V- 'I'“ ’I' W: Hill I I ll 034 ‘ N4> mm I I TH _ MORPHOLOGICAL, PHYSIOLOGICAL AND BEHAVIORAL CHANGES OCCURRING IN THE PRAIRIE DEER MOUSE, fEROMYSCUS MANICULATUS BAIRDII, DURING THE PROCESS OF DOMESTICATION Thesis For the Dogma of M... Sc MICHIGAN STATE UNIVERSI‘IY Edward 0. Price 1963 THESIS LIBRARY Michigan State University ABSTRACT MORPHOLOGICAL, PHYSIOLOGICAL AND BEHAVIORAL CHANGES OCCURRING IN THE PRAIRIE DEER MOUSE, PERONYSCUS MANICULATUS BAIRDII, DURING THE PROCESS OT DOMESTICATION by Edward 0; Price Body of Abstract AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1963 Edward 0. Price Three groups of Peromyscus maniculatus bairdii were employed to determine some of the morphological, physiological and behavioral changes that have occurred in the prairie deer mouse during twenty or more generations of laboratory breeding. Wild caught mice, the offspring of pregnant wild caught females born and reared in the laboratory and a semi- domesticated group were compared in regard to certain body and organ measurements, adrenal corticosterone output under stress and three behavioral tests measuring activity, gnawing and sand digging. Few definite trends were observed in the morphology of the three experimental groups other than that of body weight. In each of four cases, body weight was proportional to time spent in the laboratory with the laboratory mice exhibiting the largest mean body weight. These basic differ- ences in body weights were partly responsible for certain differences observed in organ-body weight ratios. Almost a two to one ratio was observed for the relative adrenal corticosterone output of wild caught and first genera- tion wild mice, respectively, under three hours of cold stress. The greater timidity and emotionality of mice with early experience in the wild was discussed as a possible ex- planation for this phenomenon. Wild caught mice showed significantly less activity in three behavioral tests measuring activity, gnawing and Edward 0. Price sand digging. The similarity in performance of first genera- tion wild and laboratory mice pointed to environmental factors as responsible for the relative inactivity of the wild group. MORPHOLOGICAL, PHYSIOLOGICAL AND BEHAVIORAL CHANGES OCCURRING IN THE PRAIRIE DEER HOUSE, PEROMYSCUS MANICULATUS BAIRDII, DURING THE PROCESS OF DOMESTICATION By Edward 0. Price A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1963 ACKNOWLEDGMENTS The author is extremely grateful to Dr. John King for instituting this study and for his advice and encourage- ment throughout its tenure and in the preparation of the manuscript. Appreciation is also expressed to Doctors Rollin Baker and Herman Slatis for their careful appraisal of this dissertation and to Dr. Phillip Clark for his advice concerning the statistical treatment of the data. Thanks also go to my wife, Mabell, who so willingly gave of her time to assist in collecting the morphological data and in typing the rough draft of this paper. The assistance of Mrs. B. Henderson in obtaining various materials for this study was also greatly appreciated. ii TABLE OF LIST OF TABLES . . . . LIST OF ILLUSTRATIONS. I. II. INTRODUCTION . 0 III 0 IIIETHODS o o o o 0 Subjects . . . LITERATURE REVIEW. Care and Handling. Morphology . . Physiology . . Behavior . . . Activity . . Gnawing. . . Sand Digging .3 IV. RESULTS. . . . . Morphology . . Physiological Analysis . . . . . . . . . . . Behavior . . . Activity . . Gnawing. . . Sand Digging V. DISCUSSION . . . Morphology . . Physiology . . Behavior . . . V I o S UI'IIVIA RY o o o o o LITERATUR CITED . . . CONTENTS Page . . . . . . . . . . . . ii . . . . . . . . . . . . iv . . . . . . . . . . o . V . . . . . . . . . . . . l . . . . . . . . . . . . 12 12 12 O O O O O O O O O O O 0 l3 0 O O O O O O O O O O 0 11+ 0 O O O O O 0 O O O O O 16 O O O O O O O O O O O 0 19 O O O O O O O 20 20 21 23 23 31 O O O O O O O O O O C 0 31 31 32 33 3H 3H 0 C 0 O O O O O O O O O 36 O O O O O I O O O O O O 37 40 #2 iii LIST OF TABLES Table Page 1. Morphological Measurements Collected (Indicated by X) According to Group, Sex, and Age in Months. . . . . . . . . . . . . . 1H 2. Numbers and Ages (In Months) of Mice, Employed in Three Behavioral Tests. . . . . . . . . . l9 3. Means and Standard Deviations for Body and Organ I‘IeasureInents o o o o o o o o o o o o o 23 u. Sex.Differences Within Treatment Groups for 'Body and Organ Measurements ("P" Ratios) . . 25 5. Age Differences Within Treatment Groups for Body and Organ Measurements ("P" Ratios) . . 27 6. Differences Between Treatment Groups, Keeping Ages and Sexes Separate. . . . . . . . . . . 29 7. Intergroup Differences for Those Measurements Having Significant "P" Ratios in Table 6 . . 30 8. Concentration of Corticosterone in Blood Plasma of Wild Caught and First Generation Labora- tory Stocks of P.m. bairdii. . . . . . . . . 31 9. Ranges, Means and Standard Deviations for Scores Obtained in a Tilt-Box Test for ACtiVity O O O C O O O O O O O O 0 O O O O O 32 10. Mean Number of Sticks Gnawed by Three Treat- ”Lent Groups 0 o o o o o o o o o o o o o o o o 32 11. Mean Ounces of Sand Dug by Three Treatment Groups 0 O O O O O O O O O O O O O O O O O O 33 iv Figure 1. LIST OF ILLUSTRATIONS Apparatuses Used in Three Tests of BehaVior. O O O O O O O O O O O Literature Review The practice of domesticating wild animals dates back to the time man began cultivating the soil (Dyson, 1953; Zeuner, 1956). Since that time a great variety of animals have been bred and reared in captivity for religious sacri- fices, food, draft purposes, pets, decoy hunting and more recently for scientific investigation. During the process of domestication, changes have occurred in the morphology, physiology and behavior of those animals involved. Breeding for size as in dogs and beef cattle has produced gross changes in the morphology of these animals when compared with their wild ancestors. Selection for the most gentle and productive females has resulted in as increase in fertility in many of our domesticated species (King, 1939; Richter, 1959; Castle, 19u7). Another change is the general lack of fear shown by domesticated species toward human beings (Hediger, 195%). Although tameness is essentially acquired, the process of domestication is basically a genetic alteration (Hale, 1962; Hediger, 195%; Richter, 195”) affect- ing the genotype and phenotype of organisms by way of evolu- tionary processes. Much is known concerning the genotype and phenotype response of animals to specified types of artificial selection (Lerner, 1958), but the effects of relaxed selections I 2 have been examined only in captive groups far removed from their wild counterparts. A change in selective pressure occurs when a species is removed from its natural habitat and placed in an artifi- cial environment. From the species standpoint this transfer is detrimental in that the stability attained by natural selection in the wild is broken down changing both the popula- tion structure and ultimately the genetic constitution of the species. Spurway (1955) has stated that in an artificial environment three types of selection pressures exist which are essentially unalterable: (1) selection exerted on the population by unstabilized patterns of development, (2) selection exerted by a reduced number of intraspecific mating partners, and (3) purposeful or unconscious selection exerted by man. Selective advantage will go to those individuals which show the greatest "fitness" to their new environment and only as the species approaches a new adaptive peak will stability again be attained. Without variation new adaptive peaks could not be achieved and it was Darwin (1875) who first wrote concerning the variation prevalent among domestic plants and animals. He ascribed the variability to: (I) changed conditions of life, (2) crosses between already existing breeds, and (3) selection by man, and concluded that variation and selection alone were responsible for the changes seen in domestic ani- mals over their wild counterparts. Darwin was a pioneer in the study of the anatomical 3 differences between domestic animals and their wild counter- parts. He stated (1875) that domestic rabbits have smaller brains per unit body size than wild species. In comparing wild and domestic Norway rats, C. P. Richter (1959), Donald- son and Hatai (1931), and King and Donaldson (1929), found that the relative brain weights of domestic rats are one- tenth to one-eighth smaller than those of their wild counter- parts. Herr (1955), in examining wild and domestic ducks found that the domestic strains invariably have the smaller brains, attributable to smaller cerebrums. Leopold (194u), reported that the brain of the wild turkey is 37% larger than that of the domestic bird. Agreement is found concern- ing the fact that domestic animals are usually larger in body size than their wild relatives, probably resulting from better living conditions and selection for size by humans. Darwin (1875) reports a smaller body size for wild rabbits and Richter (195”), and King and Donaldson (1929) found that wild Norway rats also tend to be smaller than the domestic strains. LeOpold (1949) observed that domestic turkeys are considerably heavier than either wild or hybrid birds. However, Zeuner (195”) reports a reduction in size of large and hard to manage domestic animals because of selection for tractable charac- teristics. He further states that changes in the skeleton of both body and limbs are generally species-Specific and that changes in the soft body parts occur mainly in the skin and in the length and texture of the hair. Another point which Zeuner Inakes is that domestication generally involves a reduction in u musculature, such as chewing muscles in domesticated carni- vores, and an accumulation of body fat which may partially account for the greater size of domestic animals. Relative size of adrenal glands in wild and domestic animals has received much attention in the literature and all reports agree that the wild strain has larger adrenals. Watson (1907) was the first to observe this difference and reported a three to one ratio in adrenal weight. King and Donaldson (1929) observed a 21% and 32% decrease in adrenal weight of male and female Norway rats, reSpectively, during ten generations of laboratory breeding. Donaldson (1928) and Richter (195%) found that the adrenal weights of domestic Norway rats were from one-half to one—fifth smaller than those of their wild counterparts and that most of this reduc- tion occurred in adrenal cortical tissue. That the wild rat adrenal is superior in secretory activity has been shown by Mosier (1957) who found a greater concentration of lipid, aldehyde, ketoniccarbonyl groups and a richer blood supply in the wild rat adrenal. Woods (1957) also demonstrated that metabolic depletion of the adrenal cortex was lacking in wild rats under conditions of acute stress which regularly produced a depletion of adrenal ascorbic acid and sudano- philic lipid in domestic rats. Leopold (iguu) observed that the adrenals of wild turkeys were twice the size of those of domestic turkeys. Crile (19u1) reported 25% larger adrenals in wild lions as compared with those of captive lions of the same body weight. 5 In regard to other morphological features, King and Donaldson (1929) found that in ten generations of laboratory breeding of the wild rat the thyroid decreased in weight while the hypophysis weight increased. Richter (195”) made similar observations when comparing wild caught Norway rats with a captive albino strain. They disagree, however, con- cerning the gonads in that King and Donaldson report 17% larger testes and 101% larger ovaries in wild rats when com- pared with albinos many generations removed from the wild while Richter failed to find any differences. Richter (1959) also demonstrated that the wild rat possesses the larger spleen, heart, kidneys, liver, pancreas, seminal vescicles, prostate and preputials while the domes- tic rat has the larger thymus and uterus. No differences were found in regard to the lungs. Leopold (lean) noted that the pituitary of the wild turkey is 50% larger than that of the domestic fowl, an observation supported by Richter (1959) in the Norway rat. Certain changes in the physiology of the adrenal gland due to domestication have been mentioned previously (Mosier, 1957; Woods, 1957). Nichols (1950) found twice as much choles- terol per unit of adrenal tissue in wild rats as in domestic rats. Immediately after capture, the adrenal of the wild rat lindergoes hypertrophy with a loss of cholesterol. After 24 Puours, the cholesterol content has returned to normal but tidere is more cholesterol per unit body weight in domestic rats tlian in wild ones. The gland finally returns to normal size a13ter ten weeks of captivity. Mosier and Richter (1958) 6 observed that whereas both wild and domestic Norway rats remained in good health when fed sodium chloride diets from .5% to 25%, both groups died on salt concentrations above 35% due to their inability to ingest sufficient food and water. On a low salt diet, the adrenals of the domestic rats showed a definite hyperplasia while no change was ob- served in the adrenals of wild rats, possibly because only low salt diets were available to rats in the wild state. A high salt diet resulted in atrophy of the zona glomerulosa in the adrenals of both strains. The loss of lipids and aldehydes was complete in domestic rats while wild rats showed only a partial loss. However, no differential reSponse to a high salt diet occurred in the zona fasiculata and zona reticularis, both showing an increase in lipids and aldehydes in both strains. Richter, Rogers and Hall (1950) noted that the response to adrenalectomy in both groups on "salt poor" diets was the same; both groups survived for only eight or nine days. With salt replacement after ad- renalectomy 87% of the domestic rats survived and all but 2% of the wild group died. The authors were in agreement that the wild rat is much more dependent on adrenal secretion than its domestic counterpart. Woods (1957) found that the adrenals of wild rats showed no hypertrOphy when exposed to 26 days of freezing temperatures while the adrenals of the domestic group increased 35% in weight. No change was observed in the as- cxorbic acid content of either strain. A differential response to gonadectomy was observed ifl wild and domestic Norway rats by Richter and Uhlenhuth 7 (195”). The domestic group exhibited a decrease in food and water consumption and an increase in body weight (due to in- activity) while little or no change occurred in the wild strain. Gonadectomy also produced a rise in the adrenal weights of the wild males and a decrease in the adrenals of the domestic females while the adrenals of the domestic males and wild females remained constant. The experimenters further noted that gonadectomy brought about almost total inactivity in the domestic rats but had little effect on the running activity of wild Norways. This they attributed to a greater dependence of the domestic rats on gonadal secretions. Griffiths (19””, l9”7) and Richter (195”) point out that domestic Norway rats are more likely to show convulsive seizures when exposed to auditory stimulation than are wild rats. Furthermore, both wild and domestic strains show fits when on a magnesium deficient diet but the wild group do not die as a result. In regard to the heart rates of gentled and non-gentled albino rats, Marcuse and Moore (19”3) found that the non- gentled group had a significantly faster heart rate than the gentled group in three out of four cases. Richter (195”), however, observed a marked slowing of the heart beat of wild rats when restrained while domestic rats showed little or no change. Richter (195”) also points out that the domestic Nk>rway rat has a lower metabolic rate than the wild rat as STIown by its lower food and water intake per unit body size. Hatai (19”5), studying the effect of long continued 8 exercise on certain body organs of the albino rat, found that the following organs were heavier in the exercised group, relative to body length: heart - 23.5%; kidney — 19%; liver - 17.5%; testes - 12.5%; ovaries - 8”.5%; and brain - ”.02%. The spleen, thyroids and eyeballs were heavier in the non-exercised group while no change in size was observed in body weight and length, alimentary tract and spinal cord. Hatai's findings agree closely with those of Richter (195”) in regard to the morphology of wild and domestic Norway rats. Wild rats presumably exercise more in their natural habitat than do strains in captivity. The fact that the various organ weights of wild rats closely parallel the weights ob- tained for the exercised group relative to corresponding values for the domestic and non-exercised groups, respectively, may represent a possible clue to the factors responsible for certain changes in the domestication process. A study by Richter and Rice (195”) on a closely related problem indicated that fasting produced a 1”2% increase in activity of wild rats on an activity wheel while the activity of the domestic group increased only 32%. From the standpoint of survival, these findings are quite pertinent in that selec- tion would favor the more active wild rat (under conditions of low food supply) since it would be the most likely to locate food. In regard to the effects of domestication on the Processes concerned with reproduction, Donaldson and Hatai (1911) state that the wild Norway rat breeds later, has larger litiers and a longer sex life than the domestic albino rat. 9 King (1939), however, found that after 25 generations of laboratory breeding the average reproductive period for the Norway rat was increased 8 months due to earlier breeding and a continuation of reproductive activities to an older age. Furthermore, fertility gradually increased from 3.5 young per litter by the first generation females to 10.18 young per litter by females of the 19th generation. In support of these findings, Richter (1959) points out that the reproduc- tive tract of the domestic rat begins to function earlier, the vagina Opens earlier and the estrous cycles are more regular in the females. This increase in fertility is no doubt favored by an either conscious or unconscious selec— tion for the most gentle and productive females. Castle (19”?) regards these changes as the consequences of muta- tions affecting behavior either directly or by way of changes in the endocrine system. Many wild animals are difficult to breed in captivity if breeding is accomplished at all. The failure of wild pintail ducks to breed in captivity led Phillips and Van Tienhoven (1960) to study the gonadal development of ducks caught in the wild when young and those reared from the eggs of wild parents. The arresting of gonadal development in the wild caught birds was found to be due to a lack of gonado— trophic hormones from the pituitary. This was confirmed by the fact that injections of chicken pituitaries produced normal ovarian development. Furthermore, gonadal development and pituitary gonadotrOphin content was greater in birds hand Feared from eggs of wild parents than in the wild caught birds, 10 indicating that early behavioral experiences may be involved in the reproductive failure of the captives. Leopold (l9””) found that wild turkeys seldom breed their first year while first-year domestic birds were con- sidered the most vigorous breeders. Furthermore, domestic turkeys started breeding activities two months before the wild birds in spring while hybrids followed domestic turkeys by only a month. Although no significant differences were found between the three strains in regard to clutch size, egg fertility or hatching success, the wild turkeys were more successful in rearing young in the wild. Wild females showed a greater ability to conceal their young, their young froze upon the approach of danger while hybrid young fled, and wild birds nested during more favorable weather than did domestic birds. The act of removing an animal from its natural habitat and placing it in a strange artificial environment invariably results in some form of psychological stress. The effect of this stress upon the physiology and behavior of the animal will depend on its ability to adapt to its new environment. The hyperexcitability of most wild creatures in captivity is evidenced by their wildness and timidity (Hediger, 195”). The Norway rat is no exception to this rule. Richter (195”) mentions that while domestic rats show only a mild reaction to handling, wild rats become extremely emotional often resulting in death. Wild rats were found to be much more cautious than domestic rats in accepting a change in diet and would often starve to death than eat poisoned food. 11 He goes on to state that when two wild rats are shocked they will immediately attack one another, while shocking two domestic rats results only in escape behaviour. Richter (195”) and Barnett (1960) found that wild rats attack and kill strange rats and mice introduced into their cages while albino rats show little aggression toward strange animals. Barnett also pointed out that hybrid rats, obtained from an albino-wild cross also showed conflict under similar conditions but of a lesser intensity. The albino rats appeared to lack the repertoire of aggressive and "amicable" social signals common to the wild rats. Hediger (195”) points out that the process of domestica- tion necessarily involves hereditary factors while tameness is merely an acquired behavioral trait. By a series of be- havioral tests Yerkes (1913) and Coburn (1922) were able to demonstrate in rats and mice, respectively, that savageness, wildness and timidity are heritable behavior complexes. In each successive generation these characteristics showed a definite drop in intensity. A decrease in savageness, wild- ness and timidity also accompanied repetition of the tests administered, but rearing by a wild mother and tame father or vice versa had little effect on the inheritance of these traits. Furthermore, if both parents were wild, the majority of the offspring would be wild while the majority of offSpring born to tame mice would be tame. King (1939) pointed out that rats did not lose their high nervous tension and fear of man until the 25th generation removed from the wild. Dawson (1932), experimenting with wild and domestic mice, found that 12 the wild mice traversed a given runway in the shortest amount of time. He further observed that the offspring of a wild and tame cross were nearly as fast as the wild mice, suggest- ing that he was dealing with a heritable behavioral response. Leopold (l9””) found the wild turkey extremely wary and much less tolerant of disturbances than either hybrid or domestic birds. He also discovered that in the process of domestication, hybrid and domestic juveniles lost their ten- dency to "freeze" or hide in the face of natural enemies, a response indicative of relaxed selection pressures. Introduction A review of the literature pointed out that certain changes in the morphology, physiology and behavior of animals occurs during the process of domestication. The purpose of the present study was to determine what changes have occurred during approximately 20 generations of laboratory breeding of the prairie deer mouse, Peromyscus maniculatus bairdii. Com- parison was made between wild caught mice, first generation laboratory mice (the laboratory raised offSpring of pregnant wild caught females), and semi-domestic mice about 20 genera- tions from the wild. The first generation laboratory group was employed as a measure of the relative importance of genetic versus experiential factors contributing to the differences observed. Methods Subjects Wild Caught. Seventeen male and twelve female deer mice were l3 live-trapped in October and November, 1962, four miles south of East Lansing in Ingham County, Michigan. In January and March, 1963, ten male and ten female P.m. bairdii were kill-trapped five miles south of Okemos, Michigan, also in Ingham County. First Generation Laboratory. Twenty male and twenty-four female offspring of pregnant wild caught females were born in the laboratory and raised by their wild mothers until weaning at 21 days of age. The parent mice were caught in live-traps during April, 1962, about four and seven miles south of East Lansing, Michigan. The mice in this group were chosen from approximately ten different litters. Semi-Domestic. The ancestors of the semi-domestic group were caught in the vicinity of Ann Arbor, Michigan, in 19”8. Forty-nine descendents of each sex were obtained from the colony of Dr. John King and employed in the present study (Harris, 195”). Having been reared in the laboratory for nearly fifteen years, these mice were estimated to be about 20 generations removed from the wild. Seventy-eight of the mice used in the morphological study were sacrificed as part of a mating experiment. Care and Handling The mice were housed individually in clear flastic cages (5" by 11" by 6" deep) from the time of weaning (21 days) or capture, as in the case of the wild caught group. Wood- shavings were used to cover the bottom of the cages and cotton l” was provided for nesting material. A surplus of food and water was on hand at all times. The mice remained undisturbed except when being tested or for cage cleaning about every three weeks. Handling was accomplished by means of 12" metal forceps with rubber-covered tips. Morphology Table 1 summarizes the numbers and approximate ages of the mice used in collecting the morphological data. TABLE 1 MORPHOLOGICAL MEASUREMENTS COLLECTED (INDICATED BY X) ACCORDING TO GROUP, SEX, AND AGE IN MONTHS Measurement Wild Caught lst Gen. Lab. Semi-Domestic 6 10-12 8-9 16 6-12 7-8 12-1” mgmgalga'ga' 3‘3 Body Weight X X X X X X X X X X X X Skull Length X X X X X X X X X X Brain X X X X X X X X X X Eyeball X X X X X X X X X X Lens X X X X X X X X X X Heart X X X X X X Kidney X .X X X X X Spleen X X X X X X Testis X X X Adrenals X X ' X X Total No. 10 10 17 12 10 10 10 l” 39 39 10 10 Dissections were performed on ten wild caught (snap- trapped), ten first generation laboratory and 39 semiedomestic mice of each sex. The following measurements were obtained: (1) body weight, (2) skull length, (3) brain weight, (”) eyeball weight, (5) lens diameter and weight, and (6) adrenal 15 weights. All weight measures were from fresh tissues weighed on a "Federal Pacific" precision balance. While the brain was being removed the various organs were placed in saline solution until they could be cleaned and weighed. Since the wild mice were caught during the months of March and April, their average age was estimated at six months since Howard (19”9) found that over 75 per cent of all early Spring-caught P.m. bairdii are born the previous September and October. The first generation laboratory mice were eight to nine months of age when sacrificed. The age of the semi—domestic males ranged from six to twelve months with the majority falling in the seven, ten and eleven month categories while the majority of the females were seven or eight months of age. In several cases the snap-traps damaged parts of the wild caught mice and certain measurements could not be made. If a mouse was tail-caught the adrenals were discarded and when part of the body was consumed by shrews a body weight measurement was not taken. When pregnant females were ob- tained, the adrenals were discarded and body weight was ob— tained by subtracting the weight of the fetuses from the total weight. Subsequent to this study an additional 73 mice were sacrificed to obtain blood plasma for the corticosterone lanalysis discussed shortly. These mice were also dissected ‘to increase the sample size and gather additional morphological data. Seventeen male and twelve female wild caught mice, kept in the laboratory for nine months and estimated to be ten to 16 twelve months of age, were examined in regard to: (1) body weight, (2) skull length, (3) brain weight, (”) heart weight, (5) left kidney weight, (6) spleen weight, (7) left testis weight in males, (8) right eyeball weight, and (9) right lens diameter and weight. The same measurements were obtained from ten male and fourteen female first generation laboratory mice at an age of 16 months. In addition, ten semi-domestic mice of each sex were sacrificed at 12 to 1” months of age and data was obtained on the following: (1) body weight, (2) heart weight, (3) kidney weight, (”) spleen weight, (5) left testis weight in males. Differences due to sex, age and treatment groups were tested by means of analysis of variance techniques. In most cases, the ratios of organ weight to body weight were used as the criterion for comparison except for the eye measurements where skull length was more closely correlated. Physiology A biochemical analysis of the relative corticosterone levels in the blood plasma of the three treatment groups under cold stress was made for a physiological measure. Re- cent work (Schaporo, Geller and Eiduson, 1962; Timmer, 1962; Hyde and Skelton, 1961) has shown that in animals where cor- ticosterone is the principle adrenal steroid, its concentra- tion in the blood plasma of the animal concerned is propor- tional to the amount of stress the animal is experiencing. Halberg, Peterson and Silber (1959) have demonstrated that corticosterone is the principle adrenal steroid in mice and l7 Eleftheriou (person. comm.) has confirmed this finding for the prairie deer mouse. Seventeen male and twelve female wild caught 3:2; bairdii, ten male and fourteen female first generation laboratory mice and ten semi-domestic mice of each sex were subjected to a H5°F temperature for three hours. On the day preceding their sacrifice, the animals were individually marked and weighed. Each mouse was put in an empty cage the following morning and placed in the cold room at 9:00 a.m. At 12:00 noon, three hours later, they were removed and placed in an isolated room for one hour. The purpose of the hour waiting period was to facilitate blood removal by permitting expansion of the blood vessels following a contraction period during the cold exposure. At 1:00 p.m., after the waiting period, the animals were individually re- moved from the isolated room and decapitated in less than #5 seconds. The blood was collected by means of a powder funnel coated with "Anti-foam A" silicone spray to facilitate blood flow and a 50 ml. test tube containing heparin to prevent coagulation. Following collection, the blood was immediately centrifuged and the plasma removed and frozen. The pooled blood from the complete N of each group was necessary to make one determination. Plasma aliquots were varied depending on the amount of plasma available. The technique employed for measuring the plasma corti- costerone was one established by Silber, Busch and Oslapas (1958) and modified by Peron and Dorfman (1959). Briefly, it 18 consists of: (l) washing a blank, two standards of known corticosterone concentration and four plasma aliquots (made up to two ml. with 13% EtOH) with five ml. of ligroin; (2) extracting the corticosterone with five ml. of dichloromethane; (3) washing the latter with 1.0 ml. of ice cold .lN NaOH; and (H) re—extracting the corticosterone with three ml. of 30M H280”, and reading the fluorescence after H5 minutes. Each washing or extracting step involved shaking the tubes vigorously for one minute, centrifuging for five minutes at 0°—5° C. and aspiration of thetnpmost layer. The sulfuric acid acts on the corticosterone molecule (McGowan and Sandler, 1961) inducing a fluorescence that is maximal in light with a wave length of 955 to #60 mP.(Sweat, 1959), 30-90 minutes after addition of the sulfuric acid (Silber, Busch and Oslapas, 1958). Fluorescence was read on a fluorimeter after adjusting the bhnk reading to zero. Aliquots containing .1 and .2 Pg of corticosterone were used to establish the standard curve for each determination while four aliquots of plasma up to .8 ml. were used to establish the plasma curve. The corticos- terone concentration in Pits/100 ml. of plasma was then deter- mined by extrapolation. To achieve absolute cleanliness and absence of cations, the glassware was soaked in concentrated nitric acid overnight and then rinsed in de-ionized and double distilled water. Only the purest chemicals available were used to avoid possible GPPOI‘S o 19 Reagents: - 30 N H280” (DuPont's Reagent Grade) - 8 vol. H2804 mixed with 2 vol. distilled water. - 0.1 N NaOH — .1 mol. weight in 1000 mls. water - 13% ethyl alcohol - distilled from 95% EtOH and 2,4 dinitrophenylhydrazine and redistilled (95% pure); make up 13% by volume. - Dichloromethane (Spectro Grade - Eastman Kodak) - washed with equal volume of water, dried with anhydrous NaZSOu, kept over NaOH flakes for 24 hours and distilled (b.p. uo—u1°c.) (Saffran, 1955). - Ligroin (b.p. 60°C. Eastman Kodak) - purified by permanganate. - Standard Corticosterone (U-HMBO, Upjohn, Kalamazoo, Michigan) - 10 mg. weighted out and dissolved in EtOH and diluted with water to desired concentration. Behavior” The behavioral tests employed were concerned with the areas of activity, gnawing and sand digging. The numbers and ages of the mice used are summarized in Table 2. TABLE 2 NUMBERS AND AGES (IN MONTHS) OF MICE EMPLOYED IN THREE BEHAVIORAL TESTS WiId Caught lst Gen. Lab. Semi-Domestic N Age N Age N Age Te“ 0" 9 5'89 5‘ 9 6199 a 9. a} Activit '10 10 1-6 10 10 7-10 10 10 5-9 Gnawing 7 3 M-7 5 5 10 5 H 8-10 Sand Digging 7 3 u-7 5 5 10 6 u 8-10 20 Activity. Activity tests began on wild caught mice from one week to two months after being brought into the laboratory. Nearly three months laboratory experience had elapsed before they were tested for gnawing and sand digging. Activity was measured by means of a tilt box consist- ing of a 5" by 11" by H" deep plastic container covered with l/H" wire mesh that was free to rock on a wire shaft running through the center of the box. Each time the animal moved away from the cage's center the rocking of the box depressed a microswitch connected to a counter (Figure l). Sixty mice, ten of each sex and group were tested in the apparatus. A red neon light served as the dark cycle during all but the 6:00 a.m. to 8:00 a.m. period when in- candescent lights were on. The temperature in the experi- mental room averaged slightly over 70°F. Each mouse was placed in the tilt box 16 hours (4:00 p.m. to 8:00 a.m.) for five nights without food, water or nesting material. The first test period allowed the animal to explore the apparatus and becomeencustomed to the test situation. Each night thereafter a mouse was placed in a different tilt box and its mean score for the four test nights was computed. The rotation to a different box each night was designed to eliminate any errors due to differences in sensitivity of the four experimental apparatuses. The Mann Whitney U-Test was applied to the mean scores after the sexes were combined for each group. Gnawing. A second behavioral measure was concerned with the gnawing activity of ten wild caught, ten first 21 generation laboratory and nine semi-domestic mice. The apparatus consisted of a plywood frame divided into five runways 22 1/2" long, 3" wide and H" deep. With no floor, it was possible to remove all feces, odors, etc., after each run by cleaning the surface on which the apparatus was placed. The top was covered with clear Plexiglas to admit the limited amount of light given off by the red neon light in the test room. Four 2" by H" blocks 3 inches wide and spaced H" apart were placed in each runway. In the center of each block a l 114" diameter hole was drilled and a series of fourteen one-eighth inch holes were drilled from top to bottom of each block passing through the l 1/H" hole. Balsa sticks one-eighth inch by one-eighth inch were inserted into the fourteen holes and passing through the large opening provided a barrier to reach- ing the next compartment. The number of gnawed sticks that had to be replaced from a 30-minute testing trial were recorded and the mean scores for two consecutive daily test periods were computed and applied to a Mann Whitney U—Test for signifi- cance. Sand Digging. The sand digging apparatus consisted of a clear plastic cage (5" by 11" by 6") connected by an L- shaped section of 1 1/4" diameter plastic tubing to a funnel filled with dry sifted sand. The bottom of the cage was cut out and covered with a section of 1/4" wire mesh through which sand could easily pass when dug out of the tubing. The same mice as used in the gnawing test, with one additional male semi-domestic mouse, were tested for 30 minutes on two 22 FIGURE 1 APPARATUSES USED IN THREE TESTS OF BEHAVIOR I 9 ll —) ACTIVITY m1: 301: '7 I I “HQRO- J sung» E J dmwm BLOCK +— w- SLN'D DIGGING J consecutive days. 23 Group differences in the mean ounces of sand dug per day per individual were tested with the Mann Whitney U-Test. Morphology and organ measurements are presented in Table 3. Results Means and standard deviations for the various body TABLE 3 MEANS AND STANDARD DEVIATIONS FOR BODY AND ORGAN MEASUREMENTS Wild Caught lst Gen. Lab. Semi-Domestic Age (mo.) 6 10-12 8-9 16 6-12 12-19 X’ 15.50 17.53 15.05 17.31 18.11 18.95 Body 6" 5.0. 3.00 1.75 1.99 1.35 1.75 2.83 Weight 2_ x . 19.92 15.58 15.19 15.99 15.50 18.55 570. 3.21 3.20 1.85 2.00 1.80 2.09 x 22.78 23.37 23.21 23.27 23.30 Skull 5” 5.0. 1.07 0.55 0.93 0.98 0.53 — Length g_ x 22.90 23.35 23.90 23.50 23.32 5.0. 0.59 0.73 0.75 0.57 0.57 2' 529.90 507.71 592.50 511.00 520.02 Brain 64 5.0. 95.09 27.92 38.51 35.87 30.05 Weight .9 2' 515.99 999.82 595.95 529.51 519.13 5.0. 28.27 15.18 99.28 35.80 27.55 6” 2' 35.03 29.17 39.19 29.52 28.91 Frain Wt. S.D. 5.95 2.58 9.09 2.38 2.52 Body Wt. .9 2' 35.99 32.85 39.99 2132.98 31.79 5.0. 8.62 9.98 9.78 9.57' 3.08 - 2' 39.99 97.99 99.37 99.57 50.10 Eye 6” S.D. 9.75 2.89 2.11 3.33 3.71 Weight ‘g 2' 90.77 99.70 99.92 95.55 99.72 P 5.0. 3.83 3.38 2.72 2.99 9.79 2” TABLE 3--Continued Wild Caught lst Gen. Lab. Semi-Domestic Age (mo.) 5* 10-12 849 715 5-12 12-19 X' 1275’ 2.13 2113 2.11 2.15 Ege Wt. 62 5.0. 0.21 0.20 0.10 0.19 0.15 u gt 3_ x 1.82 2.13 2.21 1.98 2.13 5.0. 0.18 0.12 0.27 0.12 0.19 x 15.92 18.92 18.55 19.37 18.35 Lens 6” 5.0. 2.82 1.05 0.95 1.37 1.95 Weight g X 15.19 19.08 18.80 18.83 17.85 5.0. 2.19 1.59 1.25 1.18 1.89 x 0.57 0.81 0.80 0.83 0.79 Lens Wt. 31 5.0. 0.10 0.09 0.09 0.05 0.08 Skull Lgth 2 0.57 0.82 0.80 0.80 0.77 5.0. 0.09 0.05 0.05 0.09 0.08 2’ 111.55 107.15 105.53 Heart 0" 5.0. 10.59 15.58 17.75 Weight ‘2 2' 100.95 108.11 105.89 5.0. 15.83 20.37 13.09 . X 5.38 5.22 5.79 Heart Wt. 51 5.0. 0.53 0.80 0.50 Body Wt. p, x 5.55 5.75 5.77 5.0. 0.71 0.55 0.38 2 119.19 111.19 121.08 Kidney 61 5.0. 15.30 19.90 20.10 Weight _g ‘2 113.50 119.19 115.99 5.0. 19.25 21.52 15.68 2 5.50 5.91 5.58 Kidney Wt.61 5.0. 0.57 0.57 0.78 Body Wt. p, 2' 7.38 7.15 5.31 5.0. 1.19 0.98 0.57 x 23.52 18.08 21.79 Spleen 57 570. 9.53 9.33 7.27 Weight 3_ Y 19.57 22.05 22.92 5.0. 9.20 7.59 9.39 25 TABLE 3-—Continued Wild Caught lst Gen. Lab. Semi-Domestic Age (mo.7' 5 *10-12 8-9 15 5—12 12-19 N 1.33 1.05 1.13 Spleen Wt. 5.0. 0.99 0.28 0.38 Body Wt. 7' 1.28 1.36 1.20 S.D. 0.26 0.35 0.38 Testis R 123.92 132.36 108.56 Weight 5.0. 38.97 90.59 32.95 Testis Wt. X 7.07 9.71 5.91 Body Wt. 5.0. 2.32 9.95 1.79 X 2.80 3.74 Combined S.D. 0.54 1.09 Adrenal Weight Y' 2.53 3.32 S.D. 0.83 1.25 Sex differences for the various body and organ measure- ments were examined by an analysis of variance with a two- tailed test of significance. Table 4. The results are summarized in TABLE 4 SEX DIFFERENCES WITHIN TREATMENT GROUPS FOR BODY AND ORGAN MEASUREMENTS. ("F" RATIOS) ‘ Wild Caught lst Gen. Lab. Semi-Domestic .____nge (mo.) 6 10-12 8-9 16 6-12 12-14 B<>dy Weight .238 4.453 .024 3.039 13.228** .008 Sl