Three Types of Anomalous Vasculature in the Equine Umbilical Cord S

EQUINE VETERINARY EDUCATION / AE / March 2011 109

Case Reporteve_149 109..118 Three types of anomalous vasculature in the equine umbilical cord S. Wilsher*, J. Ousey, K. Whitwell†, J. S. Crowhurst‡, H. N. Neal‡ and W. R. Allen The Paul Mellon Laboratory of Equine Reproduction (formerly The Equine Fertility Unit); †Equine Pathology Consultancy; and ‡Newmarket Equine Hospital, Suffolk, UK.

Keywords: horse; umbilical cord; placenta; umbilical artery; umbilical vein; supernumerary umbilical vessels

Summary branches of the aorta. However, some of the blood that enters the right atrium is directed via the right ventricle to Anomalies in the number of major blood vessels in the mix with deoxygenated blood returning from the head umbilical cord are well documented in man and have and myocardium, before heading to the pulmonary trunk been associated with both placental abnormalities and where the greater volume passes through the ductus congenital problems in the neonate. This paper describes 5 arteriosus to the caudal aorta. The lesser volume supplies cases of anomalies in the number of umbilical arteries or the pulmonary vessels of the nonfunctioning fetal lungs. veins within the equine umbilical cord at term. In 4 cases, Most of the blood in the caudal aorta heads back to the anomalies affected vessels in the amniotic part of the the placenta, with arterial branches ensuring that cord, and in one case, vessels in the allantoic part. In all the thoracic and abdominal organs are oxygenated. The the cases placental morphology was essentially normal deoxygenated blood finally leaves the fetus through the and fetal health and post natal development were not paired umbilical arteries, returning to the allantochorion to compromised as a result of the unusual arrangement of become reoxygenated (Fig 1; McGeady et al. 2006). blood vessels within the cord. The equine umbilical cord develops from the convergence of the 2 fetal sacs, the amnion and allantois, Introduction around the remnants of the yolk sac and the vitelline duct to form a cord-like structure by Day 50. Embedded in the connective tissue, and formed from the allantoic In pregnant mammals, the umbilical cord provides the mesoderm, are the umbilical vessels. In ruminants there are route through which the fetal blood carries the nutrients 2 arteries and 2 veins along the whole length of the cord and oxygenated blood to, and all the gaseous and but in other species, such as equids and carnivores, the metabolic waste from, the developing fetus. Since the amniotic portion end of the cord has 2 arteries but only one fetal lungs are not functional in utero it is the umbilical veins vein. However, regardless of the species, only one umbilical which supply oxygenated blood to the fetus. This passes vein persists inside the fetus in late gestation (Fig 1; Latshaw initially through the ductus venosus, the major blood 1987). channel from the placenta through the embryonic liver, to The umbilical cord in the horse comprises a proximal the caudal vena cava (McGeady et al. 2006). This shunt intra-amniotic portion, which is covered by amnion and is remains patent throughout gestation in most domestic attached to the fetus at the umbilicus, and a distal species but, in the horse and pig, it atrophies during allantoic portion covered by allantois that is attached to gestation and blood from the umbilical vein then passes the allantochorion. The amniotic portion of the cord through the sinusoids of the liver en route to the caudal contains 2 umbilical arteries, one umbilical vein, the vena cava. From here the blood enters the right atrium of vitelline vein remnant and the urachus. In the allantoic the heart where most of it is directed through the foramen portion of the cord the 2 major umbilical arteries become ovale into the left atrium. This blood then enters the left increasingly divergent and multibranched towards the ventricle and is pumped throughout the body via the chorion: the 2 umbilical veins returning blood to the fetus aortic arch, with the cardiac musculature and brain tend to lie in close proximity to these arteries. The 2 major receiving highly oxygenated blood from the first 2 veins unite at the proximal end of the allantoic portion of the cord or just inside the amniotic cavity (Fig 2; Whitwell *Corresponding author email: [email protected] and Jeffcott 1975; Schlafer 2004). The allantoic portion of

1 42 III 8 the human umbilical cord, particularly the existence of a 3 single umbilical artery (SUA), are well documented (reviews: Baergen 2005; Fox and Sebire 2007). In this paper, 5 cases of anomalies in the major vessels within the term equine umbilical cord are reported. Two IV cases describe the atypical arrangement of the 2 arteries I 9 6 within the umbilical cord, 2 cases detail an increased 5 number of vessels in the amniotic portion of the cord, and the fifth describes an increased number of vessels in the 7 II allantoic portion of the cord. The cases are discussed in relation to the normal development of the feto-placental V circulation and compared to similar conditions in the human infant.

Case 1: Atypical arrangement of the umbilical arteries in the umbilical cord

Fig 1: A diagrammatic representation of the relationship of the Clinical history umbilical vessels and the fetal circulation in utero. I: heart; II: liver; III: lung; IV: bladder; V: urachus; 1: aorta; 2: ductus arteriosus; 3: foramen ovale; 4: pulmonary arteries; 5: left umbilical vein; A multiparous, 7-year-old Thoroughbred (TB) mare foaled a 6: umbilical arteries; 7: ductus venosus; 8: caudal vena cava; healthy 52 kg colt foal without complication following an 9: portal veins. Redrawn from Rüsse and Sinowatz (1998) and uneventful gestation of 336 days. Fetal development Latshaw (1987). appeared normal as judged by foal birthweight as a percentage of maternal weight (Table 1). Gross examination of the placenta revealed only one major a) Intra-amniotic cord Intra-allantoic cord artery in the amniotic portion of the umbilical cord. Am Retrospective checking of the mare’s breeding records UA revealed that she had been mated only once during UA UV UV oestrus when she exhibited a follicle >35 mm in one ovary. UV UA UA She had subsequently undergone 5 transrectal ultrasound examinations to diagnose and monitor pregnancy on Days Normal cord 13, 19, 27, 52 and 66 after ovulation when development appeared normal in the singleton conceptus. b) In the 2 previous breeding seasons the mare had UV produced healthy colt foals (41.1 and 48 kg, respectively) UA after gestation lengths of 334 days in both instances. The same TB stallion had sired all 3 foals.

Normal cord UA Gross placental findings

Fig 2: a) Normal morphology of the equine umbilical cord. Note Linear placental measurements fell within the normal that there are 2 umbilical arteries (UA) and one umbilical vein (UV) ranges for TB placentae as reported by Whitwell and in the amniotic portion of the cord, but 2 arteries and 2 veins in the Jeffcott (1975). The placenta was presented with the allantoic portion. A white arrow indicates the urachal opening into the allantoic cavity. The amnion (Am) has been cut off close to the allantoic surface outermost, which was normal in cord so the vessels can be visualised; b) the urachal lumen has appearance. The chorionic (villous) surface appeared been opened to reveal the joining of the 2 umbilical veins. The grossly normal. The placental parameters are listed and direction of flow and oxygenation status of blood in the umbilical compared in Table 1 to previously published data for TB vessels are indicated by the arrows and dashed lines (red denotes oxygenated blood and blue denotes deoxygenated blood). mares. The umbilical cord attachment site to the allantochorion was normal (i.e. between the 2 horns). The the umbilical cord also contains remnants of the yolk sac cord had ruptured naturally as indicated by protrusion of and vitelline vessels as well as a smooth walled cavity, the the wall of an artery from the fetal end of the cord. The infundibulum, a remnant of the extra-embryonic coelom. vascular pattern of the major vessels was type I (K.E. To the authors’ knowledge, apart from an isolated case Whitwell, personal communication in Rossdale and Ricketts of a single umbilical artery (Whitwell 1975), there are no 2002). That is, one vein and artery vascularised the whole other reports of anomalies in vessel number in the equine of the gravid horn while the other vein and artery umbilical cord. In contrast, anomalies in vessel number in vascularised the nongravid horn and uterine body. The

TABLE 1: Previously published placental and fetal parameters for Thoroughbred and Pony mares compared to those for Cases 1, 2, 3, 4 and 5. Where the placental parameter is outside the 95% range for Thoroughbreds (Cases 1, 3, 4 and 5) and the range† for Ponies (Case 2)from previously reported for the respective breed this is indicated by either an ↓, if the value falls below, or ↑, if the value is above

Mean Ϯ s.e. (95% range) of Mean Ϯ s.e. (range) normal TB placentae of normal Pony placentae (Wilsher and Allen 2003, (Allen et al. 2002, n = 7; n = 84; Whitwell and Case 1 Case 3 Case 4 Case 5 Whitwell and Jeffcott§ Case 2 Parameter Jeffcott 1975*, nՆ139) TB mare TB mare TB mare TB mare 1975, n = 10) Pony mare

Gestation length (days) 340.5 Ϯ 0.77 (324–359)* 336 345 327 330 324.6 Ϯ 2.97 (312–333) ↑345 Maternal weight (kg) 521.6 Ϯ 7.6 (469–577) 543 – 532 – 300.1 Ϯ 8.6 (264–326) 295 Foal birthweight (kg) 52.0 Ϯ 0.6 (44.4–61.6) 52 ↓44 58 52 24 Ϯ 1.25 (20–30) ↓18.7 Foal birthweight/maternal weight 9.9 Ϯ 0.1 (8.0–12.3) 9.6 – 10.9 – 8.1 Ϯ 0.7 (6.1–11.4) 6.3 Mass of allantochorion (kg) 3.9 Ϯ 0.07 (2.9–4.9) 3.37 ↓2.71 4.3 4.30 1.73 Ϯ 0.13 (1.4–2.3) ↓1.09 Gross area of allantochorion (cm2 x103) 12.6 Ϯ 0.2 (10.5–14.6) 12.4 14.2 12.8 14.4 8.3 Ϯ 0.35 (7.6–8.9) ↓4.2 Volume of allantochorion (l) 3.7 Ϯ 0.1 (2.7–4.7) 3.3 ↓2.60 4.26 4.2 1.75 Ϯ 0.13 (1.38–2.72) ↓1.08 Volume of chorion (Vc; l) 1.2 Ϯ 0.2 (0.9–1.6) 1.48 1.00 ↑1.75 1.59 0.6 Ϯ 0.04 (0.48–0.77) 0.44 Depth of allantochorion (mm) 2.93 Ϯ 0.05 (2.3–3.7) 2.66 ↓1.83 3.33 2.92 2.12 Ϯ 0.14 (1.74–2.72) 2.57 Height of microcotyledons (mm) 0.99 Ϯ 0.02 (0.74–1.3) 1.19 ↓0.70 ↑1.37 1.10 0.76 Ϯ 0.04 (0.62–0.89) ↑1.07 Microcotyledon surface density (Sv;/mm) 0.036 Ϯ 0.0004 (0.030–0.042) 0.033 0.036 0.036 0.033 0.030 Ϯ 0.001 (0.027–0.033) ↑0.035 Total microscopic area of fetomaternal 44.8 Ϯ 1.0 (31.4–63.2) 48.8 36.0 62.7 51.7 18.7 Ϯ 1.3 (14.7–23.3) 15.4 2 contact (Sv xVc;m) Foal wt per m2 of placenta (kg/m2) 1.2 Ϯ 0.02 (0.9–1.5) 1.07 1.22 0.93 1.01 1.3 Ϯ 0.1 (1.0–1.6) 1.21 Mass of amnion (kg) 1.85 Ϯ 0.04 (1.1–2.8)* 1.59 1.12 2.26 1.71 0.65 Ϯ 0.11 (0.3–1.4)§ 0.68 Mass of cord (g) 246.5 Ϯ 5.54 (155–400)* 279 222 216 215 93.5 Ϯ 9.53 (50–138)§ 99 Total cord length (cm) 55 Ϯ 0.9 (36–83)* 61 37 42 55 41 Ϯ 1.74 (30–48)§ ↓29 Amniotic cord length (cm) 26 Ϯ 0.96 (18–41)* 38 24 28 26 – 17 Cord weight/length 4.5 Ϯ 0.06 (3.2–6.3)* 4.6 ↑9.25 5.14 3.9 2.3 Ϯ 0.18 (1.2–3.0)§ ↑3.41

† The complete range, rather than the 95% range, is reported for Pony mares since the n values are low for the data sets.

total cord length was 61 cm, with a 38 cm intra-amniotic any associated congenital anomalies existed. There was portion and a 23 cm allantoic portion. The allantoic portion a large discrepancy in the diameter of the 2 umbilical of the cord showed normal morphology in that 2 major arteries; the left artery was narrow whereas the right one veins and 2 major arteries were present. In the amniotic was of normal diameter (Fig 4a and b). The spatial portion there was one large artery and a second arrangement of the internal and external iliac arteries as thread-like artery. Unusually the larger artery divided at they branched from the aorta was asymmetrical (Fig 4a). the amniotic-allantoic junction into 2 well developed No abnormalities were noted in the right and left kidneys branches; each one running parallel with, and or in their renal arteries. The left kidney was smaller than accompanying, one of each of the umbilical veins. The the right kidney (669.2 vs. 738.6 g) but this was not tiny second artery fused/anastomised with one of the large considered an unusual finding. The other major arterial branches a short distance from the arterial abdominal organs, including the spleen, pancreas, liver, bifurcation (Fig 3a and b). The umbilical cords from the 2 heart, lungs and intestines all appeared grossly normal. previous pregnancies sired by the same stallion had shown Thus, in summary, the post mortem examination no abnormalities. confirmed asymmetry of the internal umbilical arteries with no associated abnormalities in kidney structure or Histological and stereological findings vasculature.

Histological and stereological analyses of 10 random Case 2: Atypical arrangement of the biopsies of the placenta, as described previously by Allen umbilical arteries in the umbilical cord et al. (2002) and Wilsher and Allen (2003), revealed no abnormality in structure (surface density; Sv)ofthe Clinical history microcotyledons. A healthy 1 m (10 hh), maiden, 12-year-old Pony mare Follow-up on the foal weighing 295 kg was inseminated once with pony semen during oestrus. She subsequently underwent 4 transrectal The foal experienced no health problems in the post natal ultrasound examinations on Days 12, 15, 29 and 44 after period or subsequently. At 4 years of age the animal was ovulation, which showed a singleton conceptus to be destroyed due to a tendon injury. developing normally. The mare produced a healthy 18.7 kg colt foal after 344 days of gestation, the Post mortem findings birthweight of which was lower than previously reported (Table 1). However, when the birthweight was expressed A post mortem examination was carried out to examine as a percentage of maternal weight it fell within the the umbilical arteries within the abdomen and ascertain if expected range (Table 1).

a) Intra-amniotic cord Intra-allantoic cord UV UV UA

UA UV Thread-like * UA UA Case 1

b) UA Thread-like UA UA UV

UV UV Case 1

c) UA UV

UV UA Case 2 UV * d) Thread-like UA UA

Case 2

Fig 3: a) The umbilical cord in Case 1, opened to show the arrangement of the umbilical veins (UV) and arteries (UA). Note the thread-like UA which fuses with one of the major UA branches in the allantoic portion of the cord (asterisk); b) The umbilical cord of Case 1 flipped-over to show bifurcation of the large UA into 2 intra-allantoic arteries as it leaves the amniotic portion of the cord; c) The umbilical cord of Case 2 showing the same vessel arrangement as Case 1; d) The umbilical arteries of Case 2 dissected out to show the thread-like arterial vessel.

Gross placental findings Histological and stereological findings

The cord was attached at the bifurcation of the 2 uterine Histological analysis of 10 random biopsies of the horns and the vascular pattern was type I, as described allantochorion revealed no pathological changes. above in Case 1. One worm-like artery wall protruded from Stereological measurements showed that both the height the end of the artery where it had ruptured naturally. The and plexiform nature of the microcotyledons (Sv) were length of the cord was less than previously reported values greater than previously reported. Sectioning of the for ponies (Table 1), as were the mass, gross area and amniotic artery and the associated thread-like vessel volume of the allantochorion (Whitwell and Jeffcott 1975; showed no abnormality in structure of either vessel (Fig 5). Allen et al. 2002). All placental and fetal parameters are Blood cells were noted within the thread-like vessel detailed and compared in Table 1. The membranes were demonstrating its patency. grossly unremarkable apart from the umbilical cord, which showed the same vessel arrangement as in Case 1: there was a major disparity in the size of the arteries in the Follow-up on the foal amniotic portion of the cord, bifurcation of the larger artery at the amniotic-allantoic junction and fusion of the The foal showed normal development and behaviour fine thread-like artery with one of these arterial branches post partum. Following weaning at approximately 6 (Fig 3c and d). Two arteries and 2 veins were present in the months of age the colt was sold and subsequently lost to allantoic portion of the cord. follow-up.

a) Caudal mesenteric arteries R. deep circumflex L. deep circumflex iliac artery Aorta iliac artery

Internal iliac arteries

L. external iliac R. external iliac

L. internal pudendal artery R. umbilical artery

R. internal pudendal artery Case 2

Fig 5: A histological section of the major umbilical artery and the Bladder associated thread-like vessel in the intra-amniotic portion of the umbilical cord of Case 2. (Scale bar = 5 mm)

L. umbilical artery Case 1 the linear dimension of the allantochorion fell within the normal ranges reported for TB placentae, with the b) exception of the width of the posterior and middle body Left UA (33 and 34 cm, respectively), which were below Right UA Bladder expected values (range 37–60 and 37–55 cm, respectively; Whitwell and Jeffcott 1975). However, for a maiden mare this shortfall would not be an unusual finding. In addition, and typical of many maiden placentae, the allantochorion was noted to be light in Case 1 weight in relation to its size. The allantoic and villous Fig 4: a) The dissected vasculature associated with the umbilical surfaces were unremarkable and the placental pole arteries (viewed from the ventral side), recovered at post mortem had ruptured normally. The vascular pattern of the from Case 1. Note the disparity in diameter of the umbilical arteries; allantochorion was type I. The placental parameters for b) the umbilical arteries (UA) as they run along the surface of the this case are listed in Table 1 for comparison with bladder. The portion of the umbilical artery that returned to the umbilicus and placenta during gestation becomes the round previously published parameters for TB mares. ligament of the bladder in the adult (not shown). The umbilical cord was attached at the bifurcation of the 2 placental horns and was 37 cm in length. Two Case 3: Supernumerary vessels in the arterial walls extended from the end of the cord, amniotic portion of the umbilical cord indicating its natural rupture. However, notable parturient haemorrhage had occurred at the fetal end of the cord, Clinical history causing blood to seep back between the umbilical vessels and amniotic covering of the cord for A maiden, 5-year-old, TB mare foaled a small 44 kg colt approximately 13 cm (Fig 6a). The cord weight was within foal after a gestation of 345 days. The foaling was the normal range for TBs, but the cord index (weight/ uneventful, except for rupture of the umbilical cord when length) was higher than normal (Table 1). Unusually, the the foal’s hindlimbs were still in the mare’s vagina. The foal umbilical veins had not joined near the amniotic-allantoic stood unaided and sucked within 90 min after birth. On junction so that 2 veins and 2 arteries were present along examining the placenta the attendant noted that the its entire length (Fig 6a and b). umbilical cord looked unusual due to the presence of a large blood clot (Fig 6a). Histological and stereological findings Gross placental findings Histological examination of 10 random biopsies of the The allantochorion was complete and was presented placenta showed no abnormality in structure and with the allantoic surface outermost. For the most part, stereological analyses of these biopsies showed the

: Supernumerary vessels in the a) Case 4 amniotic portion of the umbilical cord

Clinical history

A 17-year-old, multiparous TB mare foaled a 58 kg singleton colt after a normal gestation of 327 days. During Case 3 second-stage labour the unruptured allantochorion appeared in the vagina (a ‘red-bag’ delivery). The Intra-amniotic cord Intra-allantoic cord b) attendant ruptured the allantochorion manually and UA assisted the foal’s passage through the birth canal. The placenta was passed immediately after delivery of the foal. Initially the foal was weak and slow to stand so UV colostrum was administered via a stomach tube. By 24 h post partum the foal was standing and suckling unassisted and showed no further problems during its first week of life. UV UA Gross placental findings Case 3 The placental membranes were complete, had not c) inverted and were presented with the chorionic surface outermost. All the linear and gross measurements of the placenta fell within the ranges expected for a TB mare (Table 1). The umbilical cord was attached at the bifurcation of the horns and the vessels radiated out from Case 4 this attachment in a type I vascular pattern. Unusually, however, 2 veins and 2 arteries existed along the full length d) of the umbilical cord as described in Case 3 (Fig 6c and d). UA UV Histological and stereological findings

Histological and stereological analyses of 10 random biopsies of the placenta revealed no abnormality in Ur structure (Table 1). Histological examination of the UV UA umbilical cord likewise demonstrated no abnormalities in the morphology of the 4 umbilical vessels in both the Case 4 amniotic (Fig 7d) and allantoic portions of the cord

Fig 6: a) The umbilical cord of Case 3 showing the large blood clot at the fetal end of the cord. b) The same cord dissected to show the Follow-up on the foal presence of 2 umbilical arteries (UA) and 2 veins (UV) along the entire length of the cord. c) The umbilical cord of Case 4.d)A No problems were reported with the foal prior to it passing histological section of the amniotic section of the umbilical cord of through the sales ring as a yearling, where it was Case 4 (UV = umbilical vein; UA = umbilical artery; Ur = urachus; purchased by an overseas buyer and lost to further Scale bar = 5 mm). follow-up. feto-maternal contact at the placental interface was Case 5: Supernumerary vessels in the within normal limits despite a shortfall in the height of the allantoic portion of the umbilical cord microcotyledons. Clinical history

Follow-up on the foal After an uneventful gestation of 330 days, a multiparous 9-year-old TB mare foaled a 52 kg single colt foal without No problems or complications were reported in the foal’s complications. The placenta was expelled 40 min after post natal development or health. The colt was sold foaling and the attendant noted a yolk sac remnant in the through the sales as a yearling and entered training. He amnion and therefore submitted the placenta to the subsequently raced as a 2- and 3-year-old, starting in 6 authors’ laboratory as part of a study being undertaken on races and being placed twice. abnormal placentation.

Histological and stereological findings a) Intra-amniotic cord

Microscopic examination of 10 random biopsies of the UA allantochorion showed no pathological changes in the UV structure of the microcotyledons or the underlying UA UV allantois. UA UV UV Follow-up on the foal UA The foal experienced no problems during the post natal period. Further history was collected retrospectively and no Case 5 problems or complications were reported with the colt, which is currently in training.

b) UV Discussion UV UV Cases 1 and 2

UA UA There is only one previous report of an anomaly in the number of major vessels in an equine umbilical cord; namely, the complete absence of one umbilical artery (Whitwell 1975). The affected foal was carried to term but showed serious defects, including the absence of a kidney and its renal artery. In contrast, in Cases 1 and 2 Case 5 reported above the amniotic cord contained one major umbilical artery, which divided into 2 near the Fig 7: The umbilical cord of Case 5, shown (a) grossly and (b) microscopically. Note the 3 umbilical veins (UV) in the amniotic-allantoic junction. The second artery was intra-allantoic portion of the cord that unite to form the solitary UV thread-like and anastomosed with one of the branches of in the intra-amniotic portion (UA = umbilical artery; Scale bar = the major artery. 5 mm). The arrangement of the blood vessels in the human umbilical cord differs from the horse in that 2 arteries and one vein exist over its entire length. Furthermore, an allantoic cavity, and hence allantoic portion to the Gross placental findings umbilical cord, is not a feature of human placental membranes. Nevertheless, some human cord vessel The fetal membranes were complete and had been abnormalities do show similarities with the cases detailed presented with the villous surface outermost. The above. The most common of these anomalies in the allantochorion was unremarkable and its linear and gross human infant is the presence of a single umbilical artery parameters were within normal limits (Table 1). An ossified (SUA), which is well recognised and documented (reviews: yolk sac remnant 6 cm in diameter was present within Baergen 2005; Fox and Sebire 2007). Indeed, SUA is the the allantoamnion ‘sandwiched’ between the 2 most common human congenital anomaly with a component layers. The umbilical cord was attached reported incidence of 0.5–1% in singletons and 8.8% in twins to the allantochorion within the nongravid horn, (Baergen 2005). SUA can also result from aplasia or atrophy approximately 15 cm from the entrance to the horn. The of one artery: histological examination sometimes reveals amniotic portion of the cord had the usual configuration a remnant of the second vessel. of 2 arteries and one vein, whereas the allantoic portion It would appear that the thread-like umbilical artery in of the cord consisted of 2 arteries and 3 veins, one the amniotic portion of the cord in the 2 equine cases of which was noticeably larger than the other 2 described above was either aplasic or atrophic. In (Fig 7a and b). All 3 veins converged near the attempting to explain atrophy of an umbilical artery in amniotic-allantoic junction to form the single amniotic human pregnancy, which is believed to be more common vein. than aplasia, Baergen (2005) suggested that it occurs The vascular pattern of the allantochorion was when a portion of the placenta atrophies and one atypical: one vein and artery vascularised part of the umbilical artery loses its ‘territory’. Hence, the condition is nongravid horn and the other pair of vessels vascularised often associated with placental shape aberrations or with the remainder of the allantochorion and part of the abnormal insertion of the cord. This was not the situation in nongravid horn. The additional vein returned blood from Cases 1 and 2 since both the position of the cord on the part of the body of the chorion. allantochorion, the vascular pattern and the morphology

of the latter were normal. A disparity in the size of the Cases 3, 4 and 5 umbilical arteries in mares has been noted by several authors, occurring as a consequence of variability in the Cases 3 and 4 showed supernumerary vessels in the area of the allantochorion each vessel is associated with amniotic portion of the cord while Case 5 showed (Whitwell 1975; Whitwell and Jeffcott 1975; Wilsher et al. supernumerary vessels in the allantoic portion. More than 3 2009). However, this is an unlikely explanation since a umbilical vessels in the human umbilical cord is considered normal vascular pattern (type I) was seen in both cases, so to be a rare finding by some authors (Schimmel and the area of allantochorion each artery was associated Eidelman 1998; Baergen 2005), although Fox and Sebire with was not dissimilar. (2007), while noting that the condition had attracted little In Case 1, in which a post mortem could be scientific attention, believed it to be more common than undertaken, it became apparent that the left SUA. Indeed, Meyer et al. (1969) found an accessory 4th intra-abdominal umbilical artery was significantly reduced vessel in 16 of 310 (0.05%) cords examined and Ogino et al. in size. Although it was not possible to determine which (1985) reported supernumerary vessels in 5/509 (1.0%) of umbilical artery was affected in Case 2, in approximately the cases they examined. In another study, Gupta et al. 70% of human infants with SUA it is the left artery that is (1993) noted the presence of >3 vascular profiles in 40 (6%) absent, aplasic or atrophied (Geipel et al. 2000; Baergen of 644 cross sections of human umbilical cord from which 2005). It could not be determined if the asymmetrical samples at the fetal, mid-portion and placental ends of spatial arrangement of the left iliac arteries in Case 1 may each cord were examined. Some of the vascular profiles have contributed to the reduced diameter of the left were found to be due to branching rather than the umbilical artery; however, in man (Hartwig 2007) and presence of true supernumerary vessels. Careful horses (Mark Hillyer, personal communication) the examination of the umbilical cord in the equine cases arrangement of the internal and external iliac as they reported above showed clearly that the extra vessel in branch from the aorta is extremely variable, so this was not Cases 3 and 4 was an umbilical vein that persisted along an unusual finding. the entire length of the cord rather than fusing with the Baergen (2005) reported that placental abnormalities other one. On the other hand, the extra umbilical vein in are detected in around 16% of human infants with SUA the allantoic portion of the cord in Case 5 had arisen from and in 30–45% of such cases other congenital anomalies nonfusion of umbilical vein branches. are seen, with renal anomalies being particularly The length of the umbilical cords in Cases 3 and 4 were common. However, this author and others (Froehlich and at the lower end of the normal range (Whitwell and Fujikura 1973) observed that an isolated SUA without Jeffcott 1975). The significance of this finding in relation to other anomalies is often found in infants that are supernumerary umbilical vessels is unclear, especially as perfectly healthy. Likewise, the unusual vessel the human literature does not relate cord length to arrangement in Case 1 was not associated with other supernumerary vessels. In Cases 3 and 4 it could not be organ anomalies and the foals from both Cases 1 and 2 determined if the umbilical veins joined inside the fetus, appeared normal and healthy. Furthermore, the cord prior to the entry of the left umbilical vein into the liver, or if anomaly did not appear to adversely affect umbilical the 2 vessels persisted within the fetus. McGeady et al. blood flow or fetal growth since placental and foal (2006) describes that in the normal course of fetal birthweights were within the normal range for their development in domestic animals 2 umbilical veins do respective breeds. indeed exist within the fetus but, as the liver expands, they No consistent change in placental architecture could become subdivided into cranial, middle and caudal be discerned between Cases 1 and 2. The shortfall in segments. The middle portions of the veins become placental mass, area and volume recorded in the Pony incorporated into the hepatic tissue while the cranial mare in Case 2 was probably due to the its smaller size segments of both the left and right veins atrophy. Fusion of compared to the Pony mares reported by Allen et al. the left and right umbilical veins occurs at the umbilicus (2002). However, the increased height and complexity and, subsequently, the caudal segment of the right (Sv) of the microcotyledons over expected values in this umbilical vein atrophies to leave just the enlarged left animal cannot be explained, especially as maiden mares umbilical vein within the fetus (Fig 1). The remnant of the tend to have lower, rather than higher, Sv values (Wilsher left umbilical vein persists in the adult in the form of the and Allen 2003). Hence, in these 2 cases an atypical round ligament of the liver situated within the falciform arrangement of the arteries in the umbilical cord did not ligament (McGeady et al. 2006). cause placental abnormalities or result in neonatal In human infants an additional umbilical vein in the problems. The reason this anomaly occurred is unclear cord often denotes persistence of the right umbilical vein but, perhaps, the unusual bifurcation of one umbilical within the abdomen (review: Jeanty 1990). Congenital artery represents a compensatory response to the one problems in infants associated with persistence of both hypoplastic or atrophied artery thus ensuring that each of umbilical veins include cardiovascular and gastrointestinal the 2 umbilical veins were accompanied by one anomalies (Kaufman and Weisser 1964; Painter and Russell substantial artery. 1977). However, persistence of the right umbilical vein

associated with a 4-vessel umbilical cord is not always an practitioner to consider that other development ominous finding and several authors have reported no abnormalities may be a possibility when presented with a congenital abnormalities in babies with this condition sick newborn foal with anomalies in the umbilical vessel (Murdoch 1966; Rodriguez 1984; Schimmel and Eidelman number. 1998). Likewise, in the equine cases reported here, supernumerary umbilical vessels presenting as an Acknowledgements additional umbilical vein in either the amniotic or allantoic portions of the cord were not associated with any post The authors are grateful to the Newmarket studfarms that natal problems for the foal. provided the placentae described in Cases 3, 4 and 5.Mr Supernumerary vessels in the human umbilical cord are John Fuller kindly drew Figure 1. The normal umbilical cord thought by some to occur as a consequence of splitting of pictured in Figure 2 was kindly provided by Cheveley Park the umbilical vessels between the third and fifth week of Stud. development (Nyberg et al. 2002). While anomalies in the number of vessels present in the equine umbilical cord are References probably the result of developmental oddities or the persistence of an earlier embryonic phenotype, precise Allen, W.R., Wilsher, S., Turnbull, C., Stewart, F., Ousey, J., Rossdale, P.D. information on the timing and physical changes involved in and Fowden, A.L. (2002) Influence of maternal size on placental, the development of the equine umbilical vessels has not fetal and postnatal growth in the horse. I. Development in utero. Reproduction 123, 445-453. been reported. Baergen, R.N. (2005) Pathology of the umbilical cord. In: Manual of When an additional vessel is present in the cord, it may Benirschke and Kaufmann’s Pathology of the Placenta, Ed: R.N. be a remnant of the vitelline vein that once vascularised Baergen, Springer, New York. pp 268-270. the yolk sac, rather than the right umbilical vein. In man, De Bosschere, H., Simeons, P. and Ducatelle, R. (1999) Persistent vitelline these vitelline vessels are smaller than normal umbilical vein in a foal. Vet. Rec. 145, 75-77. veins, they have no blood flow and they are more Fox, H. and Sebire, N.J. (2007) Pathology of the umbilical cord. In: Pathology of the Placenta, Eds: H. Fox and N.J. Sebire, Saunders common at the fetal end of the cord (Jeanty 1990). In the Elsevier, Philadelphia. pp 477-482. horse, persistence of an intra-abdominal remnant of the Froehlich, L.A. and Fujikura, T. (1973) Follow-up of infants with single vitelline vein has been reported in a 3-day-old foal, which umbilical artery. Pediatrics 52, 22-29. resulted in intestinal strangulation (De Bosschere et al. Geipel, A., Germer, U., Welp, T., Schwinger, E. and Gembruch, U. (2000) 1999). Gross and histological examination of the extra Prenatal diagnosis of single umbilical artery; determination of the umbilical vessels in all 3 cases in the present study showed absent side, associated anomalies, Doppler findings and perinatal outcome. Ultrasound Obstet. Gynecol. 15, 114-117. morphology typical of an umbilical vein and blood cells Gupta, I., Hillier, V.F. and Edwards, J.M. (1993) Vascular branching in the present within the lumenae. Furthermore, in all 3 cases the umbilical cord: An indication of maternal smoking habits and extra vessel was not associated with the yolk sac remnant intrauterine distress. Placenta 14, 117-123. and was associated with vascularisation of the Hartwig, W.K. (2007) Cardiovascular system. In: Fundamental Anatomy, allantochorion. Hence, it can be safely assumed that these Ed: W. Hartwig, Lippincott, Williams and Wilkins, Philadelphia. pp 47-106. accessory vessels did not represent a remnant of the Jeanty, P. (1990) Persistent right umbilical vein: An ominous prenatal vitelline vein. finding? Radiol. 177, 735-738. Any association between the presence of the ossified Kaufman, H.J. and Weisser, K. (1964) Neonatal trans-umbilical yolk sac remnant and the anomalous umbilical vessel angiography: A preliminary report. Ann. Radiol. 7, 437-441. arrangement in Case 5 is unknown. Latshaw, W.K. (1987) Embryonic membranes and placentation. In: Veterinary Developmental Anatomy: A Clinically Orientated Approach, Ed: W.K. Latshaw, BC Decker, Philadelphia. pp 49-74. Conclusions McGeady, T.A., Quinn, P.J., FitzPatrick, E.S. and Ryan, M.T. (2006) Cardiovascular system. In: Veterinary Embryology, Ed: T.A. McGeady, Blackwell Publishing, Oxford. pp 126-127. With the exception of Case 3, the anomalies in vessel Meyer, W.W., Lind, J. and Moinian, M. (1969) An accessory fourth vessel number within the umbilical cord in the present study of the umbilical cord: A preliminary study. Am. J. Obstet. Gynaecol. came to light as incidental findings when examining the 105, 1063-1068. placentae for other reasons. Thus, it is likely that such vessel Murdoch, D.E. (1966) Umbilical-cord doubling: Report of a case. Obstet. Gynaecol. 27, 555-557. anomalies may go unnoticed during routine or cursory Nyberg, D.A., McGahan, J.P., Pretorius, D.H. and Pilu, G. (2002) The examinations of the placenta. Although no placental or placenta, umbilical cord and membranes. In: Diagnostic Imaging neonatal problems were associated with the 5 cases of Fetal Anomalies, 2nd edn., Lippincott, Williams and Wilkin, reported in the present paper, in view of the known Philadelphia, PA. pp 85-132. increased risk of congenital anomalies in human infants Ogino, M., Jimbo, T., Mizuno, M. and Sakamoto, S. (1985) Study on the with SUA and supernumerary vessels careful examination histopathological abnormalities of the umbilical cord. Nippon Sanka Fujinka Gakki Zasshi 37, 329-335. of the equine umbilical cord to ascertain the number of Painter, D. and Russell, P. (1977) Four-vessel umbilical cord associated vessels present along its length should be undertaken with multiple congenital anomalies. Obstet. Gynaecol. 50, 505- routinely. It would be prudent for the studfarm veterinary 507.

Rodriguez, M.A. (1984) Four-vessel umbilical cord without congenital D. Furry and G. Hale, Kentucky Agricultural Experimental Station, abnormalities. S. Med. J. 77, 539 (Letter). Lexington. pp 92-99. Rossdale, P.D. and Ricketts, S.W. (2002) Evaluation of the fetal Whitwell, K.E. (1975) Morphology and pathology of the equine umbilical membranes at foaling. In: Reproduction: Foaling Part 1: Maternal cord. J. Reprod. Fert., Suppl. 23, 599-603. Aspects, Eds: P.D. Rossdale, T.S. Mair and R.E. Green, Equine Whitwell, K.E. and Jeffcott, L.B. (1975) Morphological studies on the fetal Veterinary Journal Ltd, Newmarket. pp 78-82. membranes of the normal singleton foal at term. Res. vet. Sci. 19, Rüsse, I. and Sinowatz, F. (1998) Lehrbuch Der Embryologie Der 44-55. Haustiere, 2nd edn., Parey Buchverlag, Berlin. Wilsher, S. and Allen, W.R. (2003) The effects of maternal age and parity Schimmel, M.S. and Eidelman, A.I. (1998) Supernumerary umbilical vein on placental and fetal development in the mare. Equine vet J. 35, resulting in a four-vessel umbilical cord. Am. J. Perinatol. 15, 299-301. 476-483. Schlafer, D.H. (2004) The umbilical cord – lifeline to the outside world: Wilsher, S., Ousey, J.C. and Allen, W.R. (2009) Abnormal umbilical cord structure, function, and pathology of the equine umbilical cord. In: attachment sites in the mare: A review illustrated by three case Proceedings of A Workshop on the Equine Placenta, Eds: D. Powell, reports. Equine vet. J. 41, 930-939.

nearest the barrel is at the appropriate notch. Rotate the plunger ring 1/4 turn to lock it in place and ensure it is locked. Make sure the severity. These trials included horses of various breeds and under different management conditions, and included horses in race or show horse’s mouth contains no feed. Remove the cover from the tip of the syringe, and insert the syringe into the horse’s mouth at the training, pleasure horses, and foals as young as one month. Horses enrolled in the efficacy trials were healthy animals confirmed to have interdental space. Depress the plunger until stopped by the knurled ring. The dose should be deposited on the back of the tongue or deep gastric ulcers by gastroscopy. In these field trials, horses readily accepted GASTROGARD Paste. There were no drug related adverse into the cheek pouch. Care should be taken to ensure that the horse consumes the complete dose. Treated animals should be observed reactions. In the clinical trials, GASTROGARD Paste was used concomitantly with other therapies, which included: anthelmintics, antibiotics, briefly after administration to ensure that part of the dose is not lost or rejected. If any of the dose is lost, redosing is recommended. non-steroidal and steroidal anti-inflammatory agents, diuretics, tranquilizers and vaccines. • If, after dosing, the syringe is not completely empty, it may be reused on following days until emptied. Replace the cap after each use. Diagnostic and Management Considerations: The following clinical signs may be associated with gastric ulceration in adult horses: inappetence or decreased appetite, recurrent colic, intermittent loose stools or chronic diarrhea, poor hair coat, poor body condition, or poor Oral Paste for Horses and Foals Warning performance. Clinical signs in foals may include: bruxism (grinding of teeth), excessive salivation, colic, cranial abdominal tenderness, NADA 141-123, Approved by FDA Do not use in horses intended for human consumption. Keep this and all drugs out of the reach of children. In case of ingestion, contact a physician. Physicians may contact a poison control center for advice concerning accidental ingestion. anorexia, diarrhea, sternal recumbency or weakness. A more accurate diagnosis of gastric ulceration in horses and foals may be made if Caution ulcers are visualized directly by endoscopic examination of the gastric mucosa. Adverse Reactions Federal (USA) law restricts this drug to use by or on the order of a licensed veterinarian. Gastric ulcers may recur in horses if therapy to prevent recurrence is not administered after the initial treatment is completed. Use In efficacy trials, when the drug was administered at 1.8 mg omeprazole/lb (4 mg/kg) body weight daily for 28 days and 0.9 mg omeprazole/lb Description GASTROGARD Paste at 0.9 mg omeprazole/lb body weight (2 mg/kg) for control of gastric ulcers following treatment. The safety of (2 mg/kg)body weight daily for 30 additional days, no adverse reactions were observed. Chemical name: 5-Methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinyl) methyl]sulfinyl]-1H-benzimidazole. administration of GASTROGARD Paste for longer than 91 days has not been determined. Precautions Empirical formula: C17H19N3O3S. Molecular weight: 345.42. Structural formula: Maximal acid suppression occurs after three to five days of treatment with omeprazole. The safety of GASTROGARD Paste has not been determined in pregnant or lactating mares. Safety OCH Clinical Pharmacology 3 • GASTROGARD Paste was well tolerated in the following controlled efficacy and safety studies. H C CH Mechanism of Action: Omeprazole is a gastric acid pump inhibitor that regulates the final step in hydrogen ion production and blocks gastric 3 3 • In field trials involving 139 horses, including foals as young as one month of age, no adverse reactions attributable to omeprazole O OCH3 acid secretion regardless of the stimulus. Omeprazole irreversibly binds to the gastric parietal cell’s H+, K+ ATPase enzyme which pumps treatment were noted. hydrogen ions into the lumen of the stomach in exchange for potassium ions. Since omeprazole accumulates in the cell canaliculi and is • In a placebo controlled adult horse safety study, horses received 20 mg/kg/day omeprazole (5x the recommended dose) for 90 days. H irreversibly bound to the effect site, the plasma concentration at steady state is not directly related to the amount that is bound to the enzyme. The relationship between omeprazole action and plasma concentration is a function of the rate-limiting process of H+, K+ ATPase activity/ No treatment related adverse effects were observed. How Supplied turnover. Once all of the enzyme becomes bound, acid secretion resumes only after new H+, K+ ATPase is synthesized in the parietal cell • In a placebo controlled tolerance study, adult horses were treated with GASTROGARD Paste at a dosage of 40 mg/kg/day (10x the recommended dose) for 21 days. No treatment related adverse effects were observed. GASTROGARD® (omeprazole) Paste for horses contains 37% w/w omeprazole and is available in an adjustable-dose syringe. Each syringe (i.e., the rate of new enzyme synthesis exceeds the rate of inhibition). contains 2.28 g of omeprazole. Syringes are calibrated according to body weight and are available in boxes of 7 units or 72 units. Pharmacodynamics: In a study of pharmacodynamic effects using horses with gastric cannulae, secretion of gastric acid was inhibited in • A placebo controlled foal safety study evaluated the safety of omeprazole at doses of 4, 12 or 20 mg/kg (1, 3 or 5x) once daily for 91 days. Foals ranged in age from 66 to 110 days at study initiation. Gamma glutamyltransferase (GGT) levels were significantly elevated in horses Storage Conditions horses given 4 mg omeprazole/kg/day. After the expected maximum suppression of gastric acid secretion was reached (5 days), the actual secretion of gastric acid was reduced by 99%, 95% and 90% at 8, 16, and 24 hours, respectively. treated at exaggerated doses of 20 mg/kg (5x the recommended dose). Mean stomach to body weight ratio was higher for foals in the 3x Store at 68°F – 77°F (20-25°C). Excursions between 59°F – 86°F (15-30°C) are permitted. Pharmacokinetics: In a pharmacokinetic study involving thirteen healthy, mixed breed horses (8 female, 5 male) receiving multiple doses and 5x groups than for controls; however, no abnormalities of the stomach were evident on histological examination. Indications of omeprazole paste (1.8 mg/lb once daily for fifteen days) in either a fed or fasted state, there was no evidence of drug accumulation in the Reproductive Safety For treatment and prevention of recurrence of gastric ulcers in horses and foals 4 weeks of age and older. plasma when comparing the extent of systemic exposure (AUC0-∞). When comparing the individual bioavailability data (AUC0-∞, Cmax, In a male reproductive safety study, 10 stallions received GastroGard Paste at 12 mg/kg/day (3x the recommended dose) for 70 days. Dosage Regimen and Tmax measurements) across the study days, there was great inter- and intrasubject variability in the rate and extent of product No treatment related adverse effects on semen quality or breeding behavior were observed. A safety study in breeding mares has not For treatment of gastric ulcers, GASTROGARD Paste should be administered orally once-a-day for 4 weeks at the recommended dosage of absorption. Also, the extent of omeprazole absorption in horses was reduced by approximately 67% in the presence of food. This is been conducted. 1.8 mg omeprazole/lb body weight (4 mg/kg). For the prevention of recurrence of gastric ulcers, continue treatment for at least an additional evidenced by the observation that the mean AUC0-∞ values measured during the fifth day of omeprazole therapy when the animals were For More Information Please call 1-888-637-4251 and please visit our web site at www.gastrogard.com. 4 weeks by administering GASTROGARD Paste at the recommended daily maintenance dose of 0.9 mg/lb (2 mg/kg). fasted for 24 hours was approximately three times greater than the AUC estimated after the first and fifteenth doses when the horses were Marketed by: Merial Limited Directions For Use fed hay ad libitum and sweet feed (grain) twice daily. Prandial status did not affect the rate of drug elimination. The terminal half-life estimates Duluth, GA • GASTROGARD Paste for horses is recommended for use in horses and foals 4 weeks of age and older. The contents of one syringe will (N=38) ranged from approximately one-half to eight hours. 30096-4640 dose a 1250 lb (568 kg) horse at the rate of 1.8 mg omeprazole/lb body weight (4 mg/kg). For treatment of gastric ulcers, each weight Efficacy marking on the syringe plunger will deliver sufficient omeprazole to treat 250 lb (114 kg) body weight. For prevention of recurrence of gastric Dose Confirmation: GASTROGARD® (omeprazole) Paste, administered to provide omeprazole at 1.8 mg/lb (4 mg/kg) daily for 28 days, Merial Limited, a company limited by shares registered in England and Wales (registered number 3332751) with a registered ulcers, each weight marking will deliver sufficient omeprazole to dose 500 lb (227 kg) body weight. effectively healed or reduced the severity of gastric ulcers in 92% of omeprazole-treated horses. In comparison, 32% of controls exhibited office at PO Box 327, Sandringham House, Sandringham Avenue, Harlow Business Park, Harlow, Essex CM19 5QA, England, • To deliver GASTROGARD Paste at the treatment dose rate of 1.8 mg omeprazole/lb body weight (4 mg/kg), set the syringe plunger to the healed or less severe ulcers. Horses enrolled in this study were healthy animals confirmed to have gastric ulcers by gastroscopy. Subsequent and domesticated in Delaware, USA as Merial LLC. appropriate weight marking according to the horse’s weight in pounds. daily administration of GASTROGARD Paste to provide omeprazole at 0.9 mg/lb (2 mg/kg) for 30 days prevented recurrence of gastric ulcers US Patent: 4255431 and 5708017 • To deliver GASTROGARD Paste at the dose rate of 0.9 mg/lb (2 mg/kg) to prevent recurrence of ulcers, set the syringe plunger to the in 84% of treated horses, whereas ulcers recurred or became more severe in horses removed from omeprazole treatment. Copyright © 2005 Merial Limited. weight marking corresponding to half of the horse’s weight in pounds. Clinical Field Trials: GASTROGARD Paste administered at 1.8 mg/lb (4 mg/kg) daily for 28 days healed or reduced the severity of gastric All rights reserved. Rev. 08-2005 • To set the syringe plunger, unlock the knurled ring by rotating it 1/4 turn. Slide the knurled ring along the plunger shaft so that the side ulcers in 99% of omeprazoletreated horses. In comparison, 32.4% of control horses had healed ulcers or ulcers which were reduced in ®GASTROGARD is a registered trademark of the AstraZeneca Group of Companies.