Twin reduction techniques

Introduction

We have known three things about twins for a long time. Firstly twins are repeatable within the same mare, secondly twinning rate varies according to breed and thirdly, the more fertile the stallion the more twins he is expected to achieve.

Twins are avoidable and should not occur on well managed breeding farms. Clients understand this and are demanding the most sophisticated management programs available. Presented below are the management techniques in use at the Goulburn Valley Equine Hospital. The hospital is a major referral centre and each year is expected to handle multiple referral cases of twin pregnancy that are diagnosed after cessation of the mobility phase.

 Historically twins have been the single most important cause of abortion in Thoroughbreds ^1,2 . As most twin pregnancies terminate in early foetal resorption or loss, late term abortions, or the birth of small growth retarded foals and mares aborting twins in late gestation frequently have foaling difficulties, damage their reproductive tracts and are difficult to rebreed, twins are disastrous financially. If foals born alive they are frequently small, have intra-uterine growth retardation and a poor survival rate with many needing expensive sophisticated critical care.

Origin, associations, and a general overview of twinning in the horse has been published elsewhere ^3,4 .

It is our responsibility to successfully manage early pregnancies such that no mare delivers or aborts twin foals. In consultation with farm managers, owners and clients we must to utilise available equipment and technology commensurate with economic constraints and other owner/manager preferences to diagnose twin pregnancies as early as practically possible. It is also the responsibility of a veterinary profession to adequately inform owners/managers/clients of reasons why twins may be not diagnosed.

Twins are still occasionally missed despite multiple examinations. Reasons why twins may not be detected, despite repeated examination are: 1) difficulty distinguishing structures (this may be related to a poor examination environment i.e., too much light, poor display characteristics of the ultrasonographic unit, mare movement and/or lack of restraint) 2) variable growth patterns 3) inability to detect heart beats of adjacent embryos 4) operator experience and 5) resolution of the equipment. The most common cause of misdiagnosing the presence of twin pregnancies is examination prior to the time period that a second pregnancy (asynchronous ovulation) may be reasonably expected to be detected. Another reason appears to be scanning too quickly.

The incidence of abortion has been reported as decreasing ⁵ and in the German Thoroughbred industry has almost halved since the event of ultrasonography ⁶

Origin of twins

Twins in the horse are unlikely to be identical. Almost all cases of twin pregnancy are expected to be related to multiple ovulation, although at least two occurrences of monozygotic twin pregnancies have been suspected ^7,8 .  The reason for lack identical twin formation in the horse is probably related to the capsule ⁹ . The capsule forms in equine embryos aged around 6 days, shortly after their entry into the uterus ^10,11 . When embryos were cultured prior to the formation of the capsule, hatching occurred similar to as occurs in the bovine ¹¹ , however when embryos were cultured after formation of the capsule the zona pellucida continued to become progressively thinner and finally fell away from the developing conceptus. Other species that do not have a capsule, such as sheep, cattle and humans, all have the ability to routinely give birth to identical twins which apparently form from pinching of a hatching embryo by the zona pellucida.

It has been shown that twins in mares are as likely to result from synchronous versus asynchronous ovulation ¹² and that pregnancy rate per follicle was identical for double ovulations on opposite ovaries to that obtained from single ovulations per cycle but was higher than the pregnancy rate per follicle when double ovulations occurred on the same ovary ¹³ .

The outcome of twin pregnancies

Understanding the outcome if twin pregnancies are left to develop is of great importance to veterinarians, farm managers and owners.  Also important is when to intervene and what probability of success such interventions may be expected to achieve.

What happens if we do nothing?

The mare is very efficient at reducing twins to a single pregnancy. This is done by a competitive absorption of nutrients that is related to size and position of the early pregnancy and later to orientation of the embryo proper within the developing conceptus ¹⁴ .

However the initiation of any non-intervention program depends on the age of identification, the orientation of the vesicles and any disparity in size.

Recognition ≤16 days from ovulation.

Embryo reduction before or on the day of fixation is not considered an important aspect of the natural correction of twins ¹⁵ . The probability of a mare loosing one or both vesicles of a set of twins from identification prior to fixation is minimal and approximates that of  early embryonic death for the same time period (per vesicle). The recognition of twin pregnancies prior to fixation day (day 16) is dependant on the day of examination relative to the day of ovulation. Asynchronous ovulations occasionally result in a gross disparity in vesicle size, sometimes as much as 4-5 days i.e. identification of a day 11-12 and a day 16 vesicle concurrently (Fig 1a). In instances such as this, examination one day earlier may have failed to detect the younger of the two pregnancies. Recognition that all twin pregnancies occur from multiple ovulation dictates mandatory re-examination of all mares that have two CL’s and only a  single vesicle detected prior to fixation (day 16). Recognition prior to fixation is also dependent on operator experience, resolution of the equipment (≥ 5 MHZ preferred), monitor capabilities, restraint and other facilities (ability to darken the environment), the presence of uterine cysts and the skill of the examiner.

Recognition after fixation (after day 16)

The recognition of unilaterally fixed twins from day 17 through to 21 (prior to clear recognition of the developing foetus within the vesicle) may be the most difficult time to determine if there are twins present. Ultrasonicgraphicaly, all that is detectable is a thin line (the apposition of the two yolk sacks) running approximately in the middle of a slightly over-sized vesicle (Fig 1b). Recognition of the fetus(es) within the vesicle a few days later makes differentiation easier(Fig 2). Occasionally an inexperienced operator may confuse an abnormally orientated 28 to 30 day single pregnancy with 17 to 20 day unilaterally fixed twins (Fig 3). From days 22 to 60 the presence of multiple foetuses, umbilical cords and general excess in the number of visible membranes should alert the practitioner to the likelihood of more than one pregnancy (Fig 4) . The junction between two developing foetuses (after 30 days) between the two allantochorions results in a common membrane from the area of apposition. This common membrane has been referred to as the twin membrane  (Fig 4) ¹⁶ and has diagnostic potential, particularly late in pregnancy when it might not be possible to view both foetuses transrectally (>100 days). After 100 days, careful transabdominal ultrasonography may be necessary to determine the presence of twins.

Days 17 to 40

The outcome of pregnancies post fixation is dependent upon their size (diameter) and the nature of their fixation. Unilateral (both fixed together at the same corpus cornual junction) fixation reduction is much higher than bilateral (one on each side) fixation reduction. Fortunately, unilateral fixation is much higher (approximately 70%) compared to bilateral (30%) ¹⁴ . In 28 mares with known ovulatory patterns, synchronous ovulations did not affect the type of fixation (9/17 unilateral , 8/17 bilateral). However for asynchronous ovulation the frequency of unilateral fixation (10/11) was greater (p< 0.01) than the frequency of bilateral fixation (1/11). The incidence of embryo reduction was greater (p< 0.01) for unilateral fixation (14/19) than for bilateral fixation (0/9) and was greater (p< 0.05) for asynchronous ovulation (9/11) than for synchronous ovulation (5/17) ¹⁴ . Practically speaking this means that if asynchronous ovulations have resulted in significant age differences between vesicles (i.e. > 3 mm diameter at day 15) then rate of embryonic reduction is very high ¹⁴ . In cases of unilateral fixation, 22 of 22 mares with vesicles of dissimilar size had reduction compared to 19 of 26 (73%) with vesicles of similar size ¹⁷ . As a result of work studying reduction of unilateral versus bilateral twin pregnancies in mares  from days 17 to 40 Ginther proposed that the nutrient intake from the larger vesicle (before the foetus was present) prevented adequate nutrition of the smaller vesicle. Later the position of the foetus proper and its emerging allantoic sac seemed to determine whether a given conceptus survived or underwent late reduction. The foetus, the vascularised wall of the yolk sac adjacent to the foetus and the emerging allantoic sac were exposed to the endometrium (uterine lumen) in the surviving vesicles. In the vesicles that underwent reduction, much of the corresponding area of the vesicle wall was covered by the wall of the adjacent survivor and is thus deprived of adequate embryonal-maternal exchange and therefore regresses.

 In summary, dissimilarity in diameter increases the likelihood of unilateral fixation, increases the incidence of reduction for unilateral fixed vesicles, hastens the day of occurrence of reduction and shortens the interval from initiation to completion of reduction.

The incidence of reduction for bilaterally fixed vesicles is negligible and approximates that of standard early embryonic death in this period.

Of the 85% of reductions by day 40 in cases of unilateral fixed twin pregnancies, 59% of reductions had occurred between day 17 and 20, 27% between day 21 and day 30 and 14% between days 31 to 38. The majority of early reductions occurred spontaneously (by day 20) as compared to reductions after day 20 that were proceeded by a gradual decrease in size of the eliminated vesicle. In addition when twins were dissimilar in diameter (4mm or more) they were more likely to undergo reduction by day 20 ¹⁷ . Other studies have demonstrated similar results. The hypophysis of an early embryonic reduction mechanism for elimination of excess embryo in mares was not new and had been suggested as early as 1982. However, ultrasonography was necessary to adequately document the occurrence and nature of the reduction ¹⁸ .

Day 40 onwards

Ginther and Griffin ¹⁶ examined the natural outcome of bilateral twins (one in each horn) that were viable on day 40 in 15 pony mares. Readers should be aware that pony mares are not necessarily a good model for larger breeds, as the incidence of twins is low and evidence is suggestive that the larger the breed (Draught, Thoroughbred and Warmblood) the higher the probability of maintaining twins. Fifteen pony mares were monitored by ultrasonography until the outcome of the pregnancy was determined. Sixty six % (10/15) of the pregnancies resulted in either death of both (80%) or death of one (20%) during months two or three. Nothing occurred from then until month 8. Between months 8-11, two mares lost one foetus (foetal death was associated with mummification) and two mares lost both. The two mares that lost one pregnancy both delivered undersized weak foals at birth. One mare (7%) delivered live twins at term and two normal foals were born from mares loosing the one pregnancy (absorption of the foetus rather than mummification) in month two. In this study six live foals were born (2 of normal size), from a total of 15 mares and 30 foetus. This incidence is similar to previous reports wherein of 130 pregnant mares with twins, only 17 live foals (13%) were produced ¹⁹ . An interesting observation from the later report was that from the 102 mares that delivered live or dead twins in the previous year, only 37 produced live foals the next seasons and thus over two seasons there was an average of 23% producing live foals ¹⁹ . An earlier study ²⁰ , was extremely useful in categorising outcome of twins that managed to survive to later pregnancy. Twinning accounted for 22% of the cases of abortion and still-birth between 1967-1970. Sixty two sets of twins and their placentas were examined from Thoroughbred mares. All were considered to be dizygous. Abortion or still-birth of both twins from 3 months of gestation to term occurred in 64.5% of mares, although most (72.6%) slipped from 8 months to term. In the remaining cases one twin (21%) or both twins (14.5%) were born alive. Most foals at term were stunted and emaciated and of the 31 alive at birth only 18 had survived to 2 weeks of age ²⁰ . In this study twin placentation was divided into three morphological groups according to the disposition of the chorionic sacks within the uterus. Type A placentation was seen in 79% of cases (48 sets of twins). One foetus occupied one horn and most of the body (mean 68% of the total functional surface area) while the other twin occupied only one horn and usually only a small part of the adjacent body. Where the chorions abutted there was a variable degree of invagination of the smaller chorion into the allantoic cavity of the larger twin. These pregnancies frequently ended in abortion or stillbirth of one or both twins. In this group 31/48 lost their pregnancies between 3 and 9 months (64.5%). The gestation length in this group was frequently shorter and at birth the larger twin had a much greater chance of survival than the smaller one. In this group only 6 foals of 48 sets were born alive. Of the 6 foetuses born alive, 5 were the larger twin. Type B placentation occurred in 11% of cases (7 sets) and the placentas were orientated such that the villous surface areas were more or less equally divided and each foetus occupied one horn and half of the body. Both foals were usually similar in size and were usually born alive. Nine foals survived to 2 weeks from 6 sets of twins that made it to term. In this group (7 total) one aborted at 7 months. Type C placentation was seen in 10% of cases (6 sets of twins). In this group there was a greater disparity between the surface area of the 2 chorions. The smaller twin occupying only part of one horn, died earlier on and became mummified. The larger twin was usually born alive and a fair chance of survival. In this group 3 foals were born alive from 6 pregnancies at term ²⁰ . The authors attributed the loss of twin foetuses and poor survival rates to placental insufficiency.

It should be clear that our philosophy is that non intervention is only acceptable when twins are diagnosed as a unilateral occurrence between days 17 and day 40 and then the decision depends on factors such as the value of the foal, the potential for rebreeding and the ability of the veterinarian to manually intervene. Intervention in twin pregnancies is strongly recommended in all other circumstances (see below).

When and how should we intervene?

Recognition ≤16 days from ovulation.

The first technique for manual crush of  the conceptus during the mobility phase utilised manual reduction with good results ²¹ and was a variation from previously reported techniques for twin pregnancies ^22,23 . The technique involved gentle manipulation of the embryonic vesicle to the tip of one uterine horn and manual rupture. When applied to single pregnancies it resulted in pseudopregnacy and when applied to twin pregnancies it resulted in a single pregnancy in 7 of 8 attempts ²¹ . Later utilising the same techniques, mares were treated with single or multiple progestagen administration (hydroxyprogesterone caproate), an anti-prostaglandin (flunixin meglumine) plus progestagen or given no treatment prior to manual embryonic rupture in the mobility phase ^24,25 . Results were 10/10 (100%) mares maintaining pregnancy in the control group (no treatment, just manual rupture) and 37/40 (92.5%) for treated mares. The amount of PGF_2a released was directly correlated with the pressure required to cause embryonic rupture. Flunixin meglumine inhibited PGF_2a release after embryonic rupture. Treatment with progestagen plus flunixin meglumine or progestagen singly or multiply was not better than no treatment at all (although it was subsequently shown that the progestagen chosen had no ability to maintain pregnancy in ovariectomised mares and did not bind to progesterone receptors in the horse ²⁶ ). Another report ²⁷ demonstrated that 60 of 66 mares (90.9%) maintained a single vesicle after manual reduction was attempted prior to fixation. Five of the six mares in which the procedure was not successful subsequently conceived. Since 1984 ²⁸ we have used a modification ⁴ of the technique described originally ²¹ . With this technique the ultrasound probe is used to manipulate the vesicles while keeping one or both vesicles in view during the manipulation and more importantly the crushing or rupture of the vesicle (Fig 5). Utilising this technique it is possible to more accurately and quickly separate vesicles. It was original proposed ²¹ that when vesicles were in apposition mares be re-examined approximately one hour later. By utilising the probe to manipulate vesicles, separation is achieved (pre-fixation) very quickly in most instances. Commonly the smaller foetus is destroyed despite the lack of evidence to support pre-fixation reduction. On occasion it is necessary to revisit the mare 24-48 hr after the original evaluation if the smaller of the two vesicles is less than 1 cm in diameter as sometimes these can be more difficult to destroy.

Separation of vesicles should always be possible if the vesicles are still able to be identified as two spherical non-coalesced structures. Briefly, the technique involves separation of the vesicles using the probe. A finger is placed on either side of the probe to help stabilise the vesicle to be moved. Gentle back and forth movement of the probe with pressure results in the two vesicles becoming separated (Fig 6). The separation is identified by lack of a vesicle under the probe. The vesicle can be crushed as close as 0.5 cm from the other but it is generally best to separate them at least 2 cm. This is in case the mare moves at the time of increasing pressure. The vesicle is crushed by gradually increasing the pressure using the probe. Occasionally refractory cases may need a sudden increase in pressure much like a quick flick of the end of the probe. This later technique is quite useful for smaller (day 11-13) vesicles.

When the vesicle is crushed it is not uncommon for fluid to surround the other. This is not a problem at this stage of pregnancy. Later (≥day 25) fluid surrounding then other vesicle is thought to be a potential problem.

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