J . small Anim. Pract. (1971) 12, 179-184.

Hyperthermia and disturbances of prenatal development*

M . J . E D W A R D S

Department of Veterinary Medicine, University of Sydney, Australia

A B S T R A C T

Pregnant guinea pigs at all stages of gestation were exposed for 1 hr daily on 1, 2, 4 or 8 days to temperatures of 42-43' C. 'High doses' of heat stress resulted in maternal death ; progressively smaller doses caused foetal death, abortion, foetal malformations or no effect. The commonest foetal abnor- malities were micrencephaly, hypoplasia of digits, umbilical hernia, club- foot and arthrogryposis. Proliferating neuroblasts were especially suscept- ible to heat between days 20 and 23.

I N T R O D U C T I O N

I n Australia (as in other countries) enormous losses amongst farm livestock are associated with reproductive failure. For example, in the mid-1960s from 60 million ewes mated, only 40 million lambs were weaned. The losses in pigs are similar to those of other countries; about 40% of ova shed are not represented by newborn piglets and 10-25% of piglets born do not survive to weaning. I n general the infectious causes of infertility are becoming less important as control measures are applied. However, there is evidence that heavy losses still occur and it is probable that many different factors each contribute to them.

Much of the research effort of the Department of Veterinary Medicine at the University of Sydney has been directed to the effects of adverse environment, particularly in feeding and climate, on reproduction in the economic species. My own interest has been in reproductive diseases of cattle, and quite a spectacular condition affects calves particularly in the eastern seaboard cattle breeding areas

* This was a guest-lecture delivered at the Royal Veterinary College, London on 7 October 1970. Although the experimental work in guinea-pigs which is described was motivated by natur- ally occurring congenital defects in cattle, the Editor believes that the results obtained have such wide biological and clinical implications that readers of this journal would like to be aware of them.

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of New South Wales. The condition has been termed 'arthrogryposis and hydran- encephaly'. I t affects about 0.1% of calves born in the area in most years, but occasionally occurs as an epidemic affecting up to 2% of all calves born and as much as 30% of calves in individual herds. I n 'epidemic' years, calves with arthrogryposis are born early in the calving season, in mid-season affected calves commonly have both arthrogryposis and hydranencephaly while hydranencephaly alone is common late in the calving season.

Calves affected with arthrogryposis have the joints of the limbs and/or spine fixed and immobile, and there is wasting of muscles in the affected area which appears to result from loss of motor nerve cells in parts of the spinal cord which innervate the affected muscles. A calf weighing 80 Ib with arthrogryposis and causing dystocia presents a formidable obstetric problem. Calves with hydranence- phaly have lost most of the cerebral hemispheres which are replaced by fluid-filled sacs. These animals, while with the mother, frequently appear fairly normal; they can be taught to drink and will walk alongside the cow. However, when they are separated from the cow they blunder into fences, dams, etc. due to blindness. The visual cortex is absent.

Some years ago towards the end of an epidemic of arthrogryposis and hydran- encephaly in calves there was a period of 3-4 days in which air temperatures dur- ing the day rose to about 100-104" F. Shortly afterwards abortion occurred in a number of guinea pigs in a breeding colony which was kept in the department in an iron shed; 3-5 weeks later young guinea pigs were born with leg deformities typical of arthrogryposis ,and a number of affected young also had hydranence- phaly. On reflection, it appeared that the abortions, followed by the birth of malformed guinea-pigs were associated with the hot environment. Pilot experi- ments which involved placing pregnant guinea-pigs for short periods into in- cubators at about 106-109" F supported this association, as in each case, limb malformations, and occasionally hydranencephaly, occurred in the young of these guinea-pigs.

This offered a promising model for the study of the condition in cattle and experiments were conducted to determine the tolerance of guinea-pigs to heat and the variations in deep body temperatures which occurred during exposure to heat.

E X P E R I M E N T A L R E S U L T S

It was found that guinea-pigs tolerated exposure to air temperatures of 42-43" C (107-6-109-4" F) for 1 hr quite well. The time interval of 1 hr was chosen simply because it was a convenient amount to measure; the temperature of exposure was selected because it appeared to be tolerated with good survival in exposures of 1 hr duration.

The temperature of the mother, measured with clinical thermometers as a deep rectal temperature, showed a gradual elevation for about 30 min after the begin- ning of the exposure and a more rapid elevation during the last 30 min. The

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rectal temperature fell rapidly following removal from the incubator. During each exposure it was above normal for about 90 min and was elevated by more than 2.5" C (4.5" F) for about 20 min. The foetal temperatures, or temperatures of the embryonic fluids, were slightly less than that of the mother before, during and after exposure. Here, it should be stated that the temperature elevations employed were within the ranges expected for temperature responses to infections, or hot weather. Female guinea-pigs at all stages of gestation were exposed to these con- ditions once daily on 1, 2, 4 or 8 days. The normal gestation period in this colony is 68 days.

I n general the dose response to heat was similar to that of other noxious agents. High doses resulted in maternal death. Progressively smaller doses resulted in foetal death, abortion, foetal malformation or no ill-effects respectively. The evidence suggests that very susceptible mothers die or abort and that the less susceptible mothers (those more resistant to excess body temperature elevations) produce malformed young. This illustrates a principle in teratology-agents which result in foetal malformation frequently have little, if any, effect on the mother. If the mother is severely affected she dies or aborts, but no malformed young result.

I n this investigation a wide spectrum of abnormalities has been noted following maternal heat-stress during gestation. The most common were micrencephaly (100% between days 18 and 25 of gestation), abortion (60% between days 11 and 18), hypoplasia of digits (60% between days 18 and 25), umbilical hernia (40% between days 18 and 25), clubfoot (30% between days 18 and 25) and arthro- gryposis (35% between days 35 and 43). The effects seen after heat-stress between days 4 and 67 of gestation included :

Abortions particularly to exposure between days 11 and 18. Microphthalmia and coloboma (failure of fusion of the ventral part of the iris)

Cataract involving the central part of the lens occurred following heat-stress at

Hypoplasia of the incisor teeth and of the lateral digits of the forelimbs followed

Renal agenesis and exomphalos (congenital umbilical hernia) and clubjoot (talipes)

Arthrogyposis was most commonly encountered when the mother was heated

Micrencephaly occurred after heat-stress at three stages of gestation, between days

Most malformations followed damage by heat at fairly specific and restricted stages of gestation. Heat-stress between days 18 and 25 had a devastating effect on the embryo (13% with cataract, 18% with hypoplasia of incisors, 38% with exomphalos, 15% with renal agenesis, 60% with hypoplasia of digits, 28% with clubfoot and 100% with micrencephaly). At this stage the affected organs are

following heat-stress between days 11 and 18.

most stages of gestation.

heat-stress between days 18 and 32.

followed exposure between days 18 and 25.

between days 35 and 45.

15 and 32, days 39 and 46 and days 53 and 60.

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undergoing rapid proliferation and differentiation. This illustrates another general principle in teratology-an organ is most susceptible to damaging agents a t the stage of its most rapid growth and differentiation.

All the conditions described occur sporadically under normal breeding con- ditions as ‘spontaneous defects’, i.e. there is no detected cause. There is a genetic tendency toward the condition which is ‘triggered’ by the environmental influence, acting, as it were, on the genetic weak link. Thus, many different agents applied at a certain stage of gestation can produce the same type of foetal abnormality.

It was interesting to note that abortions following exposure in early gestation frequently occurred at about day 31-35 whether the exposure was of 4 or 8 days and whether the exposure occurred between 11 and 18 days or 26 and 30 days. Thus, the initiating cause of abortion was not related closely in time to the sub- sequent act of abortion. The abortions might have been due to any one or to a combination of malformations incompatible with continuing development, or to placental damage.

I t was quite obvious that the central nervous system is most susceptible to damage by elevated temperatures and my effort has been concentrated on this system. Central nervous system anomalies have been commonly associated with skeletal or brain abnormalities, and the susceptibility of the developing brain in early gestation, and of the developing spinal cord in later gestation has already been indicated.

Clubfoot can affect the forelimbs or hindlimbs and follows heat exposure be- tween days 18 and 25 of gestation. Typically, when the hindlimbs are involved, the tarsal joint (only) is immobile and distorted with the foot turned medially; the fibula frequently is missing, the gastrocnemius and plantaris muscles appear con- tracted and the tibia is frequently bowed. Histological examination of serial sections of the spinal cord have shown areas of dysplasia, including duplication of the central canal and an abnormally wide grey commissure; a displaced and dilated central canal, or absence of the canal in the cervical, thoracic or lumbar regions, or in all regions. At present it appears that the foot deformity is caused by spasticity of the gastrocnemius and plantaris muscles due to defective innervation from the malformed spinal cord.

In animals with arthrogryposis, a number ofjoints are fixed in various positions of flexion or extension. Arthrogryposis affecting the forelimbs, neck and muscles of the thorax and abdomen follows heat exposure between about days 27 and 31. The muscles of the forelimbs are atrophied or absent and the intercostal and abdominal muscles are very wasted. The neck joints also may be involved and result in a ‘wry neck’. The mortality in these animals obviously is high. At post- mortem examination there is a syringomyelia of variable extent. When the hind- limbs are involved, similar defects of muscles and joints are seen at post-mortem examination and the spinal cord in segments innervating affected areas is reduced in size. On histological examination, loss of motor neurones is evident in the affected areas of cord and a denervation-type atrophy affects the muscles, often

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with replacement by an adipose-type tissue. Thus the pathogenesis of the condition appears to be a denervation atrophy of muscle, with fixation of joints due to the prolonged prenatal immobilization.

Frequently, moulding deformities are seen in domestic animals ; twisted noses of foals and calves, crooked legs in foals. If an extremity be immobilized in utero, the unaffected extremities, by their unopposed action, would tend to push the immobile part against the uterus and amnion and hold it there. This might be the mechanism by which these parts have a rounded and moulded appearance. The moulding represents a secondary deformation resulting from a primary defect of the spinal cord. I t is obvious that effective surgical correction of these defects would depend on knowledge of the primary cause of the condition.

Development of the brain was disturbed following maternal heat-stress at three stages of gestation. During early gestation (days 16-30) brain development was extremely sensitive. At this stage there is rapid neuroblast proliferation taking place. During mid-gestation (days 39-46) and late gestation (days 53-60) develop- ment was less susceptible to heat. At these stages dendritic growth and neuronal maturation and myelination respectively are taking place. Brain development was not affected in the intervening stages of gestation.

In this study my attention was concentrated on the effects of heat on the developing brain during early pregnancy because a t this stage small 'doses' of heat for a number of days retards brain growth in all embryos and up to a 30% reduct- ion in wet weight at birth can be produced. In a number of experiments it was found that the most susceptible stage was between days 20 and 23 of gestation and that following one exposure on day 21, if the maternal temperature exceeded 2.5" C (4.5" F) above normal, the brain growth of the embryo and foetus was retarded. For each 1" C elevation above this level there was a deficit of 8.5% in brain-weight at birth (1" F, 5%). Recent work indicates that the smaller brains have fewer cells and that the loss of cells results from death of proliferating neuroblasts. Newly born guinea-pigs with severe deficits in brain size show obvious abnormalities of behaviour. They are dull, slow to move, unresponsive, frequently do not form a bond with the mother or learn to drink and are clumsy or unable to stand.

C O M M E N T

Heat-stress has been shown to affect prenatal development in other species. In the rat the effects resemble prenatal exposure to ionizing radiation-anophthalmia, anencephaly, tail malformations, clubfoot, harelip, growth retardation. I n the developing chicken exposure to high incubation temperatures results in defects, particularly of the central nervous system. This is of interest since the action must be directly on the developing organ and not mediated by changes in the maternal metabolism due to the heat-stress. I n sheep exposure to high temperatures in mid- gestation has been followed by extensive cavitation of the white matter of the

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brains of lambs at birth. I n the human, there appears to be an association be- tween congenital umbilical hernia (exomphalos) and a fever between the eighth and twelfth weeks of gestation.

In cattle the effect of gestational hyperthermia is unknown at present. However, it is doubtful whether it contributes to any extent to the outbreaks in New South Wales of arthrogryposis and hydranencephaly investigated. Hyperthermia due to infection or environment would be relatively common during gestation and, depending on the stage of development of the embryo or foetus, seems to be a par- ticularly potent agent, in the guinea-pig at least, in the causation of birth defects.

RCsumC. Des cobayes enceintes a tous les stades de la gestation ont CtC exposCes pour une heure par jour pendant 1, 2, 4 ou 8 jours a des temptratures de 4243” C. De ‘hautes doses’ de temps de chaleur ont eu pour resultat la mort de la mtre; des doses progressivement plus petites ont causC la mort du fetus, l’avortement, la malformation du fetus ou n’ont eu aucun effet. Les anomalies fetales les plus frkquentes Ctaient la microcCphalie, l’hypoplasie des doigts, l’hernie ombilicale. le pied bot et l’arthrogrypose. Des neuroblastes proliflres Ctaient particulitrement sensibles a la chaleur entre les 20ilme A 23ilme jours.

Zusammenfassung. Trachtige Meerschweinchen in allen Stadien der Trachtigkeit wurden 1 ,2 ,4 oder 8 Tage eine Stunde taglich Temperaturen von 4243” C ausgesezt. ‘Hohe Dosen’ der Warmebelastung fiihrten zum Tode des Muttertiers; zunehmend kleinere Dosen fiihrten zum Tod des Fetus, zur Fehlgeburt, zu fetalen Missbildungen oder sie blieben wirkungslos. Die haufigsten fetalen Anomalien waren Mikrenzephalie, Hypoplasie de Zehen, Nabelbruche, Klumpfuss und Arthrogryposis. Proliferierende Neuroblasten waren besonders vom 20. bis 23. Tag empfindlich gegen Warme.