Twinning as a cause of foetal and neonatal loss in the Thoroughbred mare

J. COMP.

PATH.

1973.

VOL.

TWINNING LOSS

!)I

83.

AS A CAUSE OF FOETAL IN THE THOROUGHBRED

AND NEONATAL MARE

BY

L. B.

JEFFCOTT

and

E.

KATHERINE

Equine Research Station, the Animal

Health

LVHITWELL

Trust, Xewmarket,

Suffolk

INTRODUCTIOK

Foetal and perinatal mortality following twin conception in mares forms a major source of loss to the Thoroughbred breeding industry in this country (Hancock, 1948; Mahaffey, 1960, 1968; Rossdale, 1968). The situation is apparently similar in the U.S.A. (Rooney, 1970), in Australia (Bain, 1969), Germany (Kudlac, Vorba and Munk, 1969), and in Russia (Korol’kov, 1939). The mortality of twins in this breed enormously exceeds that observed in any other domesticated mammal. There seems to be some breed variation in the incidence of twinning in the horse, as it is rare in pony and native breeds compared with the Thoroughbred. In a small herd of 25 to 30 crossbred pony mares kept by the Equine Research Station, a set of twins has not been recorded in the last 19 years. Arthur and Allen (1972) also report twinning as being very rare in Welsh Mountain ponies, and David ( 197 1, personal communication) finds that twin ovulation in ponies is very uncommon. Vandeplassche, Podliachouk and Beaud (1970) report that the incidence in Belgian draft and crossbred horses is probably similar to that in the Thoroughbred, but that survival rates are considerably higher. Equine twins are almost exclusively the product of 2 fertilized ova and therefore dizygotic. One case of homozygous or identical twins has been recorded (Rooney, 1970), but none have been seen in 5 years at the Equine Research Station. Twin ovulation, based on the postmortem evidence of Arthur ( 1958, and Osborne (1966), is fairly common, but in most instances one or both embryos undergo early embryonic death or are resorbed (Vandeplassche et al., 1970). Those that continue to develop are prone to abortion, mummification, or if born alive? neonatal death. MATERIALS

AND

METHODS

Material submitted to the Equine Research Station from 62 pairs of twins from 5~ Thoroughbred mares was examined at different stages of development from 3 months to term. The foals born alive were examined and wherever possible weighed within a few hours of birth. A detailed postmortem examination was carried out on all foetuses and foals presented, except where marked autolytic changes made this impossible. It was considered that in all cases the twins were dizygotic by virtue of their differing sex, markings and/or type of placentation. Examination of the foetal membranes to assess the type of placentation was carried out in 61 cases. The total weight and approximate surface area of all allantochorions were measured in 28 cases from 3 months gestation to term. The separated chorions were spread flat on the floor and the avillous in-contact areas removed. Each villous

92

L.

B. JEFFCOTT

AND

KATHERINE

E.

WHITWELL

chorion was opened out and arranged to form, as near as possible, single thickness rectangles, which were then measured. The aviilous areas were similarly opened out, arranged into rectangles and measured. The villous or functional surface area of each of the ‘2 chorions was expressed as a percentage of the total functional surface area. An examination for evidence of vascular anastomoses of blood vessels was carried out in 5 cases by injection of Indian ink or methylene blue into a large vessel of 1 chorion and the dye followed to blood vessels of the other. In addition, the placentae of 30 cases were examined histologically. Sections were stained with haematoxylin and eosin, von Kossa, Weigert’s fibrin stain, P.T.A.H., and methyl green pyronin. A number of factors were investigated. (1) the types of placentation encountered; (2) the areas where the 2 chorions were in contact; (3) the date of twin conception; (4) the age of the mare; (5) the length of gestation to which twins were usually carried; (6) the outcome of the twin pregnancies; (7) the body weight of twins at parturition. Throughout this paper the term allantochorion will be abbreviated to chorion. Twin 1 is defined as the foal or foetus which occupied the chorion with the larger surface area. RESULT5

The incidence of twinning in the Thoroughbred mare obtained from statistics published in the General Stud Book from 1929 to 1968 was between 1 to 2 per cent. of recorded conceptions. This figure is inaccurate as it does not take into account many early abortions, those not witnessed and those not reported. Twin abortions comprise the largest single category of foetal mortality in an analysis of material submitted to the Equine Research Station between 1967 to 1970 (Table 1); the majority of this material was Thoroughbred in origin. TABLE ANALYSIS

OF CAUSES

0~

ABORTION RESEARCH

1

AND s~~m,*uvr~ STATION 1967-1970

n-4 MARES

AT

THE

EQUINE

No. of cam examined

Percentage

of total

Category

Non-infective (unspecified cause) Twinning Foetal anomaly Fungal Bacterial Viral (Rhinopneumonitis)

< >

lO&mth. 104 mth.

1969

1970

11

27 13

30 12

105 45

41 17

5

14

15

22

56

22

41

1% 4 1

i

2: 17 6

:

1967

1968

20 9

28

: 3 5

1967-70

no. of cases

:

Tykes of Placentation Encountered All the twin placentae examined in this series were bicornual, dichorionic and diamniotic. From examination of the chorions from 61 sets of twins it was possible to divide them into 3 categories (see Figs. 1 and 4 to 6). Quantitative appraisal by measurement of the chorionic surface areas (Table 2) confirmed the visual appraisal of the placentation types. In the first, type A, which was encountered in 79 per cent. of twin pregnancies, one foetus (twin 1) occupied one

TWINNING

horn and most area) while the of the adjacent of invagination

of the other body. of the

IX

MARE:

FOETAL

AND

NEONATAL

!K3

LOSS

body (a mean 68 per cent. of the total (twin 2) occupied one horn and usually Where the 2 chorions abutted there was smaller chorion into the allantoic cavity

functional surface only a small part a variable degree of the larger twin.

’ pregnant’ horn of chorlon

horn

in amnkon

-

body

of chorion

invagmated of avillus

Oreo chortor

of chorlon

-_Stnqleton _Type-

lypfi

Fig.

- twins

about

1. Diagrammatic

equal

size

representation

--Type

twms

C - one

of the different

dlsslmllar

twin undergolng

types of twin

sze

mummification

placentation.

In the second type of placentation, type B, which comprised 11 per cent. of all cases, there was more or less equal division of the total functional surface areas of the chorions between the 2 foetuses. Each chorion occupied one horn and approximately half of the uterine body and there was little or no invagination where the chorions were in contact. The third category, type C, seen in 10

94

L.

B. JEFFCOTT

AND

KATHERINE

m

Mares

conceiving

twins ( Total 55)

D

Mares

produng

live smgleton

;

Mares

twin ovulating

Feb

2. Month

Mar

of twin

ovulation

foals

(expressed

ovulations

May

per month)

June

of conceptlon

and conception

IO

854)

as % of total

in Thoroughbred

------Twmrvng

5

WHITWELL

(Total

Apr. Month

Fig.

E.

15

20

mares.

mares

25

30

Age (yrs) Fig.

3. Ages of twinning (1968).

mares

compared

with

11,680

entered

in the General

Stud Book,

Vol.

36

per cent. of twin pregnancies, was essentially similar to the first but there was a far greater disparity between the sizes of the 2 chorions. The chorion of twin 1 occupied all of one uterine horn, the body and part of the other horn, a mean

TWINNING

Fig.

Fig.

IN

MARE:

4. Type A---at term. Twin chorions large invagination of in contact The scale is in centimetres.

6. Type B-at term. Separated chorionic surface area.

twin

FOETAL

AND

with area.

Fig.

chorions,

showing

NEONATAL

5. Type

LOSS

C

snowmg in its foetal

m..mm;f

approximately

95

equal division

of the villous

85 per cent. of the total functional surface area. Twin 2 with most of its foetal membranes had sunk down into the large invagination. The foetus was mummified and the membranes grossly autolysed and necrotic. There was an absence of foetal fluids; hence the avillous portion of the chorion of twin 1 tightly enclosed the foetus and membranes. A comparison of the chorionic surface area of normal singleton foals with that from 15 cases of twin pregnancy at term is given in Table 3. The combined villous chorionic surface area for both twins of all placentation types fell within

96

L.

B. JEFFCOTT

AND

KATHERINE

TABLE

E.

WHITWELL

2

PARTITION OF FUNCIONAL CHORIONIC SURFACE BETWEEN ACCORDINGT T O THE TYPE OF PLACENTATION (28

Category

cases)

Percentage of thetotal functional surfue area (mean and range)

No. of twin pregnancies examined

of placentation

month)

TWINS

Type

A (3 month-9

Type

A (10 month-term)

8

Type

B (7 month-term)

7

Type

C (term)

2

Chorion

I

Chorion

2

11 (6055)

(253-243)

(5&O)

(2&l)

(5~k)

(42H)

(77.F-92)

(8-E.5)

the range of that for 144 singleton foals (Whitwell and Jeffcott, unpublished). The mean total surface area for types A and B chorions was somewhat larger than the mean for the singletons of 16 700 cm.2 whereas type C was smaller. The largest total chorionic surface area was seen in the type B placentae and this enlargement appeared to be due mainly to an increase width of the body of the chorion. Only in this group did both twins occupy an appreciable part of the uterine body (Fig. 6). TABLE MEASUREMENT

From

0F

THE

TOTAL

(kg)

WEIGHT

15 sets of twins delivered

AND

at term

3 SURFACE

AREA

and 144 normal

(cm")

OF

singleton

Twin Singleton chorions t

No. of pregnancies examined Mean total weight of chorion(s) (kg.) Mean total functional surface area of chorion (cm*) Mean functional surface area of chorion 1 (cm”) Mean functional surface area of chorion 2 (cm*) Mean surface area of avillous area (cm”)

t Whitwell

and Jeffcott,

144

THE

CHORIONS

foals

chorions Trpe B

7

6

2

3.6&0.05

5.87+0.51

4.94+025

3.67

16 700+154

18 116*906

19 7801967

14 420

-

12 64Oh650

10 711+514

12 204

1972 unpublished.

-

5476 &682

9069&508

2216

-

5068 5496

2467&458

1610

TWINNING

IN

MARE:

FOETAL

AND

NEONATAL

07

LOSS

Area Where the Two Chorions Were in Contact Particular attention has been paid to the area where the twin chorions abutted. There was an absence of chorionic villi over this region and an invagination of chorion 2 into the allantoic cavity of chorion 1 was seen in types A and C, but not in type B. This region was largest in the type A chorions, mean 4068 cm.2, in which the greatest amount of invagination occurred. In type B the avillous area was a mean 2467 cm.2, and in type C only 1610 cm.:! Many placentae were presented for examination with the 2 chorions separated and often there was evidence of small haemorrhages on the in-contact areas. Of those submitted in-contact, some were fairly loosely attached and required only light tension to separate them, while others were firmly adherent to one another. On stripping them apart, numerous fibrous bridging areas were detected and often small blood vessels were seen to cross from one chorion to the other. Occasionally a larger vessel was detected (Fig. 7), but there was no evidence of widespread anastomosis of these vessels nor of appreciable sharing of blood supply by the twins. In 5 cases where the chorions were presented still adherent, Indian ink or 2 per cent. methylene blue solution was injected into one of the allantoic vessels of chorion 1. In all cases dye was detectable in the small vessels of the avillous area of chorion 2, and this was confirmed by histological examination (Fig. 8). Where there was adhesion the chorions were usually thickened. often with small cysts at the interface, focal areas of calcification and cicatrization. The chorion of twin 2 in placentations types A and C was often severely

Fig.

7. Blood

Fig.

8.

G

vessel

crossing

the

in-contact

area

in a Type

A placenta.

Point of adhesion, in Type A placenta, showing small blood vessels ing from one chorion to the other. Deposits of fibrin matwial chorions and some inflammatory reactions. H.E. x 30.

containing seen at

the

Indian ink crowsinterface of thr.

98

L.

B.

JEFFCOTT

AND

KATHERINE

E.

WHITWELL

autolysed, the only areas remaining vitalized being small islands in the avillous part where fusion had occurred with twin 1 before the death of twin 2. The histological appearance of the area of adherent chorions was somewhat variable. In all 30 cases examined there was an absence of chorionic villi and fusion of the adjacent surface epithelial layers. The demarcation between villous chorion and avillous was sharp. In many cases at the periphery of the avillous area there was a narrow band of necrotic villi. At the interface there was a variable amount of densely eosinophilic fibrin-like material. In some areas a rather loose attachment was noted with little thickening or reaction of either chorion (Fig. 9). However, in 2 1 cases, more particularly towards the periphery of the invagination, considerably more reaction was seen. Hyperaemia and some other oedema with local infiltration of inflammatory cells, particularly plasma cells occurred (Fig. 10). Quite large amounts of fibrin were deposited between the 2 layers of chorion.

9 Fig. Fig.

10 9. In-contact area (Type A placenta) showing evidence of hyperaemia matory reaction at the interface. H.E. x 30. 10. Aggregations of plasma cells on the in-contact chorion in a Type

but

minimal

A placenta.

H.E.

inflamx

520.

In one case (H8/70), where both twins were aborted at 9& months gestation, even at the base of the invaginated portion there was considerable hyperaemia at the line of fusion with infiltration of round cells, particularly plasma cells. Near the periphery of the invagination there was a number of apparently degenerate villi with infiltration of plasma cells and local congestion; also there was the formation of numerous small fibrous cysts containing dense eosinophilic material and scattered inflammatory cells. At a number of points of attachment there was fusion of the 2 chorions by fibrous tissue, and blood vessels were seen to cross from one chorion to the other (Fig. 8). In some areas of 1 case (H20/70)

TWINNING

IN

MARE:

FOETAL

AND

NEONATAL

LOSS

99

there was extensive necrosis of tissues at the interface of the 2 chorions with considerable secondary calcification. Much local hyperaemia had occurred and very large numbers of haemosiderin-laden macrophages were present. In another case (H44/70) quite marked haemorrhage was seen between the 2 chorions. The livers of aborted and stillbirth twin foetuses revealed a variable number of small foci of inflammatory cells. These were usually in the periportal areas and were not accompanied by necrosis or degeneration of the parenchymal cells. On culture, no evidence of bacterial infection was detected from any case and virological examination of 2 cases was also negative. Date of Twin Conception The covering season for Thoroughbreds in this country up to the end of 1970 was from February 15th to June 30th. (It has since been lengthened to July 15th). The month of conception for the 55 mares which had twins compared with 854 mares which produced singletons on 12 studs in the Newmarket area between 1968-70, is shown in Fig. 2. The peak number of conceptions for the mares producing singletons was April (35.1 per cent.), whereas for the twinning mares it was May (35.5 per cent.). The percentage for the singleton mares for mares it was May and June together was 38.6 per cent., but for the twinning considerably higher, 61.3 per cent. Also presented in Fig. 2 is the incidence of twin ovulations, as palpated per rectum in 259 Thoroughbred mares on 1 stud for 1969-71. Records from 478 oestral periods have been included. Twin ovulations were recorded in approximately 12 per cent. of ovulations until the end of May, but in June the figure rose to 19 per cent.

The ages of 60 mares that twinned was compared with the ages of Thoroughbred mares entered in Volume 36 of the General Stud Book (1968). Mares that had no return recorded have not been included. Two superimposed histograms I Fig. 3) show the percentage distribution of mares in yearly age groups from 3 to 3 1 years. The mean age of the mares in the G.S.B., was 11.7 years and for the twinning mares was 9.6 years. A very high incidenct of twinning was seen in the 4th or 5th year of life which is usually the first breeding season. Twinning in mares of 14 years or over was not common. Three of the mares twinned in successi\.e breeding seasons. Length of Gestation to Which Twins are Usually, Carried The time of abortion or birth of 61 sets of twins from the date of last service in this series, is shown in Table 4. There were relatively few cases that were presented before 7 months gestation and the majority (72.6 per cent.) were seen from 8 months to term. In the 2 cases which occurred before 5 months, abortion took place at 3 months (88 days) and 4 months (120 days) respectively. The mean length of gestation in 2 1 mares which produced one or both live twins was 329.5 days from the date of last service. This is somewhat shorter than that

100

L.

B.

JEFFCOTT

AND

KATHERINE

E.

WHITWELL

recorded for mares producing a normal singleton foal (340.5 days), (Whitwell and Jeffcott, unpublished). In 5 cases, where one foal was born alive and the other twin was mummified, the gestation length was 342.4 days (Table 5). TABLE ORDER

OF PRECEDENCE

OF

4

INTRAIJTERINE

FOETAL

DEATH

IN TWINS

Both

Duration pregnancy

of

hb. of twin pregnancies

10 months-term 9 months 87 months 6 months 5 months Total

Twin 2 predeceasing

29 1:

16 2

9 F

5 4

Twin I predeceasing

Both twins autolysed

2 2 i

11

2 2

-

-

(2&i

(2:;)

1 -

1

61 (2:;;)

Twin

fwins

born alive or only recently dead

1 = Foetus

or foal which

occupied

the larger TABLE

GESTATION A

LENGTH AND COMPARISON

One or both

BODY WITH

twins

surface

area of chorion.

5

WEIGHT NORMAL

OF TWINS SINGLETON

in each set were

born

AT FOALING FOALS

AND

alive

Twin placentalion qpe

Gestation length (day4

No. of pregnancies Mean Range

.4

322.:0,,.5 308-333

rvpe B

6 330.0+3.8 317-341

Type C

5 342.4*6.0 333-353

&xmaE singletont

145 340.5 iO.8 3 16-364

Body weight Fg-)

No. of pregnancies Twin 1

7 (27Ti6.5)

3 :9 (20.5533.5)

(1237)

24.5 (14.5534.5)

1.5 (0.7-3.2)

145 51

Twin

2

(13~:7.5)

(42-60) Both twins

t Whitwell

and Jeffcott,

51

53.5

30.5

1972 unpublished.

Outcome of the Twin Pregnancies The results of the twin pregnancies examined in this series are shown in TabIe 6. In 64.5 per cent. of mares both twins were aborted or born dead, in 21 per cent. only 1 twin was born alive and in the remaining 14.5 per cent.

TWINNING

IN

MARE:

FOETAL

OF

62

CASES

KEOXATAL

EQUINE

TWINNING

101

LOSS

6

TABLE OUTCOME

AND

OF

3 months-term

Percentage

l3ftotal

.?‘umbe,

cases

I. Total (a) (b) ir)

number of cases of twinning Both twins born dead One twin born alive Both twins born alive

examined

62 40 I3 9

I 00 fi4..i ‘1.0 14.5

2. Total (a) (b) (cl

number of foetuses Prenatal mortality (3 months-term) Postnatal mortality (birth to 2 weeks) Foals surviving beyond 2 weeks

124 93 13 18

100.0 7.5.0 10.5 14.5

3. Total number of foals alive at birth (a) Postnatal mortality (birth to 2 weeks) (b) Foals surviving beyond 2 weeks

31 I :1 18

100.0 4’2.0 58.0

both twins were born alive. Out of the possible 124 foetuses, the total number of foals born alive was 3 1 and only 18 of these survived to 2 weeks of age. Some of these foals succumbed to disease, but many were killed because of weakness, poor conformation or their small size. One feature of the twin pregnancies of placentation types A and C was that usually one twin was either born alive or appeared fresh at postmortem, while the other was severely autolysed or even undergoing mummification. Yostmortem appearances would indicate that in 59 per cent. of all pregnancies 7

TABLE “I-TCOME

OF

So. of Win pregnancies ‘l’otal no. of foetuses Icoals born alive Foals survived 2 weeks age

Twin Twin Total to Twin of Twin Total

I (‘I,,)

of

total

61 T\VIN

PREGNANCIES

31 62

17 34

1 2 (“O)t

z

8

0

13 $3:;)

1 2

0 0

5 1

(q,)t

0

no. of foetuses.

6 (18%)

ACCORDINO

0

TO

TYPI.

THE

OF

PLACONTATION

6 12

Ii I:!

61 122

6 6 12 (loo”,,

Ii

20

II 31 (P.i”,,j

j

0 0

5 4

3 0

0

9 (75”,,j

3 (25”,)

13 .5 18 (15”,,l

102

L.

B.

JEFFCOTT

AND

KATHERINE

E.

WHITWELL

examined twin 2 had pre-deceased twin 1, whereas in only 20 per cent. of pregnancies had twin 1 died first (Table 4). In 12 pregnancies, 6 of type B and 6 of type A placentation, both twins were born alive or appeared recently dead. In only 1 case aborted at 9 months gestation were both twins severely autolysed at postmortem (type A). The survival of twin foals varied considerably according to the type of placentation (Table 7). In 17 near-term pregnancies of type A 13 foals were born alive and of these only 6 survived to 2 weeks of age. These surviving foals were made up of 5 which were twin 1 and only one which was a twin 2. In 6 pregnancies of type B placentation all 12 foals were born alive and 9 of these survived to 2 weeks. Out of 6 mares that had type C placentation 6 foals, all twin 1 foals were born alive and 3 of these survived to 2 weeks. Body Weight of Twins

at Parturition

The birth weights of twin foals have been compared with the birth weights of 145 normal singleton foals (Table 5). The twins weighed considerably less than the singletons, in fact the weight of both twins was comparable to the mean weight for singletons. There was little difference between the weights of twin I in all 3 placental types. Twin 2 was largest in the type B cases, and by far the smallest in type C where it was markedly decomposed and mummified. DISCUSSION

The results presented here confirm the high incidence of losses due to twinning in Thoroughbred mares in this country. The percentage of pregnancies giving birth to 2 live foals was only 14.5 per cent. of the total number conceived. Foals which did survive to term were usually of small size and body weight, emaciated and had a very high rate of mortality (42 per cent.) in the neo-natal period as compared to singleton foals. Platt (1972, personal communication) found an incidence of neo-natal mortality (birth to 7 days) of 0.9 per cent. in a survey of 1665 Thoroughbred singleton foalings. The high incidence of twinning in Thoroughbreds may be attributable either to a genetic effect, or to environmental factors such as the more intensive methods of husbandry for Thoroughbreds. Ovarian activity varies in the mare throughout the year. Arthur (1958) and Osborne (1966) have shown that there is increasing activity, with a higher incidence of twin ovulations, towards the end of the Thoroughbred breeding season. This was confirmed by the records, from a 3-year period, of the ovarian palpations of 259 lactating mares on one stud in Newmarket. Our series of 61 twinning mares also had conceived late in the covering season. The incidence of twinning was particularly high in mares in their first covering season. This has also been shown to occur in Rhenish mares by Bergmeister (1951). The reason for this is not completely clear, but it may be associated with increased ovarian activity in mares which have recently come out of training when first going to stud. The attachment of the blastocyst to the endometrium does not take place in the mare until 5 to 7 weeks after conception and is completed by 14 weeks (Amoroso, 1952). On the other hand Van Niekerk (1965) has shown that it

TWINNING

IN

MARE:

FOETAL

AND

NEONATAL

LOSS

103

begins as early as 25 days after ovulation in the form of a band which develops on the advancing allanto-chorion adjacent to the yolk-sac. According to Zietzchmann (1924) the blastocyst remains oval or spheroidal for a period of at least 4 weeks before elongating. In the case of twins, each blastocyst becomes anchored at the base of one horn of the uterus and develops during the spheroidal stage without interfering with the other. The allanto-chorions then elongate each with apparently equal right to contact with the entire endometrium. They abut against each other and the area of contact between them remains devoid of placental villi. Placentation of equine twins has been studied by Errington and LV’illiams i 1933)) Errington ( 1942) and Williams ( 1943)) who examined the foetal membranes after abortion or birth of twins, and by Vandeplassche (1957) who studied placentation in utero. The results presented here confirm and extend The Thoroughbred mare seems to be incapable of inthese investigations. creasing the size of the placenta or uterus sufficiently to nourish 2 foetuses. Tllc overall villous chorionic area of both twins was only slightly greater than that for normal singleton foals. The disposition of the twin chorions in utero varied considerably and 3 types of placentation were recognized. The relative sizes of the 2 chorions are determined by 3 factors, namely synchrony of fertilization of the twin ova, synchrony of implantation and the respective growth rates of the conceptuses and their membranes. Synchronization of fertilization is dependent upon the time interval between ovulations, the number of times the mare is covered, the time for sperm capacitation and finally, sperm longevity. Synchronization of’ implantation is, of course, closely related to the timing of fertilization as well as to the respective times taken for tubal and uterine transport of the fertilized ova. The relative growth rates of the conceptuses and membranes may be a genetic effect or an indication of local placental sufficiency. The differences in placernation had a significant bearing on the viability and survival rates of the twins. The most successful group was that of type B, where both foetuses prysumably developed simultaneously and there was a more of less equal division of thr a\,ailahle chorionic area. The combined chorionic surface area was largest in this group and about 4000 cm.2 greater than the mean for the singlcton placentae. This was mainly due to an increase in width of the part corresponding to the uterine body. The majority of pregnancies examined were of‘ placentation type A and here the larger twin had a far greater chance of survi\,al than the smaller one. In type C the larger twin had gained an even greater proportion of the available endometrium than in type ;I, presumably by starting implantation earlier than the other twin or possibly even as the result of supcrfoetation. It is possible that type A placentation had existed at one time, and that the deep invagination cf twin 2 into the chorion of twin 2 was the combined result of the resorption of foetal fluids in the mummifying foetal membranes, and the continued extension of twin 1 chorion. Placental insufficiency resulting in inadequate nourishment, foetal anoxia and ultimately death of one twin does not explain fully all the foetal losses and abortions. In some 20 per cent. pregnancies of type A placentation, the small twin (twin 2) was born alive or was only recently dead at postmortem and the

104

L.

B. JEFFCOTT

AND

KATHERINE

E.

WHITWELL

larger one with a greater area of villous chorion, had become autolysed. Examination of the area where the chorions abut may show quite a considerable chronic inflammatory reaction. The presence of large numbers of plasma cells in some cases may indicate some kind of immunological reaction taking place between the tissues of the 2 twins, the result being death or damage to one twin. The foci of inflammatory cells in the livers of twins may also reflect this, as they are not seen in other late abortions or stillbirth of non-infective origin. Further work on the serum protein profiles of foetal equine twins to investigate the possibility of immunological reactions is at present in progress. The presence of anastomoses of small blood vessels across the avillous area has been detected in many cases. Occasionally a larger vessel was seen, but there was no evidence of a useful sharing of placental blood supply by the twins to help counteract the disparity of the available chorionic area. Although small amounts of blood must pass from one twin’s circulation to the other, the occurrence of freemartins in twins of differing sex is not usually seen. In cattle, the condition is very common (Lillie, 1916). Only 1 report of a case of possible freemartin in equine twins has been found (Freudenberg, 1960). In this series no gross abnormalities were seen at postmortem in the reproductive tracts of female twins of heterosexual pairs. Further evidence of small vascular anastomoses between the 2 chorions is indicated by the high incidence (44 per cent.) of red cell chimaerism diagnosed in equine twins (Vandeplassche et al., 1970). Minor vascular anastomosis is seen in multiple pregnancy in sheep (Mellor, 1969) but although twinning is very common in this species, chimaerism and freemartins are rarely reported. In sheep the tips of adjacent chorions are fused and show the presence of sutures along the line of apposition (Mellor, 1969). Suture formation is not seen in cattle and no evidence was seen in the cases examined in this series of mares. The high frequency of freemartins in cattle may possibly be accounted for by the time of vascular anastomosis or to the degree of mixing of the 2 circulations. Unicornual twin pregnancy in cattle is a frequent occurrence (Williams, Gordon and Edwards, 1963), and the 2 allantochorions presumably become into closer and earlier contact than in bicornual pregnancy. The gestation length of twins with placentation types A and B was shorter than that for normal singleton foals. This fact has been recorded before by Mauk (1939) and Kudlac et al. (1969) and has also been reported in man (McKeown and Record, 1952). In the 5 cases with type C placentation the mean gestation length was similar to that of the singleton foals. This may suggest that it is time of death of the smaller twin that determines whether the larger one will survive. SUMMARY

A series of 62 sets of twins and their placentae were examined from Thoroughbred mares. All were considered to be bicornual pregnancies and dizygous. The majority (64.5 per cent.) of these pregnancies terminated in abortion or stillbirth of both twins from 3 months gestation to term, although most foetuses (72.6 per cent.) were slipped from 8 months to term. In the remaining cases

TWINNING

IN

MARE:

FOETAL

AND

NEONATAL

LOSS

I 05

either one (21.0 per cent.) or both twins (14.5 per cent.) were born alive. Foals at term were stunted and emaciated, and of the 31 foals alive at birth only 18 survived to 2 weeks of age. Twin placentae were divided into three morphological groups according to the disposition of the chorionic sacs within the uterus. Type A was seen in 79 per cent. of cases. The chorion of the larger twirl occupied all of one horn of the uterus and most of the body, leaving the smaller twin only a small portion of the body and the other horn. These pregnancies frequently ended in abortion or stillbirth of one, or both twins. The larger twin had a much greater chance of survival than the smaller one. Type B occurred in 11 per cent. of cases. The villous surface areas was more or less equally divided, so that each foetus occupied one horn and half of the body. Both twins were usually born alive. Type C was seen in 10 per cent. of cases. Here there was much greater disparity between the surface areas of the 2 chorions. The smaller twin, occupying only part of one horn, died early on and became mummified. The larger twin was usually born alive and had a fair chance of survival. The loss of twin foetuses and the poor survival rates was attributed chiefly to placental insufficiency. The combined foetal surface area of the chorions of both twins was only slightly larger than that for a normal singleton foal. The presence of small vascular anastomoses was seen in many cases across the area that the 2 chorions were in contact. In some instances a chronic inflammatory reaction was seen in this area and it is suggested that this might Ix the result of an immunological reaction between the tissues of 2 foetuses. ACKNOWLEDGMENTS

WC: wish to thank the many veterinary surgeons who referred cases of twinning to us. We are particularly grateful to Mr D. J. Simpson, B.Vet.Med., M.R.G.V.S. of Messes Day, Growhurst, Simpson and McEnery who carried out the ovarian palpations, and to the National Stud for allowing us access to their records. Finally we acknowledge the support of the Horserace Betting Levy Board who defrayed part ot the cost of this investigation. REFERENCES

Amoroso, E. Cl. ( 1952). Placetztation. In Marshall’s Pkysiolo2-y oj Rejwoduction. 2. 3rd c.dition, p. 117-85, Edited by A. S. Parkes, Longmans Green; London. Arthur, G. H. (1958). I/et. Rec., 70, 682. Xrthur, G. H., and Allen, W. E. (1972). Equine vet. J., 4, 109. Bain, A. M. (1969). Jv.<. Vet. J., 17, 155. Bergmeister, E. (1951). Abstract in Anim. Br. Abstr. (1953), 21, 1591. Errington, B. .J., and Williams, W. L. (1933). Cornell I’fr., 23, 354. Errington, B. ,J. ( 1942). Ibid., 32, 367. Freudenbcrg, F. (1960). Deuts. tierarztl. Wschr., 67, 2 14. Hancock, J. L. (1948). Vef. Rec., 60, 679. Korol’kov, V. (1939). h-onevodstvo, 5, 31. Kudlac, E., Vorba, Z., and Munk, Z. (1969). Abstract in A&n. Br. -4bstu. (197Oj, 38, 210. Lillie, F. R. ( 1916). Science,43, 6 I I. McKeown, T., and Record, R. G. (1952). 3. Endocr., 8, 386. Mahaffcy, L. W. (1960). J. R. Arrnjj vet. Cps., 31, 19; (1968). Vet. Rec., 82, 681.

106

L.

B. JEFFCOTT

AND

KATHERINE

E.

WHITWELL

Mauk. (1939). Cited by Korol’kov. (1939). Mellor, D. J. (1969). Res. vet. Sci., 10, 361. Niekerk, van C. H. (1965). 3. 5’. Afr. vet. med. Ass.? 36, 483. Osborne, V. E. (1966). Aus. vet.J., 42, 149. Rooney, J. R. (1970). Autopsy of the Horse, 1st ed., p. 128, Williams and Wilkins Co ; USA Rossdale, P. D. (1968). I&t. Rec., 82, 687. Vandeplassche, M. (1957). Meded. V eearts-Sch. Rijksuniv. Gent, 1, 36. Vandeplassche, M., Podliachouk, L., and Beaud, R. (1970). Canad. 3. comb. Med., 34, 218. Williams, E., Gordon, I., and Edwards, J. (1963). Brit. vet. J., 119, 467. Williams, W. L. (1943). In The Diseases of the Genital Organs of Domestic AnimaZs, 3rd ed., p. 49, Balliere, Tindall & Cox; London. Zietzchmann, 0. (1924). Cited by E rrington, B. J., and Williams, W. L. ( 1933). Cornell Vet., 23, 354. [Received

for publication,

April

1Oth, 1972 J