Identical twins

Keywords: identical, twins, amnion, chorion, embryo, canine

A rare image of monochorionic/diamnionic identical twin fetuses. This was found in a Great Dane bitch approximately 27 days after ovulation (~23 days old). The fate of the twins at parturition was uncertain; ten fetuses were seen during this examination but only 9 puppies were born. Fetal membranes were not examined in detail.

Image size: 1186 x 668px. Copright: Dr Cathy Gartley. cgartley@uoguelph.ca

Identical twinning is complex. To give the reader a better understanding of the explanations that follow, a diagram of normal fetal-placental anatomy is shown below.

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Additional information on the canine exocelom can be found in this LORI entry.

With regard to any discussion on identical siblings in any animal (including humans) it is valuable to review the various forms that identical siblings can take. As is often the case, the situation is best understood in humans, where identical twins have been well studied. 

In reality, no twin is identical to its co-twin. Therefore the term "identical" should be viewed as approximate in all cases, even when embryos are split surgically to provide cloned siblings.

All identical twins start as a single zygote; the embryo then divides (often, still within the zona pellucida) and forms identical twins.

In some cases, the morula splits into two embryos very soon after the first divisions of the zygote. This gives rise to two completely separate individuals, with separate chorions. Within each of those chorions there is a single amnion containing a single embryo. Such twins are known as dichorionic/diamnionic, identical twins. That situation is illustrated below.

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When these twins are born, they have completely separate placentas and are born at slightly different times. Therefore in a polytocous species, dichorionic/diamnionic, identical twins can not easily be identified. Even though siblings may look similar, only microsattelite DNA analysis can establish their homozygous origin conclusively. In practice, this is seldom if ever performed. As a result, the incidence of dichorionic/diamnionic, identical twins in polytocous species is unknown. In animals that are usually monotocous, dichorionic/diamnionic twins are only likely to be recorded if embryos are split artificially. Otherwise, there too, the incidence is unknown.

Although identical twins are very similar to one another, there are usually differences between them, sometimes subtle, sometimes obvious; both physical and psychological. These differences arise from epigenetic effects i.e. differing expression of the same DNA in neighboring cells. For example, some cells will be destined to develop into limbs, others into lungs, yet both have the same DNA. Although the DNA will produce cells with virtually the same expression in both of the twins, some will differ because of the imperfect manner in which the embryo was split. This brings about differences between the morulas that manifest themselves immediately after the zygote has split into twins. Therefore, if their mutual zygote splits earlier than most,  twins will be very close to identical because they start from relatively undifferentiated blastomeres.

There comes a point when embryos split so late that a mutual chorion has already formed i.e. the two embryos share a single chorion. However, inside that chorion, each embryo has its own amnion. These twins are known as monochorionic/diamnionic identical twins. That situation is illustrated below. 

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The incidence of monochorionic/diamnionic identical twins in domestic species is unknown but the example provided by Dr Gartley (and reported by others) illustrates that it does indeed occur.

Interestingly, spontaneous monochorionic, triamnionic triplets have been reported in a mares so it is likely that monochorionic identical twins and triplets occur in all animals. Unfortunately, ruminants and carnivores usually eat their fetal membranes, precluding routine postpartum examination. Also, during cesarean sections in carnivores, insufficient attention is paid to the subtleties of placentation in efforts to obtain live neonates. In farm animals too, fetal membranes only receive cursory examinations and are often discarded with no examination at all. Therefore, little is known about the incidence of various forms of identical twins and triplets in domestic animals. The situation is further complicated in ruminants by the fact that co-twins/triplets and quads usually have fused chorionic membranes anyway, even if they are not homozygous.

Data on human reproduction shows convincingly that two different oocytes can each be fertilized by different sperm, then go on to form monochorionic/diamnionic heterozygotic twins i.e non-identical twins.  Accordingly, these twins have a single chorion and each has its own amnion. In the numerous cases described in humans, all are chimeras and are of course, some are of different sexes. If this sounds familiar to veterinarians, it should; it is akin to the freemartin situation in ruminants. Unlike freemartins however, monochorionic/diamnionic heterozygotic human twins do not usually experience abnormal genital development and function. In this regard, the author recalls a case reported in the lay press some years ago where a paternity suite could not be settled following the birth of monochorionic/diamnionic twins. In that case the complication arose when each baby was shown to have a different father!

If the embryo splits even later than previously described, its amnion will develop even before the embryo itself splits. When that occurs, the twin embryos not only share a single chorion but the same amnion as well. Accordingly, these are known as monochorionic/monomnionic twins and are even less similar to one another than other forms of identical twins. That situation is illustrated below.

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In the case of monochorionic/monomnionic  twins, when the embryo does indeed begin to split, it may be so late in its development that the embryo fails to split completely. This results in conjoined twins, with mirror imaging and partial or complete situs inversus.

The incidence of monochorionic/monomnionic  twins in domestic species is also unknown. However, conjoined canine and feline fetuses are not rare, suggesting that  monochorionic/monomnionic have at the least, an equal occurrence to that abnormality (considering that at least some monochorionic/monomnionic twins may develop normally). Indeed, conjoined fetuses are not rare in any domestic animal except horses and even in horses, conjoined fetuses have been described. Therefore it is probably safe to assume that monochorionic/monomnionic twins occur in all species.

Selected references:

Binati, B. et al. 2012. Thoraco-omphalopagus conjoined twins in chamois-colored domestic goat kids. Anat. Histol. Embryol. 41: 159–162

McNamara, H.C. 2016. A review of the mechanisms and evidence for typical and atypical twinning. Am.J. Obstetrics and Gynecology. 214:172-191

Meadows S.J. 1995. Identical triplets in a thoroughbred mare. Equine Vet J. 27:394-397.

Pregnancy; 25 to 27 days

Keywords: canine, pregnancy, 25, 27, diagnosis, surgery, polyovular, fetus,embryo,placenta

The image shows a canine pregnancy estimated to be 25 to 27 days of gestation. This estimation was based on the crown rump length of the embryos and the size of the embryo-placental bulges. There is little information on actual crown-rump length in postmortem specimens of known gestation, therefore estimates were extrapolated from ultrasonographic data. The size of the bitch was unknown and could have belonged to a small or giant breed, another factor that could contribute to inaccuracy of this estimation.

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The author repeatedly refers to embryos in this entry, not fetuses. That is because organogenesis is not yet complete and the embryo is not yet recognizable as the species of interest; two factors occasionally used to define the transition between an embryo and a fetus. In fact, there is no gestational age when organogenesis is indeed complete (CNS development continues even after birth) and recognition of the species of the embryo-fetus lies in the eye of the beholder. The reader is left with that dilemma. Certainly, refereed data do not suggest firm opinions on this matter.

Several items are of note in this specimen. 

1. The ovaries were carefully dissected and only five corpora lutea were present; yet there were six embryos. This is to be expected in bitches, where a single Graafian follicle can contain several oocytes i.e. Canis familiaris is a polyovular species.  

2. A reminder that bitches (and queens) do not have major (middle) uterine arteries such as those present in ruminants and horses. Therefore in young, non pregnant bitches that do not have excessive abdominal fat, little potential exists for severe hemorrhage when the mesometrium is transected during ovariohysterectomy.

3. Ultrasonography has largely rendered transabdominal palpation for pregnancy redundant. However, palpation of  embryonic-placental bulges are potentially important when ultrasonography is not available. In this regard, the author developed a mantra for students on how to recall when palpation was valuable and for what reason. It also included the advent of mammary development and was known as the "25, 35, 45, 55, 65" guide. At its best it is only approximate, but still potentially valuable. Timing was based on the occurrence of the LH surge and it read as follows:

25 to 35 days: embryonic-fetal bulges are palpable except in obese bitches or those that are extremely muscular. 

Note: After 35 days, the embryonic-fetal bulges lose tone and become more difficult to palpate.

45 days: mammary development is first noted in maiden bitches (Colostrum can only be expressed during the last 2 to 3 days of gestation).

55 days: the fetuses themselves become palpable.

65 days: parturition.

4. Carnivora have intimate (endotheliochorial) placentation, arranged in a band around the embryo/fetus. This is called zonary placentation. The margins of zones of placentation in bitches develop hematomas where heme breakdown products facilitate iron transport into the conceptus. One of these products is biliverdin,  the bright green pigment known as uteroverdin in this context. That pigment is not yet visible in a 25 to 27 day pregnancy but becomes a remarkable feature of late gestation, signaling the onset of whelping and in some cases, retention of the placenta after whelping is complete.

Selected references:

Yeager, A.E. and Concannon, P.W. 1990. Association between the preovulatory luteinizing hormone surge and the early ultrasonographic detection of pregnancy and fetal heartbeats in Beagle dogs

Theriogenology 34:655-665

Michelle A. Kutzler, M.A. 2003. Accuracy of canine parturition date prediction using fetal measurements obtained by ultrasonography. Theriogenology. 60:1309-1317

Lopate, C. 2008 Estimation of gestational age and assessment of canine fetal maturation using radiology and ultrasonography: A review. Theriogenology. 70:397-402