The maternal pheromone and deoxycholic acid in the survival of preweanling rats

Physiology & Behavior, Vol. 33, pp. 931-935. Copyright©Pergamon Press Ltd., 1984. Printed in the U.S.A.

0031-9384/84 $3.00 + .00

The Maternal Pheromone and Deoxycholic Acid in the Survival of Preweanling Rats I THERESA

M. L E E A N D H O W A R D

MOLTZ z

C o m m i t t e e on Biopsychology, The University o f Chicago, Dept. Behavioral Science, Green Hall 5848 South University Avenue, Chicago, I L 60637 R e c e i v e d 12 A p r i l 1984 LEE, T. M. AND H. MOLTZ. The maternal pheromone and deoxycholic acid in the survival of preweanling rats. PHYSIOL BEHAV 33(6) 931-935, 1984.---Preweanling rats selectively approach and consume pheromone-containing maternal feces. This selectivity suggests that the consumption of maternal feces might be important for the growing pup. We tested the hypothesis that such feces, because of their high deoxycholic acid content, may protect against acute enteritis. A series of experiments was carded out in which pups were denied access to maternal feces. These pups died more often from acute enteritis than control pups. Additional confirmation of the hypothesis was obtained when fecesdenied young fed deoxycholic acid showed a significantly lower mortality than feces-denied young fed laboratory chow alone. Maternal pheromone

Deoxycholic acid

Acute enteritis

A C U T E enteritis is an inflammatory disease of the gastrotestinal tract characterized by gaseous abdominal distension, diarrhea, and blood in the stool. Death may be due to dehydration and electrolyte imbalance resulting in cardiac failure, or to perforation of the ileum and colon resulting in overwhelming sepsis. Acute enteritis is often seen in neonatal mammals: piglets, for example, frequently suffer from a disease called colibacillosis [21], and babies from "necrotizing enterocolitis" [7]. Acute enteritis has also been observed in the preweanling laboratory rat [1,2]. We think that the rat, unlike the baby [20], and perhaps the piglet as well, has developed a defense against the disease. At about 14 days of age, rat young begin to consume solid food, and we know from the research o f Lee and Moltz [18] that solid food escalates the bacterial challenge to the infant gut. Specifically, it increases the population of potentially pathogenic Escherichia coli relative to that of such protective microorganisms as Lactobacillus/Bifidobacterium. Moltz [25], Moltz and Kilpatrick [26], and Moltz and Lee [27,28] have suggested that the preweanling pup defends itself against E. coli, and hence acute enteritis, by consuming the feces of its mother. Lactating rats excrete a pheromone in their feces that strongly attracts preweanling young. This pheromone is first emitted 14 days postpartum and continues to appear in the feces o f the female until she weans her young, typically at 27 days postpartum. Pups become responsive to the pheromone during the period in which the pheromone is being emitted; in other words, they approach pheromone-containing feces

Preweanling rat

when they are between 14 and 27 days of age. That they characteristically respond only during this two-week period has been determined experimentally by making the pheromone available before 14 days and after 27 days [23]. Pups 14-27 days are not only attracted to pheromone feces, but prefer to consume such feces [16]. That is, when given a choice between anal excreta taken from a pheromone-emitting female and a virgin female, they ingest significantly more of the former. This selectivity of both approach and ingestion suggests that something in maternal feces is important for pup development: Moltz and Lee [27,28] have hypothesized that it might be deoxycholic acid. Deoxycholic acid is a secondary bile acid synthesized in the cecum from cholic acid, a primary liver steroid. Kilpatrick, Lee, and Moltz [16] recently measured deoxycholic acid in feces and found the concentration as well as the output of the steroid to be significantly higher in maternal than in nonmaternal feces. They also found the output of deoxycholic acid in the pup to be well below adult levels until day 28, which is approximately the day on which pups stop responding to the pheromone. There is evidence that deoxycholic acid offers protection against acute enteritis, or at least against the endotoxin of E. coll. When the endotoxin of E. coil was extracted with phenol and treated with sodium deoxycholate, the toxic units were split into smaller, non-pyrogenic structural elements [30]. The E. coil endotoxin incubated with sodium deoxycholate and administered parenterally produced no pathogenic effects; without prior incubation overwhelming sepsis oc-

'The present research was supported by Grant HD 06872 and by Biomedical Research Support Grant PHS S07RR0729 to Howard Moltz. 2Requests for reprints should be addressed to H. Moltz.

931

932 curred 14,17]. When the E. coli endotoxin was administered orally to intact rats in a dosage 500-3000 times the parenterally lethal dose, the animal remained asymptomatic; however, when the bile duct was cannulated to prevent bile from reaching the intestinal tract, oral administration rapidly resulted in death [3,4]. The present series of experiments is designed to test the hypothesis that the ingestion of deoxycholic acid through pheromone-containing feces shields preweanling young from acute enteritis. To promote enteritis we subjected our pups to periodic cold shock. Cold, like other forms of stress, is known to shunt blood from the mesenteric vascular bed to the heart and brain [11]. This redistribution of blood produces splanchnic ischemia, leaving the mucosa of the gut susceptible to bacterial adherence and subsequent bacterial invasion [lO, 24, 33, 34]. We expected that pups stressed and denied access to pheromone-containing feces would have a high mortality, succumbing specifically to acute enteritis. We also expected that feeding deoxycholic acid to such young would significantly reduce their mortality. EXPERIMENT 1: DENYING YOUNG ACCESS TO THE FECES OF THEIR MOTHERS In this experiment we wanted to determine whether stressed young denied access to maternal feces are more likely to die from acute enteritis than stressed young allowed access to maternal feces. We also asked whether age at the time of stress affects the incidence of the disease, and hence mortality. Method

Experimental young were denied access to maternal feces using the method described previously by Lee and Moltz [19]. Briefly, it involved a cylindrical "cuff," fitted to the tail of the mother and positioned just under anus, which prevented all excreted boli from being deposited in the cage. The general health, maternal behavior and milk production of the mother were unaffected. The tail-cuff was fitted on day 10 postpartum and, except for daily emptying, remained in place until day 21 postpartum. A total of 48 litters was used, each consisting of six young. Half the litters were reared by tail-cuffed mothers and half by mothers that were simply handled for 5 min each day. Except for access to maternal feces, experimental and control litters were treated identically. Each litter was allowed to nurse from its mother and each was given unrestricted access to laboratory chow and water. The experimental and control litters were divided into three subgroups. Each subgroup contained eight litters. The fh'st subgroup was not stressed; the second was stressed daily between 15 and 21 days of age; the third between 21 and 29 days of age. Stress was applied by placing a litter at -20°C for 5 min per day. Every pup was examined daily for abdominal distension, diarrhea and blood in the stool, signs of acute enteritis. When a pup died, it was autopsied, and its large intestine was inspected for the presence of gross hemorrhagic sites. Each surviving pup was weighed at 15, 21, and 29 days of age. It was then sacrificed, and the weights of the stomach and cecum were recorded as an index of food consumption. All litters subjected to cold-stress were weaned at 21 days of age and placed in individual cages. Obviously none of the litters had access to maternal feces after separation from the mother.

LEE AND MOi~I'Z TABLE I PERCENT PUP MORTALITY No Stress

Stress 15-21 Days

Stress 21-29 Days

No Tail-Cuff

8.3

6.2

10.4

Tail-Cuff

2. I

22.9*

29.2t

*Significantly greater than corresponding subgroup at p~.0.05. tSignificantly greater than corresponding subgroup at p~<0.001.

Differences between the number of young that died in each subgroup were assessed statistically using the program of Bock and Yates [5] for log-linear analysis of mortality data. Weight measures were assessed using the multivariate programs of N i e e t al. [29] and Hull and Nie [13], with the litter serving as the unit of analysis. A multivariate approach was necessary since our weight measures were drawn from the same litters. Results and Discussion

Table 1 shows the percent young in each subgroup that died prior to 29 days o f age. Stress alone did not increase mortality: litters stressed but allowed access to maternal feces suffered a mortality no greater than litters that were not stressed. Nor did litters that were only denied access to maternal feces show an increase in mortality: in the absence of stress only 2.1% of the young died. What did increase mortality significantly was stress in conjunction with the denial of maternal feces. More than 22% of the stressed pups reared by tail-cuffed mothers died, while the mortality among pups that were stressed and allowed access to maternal feces was less than 11%. Just when the stress was applied evidently made little difference: among litters denied access to maternal feces, those stressed between 15 and 21 days of age had a mortality of 22.9%, while those stressed between 21 and 29 days of age had a mortality of 29.2%. The difference is not statistically significant (p>0.05). We observed such symptoms of acute enteritis as abdominal distension, diarrhea and blood in the stool among litters that were cold-stressed and denied access to maternal feces. The occurrence of these enteric symptoms was significantly higher than in any of the remaining subgroups (p<0.001). Moreover, autopsy of cold-stressed, feces-denied pups frequently revealed macroscopic hermorrhagic sites, further confirmation that death was the result of acute enteritis. But even so, might the high mortality among coldstressed, feces-denied young have been due to inanition rather than enteritis? This was clearly not the case. Although pups that were cold-stressed weighed significantly less than pups not cold-stressed (p<0.01), in only one instance did cold-stressed, feces-denied pups weigh significantly less than cold-stressed pups allowed access to maternal feces (Table 2). The fact that the former suffered a mortality of approximately 25% while the latter suffered not only a significantly lower mortality, but a mortality equivalent to control young, argues against death by inanition. So, too, do the weights of the cecum and stomach as measures of food intake. At autopsy on day 29, surviving cold-stressed, fecesdenied young had stomachs and ceca as heavy as young cold-stressed alone (p>0.05).

MATERNAL PHEROMONE AND SURVIVAL

933 TABLE 2 BODY WEIGHT

Treatment

Tail-Cuff

No Stress

No Yes No Yes No Yes

Cold Stress (15-21 Days) Cold Stress (21-29 Days)

Day 15¢ 32.7 26.0 28.5 25.2 29.8 28.3

___0.8 _+ 1.4" _+ 1.0 _+ 1.1 ___ 1.6 _+ 2.1

Day 21¢ 46.8 33.0 37.3 30.4 40.6 35.6

_+ 1.2 _+ 3.7* _+ 1.2 _+ 1.5" _+ 2.7 _+ 2.8

Day 29¢ 77.6 55.5 59.7 59.7 65.8 65.0

_+ 2.2 _+ 5.0* _+ 4.7 ___3.2 _+ 5.5 _+ 4.8

*Significant difference within treatments, p~<0.01. ¢All subgroups differed significantly from young that were neither stressed nor denied access to maternal feces.

It is evident that death occurred from stress-induced enteritis and that the consumption of maternal feces was protective. We have yet to demonstrate, however, that maternal feces are more protective than nulliparous feces. EXPERIMENT 2: FEEDING EXPERIMENTAL YOUNG FECES FROM EITHER MATERNALOR NULLIPAROUS FEMALES In the previous study, experimental young were not only denied access to maternal feces, but were denied access to "nulliparous feces" as well. However, nulliparous feces contain many of the same compounds as maternal feces, including deoxycholic acid. Although deoxycholic acid is present in nulliparous feces at significantly lower concentrations than in maternal feces [16], the fact that the former contains the steroid at all raises an important question: will the ingestion of nulliparous feces protect against acute enteritis as effectively as maternal feces? If it does, then it is difficult to see what enteric advantage the young gain by selectively consuming maternal feces. In the present experiment we gave cold-stressed young either maternal or nulliparous feces and recorded death from acute enteritis.

Results and Discussion

Litters that were stressed and given nulliparous feces suffered a mortality of 13.3%; in contrast, the mortality among litters that were stressed and given maternal feces was only 3.3%. The difference is highly significant (p<0.01). There were no significant differences in body weight on days 15, 21, and 29 between pups that consumed maternal feces and those that consumed nulliparous feces. Moreover, the weights o f the stomach and cecum did not differ at autopsy on day 29. It is evident here, as well as in the previous experiment, that inanition cannot explain the observed differences in mortality. Increased vulnerability to acute enteritis is a more likely explanation; all but one of the animals that died showed abdominal distension, diarrhea and blood in the stool. We now know that what protects against acute enteritis is not fecal consumption as such, but the consumption of maternal feces, whether from own mother or from other lactating females. It remains to be determined whether the effective ingredient in maternal feces is deoxycholic acid. EXPERIMENT 3: FEEDING DEOXYCHOLIC ACID TO COLD-STRESSED, FECES-DENIEDYOUNG

Method

A total of 10 litters was used. Each litter was coldstressed for 5 min per day from day 15 to day 29 of age. Each litter was also reared by a tail-cuffed mother. As in the previous experiment, the tail-cuff was fitted on day 10 postpartum and remained in place until day 21 postpartum. On day 21 mother and litter were separated. All pups had unrestricted access to laboratory chow and water. One half of the litters was offered maternal feces, that is, feces collected from colony females lactating from 16-21 days. The other half was offered nulliparous feces collected from colony females that had never been mated. Each type of feces was presented twice daily in dishes containing 810 g. We know from the work of Lee and Moltz [19] that when pups are not given a choice they will consume nulliparous feces in amounts equivalent to maternal feces. Thus any difference in mortality in the present experiment can be attributed to what the litters ate rather than to how much they ate. Each litter was weaned at 21 days of age, but continued to receive either maternal or nulliparous feces until 29 days of age. Pups that were alive on day 29 were sacrificed to obtain cecum and stomach weights.

There are many reports which address the question of whether bile acids inhibit the growth of aerobic and anaerobic bowel flora (e.g., [35,37]). Although no definitive answer has emerged, we agree with Brown et al. [6] that bile acids probably do not suppress enteric bacteria. However, our hypothesis does not relate deoxycholic acid to bacterial inhibition. Rather, it credits the steroid with the capacity to inactivate the endotoxins involved in the pathogenesis of acute enteritis. We expect, in other words, that oral administration o f deoxycholic acid will provide a "physicochemical" defense [4] in that it will split the toxic polymers of such microorganisms as E. coli into smaller, nontoxic structural elements [3]. Thus protected, the preweanling rat should be less vulnerable to acute enteritis. Method

A total of 24 litters was used, each reared by mothers fitted with tail-cuffs on day 10. As in Experiments 1 and 2, the tail cuffs remained in place until day 21. The mother was removed from the litter on day 21, so there was no time at which the young had access to maternal feces.

934

LEE AND MOLTZ

TABLE 3 EFFECT OF DEOXYCHOLIC ACID ON PERCENT MORTALITY AMONG COLD-STRESSED, FECES-DENIEDYOUNG Treatment No Dietary Deoxycholic Acid Dietary Deoxycholic Acid

Cold-Stressed 15-21 Days

Cold-Stressed 21-29 Days

4.2*

5.6*

1.4

1.4

*Significant difference within subgroups, p~O.05.

One half of the litters were cold-stressed for 5 min per day from 15-21 days. The remaining half were cold-stressed from 21-29 days. The two cold-stressed groups were divided into two subgroups. One subgroup was fed a wet mash of Teklad chow, the other a wet mash of Teklad chow to which 0.01% deoxycholic acid had been added. This dose of deoxycholic acid is identical to that used by Diaz et al. [8] in hand-rearing rat young. It also approximates the level of deoxycholic acid measured by Kilpatrick, Lee and Moltz [16] over 24 hr in the bile of normal colony young 14-26 days of age. Results and Discussion

Table 3 shows the percentage of young in each subgroup that died prior to 29 days of age. There is no doubt that dietary deoxycholic acid significantly reduced mortality among feces-denied young that were cold-stressed either from 15-21 days or from 21-29 days. As in Experiments 1 and 2, the observed differences in mortality cannot be accounted for by differences in food intake. Young fed deoxycholic acid were equivalent in body weight to young fed wet mash alone. They were also equivalent in the mean weights of the stomach and cecum. However, they were not equivalent in the occurrence of abdominal distension, diarrhea and blood in the stool, so that once again differential vulnerability to acute enteritis is a likely explanation of the observed difference in mortality. GENERAL DISCUSSION We began with the well-established phenomenon that developing rat young respond to a pheromone carried in the feces of maternal females. We noted that the young consume those feces selectively, and asked what adaptive advantage the consumption of such feces might confer. We suggested that fecal consumption might protect against stress-induced acute enteritis. Experiment 1 compared the incidence of mortality among cold-stressed animals denied access to maternal feces and those permitted access to maternal feces. The consumption

of maternal feces significantly reduced mortality from acute enteritis. Experiment 2 addressed the question of the specificity of fecal consumption. Will young which ingest the feces of nulliparous females be shielded as effectively from acute enteritis as young which ingest the feces of lactating females? The data leave no doubt that what is important is the consumption of maternal feces, whether from own mother or from other lactating rats. Experiment 3 tested the hypothesis that deoxycholic acid is the agent in maternal feces that provides enteric protection. The results were unequivocal: cold-stressed, fecesdenied young given dietary deoxycholic acid showed a significantly lower mortality than those not given dietary deoxycholic acid. There is little doubt that the consumption of deoxycholic acid in maternal feces protects against acute enteritis. But why did the percentage of animals succumbing to the disease vary so widely from experiment to experiment? Why, for example, did approximately 25% of the cold-stressed, feces-denied young die in Experiment 1 when only about 5% died in Experiment 3? Acute enteritis in rats, like necrotizing enterocolitis in babies [7] and colibacillosis in piglets [121], is a contagious disease characterized by unpredictable incidence. Within a given nursery or a given farrowing pen the incidence of necrotizing enterocolitis or colibacillosis is often high at one time and low at another without any ostensible change in environmental conditions. Apparently the same is true in a rat colony with regard to acute enteritis. What is important is that despite the unpredictable nature of acute enteritis, our hypothesis was confirmed in each experiment. In another series of experiments [19,20], we demonstrated that the ingestion of deoxycholic acid through maternal feces enhances the deposition of central myelin. Maternal feces therefore appears to play at least a dual role in rat development, protecting the gut and promoting maturation of the brain. These two benefits may have put a premium on sensitivity to a pheromone that attracts the pup to deoxycholic-acid-rich maternal feces. That is, during the evolution of the rat, a genetic program may have been selected that enhanced sensitivity to a specific odorant carried in the feces of the lactating female. We have previously shown [14,15] the odorant to be a prolactin-dependent derivative of biliary cholic acid. Pheromone feces are ordinary feces to which this derivative has been added, and are as such distinct from "caecotrophe feces," with which they have often been identified (e.g., [22,36]). Caecotrophe feces are one of two kinds of feces produced by the rabbit: excreted only at night [9], this distinctive fecal material is generated through the rhythmic action of a distinctive spindleshaped structure in the rabbit's proximal colon [31,32]. The characteristic intestinal structure and rhythm of the rat [121 give us no reason to suppose it capable of producing two distinct kinds of feces, and so we would not use the term "caecotrophe'" to refer to rat feces carrying the maternal pheromone.

MATERNAL

PHEROMONE

AND SURVIVAL

935 REFERENCES

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