Tutorial Article Current Methods for the Diagnosis and Management of Twin Pregnancy in the Mare A

EQUINE VETERINARY EDUCATION / AE / september 2008 493

Tutorial Article Current methods for the diagnosis and management of twin pregnancy in the mare A. D. J. HODDER*, I. K. M. LIU† and B. A. BALL† William R. Pritchard Veterinary Medical Teaching Hospital; and †Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, California, USA. Keywords: horse; twinning; abortion; ultrasonography; embryo; pregnancy

Summary

Historically, twin pregnancy has been one of the leading causes of pregnancy loss in the mare. Numerous studies have identified factors that increase the incidence of twinning. The identification and management of twins during the mobility phase remains the most effective method to control the potentially detrimental effects of twin pregnancy. Options exist for post fixation management of twins, although these methods are generally less successful than management during the mobility phase.

Introduction Fig 2: Twin Thoroughbred foals born alive. While most late term twin pregnancies result in abortion, some mares will give birth Twinning has long been recognised as one of the most to 2 live foals. These foals are typically dysmature and have a important causes of reproductive wastage in the mare. Mares much higher neonatal mortality rate that singleton foals. carrying twins at Day 40 or later frequently lose one or both fetuses in mid to late gestation (Ginther and Griffin 1994) (Fig 1). Mares aborting in late gestation or giving birth to twins are likely to suffer dystocia (Roberts 1982; Frazer 2003; McKinnon 2007), are prone to fetal membrane retention (Frazer 2003) and experience decreased live foaling rates in the following season (Pascoe 1983), probably due to delayed involution of an oversized uterus (McKinnon and Rantanen 1998). At parturition, twin foals suffer a higher rate of stillbirth than singleton foals (Platt 1973). Twin foals born alive are frequently smaller, weaker, more susceptible to infection and slower to develop than singleton foals (Jeffcott and Whitwell 1973; Wolfsdorf 2006) (Fig 2). They also suffer a higher neonatal mortality rate (Jeffcott and Whitwell 1973). The total villous chorionic surface area of twin placentas combined is only slightly greater than that of singleton pregnancies (Jeffcott and Whitwell 1973), making it likely that the detrimental effects of twin pregnancy are mediated by Fig 1: Late term abortion due to twin pregnancy. Impending abortion due to twin pregnancy will often be preceded by relative placental insufficiency. premature mammary development. Twin aborted fetuses often Jeffcott and Whitwell (1973) found twinning to be the appear emaciated, due to placental insufficiency. largest single cause of fetal mortality, accounting for 56 of 258 abortions (22%) examined in a predominately Thoroughbred *Author to whom correspondence should be addressed. population. Whitwell (1980) stated that twinning was the 494 EQUINE VETERINARY EDUCATION / AE / september 2008

largest single cause of abortion, responsible for between 22% within certain family lines (Ginther et al. 1982). Researchers in and 29% of all abortions each season. Examination of Poland examined 12,648 pregnancies in 2033 Thoroughbred Thoroughbred breeding records in Germany revealed 703 of mares and estimated the heritability of twinning in this 1817 abortions (38.6%) were attributable to twin pregnancy population to be from 0.24–0.29, depending on the model (Merkt and Jochle 1993). The authors of this study reported a used (Wolc et al. 2006). decline in abortion due to twinning over time. A similar finding was reported by Giles et al. (1993) who reviewed Ovulatory pattern 3514 abortions over a 6 year period in central Kentucky. In this study, which included neonatal deaths up to 7 days of age, Several studies have examined the effect of ovulatory pattern 221 abortions, or 6.28% of total abortions, were due to on the occurrence of twin embryos. Unilateral double twinning. This figure was similar to that reported by Smith ovulation has been shown to result in the recovery of fewer et al. (2003), where 75 of 1252, or 6.0% of abortions were embryos in comparison to mares in which an ovulation occurs attributable to twinning. A review of abortions in Newmarket from each ovary (bilateral double ovulation). In a review of between 1996 and 2001 reported 6 of 210, or 2.9%, were 1300 double ovulatory cycles, the proportion of embryo attributable to twinning (Ricketts et al. 2003). The decline in recovery attempts yielding 2 embryos was significantly less in the reported incidence of abortion due to twin pregnancy over unilaterally ovulating mares (247/699; 35%) than in bilaterally time has largely been attributed to the widespread use of ovulating mares (290/601; 48%) (Riera et al. 2006). This was ultrasonography for early diagnosis and management of twin similar to the finding of Squires et al. (1987) who reported the pregnancy in routine studfarm practice (Giles et al. 1993; recovery of fewer twin embryos from unilateral than from Merkt and Jochle 1993; Ricketts et al. 2003; Smith et al. 2003). bilateral double ovulators. It has been hypothesised that some mechanism at the level of either the ovary or oviduct may Factors influencing the occurrence of twins interfere with ovulation or oocyte pickup in unilateral, double ovulating mares (Riera et al. 2006). Ovulation rate Reproductive status While reports of monozygotic twins (Rooney, 1970; McCue et al. 1998) and triplets (Meadows et al. 1995) exist, almost all The reproductive status of the mare at the time of breeding cases of twinning in the mare are attributable to multiple has been shown to affect the occurrence of multiple ovulations (dizygotic or fraternal twinning). Identical twins are ovulations and twin pregnancies. Barren and maiden mares thought to arise when embryo bisection occurs in association have been shown to have a greater incidence of multiple with hatching from the zona pellucida. The most likely reason ovulation and twin pregnancy in comparison to lactating for the lack of identical twin formation in the mare is the mares. It is hypothesised that this is due to a feedback effect embryonic capsule that forms around Day 6 and is thought to of nursing on the hypothalamic-pituitary axis (Perkins and prevent bisection of the embryo by the zona pellucida Grimmett 2001). Ginther et al. (1982) demonstrated a (McKinnon and Rantanen 1998). reduction in multiple ovulation rates of lactating mares by Greater rates of twin pregnancy occur in breeds with a between 42 and 67% in comparison with barren and higher incidence of multiple ovulations and the incidence of maiden mares. A later study (Ginther 1983a) demonstrated multiple ovulations has been shown to differ between breeds. double ovulations and twin embryos more frequently in A greater incidence of multiple ovulations occurs in barren mares (11 and 6%, respectively) than in lactating Thoroughbreds (19%) than in Quarter Horses (9%) and mares (5 and 1%, respectively). In West Germany between Appaloosas (8%) (Ginther et al. 1982). More recent studies 1976 and 1985, 33% of twin pregnancies occurred in report a higher incidence of multiple ovulation in lactating mares, yet lactating mares represented 58% of the Thoroughbreds, ranging from 29.3% (Davies Morel et al. broodmare population (Merkt and Jochle 1993). In a review 2005) to 31.7% (Davies Morel and Newcombe 2007). of 3373 Thoroughbred mares, Allen et al. (2007) identified a As a result of differences in the incidence of multiple significantly greater number of twin and triplet pregnancies ovulation between breeds, more twins occur in at Day 15 in maiden mares (14.4%) and barren mares Thoroughbreds, draught horses and Warmbloods than in (16.4%) in comparison to foaling mares (8.0%). Standardbreds and Ponies (McKinnon and Rantanen 1998). In a study of early pregnancies diagnosed with ultrasound Season between 12 and 20 days after ovulation, 97 of 629 (15.4%) Thoroughbred mares had twin vesicles whereas only 39 of As the breeding season progresses, there is an increase in the 634 (6.1%) Standardbred mares were pregnant with twins multiple ovulation rate and in the percentage of mares (Bowman 1986). Twins were diagnosed in 245 of 1716 cycles conceiving twins. As diagnosed via palpation per rectum during in Thoroughbred mares (14.3%) and in 46 of 1294 cycles in 478 oestrous cycles, twin ovulations increased from 12% of all Standardbred mares (3.5%) (McKinnon 2007). In addition, a cycles in May, to 19% in June (Jeffcott and Whitwell 1973). Of high degree of repeatability of multiple ovulations and twin all mares who conceived twins, 61.3% conceived twins in May pregnancies has been reported both in individual mares and and June (Jeffcott and Whitwell 1973). This seasonal effect on EQUINE VETERINARY EDUCATION / AE / september 2008 495

the incidence of twinning was also demonstrated in a review of increase in twin pregnancy rate associated with the use of German Thoroughbred breeding records. Twin pregnancy was ovulation-inducing drugs. A similar finding was reported by diagnosed in 0.9% of 3229 conceptions during February and Davies-Morel and Newcombe (2007) who reviewed the March, which was significantly different from the rate of breeding records of 1239 Thoroughbred mares obtained over 1.76% which occurred in 2157 conceptions in June and July a 3 year period. In this study, the incidence of multiple (Merkt and Jochle 1999). ovulation and subsequent multiple pregnancy was not significantly different between mares who received 750 iu Induction of oestrus and ovulation hCG subcut. (n = 536) and those that received no ovulation- inducing agent (n = 703). The effect of ovulation induction using human chorionic gonadotropin (hCG) or oestrus induction using cloprostenol on Stallion effect twinning rates has been investigated. New Zealand researchers reviewed 2119 ovulatory cycles in Thoroughbred mares over a Ginther et al. (1982) reported the results of a survey of 7 year period, and demonstrated a 3-fold increase in the odds veterinarians in which many believed that twins were more of a diagnosis of twins on ultrasound examination performed likely to occur in mares bred to highly fertile stallions. It is 14 days post ovulation following ovulation induction with hCG possible that with extended longevity of viable spermatozoa in compared to cycles in which no ovulation induction was the reproductive tract that there is a greater possibility of performed (Perkins and Grimmett 2001). A similar result was fertilisation of asynchronous ovulations (Frazer 2003). obtained in a study of 680 Thoroughbred mares in Italy, where the percentage of mares diagnosed with twins via transrectal Diagnosis of twin pregnancies ultrasound 16 days after last being mated was significantly higher in those mares treated with 5000 iu hCG i.m. (n = 221; It is important to diagnose and manage twin pregnancy as 13.1%) than untreated controls (n = 324; 6.5%) (Veronesi early as possible. More pregnancies will go to term and result et al. 2003). The increased incidence of twin pregnancy in in the birth of a single, live foal if one of the twin vesicles is hCG-treated mares is thought to be due to an increase in the eliminated sooner rather than later (Roberts 1982). While multiple ovulation rate (Perkins and Grimmett 2001). pregnancy may be detected as early as 9 days after ovulation Veronesi et al. (2003) also found an increased twinning using ultrasonography (Ginther 1986), the first pregnancy rate in mares treated with 0.5 mg cloprostenol to shorten the check is usually conducted 14–16 days after ovulation in luteal phase (n = 86; 17.4%) and in those mares who routine stud practice (Lofstedt and Newcombe 1997). This is received both cloprostenol and hCG (n = 49; 30.6%) because both singleton and twin vesicles can usually be easily compared with untreated controls (n = 324; 6.5%). In a detected at this time. Twin vesicles arising from synchronous review of 3373 Thoroughbred mares, Allen et al. (2007) ovulations (ovulations occurring within 24 h of each other) will identified a significantly higher incidence of multiple be of similar size and should be easily identified. pregnancies in mares associated with the hormonal induction Twins may also arise from asynchronous ovulations of oestrus (13.0% vs. 10.6%). These same authors found no (ovulations occurring >24 h apart). In this instance, the embryonic vesicles will be of dissimilar size and the size disparity will depend upon the time between ovulations (Fig 3). Asynchronous ovulations can occur up to 4 days apart and may result in a second embryonic vesicle that is too small to detect if early pregnancy diagnosis is conducted in relation to the time of the first detected ovulation. Examination of the ovaries for the presence and number of corpora lutea is important at the time of early pregnancy diagnosis because of the high correlation between the number of ovulations and the number of embryonic vesicles (Ginther 1987). Diagnosis of twin pregnancy after fixation can be difficult in cases of unilateral fixation. Before the appearance of the embryo proper on about Day 21, unilaterally fixed twin vesicles may appear as a single, large over-sized vesicle, with only a thin line visible in the middle (McKinnon 2007) (Fig 4). The later appearance of embryonic heartbeats, umbilical cords Fig 3: A pregnancy check conducted 14 days after a detected and excess fetal membranes will further aid in the diagnosis of ovulation. The size of the vesicle on the left is consistent with twin pregnancy. a Day 14 pregnancy, while the vesicle on the right arose from a Transabdominal rather than transrectal ultrasonography later ovulation. Asynchronous ovulations such as occurred in may be more useful for the diagnosis of twins beyond this example result in embryonic vesicles of different sizes. Vesicles of dissimilar sizes typically fix unilaterally and are more 100 days of gestation (McKinnon 2007) (Fig 5). Beyond likely to undergo natural reduction to a single pregnancy. Day 70, transrectal diagnosis and monitoring of twin 496 EQUINE VETERINARY EDUCATION / AE / september 2008

Fig 4: Triplet pregnancy. The vesicle in the image on the left has Fig 6: Transrectal ultrasound image obtained in a pregnant fixed at the base of the left horn, whereas the image on the mare at 115 days of gestation. Due to the descent of the gravid right shows 2 vesicles fixed unilaterally at the base of the right uterus over the pelvic brim, fetuses may not be visualised using horn. The unilaterally fixed vesicles are closely apposed and if this approach after Day 70. Visualisation of the twin membrane examined inadequately may appear as a single large vesicle. (arrow) should alert the examiner to the possibility of twin Note the thin membrane separating the 2 vesicles (arrow). pregnancy. pregnancy may be difficult due to the increasing volume of Twins may be missed for numerous reasons and despite allantoic fluid and to the descent of the gravid uterus into the multiple examinations (McKinnon and Rantanen 1998; abdomen. In a study that followed 15 bilaterally fixed twins McKinnon 2007). Due to the mobility of the embryonic vesicle beyond Day 40 (Ginther and Griffin 1994), the only structure in the uterus before fixation, a second vesicle may be missed indicative of twins that was consistently detected during all if the entire uterus is not examined thoroughly. Twins resulting transrectal ultrasound examinations was the ‘twin from asynchronous ovulations may also be missed, if membrane’, which represented the area of apposition examination for pregnancy is conducted before the time that between the 2 allanto-chorions (Fig 6). a second vesicle can be detected. If more than one corpus In undiagnosed cases of twin pregnancy that advance into luteum is detected at the time of early pregnancy diagnosis, a later gestation, the first indication of twin pregnancy may be second examination should be conducted 2–3 days later to premature mammary development and lactation or the examine for the presence of a second embryonic vesicle. discovery of 2 aborted fetuses. The diagnosis of abortion due Reliance on rectal palpation alone to diagnose pregnancy may to twinning is aided by placental examination. Areas where miss twins, especially in cases of unilateral fixation. The the 2 allanto-chorions were in contact with each other are presence of endometrial cysts often confuses the diagnosis of devoid of villi and appear grossly as large, pale areas sharply twins, especially if their size and location has not been demarcated from the surrounding villous chorion (Jeffcott and recorded previously. Endometrial cysts may be differentiated Whitwell 1973) (Fig 7). from embryonic vesicles by their lack of mobility, failure to increase in size during serial examinations and by their failure

Fig 5: Transabdominal ultrasound evaluation of a pregnant Fig 7: Placentas from an aborted twin pregnancy. Note the large mare. Adequate patient preparation, restraint and examination pale avillous area on the chorionic surface of both placentae environment are important to ensure optimal imaging. The (asterisk). The avillous areas represent the regions of the transabdominal approach may be more rewarding in mares chorion that were in contact with one another rather than in after 100 days of gestation. contact with the endometrium. EQUINE VETERINARY EDUCATION / AE / september 2008 497

to develop an embryo. If closely apposed, 2 embryonic vesicles separated. A vesicle may be manually reduced as close as may be mistaken for one if examination is performed too 0.5 cm from the other but it is generally best to separate them quickly. A thorough and systematic examination of the entire by at least 2 cm (McKinnon 2007). Elimination of an embryo uterus with good quality ultrasound equipment in a well is achieved by gradually increasing the pressure on the vesicle restrained mare and an optimal environment will minimise the with the probe, using the pelvis to stabilise the uterus. Smaller chances of misdiagnosing twin pregnancy in the mare. vesicles (11–13 days post ovulation) are more difficult to eliminate using this technique and may slip away when Management of twin pregnancies pressure is applied, so additional stability may be necessary. This may be achieved by placing fingers on either side of the The strategy chosen to manage twin pregnancy in the mare probe in an attempt to keep the vesicle in one position. will depend on the age of the pregnancies at identification, Alternatively, a sudden increase in pressure, as opposed to a the orientation of the vesicles and the type of fixation. It may more gradual increase in pressure, may be useful (McKinnon also be important to consider the potential for rebreeding, the 2007). Fluid from the crushed vesicle may surround the potential value of any offspring and the ability and experience remaining vesicle, but does not appear to be detrimental at of the veterinarian. this time, as opposed to later stages of gestation (Pascoe and Before the advent of ultrasound to diagnose pregnancy Stover 1989). prior to fixation, mares with 2 or more large preovulatory Manual reduction techniques have been demonstrated to follicles on palpation were typically withheld from breeding in cause the release of PGF2α for up to 90 min, with the amount an attempt to prevent twin pregnancy. This management being directly correlated to the pressure required to effect strategy however did not result in a decreased rate of twin rupture (Pascoe et al. 1987). This increase in PGF2α did not, pregnancy (Ginther et al. 1982). however, cause a significant change in plasma progesterone concentrations. Other studies have failed to demonstrate a Recognition prior to or on Day 16 significant change in PGF2α concentrations following either fixed or mobile reduction of one member of a twin set While the mare is very efficient at reducing twins to a single (Veronesi et al. 2005). While these results suggest that anti- pregnancy, the probability that a mare with twins will lose one inflammatory and/or progestin therapy are unnecessary or both vesicles before Day 16 is minimal and equates to that following manual twin reduction, some practitioners continue of early embryonic death for the same period (Ginther 1984). to utilise these treatments as part of their twin management Embryo reduction before Day 11 is not considered an protocol. important aspect of the natural correction of twins (Ginther Manual reduction of one of twin vesicles prefixation does and Bergfelt 1988). Embryo reduction was also not detected not result in a higher rate of embryonic death in the during the mobility phase (Days 11–15) or on the day of remaining vesicle than that encountered with singleton fixation (Day 16) in any of 38 mares with multiple embryos pregnancies. Twins were diagnosed prefixation in 245 of (Ginther 1984). 1716 Thoroughbred mare cycles and subjected to manual A method for prefixation embryo reduction was first reduction of one of the vesicles (McKinnon 2007). When described by Ginther (1983b) and was a variation of mares were examined between 7 and 10 days later, 10 of techniques described for the reduction of post fixation, 245 mares (4.0%) had lost the remaining pregnancy. This was bilaterally fixed embryos (Roberts 1982). This technique similar to the rate of early embryonic death in mares with a utilised ultrasound to identify the vesicles on Days 12–14 and single pregnancy diagnosed at Day 13–15 and then found to if they were sufficiently separated, one was manually have lost the pregnancy at the next examination (63/1716; ruptured. Using the thumb and index finger, the vesicle was 3.7%). Pascoe et al. (1987) reported a 96% survival rate for ruptured either in place or following movement to the tip of the remaining conceptus (up to Day 42) after manual the uterine horn. Smaller vesicles (9–11 mm in diameter) were reduction of one member of a twin set in 100 mares, using more difficult to rupture and a distinct popping sensation was either a mobile or fixed technique. felt upon rupture of the vesicle in 8 of 15 mares. For those vesicles that were together on initial examination, it was Recognition between Days 17 and 40 recommended that the mare be re-examined approximately 1 h later. A modification of this technique was later reported Fixation of the embryonic vesicle occurs at around Day 16 in that eliminated the need to wait for vesicles to separate and the mare and is thought to be due to the increasing size of the utilised the ultrasound probe to manipulate the vesicles and embryonic vesicle and to an increase in uterine tone (Ginther manually reduce one in isolation from the other while 1992). In mares with twins, the vesicles may fix either together visualising one or both throughout the procedure (McKinnon at the base of a uterine horn (unilateral fixation) or one vesicle et al. 1993; McKinnon 2007). If 2 separate, noncoalesced may fix at the base of each uterine horn (bilateral fixation). In vesicles can be visualised, then separation should be possible a study of 31 mares with twin embryonic vesicles, unilateral (McKinnon 2007). The probe is positioned where the 2 vesicles fixation was more frequent (71%) than bilateral fixation (29%) are in contact and gentle back and forth movement of the (Ginther 1989a). In 28 mares with known ovulatory patterns, probe with pressure results in the 2 vesicles becoming synchronous ovulation did not affect the type of fixation 498 EQUINE VETERINARY EDUCATION / AE / september 2008

(9/17 unilateral, 8/17 bilateral) (Ginther 1989a). However, this spontaneous reduction following bilateral fixation, it is study also found that the rate of unilateral fixation was much recommended to eliminate one of the twins immediately upon higher than bilateral fixation (10/11 and 1/11 respectively). In detection up to Day 30 (McKinnon and Rantanen 1998). The 2 studies, the incidence of embryo reduction was found to be ability of the remaining pregnancy to survive to term has been much higher for unilaterally fixed embryos than for bilaterally demonstrated to decrease if the reduction occurs after Day 31. fixed embryos (14/19 vs. 0/9 [Ginther 1989a] and 17/19 vs. 1/9 In a review of 181 mares with bicornuate twin pregnancies in [Ginther 1984]). Twins resulting from asynchronous ovulations which one was manually eliminated (Roberts 1982), the (ovulations not occurring on the same day) were also more success of the procedure before Day 31 (33/47: 70%) was likely to undergo reduction to a singleton (9/11; 82%) as considerably greater, in terms of live foals born, than that compared with those resulting from synchronous ovulations achieved after Day 31 (31/134: 23%). The author also (5/17; 29%) (Ginther 1989a). Asynchronous ovulations are reported the inability to manually reduce some twins after likely to result in embryos of different gestational ages and Day 35, even if extreme pressure is applied. The likely reason therefore different sizes. In 22 mares with unilaterally fixed for the reduced likelihood of the remaining pregnancy to twins, reduction occurred in 100% with vesicles of dissimilar survive to term following manual reduction after Day 31 was size (>4 mm difference in diameter), whereas 19/26 twin the allantoic fluid from the ruptured pregnancy surrounding pregnancies (73%) with vesicles of similar size (0–3 mm the other pregnancy and undermining the ventral surface of difference in diameter) underwent reduction to a singleton the chorioallantois, elevating it off the endometrium and (Ginther 1989b). When reduction occurs with unilateral disrupting maternal-embryonal physiological exchange. fixation, it most commonly occurs prior to detection of both Pascoe and Stover (1989) demonstrated this with the rapid embryos. Reduction was complete before detection of both death within 2–3 h of 3 pregnancies (Days 45, 50 and 55) embryos in 10/14 twin pregnancies (Ginther 1989a). Embryo following the injection of 250 ml of sterile placental fluid into reductions were complete by Day 20 in 29 of 49 (59%) the nongravid horn of the uterus. unilaterally fixed twins, with reductions occurring in one day Because of the potential to disrupt the remaining (Ginther 1989b). Reductions that occurred after Day 20 were pregnancy if manual reduction is performed after Day 30, it usually preceded by a gradual decrease in size of the vesicle has been suggested to instead disrupt one of the vesicles by undergoing reduction (Ginther 1989b). A proposed application of manual pressure and oscillation in order to mechanism for this high rate of embryo reduction in create a ‘snowflake’ effect without rupturing the unilaterally fixed twins was proposed by Ginther (1989a). The chorioallantois (McKinnon and Rantanen 1998). deprivation hypothesis proposes that when the vascularised Demonstration of this effect almost always leads to a gradual wall of an embryonic vesicle (trilaminar omphalopleure) is in loss of the affected conceptus. contact with the bilaminar wall of the other vesicle rather than Transvaginal aspiration of fetal fluids has been described as the endometrium, embryonal-maternal physiological exchange a method for reducing twin pregnancies to a singleton. Pascoe is severely reduced and the deprived vesicle undergoes (1979) first reported this technique in which fetal fluid was reduction due to an inability to exchange nutrients across the aspirated per vaginam from one conceptus in 6 mares found yolk sac placenta. to be carrying bicornuate twins at 42 days post breeding. Because the mares’ own biological reduction mechanism is While the results of this study were disappointing (all mares so efficient, no attempt is often made to reduce post fixation subsequently lost both pregnancies), better results were unilateral twins. If the individual vesicles retain their spherical obtained in subsequent studies which utilised transvaginal shape and have not coalesced, it may be possible to manually ultrasonography. Of 13 mares subjected to the procedure separate unilaterally fixed twins between Days 16 and 20 between 20 and 45 days of pregnancy, 6 of the mares (46%) (McKinnon 2007). Although the procedure was described as were found to be pregnant with a single conceptus 10 days time-consuming and tedious, 39 of 42 unilaterally fixed twin later (Bracher et al. 1993). The success rate was higher for pregnancies between Days 16 and 20 were successfully mares with bicornuate pregnancies as opposed to unicornuate reduced in this manner (Bowman 1986). Reported success pregnancies (75% vs. 33% respectively). In contrast, Mari rates for manual reduction of unilaterally fixed twins after et al. (2004) reported the loss of both fetuses in 2 of 3 mares Day 20 range from 25% (Mari et al. 2004) to 50% (McKinnon when transvaginal ultrasound-guided aspiration (TUGA) of and Rantanen 1998), which is much lower than the rate of fetal fluids was conducted on one of 2 bilaterally fixed twins natural reduction. Instead, the pregnancy is monitored and if between 40 and 50 days of gestation. Successful reduction no reduction has occurred by Day 30, both pregnancies are and birth of a single viable foal was achieved in 14 of terminated before the establishment of endometrial cups 20 (70%) unicornuate twins when the procedure was either by manual reduction or with prostaglandin. performed between 16 and 25 days gestation. Based on these The incidence of reduction for bilaterally fixed twins is results, the authors concluded that the procedure was best negligible. No reductions occurred in a review of 20 bilaterally performed before 25 days of gestation. MacPherson and fixed twin pregnancies monitored with ultrasound until Reimer (2000) suggested that the procedure is best performed Day 40 (Ginther 1989b). Only 1 of 9 (11%) bilaterally fixed prior to Day 36, particularly in cases of unilateral twins. In this embryos reduced to a singleton by Day 40 in another study study, the gestational age at the time of reduction in 7 of (Ginther 1984). Because of the very low incidence of 10 mares with unilateral twins was >35 days and all resulted EQUINE VETERINARY EDUCATION / AE / september 2008 499

in loss of both pregnancies. The successful application of transvaginal ultrasound guided aspiration of unilateral twins in the mare appears to require its use during a period in which the rate of natural reduction of twin pregnancies is high. Therefore in many cases it may be more prudent to monitor these pregnancies carefully for spontaneous reduction rather than intervene with transvaginal aspiration.

Recognition from Day 60 onward

A technique for the attempted reduction of twin pregnancies between Days 60 and 100 has been described in which the smaller of the 2 fetuses is identified and repeatedly traumatised by oscillation or percussion with the ultrasound probe, membrane slip or single digit percussion of the cranium (Rantanen and Kincaid 1988; McKinnon and Rantanen 1998). Multiple attempts at the procedure may be necessary to create sufficient damage for fetal death, and a success rate of Fig 8: Transabdominal ultrasound image obtained during a twin reduction procedure. Note the needle in the fetal thorax approximately 50% should be expected (McKinnon and (arrow). Rantanen 1998). Successful application of this technique requires that the examiner be able to identify which fetus was traumatised during earlier attempts. Transabdominal ultrasound guided twin reduction was first Recently, cranio-cervical dislocation has been reported as a described as a method for elimination of one member of a method for the reduction of twin pregnancies identified twin set in pregnancies beyond 60 days of gestation in 1988 between 60 and 110 days of gestation (Wolfsdorf et al. 2005; (Rantanen and Kincaid 1988). The procedure involves Wolfsdorf 2006). The procedure involves identification of the transabdominal ultrasonography of a sedated mare with smaller of the 2 fetuses, or the fetus that has less contact with either a 3 MHz (Rantanen and Kincaid 1988) or 5 MHz the endometrium. Isolation of the fetus may be difficult and (MacPherson and Reimer 2000) transducer to identify the uterine relaxation is considered imperative to enable accurate presence of twins and to select the most easily accessible and identification of fetal anatomy (Wolfsdorf 2006). Once the preferably smallest of the 2 fetuses for reduction (Frazer fetus for reduction is selected, the head is isolated and 2003). Sedation of the mare and the administration of stabilised between the thumb and forefinger. The head is then clenbuterol cause uterine relaxation and move the fetuses bent from side to side to damage the ligaments attaching the cranially, which may improve visualisation and accessibility head to the neck. Dislocation of the head is then achieved by (Ball 2000). The area of the mares’ abdomen adjacent to the placing the thumb at the base of the cranium and applying fetus to be reduced is surgically prepared and infiltrated with pressure proximally and dorsally. If dislocation is successful, a local anaesthetic. With the assistance of a biopsy guide distinctive pop is felt, and the thumb and forefinger is able to attached to the transducer or using a ‘free hand’ technique be placed between the head and neck. (Ball 2000), a 15–25 cm, 16 or 18 gauge needle with a stylet By performing the procedure before placentation is is passed through the skin, abdomen and uterine wall. complete, it is believed that the remaining fetus is better able Following identification of the needle on the ultrasound to grow to its full potential, thus avoiding the birth of a small image, the needle is then passed into the fetus (Fig 8). Initial and weak foal, as can be observed when twin reduction is reports of injecting air or saline into the fetal heart were performed transabdominally at a later time (MacPherson and unsuccessful at causing fetal death (Rantanen and Kincaid Reimer 2000). Cranio-cervical dislocation can be performed 1988). Subsequently, injection of the fetal heart with KCl either transrectally or via flank laparotomy. Of 8 sets of twins (Rantanen and Kincaid 1988; Rantanen 1990; MacPherson which were reduced with transrectal manipulation, 5 mares and Reimer 2000), or injection of the fetal heart, lungs or delivered a normal sized single foal, while the other 3 mares abdomen with 10–20 ml of procaine penicillin (McKinnon and aborted both fetuses between 30 and 60 days later Rantanen 1998) have resulted in rapid fetal death, although (Wolfsdorf 2006). The procedure had to be repeated on 3 of the later technique appears to take longer to cause fetal death the 8 mares before cranio-cervical dislocation was successful. than the former. Fetal death should be confirmed the Once cranio-cervical dislocation was achieved, it took from following day (Fig 9). Reported advantages of using procaine 1–3 weeks before the reduced fetus had no viable heartbeat. penicillin for transabdominal twin reduction include reducing Of the 5 sets of twins reduced via flank laparotomy, 3 mares iatrogenic bacterial contamination, ultrasonographic delivered a normal sized single foal. The authors concluded visualisation during fetal injection (Fig 10) and eliminating the that the earlier death of the reduced fetus increased the need for intracardiac placement, as fetal death can be chance of a single pregnancy going successfully to term achieved with placement in either the thorax or abdomen (Wolfsdorf et al. 2005). (McKinnon and Rantanen 1998). 500 EQUINE VETERINARY EDUCATION / AE / september 2008

Fig 11: Chorionic surface of the allantochorion from a foal born alive following transabdominal reduction of a twin earlier in gestation. Note the difference between normal chorion (a) and chorion with sparse and poorly developed microcotyledons (b).

by subsequent results in 59 mares with twin pregnancies that Fig 9: Transabdominal ultrasound images obtained one day went on to deliver 29 single live foals (49.2%) when the after a transabdominal twin reduction procedure. The fetus in the image on the right was injected with 20 ml of procaine procedure was performed after Day 115 (McKinnon and penicillin. Anatomical landmarks of the fetus on the left are Rantanen 1998). MacPherson and Reimer (2000) reported the readily apparent (arrow = fetal diaphragm, asterisk = fetal birth of 9 normal, live, single foals from 24 mares (38%) heart), whereas identification of the same structures in the following the procedure at >120 days gestation. In addition, nonviable fetus on the right is more difficult (asterisk indicates 3 of 24 foals (12%) were born alive but were weak and small. approximate position of the fetal heart). Note the increased echogenicity in the fetal thorax and abdomen, due to procaine This growth retardation has been ascribed to the lack of penicillin. effective macrovillous placentation on that portion of the allantochorion of the surviving fetus which grows to occupy much of the uterine horn previously occupied by the dead twin (Ball et al. 1993) (Fig 11). An increased success rate, measured in terms of single live foals born, was reported with the use of 10–20 ml of procaine penicillin injected into the fetal heart, thorax or abdomen (McKinnon and Rantanen 1998). Using this technique, 8 of 13 mares (62%) with twin pregnancies went on to deliver single, live foals. Follow-up ultrasonography of the remaining fetus has been recommended, although an unsuccessful outcome for the remaining fetus is usually not immediately apparent (McKinnon and Rantanen 1998). Of the mares subjected to this procedure that subsequently aborted both fetuses, most aborted 1–2 months after the procedure (MacPherson and Reimer 2000). Most reports of the procedure include post operative treatment with NSAIDs, systemic antibiotics and Fig 10: Transabdominal ultrasound image obtained following progestin therapy, although some have found no difference in intrafetal injection with procaine penicillin. Note the echogenic fetal survival rates with long-term antibiotic and Regumate procaine penicillin (arrow) in the fetal thorax outlining the fetal therapy (McKinnon and Rantanen 1998). diaphragm. Conclusion The initial report of the procedure involved 18 mares diagnosed as carrying twins between 66 and 168 days of While the incidence of abortion due to twinning has declined gestation (Rantanen and Kincaid 1988). Following the subsequent to the routine use of ultrasonography for pregnancy intracardiac injection of KCl, 7 of these mares (40%) diagnosis, the early and accurate diagnosis and management of subsequently delivered single live foals. Based on these results twins remains an important part of routine equine breeding and subsequent attempts at the procedure (Rantanen 1990), management. While options exist for the management of twins it was concluded that the procedure was best performed post fixation, more mares will give birth to a single foal if one of between 115 and 130 days of gestation. This was emphasised the vesicles is eliminated during the mobility phase. EQUINE VETERINARY EDUCATION / AE / september 2008 501

References Lofstedt, R.M. and Newcombe, J.R. (1997) Pregnancy diagnosis and subsequent examinations in the mare: when and why. Equine vet. Educ. 9, 293-294. Allen, W.R., Brown, L., Wright, M. and Wilsher, S. (2007) Reproductive efficiency of Flatrace and National Hunt Thoroughbred mares and MacPherson, M.L. and Reimer, J.M. (2000) Twin reduction in the mare: stallions in England. Equine vet. J. 39, 438-445. current options. Anim. reprod. Sci. 60-61, 233-244. Ball, B.A. (2000) How to do trans-abdominal reduction of twin McCue, P.M., Thayer, J., Squires, E.L., Brinsko, S.P. and Vanderwall, pregnancies in the mare. IVIS. Document number A0202.0400 D.K. (1998) Twin pregnancies following transfer of single embryos www.ivis.org. in three mares: A case report. J. equine vet. Sci. 18, 832-834. Ball, B.A., Schlafer, D.H., Card, C.E. and Yeager, A.E. (1993) Partial re- McKinnon, A.O. (2007) Twin reduction techniques. In: Current establishment of villous placentation after reduction of an equine Therapy in Equine Reproduction, Ed: J.C. Samper, J.F. Pycock and co-twin by foetal cardiac puncture. Equine vet. J. 25, 336-338. A.O. McKinnon, Saunders, St. Louis. pp 357-373. Bowman, T. (1986) Ultrasonic diagnosis and management of early McKinnon, A.O. and Rantanen, N.W. (1998) Twins. In: Equine twins in the mare. Proc. Am. Ass. equine Practnrs. 32, 35-43. Diagnostic Ultrasonography, Eds: N.W. Rantanen and A.O. Bracher, V., Parlevliet, J.M., Pieterse, M.C, Vos, P.L.A.M., Wiemer, P., McKinnon, Williams and Wilkins, Baltimore. pp 141-156. Taverne, M.A.M. and Colenbrander, B. (1993) Transvaginal McKinnon, A.O., Voss, J.L., Squires, E.L. and Carnevale, E.M. (1993) ultrasound-guided twin reduction in the mare. Vet. Rec. 133, Diagnostic ultrasonography. In: Equine Reproduction, Ed: A.O. 478-479. McKinnon and J.L. Voss, Lea and Febiger, Philadelphia. Davies Morel, M.C.G. and Newcombe, J.R. (2007) The efficacy of pp 266-302. different hCG dose rates and the effect of hCG treatment on Mari, G., Iacono, E., Merlo, B. and Castagnetti, C. (2004) Reduction of ovarian activity: Ovulation, multiple ovulation, pregnancy, multiple twin pregnancy in the mare by transvaginal ultrasound-guided pregnancy, synchrony of multiple ovulation; in the mare. Anim. aspiration. Reprod. Domest. Anim. 39, 434-437. reprod. Sci. In Press. Meadows, S.J., Binns, M.M., Newcombe, J.R., Thompson, C.J. and Davies Morel, M.C.G., Newcombe, J.R. and Swindlehurst, J.C. (2005) Rossdale, P.D. (1995) Identical triplets in a Thoroughbred mare. The effect of age on multiple ovulation rates, multiple pregnancy Equine vet. J. 27, 394-397. rates and embryonic vesicle diameter in the mare. Theriogenol. 63, Merkt, H. and Jochle, W. (1993) Abortions and twin pregnancies in 2482-2493. Thoroughbreds: Rate of occurrence, treatments and prevention. J. Frazer, G.S. (2003) Twins. In: Current Therapy in Equine Medicine 5, equine vet. Sci. 13, 690-694. Ed: N.E. Robinson, W.B. Saunders, St Louis. pp 245-248. Merkt, H. and Jochle, W. (1999) More twin pregnancies as season Giles, R.C., Donahue, J.M., Hong, C.B., Tuttle, P.A., Petrites-Murphy, progresses. J. equine vet. Sci. 19, 536-539. M.B., Poonacha, K.B., Roberts, A.W., Tramontin, R.R., Smith, B. Pascoe, R.R. (1979) A possible new treatment for twin pregnancy in and Swerczek, T.W. (1993) Causes of abortion, stillbirth, and the mare. Equine vet. J. 11, 64-65. perinatal death in horses: 3,527 cases (1986-1991). J. Am. vet. Pascoe, R.R. (1983) Methods for the treatment of twin pregnancy in med. Ass. 203, 1170-1175. the mare. Equine vet. J. 15, 40-42. Ginther, O.J. (1983a) Effect of reproductive status on twinning and on Pascoe, D.R. and Stover, S.M. (1989) Effect of surgical manipulation, side of ovulation and embryo attachment in mares. Theriogenol. placental fluid, and flunixin meglumine on fetal viability and 20, 383-395. prostaglandin F2α release on the gravid uterus of mares. Am. J. Ginther, O.J. (1983b) The twinning problem: From breeding to day 16. vet. Res. 50, 1505-1511. Proc. Am. Ass. equine Practnrs. 29, 11-26. Pascoe, D.R., Pascoe, R.R., Hughes, J.P., Stabenfelt, G.H. and Kindahl, Ginther, O.J. (1984) Post-fixation embryo reduction in unilateral and H. (1987) Management of twin conceptuses by manual embryonic bilateral twins in mares. Theriogenol. 22, 213-223. reduction: Comparison of two techniques and three hormone Ginther, O.J. (1986) The single embryo. In: Ultrasonic Imaging and treatments. Am. J. vet. Res. 48, 1594-1599. Reproductive Events in the Mare, Ed: O.J. Ginther, Equiservices, Perkins, N.R. and Grimmett, J.B. (2001) Pregnancy and twinning rates Cross Plains, Wisconsin. pp 195-228. in Thoroughbred mares following administration of human Ginther, O.J. (1987) Relationships among number of days between chorionic gonadotropin (hCG). N. Z. vet. J. 49, 94-100. multiple ovulations, number of embryos and type of embryo Platt, H. (1973) Etiological aspects of perinatal mortality in the fixation in mares. J. equine vet. Sci. 7, 82-88. Thoroughbred. Equine vet. J. 5, 116-120. Ginther, O.J. (1989a) The nature of embryo reduction in mares with twin Rantanen, N.W. (1990) Ultrasound guided fetal cardiac puncture for conceptuses: Deprivation hypothesis. Am. J. vet. Res. 50, 45-53. twin reduction in mares. In: Proceedings of the Annual Meeting of Ginther, O.J. (1989b) Twin embryos in mares II: post fixation embryo the Society of Theriogenology. pp 169-170. reduction. Equine vet. J. 21, 171-174. Rantanen, N.W. and Kincaid, B. (1988) Ultrasound guided fetal cardiac Ginther, O.J. (1992) Maternal aspects of pregnancy. In: Reproductive puncture: A method of twin reduction in the mare. Proc. Am. Ass. Biology of the Mare: Basic and Applied Aspects, Ed: O.J. Ginther, equine Practnrs. 34, 173-179. Equiservices, Cross Plains, Wisconsin. pp 291-344. Riera, F.L., Roldán, J.E. and Hinrichs, K. (2006) Patterns of embryo Ginther, O.J. and Bergfelt, D.R. (1988) Embryo reduction before day recovery in mares with unilateral and bilateral double ovulations. 11 in mares with twin conceptuses. J. anim. Sci. 66, 1727-1731. Anim. reprod. Sci. 94, 398-399. Ginther, O.J. and Griffin, P.G. (1994) Natural outcome and ultrasonic Ricketts, S.W., Barrelet, A. and Whitwell, K.E. (2003) Equine abortion. identification of equine fetal twins. Theriogenol. 41, 1193-1199. In: Reproduction Foaling. Part 2: Fetal and Neonatal Aspects, EVE Ginther, O.J., Douglas, R.H. and Lawrence, J.R. (1982) Twinning in Manual 6, Eds; P.D. Rossdale, T.S. Mair and R.E. Green, Equine mares: A survey of veterinarians and analyses of theriogenology Veterinary Journal Ltd, Newmarket. pp 18-21. records. Theriogenol. 18, 333-347. Roberts, C.J. (1982) Termination of twin gestation by blastocyst crush Jeffcott, L.B. and Whitwell, K.E. (1973) Twinning as a cause of foetal in the broodmare. J. Reprod. Fert., Suppl. 32, 447-449. and neonatal loss in the Thoroughbred mare. J. comp. Pathol. 83, Rooney, J.R. (1970). The fetus and foal. In: Autopsy of the Horse. Ed: 91-106. J.R. Rooney, Williams and Wilkins, Baltimore. pp 120-134. 502 EQUINE VETERINARY EDUCATION / AE / september 2008

Smith, K.C., Blunden, A.S., Whitwell, K.E., Dunn, K.A. and Wales, PGF2α, cortisol and progesterone during manual twin reduction in A.D. (2003) A survey of equine abortion, stillbirth and neonatal Thoroughbred mares. J. vet. Med. 52, 411-415. death in the UK from 1988 to 1997. Equine vet. J. 35, 496-501. Whitwell, K.E. (1980) Investigations into fetal and neonatal losses in Squires, E.L., McClain, M.G., Ginther, O.J. and McKinnon, A.O. (1987) the horse. Vet. Clin. N. Am: Large Anim. Pract. 2, 313-331. Spontaneous multiple ovulation in the mare and its effects on the Wolc, A., Bresinska, A. and Szwaczkowski, T. (2006) Genetic and incidence of twin embryo collections. Theriogenol. 28, 609-613. permanent environmental variability of twinning in Thoroughbred horses estimated via three threshold models. J. Anim. Breeding Veronesi, M.C., Battocchio, M., Faustini, M., Gandini, M. and Cairoli, Genetics. 123, 186-190. F. (2003) Relationship between pharmacological induction of Wolfsdorf, K.E. (2006) Management of postfixation twins in mares. estrous and/or ovulation and twin pregnancy in the Thoroughbred Vet. Clin. N. Am.: Equine Pract. 22, 713-725. mares. Domest. Anim. Endocr. 25, 133-140. Wolfsdorf, K.E., Rodgerson, D. and Holder, R. (2005) How to manually Veronesi, M.C., Faustini, M., Villani, M., Kindahl, H., Galeati, G. and reduce twins between 60 and 120 days gestation using cranio- Battocchio, M. (2005) Plasma concentrations of 15-Ketodihydro- cervical dislocation. Proc. Am. Ass. equine Practnrs. 51, 284-287.