leading to higher embryonic loss rates.70 However, in horses there is no clear-cut evidence of an environmental effect on early embryonic loss, because the date of ovulation/establishment of pregnancy was not different for mares that maintained pregnancies versus those that lost pregnancies.46 Hearn et al71 reported significantly higher early embryonic loss rates for mares bred in February and March versus those bred later in the season (in the northern hemisphere); however, rather than a true seasonal effect, it could reflect that a higher proportion of the mares bred early in the season were barren, which would likely include more aged mares resulting in higher loss rates.

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Transrectal Palpation/Ultrasonography Although an early report72 indicated an adverse effect of transrectal palpation on fertility in the mare, there is no evidence that routine performance of transrectal palpation73 or transrectal ultrasonography28,74 performed by an experienced clinician have any adverse effect on early pregnancy. Sire and/or Semen Processing/Handling Although an early (pre-ultrasound) study indicated the stallion could influence the incidence of pregnancy loss after day 42 of gestation,75 a subsequent study of more than 3,700 pregnant mares found that of 261 stallions examined, there was no statistically significant effect of the stallion on pregnancy loss rates between days 22 and 44.31 More recently, Allen et al6 reported that of a total of 36 stallions that mated >30 mares, the early pregnancy loss rate between days 15 and 42 for the majority of stallions (26) was between 0 to 12%; in contrast, they noted that 10 stallions were associated with ‘‘higher than normal’’ rates of pregnancy loss (13% to 23%). It is important to note that stallions can apparently have idiopathic ‘‘clusters’’ of exceedingly high pregnancy loss rates (>50%); for example, Blanchard et al76 reported that a stallion established pregnancy in 23 mares during his first year of breeding, but 14 of the mares (61%) lost their pregnancies between 2 and 4 months of gestation. Because the majority of the mares were known to be young and fertile, an evaluation of the stallion’s karyotype was performed, which showed a slight dimorphism of the centric heterochromatin of chromosome no. 1. In addition, mares were ‘‘test bred’’ for the purpose of recovering embryonic tissue for karyotype analysis. Three conceptuses were recovered between days 45 and 50 of gestation, of which, one fetal karyotype revealed slight dimorphism of the heterochromatin of chromosome no. 1. In the subsequent breeding season, the stallion impregnated 23 of 25 mares bred, of which one aborted at 8 months of gestation, which highlights the need for caution when trying to pinpoint an exact causal effect between an individual stallion and pregnancy loss. The widespread use of cooled-transported and frozen semen raises the possibility that semen processing and/or handling procedures (eg, x-irradiation of shipped semen samples) could alter the spermatozoa in such a manner as to contribute to higher embryonic loss; of those possibilities, only x-irradiation has been studied in a controlled manner. England and Keane77 found that the typical airport security screening system would expose semen samples to between 0.5 and 1.0 micro Sieverts (mSv). In a subsequent breeding trial, they bred 3 groups of 8 mares with semen from one stallion that had been exposed to xirradiation doses of 0, 1.0, or 10.0 mSv; an entire ejaculate was irradiated and inseminated into each mare once during estrus within 48 hours prior to ovulation. The day-14

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pregnancy rates for the 3 treatment groups were 0 mSv (7/ 8 mares), 1.0 mSv (8/8 mares), and 10.0 mSv (7/8 mares). Of the 22 pregnancies that were established, one was lost at day 65 (0 mSv group), while the remaining 21 mares delivered a foal at term (one died at parturition). Therefore, there was no evidence of any effect of x-irradiation of the semen on early embryonic loss rates. Gamete Handling/Manipulation There is tremendous interest in the development of new assisted reproductive techniques that may allow production of offspring from mares and/or stallions that otherwise are incapable of reproducing. Techniques being developed include oocyte transfer, intracytoplasmic sperm injection (ICSI), sexed semen, and nuclear transfer (ie, cloning). The successful application of these procedures requires that equine gametes are collected and handled appropriately. Oocytes can be collected from mature pre-ovulatory follicles or from immature follicles. Oocytes collected from small, immature follicles must undergo maturation in vitro; the development of culture systems for in vitro maturation of equine oocytes is currently an area of considerable research interest. Like the oocyte, spermatozoa must be collected and handled appropriately for use with ICSI and semen sexing procedures. It is plausible that the handling and manipulation oocytes and/or spermatozoa undergo for use in these procedures could itself adversely affect fertilization and/or early embryonic development. Currently, there is conclusive evidence that cloned equine embryos have a very high incidence of early embryonic loss (>80%) that is often characterized by lack of formation of an embryo-proper.78 Similarly, it has been reported79,80 that a high proportion (40% to 50%) of conceptuses produced using ICSI fail to develop an embryo-proper, which suggests that something related to the ICSI procedure contributed to abnormal development, because only 4.4% of equine pregnancies fail to develop an embryo-proper.41 Clearly, further work is needed to assess the potential impact of new assisted reproductive techniques on early embryonic development. Embryonic Factors As described earlier, increasing maternal age is clearly associated with decreased oocyte quality, which may reflect chromosomal and/or other inherent changes within the oocyte that apparently do not affect fertilization rates, but dramatically increase the incidence of early embryonic loss in aged mares. Similarly, handling and/or manipulation of spermatozoa, oocytes, and/or embryos during assisted reproductive procedures may adversely affect embryonic development. Further research is needed to identify specific detrimental changes that occur in oocyte quality with increasing mare age, and how to minimize

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or prevent impairment of spermatozoa/oocyte/embryo quality when performing assisted reproductive techniques. Diagnosis Early embryonic loss may or may not be accompanied by premonitory signs of impending failure of the pregnancy. Several studies have documented that embryos that are small for their day of gestation have higher pregnancy loss rates.21,49,81-84 Prior to development of the embryoproper, which usually becomes evident with transrectal ultrasonography by day 21 or 22 of gestation, pregnancy loss is generally characterized by the sudden disappearance of the embryonic vesicle between examinations without premonitory signs. After development of the embryo-proper, signs of impending loss of the pregnancy are more likely to be observed with transrectal ultrasonography. Collectively, signs of impending pregnancy loss include irregular shape of an embryonic vesicle, prolonged mobility of a vesicle (beyond day 16), excessive endometrial edema, an undersized vesicle, lack of development of the embryo-proper (ie, developing as a trophoblastic vesicle), loss of embryonic heartbeat, dislodgement of a vesicle with loss of fluid, increased echogenicity of fluid within the conceptus, and abnormal development of the embryonic membranes.15 Clinical signs such as these have been observed in association with spontaneous pregnancy loss,82 experimentally induced pregnancy loss following ovariectomy or administration of PGF2a,85 and more recently in pregnancy losses associated with newer assisted reproductive techniques such as cloning. Management In conjunction with recognizing potential causes of embryonic loss is the need to assess whether there are management changes or treatments that can be initiated to decrease the incidence of early embryonic loss and/or ‘‘rescue’’ conceptuses showing signs of impending failure. First and foremost, all broodmares should receive proper management to ensure adequate nutrition to maintain optimal body condition; appropriate vaccination/deworming and other routine health care such as dental prophylaxis prior to breeding; and to the degree possible, efforts should be made to minimize stressful situations for mares, for example, social stress due to introduction of new herd mates, competition for food sources, etc. Optimal management in conjunction with specific therapy aimed at resolving treatable reproductive abnormalities (eg, poor perineal conformation, uterine infection, etc) will increase the likelihood that mares will successfully maintain a pregnancy.86 In contrast, it is unlikely that anything can be done to overcome early embryonic loss in aged mares caused by inherently poor oocyte quality; therefore, it is important to understand that it may require multiple estrous cycles of breeding before an aged mare ovulates a ‘‘good’’ oocyte

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that will not only be fertilized and establish pregnancy, but will survive and result in the birth of a viable offspring. As discussed previously, prophylactic administration of exogenous progesterone to pregnant mares in an effort to enhance maintenance of pregnancy is a widespread practice, although conclusive evidence it can help ‘‘rescue’’ a conceptus that will otherwise fail is lacking; however, use of exogenous progesterone is clearly warranted when, as discussed earlier, there are signs a mare has undergone luteolysis in spite of the presence of a conceptus in the uterus. Similarly, use of exogenous progesterone may be warranted when faced with a conceptus that is ‘‘smallfor-date,’’ because it is plausible an undersized conceptus may be less likely to prevent luteolysis and maintain CL function. In addition, administration of exogenous progesterone is clearly indicated when there is clinical evidence, or suspicion, of systemic endotoxemia due to severe disease, because endotoxin has a deleterious effect on CL function (mediated through PGF2a release), which may lead to pregnancy failure due to insufficient progesterone levels. In pregnant mares, the fetal-placental unit begins secreting progesterone and related progestagens between days 80 and 100 of gestation, the levels of which become sufficient to maintain pregnancy after day 100; therefore, if progesterone therapy is instituted, it is generally discontinued between days 100 and 120, because there is no physiological basis for continued administration of exogenous progesterone after that time. Currently, there are no commercially available formulations of progesterone labeled for use in pregnant mares; therefore, the use of exogenous progesterone for maintenance of pregnancy is an ‘‘extra-label’’ use. Two preparations of progesterone commonly administered to pregnant mares are daily oral administration of 0.044 mg/kg (1 mL per 110 pounds body weight) of the synthetic progestin altrenogest (Regu-Mate, Intervet, Inc, Millsboro, DE) or daily intramuscular administration of 0.2 to 0.3 mg/kg (100 to 150 mg total dose) progesterone in oil (available from compounding pharmacies). Although both treatments have been shown to be efficacious by their ability to maintain pregnancy in mares without an endogenous source of progesterone, the need for daily administration is a drawback of these formulations. In an attempt to eliminate the need for daily administration of exogenous progesterone preparations, there has been interest in the use of long-acting formulations of progesterone that can be administered at weekly (or longer) intervals. There are anecdotal reports of the clinical use of long-acting synthetic progestins labeled for use in women (eg, medroxyprogesterone acetate or 17-alpha-hydroxyprogesterone hexanoate) for maintenance of pregnancy in mares; however, these formulations were unable to maintain pregnancy in mares without an endogenous source of progesterone.87,88 It was recently demonstrated that

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intramuscular administration of a compounded longacting formulation containing 1.5 grams of progesterone every 7 days provided a sufficient level of progesterone to maintain pregnancy in mares that lacked an endogenous source of progesterone;89 therefore, this compounded formulation of progesterone seems to be an efficacious and suitable alternative to progesterone formulations that require daily administration. An important aspect of the clinical management of early embryonic loss is recognition that it will inevitably occur in some mares, particularly aged mares, and an appropriate course of action is to diagnose its occurrence as early as possible in an effort to determine if there were specific factors associated with the pregnancy loss and to provide an opportunity for re-breeding the mare as soon as possible. Early detection of embryonic loss can be accomplished by performing serial examinations with transrectal ultrasonography every 10 days to 2 weeks between days 14 and 40 of gestation. Clinically, serial examinations are routinely conducted through day 60 of gestation, at which time the frequency of examination is reduced. At each examination, a complete assessment of the conceptus should be performed and recorded (ie, size, location, presence of embryonic heartbeat, etc); if the conceptus shows any signs of impending failure, the pregnancy can be monitored more closely, and a decision about the use of progesterone supplementation can be made as discussed above.

SUMMARY A multitude of factors may contribute to the occurrence of early embryonic loss in a mare. It is clear that some factors, such as increasing maternal age, are directly associated with higher embryonic loss rates, while other factors such as breeding at foal heat are less clearly linked to pregnancy loss. In addition, there may be interactions among the various factors (eg, mare age, foal-heat, lactation, nutrition, etc) that may compound their effects on embryonic loss. As important as recognizing potential causes of embryonic loss is knowing whether anything can be done to decrease the incidence of early embryonic loss in mares. At this time, it is unlikely that anything can be done to overcome early embryonic loss in aged mares that is caused by inherently poor oocyte quality. In contrast, nutritional effects on embryonic loss can be minimized if not eliminated completely, as can the adverse effects of insemination >6 hours after ovulation. Probably the most important aspect of the clinical management of early embryonic loss in the mare is to recognize that it will inevitably occur in some mares, and the most appropriate course of action is to diagnose its occurrence as early as possible in order to provide an opportunity for re-breeding the mare (if the loss occurs, and is detected, during the breeding season). Early detection of embryonic loss can be readily accomplished by performing

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serial examinations with transrectal ultrasonography every 10 days to 2 weeks during early gestation.

17. Squires EL, Barnes CK, Rowley HS, McKinnon AO, Pickett BW, Shideler RK. Effect of uterine fluid and volume of extender on fertility. Proceedings AAEP 1989;35:25–30. 18. Troedsson MHT, Alghamdi A, Laschkewitsch T, Xue J-L. Sperm

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