oLFAcmFY CONTROL OF MAFFRNAL BEHAVIOR ‘ THE EFFECTSOF EXPERIENCEAND- AGE _ - ~ INPERDMYSCUSEREMICUS . =3” ‘ " Dissertation foF the Degree of Ph. D MICHIGAN STATE UNWERSITY ‘ ' ROBERT SILBER ' 1974 » fi‘i‘fi 7 LIBRARY Michigan Stat: University This is to certify that the thesis entitled OLFACTORY CONTROL OF MATERNAL BEHAVIOR: THE EFFECTS OF EXPERIBRCB AND ASK IN PERWYSCUS EREMICUS presented by ROBERT SILBER has been accepted towards fulfillment of the requirements for rum. deg“... in zooch gfwwxa G . $7121.12. Major professor ABSTRACT OLFACTORY CONTROL OF MATERNAL BEHAVIOR: THE EFFECTS OF EXPERIENCE AND AGE IN EEROMYSCUS EREMICUS BY Robert Silber The importance of olfactory cues from Peromyscus eremicus pups with regard to maternal behavior was investi- gated via alteration of pup odor. Anise oil scenting of pups affected retrieval, weight gain and nipple attachment. Responses observed were influenced by pup age and mother's experience with anise- scenting. A series of experiments produced the following results: 1. Mothers displayed retrieval preferences for non- scented pups. 2. Early (day 1), sustained anise-scenting of pups did not alter maternal responsiveness. 3. Late (day 9), single dose scenting reduced maternal care-taking behavior as measured by pup weight changes and reduced occurrence of pup nipple attachment. Robert Silber Reduced occurrence of scented pup nipple attach- ment was due to maternal rejection and not to altered ability of pups to grasp or locate nipples. Prior experience Wlth anise-scented pups did not reduce and may have increased the deleterious effects of late anise-scenting mentioned above. OLFACTORY CONTROL OF MATERNAL BEHAVIOR: THE EFFECTS OF EXPERIENCE AND AGE IN PEROMYSCUS EREMICUS BY Robert Silber A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1974 ACKNOWLEDGMENTS I wish to thank my guidance committee for their help in the development of this thesis and in the preparation of this manuscript. Dr. J. C. Braddock offered guidance, criticism and provocative, open-minded discussion. Dr. W. E. Cooper rendered particularly rigorous criticism which greatly improved the manuscript. Dr. S. Stephenson and Dr. R. Baker also provided criticism and commentary which was most helpful. Dr. J. King generously provided me with laboratory and desk space, animals and many hours of useful dis- cussion. Dr. J. Gill kindly lent much time and aid in the preparation of the statistical analyses presented here. To my wife Georgette, whose support, help, and understanding made completion of this work possible, goes love and gratitude for which I can find no meaningful adjectives. Thank you all. Bob ii LIST OF TABLES . . LIST OF FIGURES. . PREFACE . . . . INTRODUCTION. . . Olfaction and Soc The Questions. . ANIMALS . . . . TABLE ial OF Behavior . Olfaction and Maternal Behavior. Experimental Methodology and Critique. . . EXPERIMENT l: RETRIEVAL. Materials and Methods Subjects. . . Test Apparatus. Procedure . . Measurement and Observations . Results . . . Conclusions. . EXPERIMENT 2: ALTERED SCENT, PRIOR AND PUP AGE . . Introduction . . Materials and Method Animals . . . Procedure . . Results . . . Pup Weight Chan ges iii CONTENTS Page vi 11 15 18 19 19 19 21 21 22 23 24 24 25 25 26 27 28 Page Intra-Group Comparisons: Days 1-9 . . . . . 30 Inter-Group Comparisons to WC: Days 1-9 . . . 30 Inter-Treatment Group Comparisons: Days 1—9. . 33 Intra-Group Comparisons Days 9-12 . . . . . 33 Inter-Treatment Group Comparisons: Days 9-12- . 34 Inter-Group Comparisons to WC: Days 9-12. . . 3S Pup Presence on the Nipple: Day 10. . . . . 35 Intra—Group Comparisons: Day 10. . . . . . 36 Inter-Group Comparisons: Day 10. . . . . . 38 Numbers of Animals Showing Weight Change: Days 1-2, Days 9-10 0 o o o o o o o o 40 EXPERIMENT 3: PUP SUCKLING. . . . . . . . . 43 Materials and Methods . . . . . . . . . . 44 Measurements . . . . . . . . . . . . . 44 Results 0 O O O O I O O O O O O O O 0 4S Pup Ability to Grasp Nipple: Day 9. . . . . 45 DISCUSSION 0 I O O O O O O O O O O O O 46 SUMMARY 0 O O O O O O O O O O I O O O 53 BIBLIOGRAPHY O O O O O O O O O O O O O O 54 iv LIST OF TABLES Table Page 1. Paired t-tests: Within treatment group comparisons of experimental and control pup mean differences of a.d.g. days 1-9 . . . 32 2. tBonferoni= Comparisons of mean a.d.g. of treatment group controls to WC animals days 1-9 0 o o o o o o o o o o o 32 3. tBonferoni: Comparison of mean differences of a.d.g. between experimental groups days 1-9 0 o o o o o o o o o o o 33 4. Paired t-tests: Within-treatment group com- parisons of experimental and control pup mean differences of a.d.g. days 9-12. . . 34 5. tBonferoni= Comparison of mean differences of a.d.g. between experimental groups, days 9.12. o o o o o o o o o o o 35 6. tBonferoni: (1) Comparisons of mean a.d.g. of treatment group controls to WC animals days 9-12, (2) comparison of DOB a.d.g. to DOBE a.d.g. . . . . . . . . . . . 36 7. On and off the nipple intra-group x2 analysis day 10 O O O O O O O O O O O C O 37 8. On and off the nipple inter-group x2 analysis day 10 O O O O O O O I O O O O O 39 9. Numbers of pups showing weight loss--inter- group chi square analysis days 1-2 and 9-10 0 o o o o o o o o o o o o 41 10. On and off the nipple anesthetized mothers scented vs unscented pups . . . . . . 45 LI ST OF FIGURES Figure Page 1. Retrieval test apparatus . . . . . . . . 20 2. Experimental design and comparisons . . . . 29 3. Bonferoni t-test. . . . . . . . . . . 30 4. Average daily gain data . . . . . . . . 31 vi PREFACE Much recent research has focused upon olfactory control of maternal behavior in rodents. However, the author perceives several problems with current experimental methodologies which may have generated confounded results. Presented below is a literature review of works dealing with olfaction and a variety of social behaviors; a review and critique of research in maternal behavior and olfaction follows. A series of original experiments is described and the results discussed in light of other findings. INTRODUCTION Olfaction and Social Behavior Most mammals possess an excellent capacity for per— ception of chemical cues; such cues are frequently used as signaling devices (Heidiger, 1950; Tembrock, 1968) and often play key roles in a wide variety of rodent social contexts (for review see: Schultz & Tapp, 1973; Bronson, 1971; Gleason & Reynierse, 1969; Myktowycz, 1970). Among rodents, odor cues affect aggression displayed by a resident toward a conspecific intruder (e.g., Alberts & Galef, 1973; Jones & Nowell, 1973a; Devor & Murphy, 1973; Mackintosh & Grant, 1966) and may affect interspecific aggression (e.g., Pohorecky, Larin, & Wurtman, 1969; Spector & Hull, 1972; Bandler & Chi, 1972; Ropartz, 1968). Intra-specific dominance behavior of rodents also is mediated by olfactory cues (e.g., Desjardins, Maruniak, & Bronson, 1973; Rieder & Lumia, 1973; Christenson, Wallen, Brown, & Glickman, 1973). Specific glands have been identified as sources of aggression affecting pheromones in rodents (Bronson & Marsden, 1973; Jones & Nowell, 1973b; Mugford & Nowell, 1971). Attraction to conspecific scented areas (Beauchamp, 1973; Devor & Schneider, 1974; Gregory & Pfaff, 1971; Baran & Glickman, 1970), to self-scented areas (Baran, 1973), and avoidance of conspecific scented areas (Jones & Nowell, 1973b,c; Thiessen & Dawber, 1972) have been demonstrated in rodents. Reactivity to odor has been suggested as a Species—isolating mechanism (Moore, 1965; Godfrey, 1958). Recent work has disclosed a number of relationships between odors and female rodent fertility. The odor of strange males can block pregnancy or cause premature disruption of gestation (Bronson & Eleftheriou, 1963; Bruce, 1959, 1960a,b; Bruce & Parkes, 1960a,b; Eleftheriou, Bronson, & Zarrow, 1962). Male odors can also induce estrous synchrony (Bronson & Marsden, 1964; Whitten, 1956a; Whitten, Bronson, & Greenstein, 1968) and accelerate female sexual maturation (Colby, 1972; Vandenbergh, 1969). Under crowded conditions, the odor of female mice (Mug muSCUIgg) can cause pseudo-pregnancy (Lee & Boot, 1955, 1956) or temporary anestrous (Whitten, 1957, 1959). Removal of a female's olfactory bulbs produces several, sometimes contrasting, effects. In mice, bulbectomy can end the pregnancy block effect of strange males (Bruce & Parrot, 1960), produce atrophy of ovaries and uterus (Whitten, 1956b; Gandelman, Zarrow, & Denenberg, 1972), disrupt estrous cycling (Vandenbergh, 1973; Whitten, 1956b), and lower fertility (Lamond, 1958; Vandenbergh, 1973; Gandelman, Zarrow, & Denenberg, 1972). In the rat (Rattus norvegicus), frequency of pregnancy is not affected by bulbectomy (Reiter, Sorrentino, & Ellison, 1970; Orbach & Kling, 1966), although the onset of puberty is retarded (Orbach & Kling, 1966) and copulatory behavior is modified (Moss, 1971). In the golden hamster (Mesocricetus auratus), estrous cyclicity may be upset (Carter, 1973) as may aggressive and defensive behavior patterns (Leonard, 1972). Male rodents appear, in some degree, to depend upon olfaction for sexual performance, though the picture is confused by species differences and experimental methodology (see below). Beach (1942) concluded that odor cues were important for elicitation of male mating behavior in the rat. Wilhelmson and Larson (1973) supported this conclusion, but also showed that social experience with a female prior to olfactory bulbectomy could improve male performance. In mice, simultaneous bilateral bulbectomy neutralized mating behavior in experienced males (Rowe & Smith, 1973; Rowe & Edwards, 1972). In sexually-experienced males, chemically induced peripheral anosmia and unilateral bulbectomy had no effect on mating behavior (Rowe & Smith, 1972; Rowe & Edwards, 1972). Successive bulbectomy (removal of one lobe separated by 30 days from removal of the second lobe) produced no effect, while simultaneous bulbectomy, as mentioned above, prevented mating behavior (Rowe & Smith, 1973). Sexual experience between operations did not affect the performance of operated males. In male golden hamsters, odor also appears to be important for sexual performance, but experimental results are confusing and contradictory. Vaginal secretions con- tain a substance that attracts males and stimulates mounting when applied to inappropriate objects (Johnston, 1972). Treatment of olfactory epithelium with a zinc sulphate solution can produce short term anosmia (e.g., Alberts & Galef, 1971; Takagi, 1971; Mulvaney & Heist, 1971). With regard to male hamster sexual behavior, such treatment has produced mixed results. Lisk, Zeiss, and Ciaccio (1972), Murphy and Schneider (1970), and Devor and Murphy (1973) all reported elimination of mounting behavior in treated males. However, Powers and Winans (1973) reported that both sexually naive and experienced males mated normally after zinc sulphate treatment. Anosmia produced by olfactory bulb lesions or bulb removal generates more uniform results. In addition to the studies cited above on rats, Doty, Carter, and Clemens (1971), Devor and Murphy (1973), Murphy and Schneider (1970), and Powers and Winans (1973) all reported loss of mating behavior in male golden hamsters following destruction of the olfactory bulbs. Olfaction and Maternal Behavior A female mammal with young performs a number of behavioral patterns termed "maternal" (e.g., retrieving, licking, nursing, crouching over young, nest building, defending young), but a mocher does not behave maternally towards all objects. She only behaves maternally towards those objects emitting the "correct" cue or cues. Therefore, we can try to determine what specific stimuli from young are important cues for the elicitation of maternal responses. Since olfactory cues play roles in the rodent social contexts discussed above, it is reasonable to assume that olfactory information is significant in the context of maternal behavior. A survey of the literature reveals that this is so. Among larger mammals, olfactory cues appear to play an integral role in maternal care-taking behavior. In goats (Blauvelt, 1954; Colias, 1956; Klopfer, 1971; Klopfer, Adams, & Klopfer, 1964; Moore, 1968), sheep (Moore, 1968; Lamond, 1949; Hersher, Richmond, & Moore, 1963), elk and caribou (Altmann, 1952; deVos, 1960), dogs (Johnson, Goy, & Michiels, 1962; Menzel & Menzel, 1953), and in the Alaskan fur seal (Bartholemew, 1959), olfactory cues emanating from the young mediate some aspects of the mother's behavior toward the young. Among rodents, much work has centered around olfaction and its relationship to maternal behavior. Using multi- parous rats Beach and Jaynes (1956b) performed two types CE of olfactory manipulation. Surgical disruption of the olfactory bulbs produced no upset in maternal retrieving of pups. The performance of operated mothers was as rapid and efficient as that of unoperated females. However, pups perfumed with oil of lavender induced different reactions in mothers. Some mothers severely bit lavender-scented pups, and several pups were eaten. This behavior wasnot observed in anosmic mothers. Rate of retrieval was significantly slower for scented pups (Beach & Jaynes, 1956b). Beach and Jaynes (1956a) demonstrated that rat mothers were also capable of distinguishing their own young from alien young via olfactory cues. Lactating females with cauterized olfactory bulbs tended to retrieve their own young at the same rate as foster young. Normal lactating females retrieved their own young in preference to foster young. Unfortunately, neither of the two studies mentioned above controlled for auditory cues which may vary from pup to pup (Meier and Schutzman, 1968) or be affected by genetic differences (Huff, 1973). Herrenkohl and Rosenberg (1972) extended and supported the work of Beach and Jaynes by using primiparous rats that were surgically bulbectomized, blinded, or deafened. Some deficits were noted in blind or deaf mothers but anosmic animals were not significantly different from normal controls regarding initiation or maintenance of maternal care (see also Roth, 1971). Mice (Mug musculus), on the other hand, do appear drastically to alter maternal behavior patterns when the sense of smell is surgically disrupted by bulbectomy. Gandelman, Zarrow, Denenberg, and Myers (1971) induced pup cannibalism in virgin females and in lactating females (operated after pregnancy but before birth). A11 bilaterally-operated mice killed and ate pups. Unilaterally- operated and sham-operated controls behaved normally towards pups. In a subsequent study (Gandelman, Zarrow, & Denenberg, 1971) post-natal bulbectomy also produced pup cannibalism in mouse mothers, but the percent of mothers exhibiting cannibalism declined as the day of bulbectomy advanced from the day of birth. Attenuation of cannibalism by female mice was shown to be correlated with the presence of body hair on older pups rather than with the mother's prior experi- ence with the pups (Gandelman, et a1., 1971). In rats, experience with pups can play a role as to what happens after bulbectomy. Either the experience of pregnancy and birth (Beach & Jaynes, 1956b; Herrenkohl & Rosenberg, 1972; Fleming & Rosenblatt, 1974a), or the exposure of females to young prior to bulbectomy (Fleming & Rosenblatt, 1974a) appears to prevent cannibalism after bulbectomy. Surgical procedure can also determine the occurrence of cannibalism. Successive bulbectomy (as described earlier in relation to male sexual behavior) produces different results than simultaneous bulbectomy. Virgin rats with no prior experience with pups other than their own littermates will cannibalize young pups after simul- taneous bulbectomy; successive bulbectomy produces no cannibalism. This occurs whether or not successively- operated animals are presented with pups between operations (Fleming & Rosenblatt, 1974a). A third paper supports and extends the results obtained with mice mentioned above (Gandelman, Zarrow, & Denenberg, 1972). Virgin female mice were bulbectomized before mating, and the care given to subsequent litters was studied. Bilateral bulbectomy again produced pup-killing. No attenuation of the response was seen in subsequent litters. In addition, the operation produced a lower rate of conception and reduced ovary weights, verifying Whitten (1956b). Zarrow, Gandelman, and Denenberg (1971) also showed that bulbectomy inhibited nest-building activity of pregnant and virgin females. Bulbectomy also affects the reactions of male mice toward young pups. Gandelman (1973) demonstrated that loss of olfaction will advance the age at which pup—killing will be exhibited by males. In his colony of Rockland- Swiss albino mice, about 50 percent of 78-day—old normal males killed day-old pups upon first presentation (average latency for pup killing: 53 sec.). In contrast, virtually no pup-killing or cannibalism occurred among normal 22 day males. However, roughly 50 percent of a bulbectomized 10 group of 22 day males did cannibalize day old pups. Sham- operated controls exhibited no pup killing or cannibalism. Unexpectly, loss of olfactory cues may also facilitate maternal responsiveness in virgin female rats (Fleming & Rosenblatt, 1974a,b). Both peripheral and successive central disruption of olfactory tissue decreased the latency to the onset of maternal behavior patterns in pup-naive virgins. The authors suggested that this occurred because anosmia eliminated fear or aggression-provoking stimuli. This theory gains support from the work of Mollenauer, Plotnik, and Synder (1974) who found that bulbectomy reduced timidity in rats exposed to a cat. However, the work of Noirot (1970) argues against this hypothesis. She found that exposure to pup odors can enhance maternal performance. Noirot exposed female mice, which had no prior contact with pups beyond their own littermates, to the odor of young pups and later allowed the females to physically contact the pups. Exposed females performed more pup-cleaning responses than females without prior exposure to pup odors. In rats, also, pup odors appear to facilitate some adult behavioral patterns. Smears from pups' ano-genital regions applied to drinking water generated preferences for treated water among parturant females (Charton, Adrien, & Cosnier, 1971). Ano-genital licking of pups by adults declined when pups were smeared with colloidon in the ano-genital area. It 11 was not affected by colloidon applied to the backs of control pups (Charton, et a1., 1971). Pup odors facilitate maternal retrieving in the Mongolian gerbil (Meriones unguiculatus) (Wallace, Owen, & Thiessen, 1973), but in this case, it is the odor applied to the young by the female via a ventral sebaceous gland (Thiessen, Blume, & Lindzey, 1970). The presence of a female's sebum on a pup increased the probability of retrieval of the pup (Wallace, et a1., 1973). Control of an adult's marking behavior appears, in part, to depend upon the odor of the object to be marked (Baran & Glickman, 1970). (For a review of mammalian scent-marking behavior see Johnson, 1973). Experimental Methodology and Critique The work mentioned above can be categorized by the experimental technique applied. Basically three approaches were used: a. Removal or lesion of the olfactory lobes of the brain. b. Peripheral disruption of olfactory sensory capacity. c. Alteration of what the animal smells while leaving the sensory capacity intact. The first two techniques share a common fault. They both place an animal in the strange position of having no sensory input via a modality that has significance in a 12 variety of contexts. Rodents are probably never out of olfactory communication with their environment except possibly during sleep. If one removes olfactory capabili— ties, the environment, from the animal's point of view, changes draStically. The effects of a strange environment on rodent emotionality are well documented. It is standard laboratory practice to habituate an animal to its environ- ment for some time before performing an eXperimental manipulation. If the sense of smell is removed, behavioral aberations can be expected to appear. These aberations could either result from the loss of a specific olfactory cue or from a generalized disruption of the animal's behavior as a function of a drastically altered 'merkwelt." Experimental techniques involving lesion or removal of the olfactory lobes suffer from another drawback: they disrupt the central nervous system. Therefore, behavioral changes may be due in whole or in part to C.N.S. disruption rather than to loss of olfactory cues. Several lines of evidence support this hypothesis. The limbic system is reciprocally connected to olfactory centers (Dennis & Kerr, 1968; Heimer, 1968; Raisman, 1972). Of those portions of the limbic system connected to the olfactory lobes, few have been implicated in the mediation of olfaction (Bennett, 1968; Long & Tapp, 1970). Further- more, interconnections have been demonstrated between the olfactory lobes and the hypothalamus and between the olfactory lobes and the medial forebrain bundle (Marshall & 13 Teitelbaum, 1974; Scott & Leonard, 1971; Scott & Pfaff, 1970; Scott & Pfaffman, 1967). Fleming and Rosenblatt (1974b) citing this and other evidence, argue that the olfactory bulbs provide tonic inhibition to those areas of the brain responsible for the release of aggressive behavior patterns. Several studies dealing with bulb damage and non- olfactory testing situations tend to support this argument. Bulbectomy can generate hyper-reactivity (Fleming 8 Rosenblatt, l974a,b) and increased aggression (Mollenauer, et a1., 1974; Alberts & Friedman, 1972, Bernstein & Moyer, 1970; Malick, 1970; Karli, Vergnes, & Didergeorges, 1969). Seick and Baumbach (1973) noted differences in spontaneous behavior patterns correlated with the amount of surgical damage done to the olfactory bulbs of male rats. Appetitive and aversive behaviors differed between groups in relation to the location of lesions in the olfactory lobes (Seick & Gordon, 1973). Avoidance performance of rats is affected by bulbectomy (Thomas, 1973; Brown, Harrell, & Remly, 1971); emotionality as well as open field behavior are also affected (Richman, Gulkin, & Knobloch, 1972). Bulbectomy also reduces eating, running activity, and self-stimulation, although the effect is temporary (Borer, Powers, Winans, & Valenstein, 1974). Bulbectomy also alters an animal's hormonal state. Both corticosterone and norepinephrine levels appear to be influenced by the procedure (Zarrow, Schlein, & Denenberg, 14 1971; Pohorecky, et a1., 1969). Hormonal state, in turn, affects maternal responsiveness; the rapid onset of maternal care-taking behaviors after birth is influenced by the parturant female's unique hormonal state (e.g., Terkel and Rosenblatt, 1968, 1972; Moltz, Lubin, Leon, & Numan, 1971; also see reviews by Lehrman, 1961; Moltz, 1970, 1971; Rosenblatt, 1970; Richards, 1967). Because of the problems considered above, the third technique of controlling the odor of objects perceived by the animals could be more successfully used for a study of maternal behavior. Although this seems to be a reasonable choice, few workers have used it in this context. Pups could be scented with an essential oil and the behavior of the mother observed. This method has several inherent advantages. The chance is low that other vari- ables are being simultaneously introduced to confound the experiment. Furthermore, essential oils are commercially prepared to Federal standards which, at least to some degree, guarantees the uniformity of stimulus quality. Cost of treatment is low. Treatment application is fast and easily performed. The treatment does not physically disturb the mother nor is there olfactory disruption of her entire environment since the oil is applied to a specific object. 15 The Questions Methodological problems aside, the works cited above can be cast into a theoretical framework. Beach and Jaynes (1956b) proposed a multi-sensory control hypothesis for maternal behavior. Briefly, their study led them to believe that no single sensory modality was responsible for maternal performance in the rat despite the effects of lavender-scenting mentioned earlier. Support for this hypothesis lies also in the work of Herrenkohl and Rosen- berg (1972). Noirot (1970) demonstrated that different sensory cues can operate on different aspects of maternal care-taking behavior: olfactory cues facilitate pup cleaning and auditory cues facilitate nest building. Other work has shown the importance of auditory cues to retrieval (e.g., Huff, 1973; Noirot, 1972; Sewell, 1970; Zippelius & Schleidt, 1956). Ultrasonic pup calls (above 20 kHz.) appear to enhance the probability that a pup will be retrieved. Pup calls are not, however, a requisite for retrieval as is demonstrated by retrieval of dead or anesthesized pups (Noirot, 1964; Smith & Berkson, 1973; personal observations). In opposition to the multi-sensory control hypothesis is the work of Gandelman and his associates, cited earlier. They demonstrated that loss of the olfactory bulbs invari- ably induced cannibalism of young mouse pups. Later work (Gandelman, Zarrow, & Denenberg, 1971) showed that as pups aged the appearance of hair (presumably affecting other 16 sensory modes in anosmic mothers) reduced cannibalism. This led the authors to postulate a combined uni- and multi-sensory maternal control mechanism. Briefly, they suggest that in mice olfactory cues are chiefly responsible for maternal performance in the early phase of lactation. As pups age and lactation progresses other cues come into play. Species (i.e., genetic) differences between rats and mice account for the different effects of bulbectomy and experience on maternal performance. This theoretical synthesis, while logical and con- sistent with reported results, is open to question. First, Gandelman and associates used bulbectomy to alter olfactory cues. Therefore, conclusions based upon their results are subject to the criticisms expressed above. In short, while bulbectomy produces repeatable results in mice, it is not certain that the effects are due to loss of olfactory cues (Sachs, 1971). Furthermore, Beach and Jaynes (1956b) did observe pup cannibalism in experienced rat mothers due to scenting of pups with oil of lavender. Surgical anosmia did not produce cannibalism. I know of no study confirming these results. Experience with pups appears to interact with olfactory cues in a maternal behavior context, but as mentioned earlier, the effects of experience are conflicting (Herrenkohl & Rosenberg, 1972; Gandelman, et a1., 1971; 17 Fleming & Rosenblatt, 1974a), possibly due to species and/or methodological differences. In an attempt to clarify the results of the studies discussed above, and to expand the results across taxonomic lines, a study was designed to examine the interaction of olfaction and maternal behavior by directly altering pup odor. Basically, three questions were asked: a. Does a pup's odor affect maternal behavior? b. Do olfactory cues produce changes in the mother's behavior as a function of pup age? c. Does prior experience with a novel olfactory cue effect a mother's behavior patterns? The species studied was Peromyscus eremicus, a desert- dwelling, cricetid rodent. Pilot work indicated that multiparous g, eremicus mothers were behaviorally suscep- tible to olfactory alteration of their young since daily placement of a drop of anise oil on the backs and the ano- genital areas of pups resulted in high pup mortality; 14 of 15 scented pups died. Only 2 of 9 control littermates, handled identically, but unscented, died. These mortality differences were statistically significant (x2 = 12.8; P < .005; n = 24; df = 1). ANIMALS The experimental subjects were first generation (F1) female offspring of wild-caught £3 eremicus parents. These females were mated to F1 males. Before assignment to an experimental group, all had successfully reared at least one litter of pups to weaning age (26 days). Subjects were maintained in a colony room at the Biology Research Center, Michigan State University. Their cages were of transparent plastic (11.5" x 5.25" x 6") with shaved wood bedding material and a single "Nestlet" cotton bedding square (Ancare Corp., Manhasset, N.Y.). Food (Wayne Mouse Breeder Blox, Allied Mills Inc., Chicago, Ill.) and water were available ad-lib. Room temperature was maintained at 70°F:3°F. Lights in the room were on at 8 a.m. and off at 11 p.m. (Daylight savings time) providing 15 hours of light and 9 hours of darkness. All animals used in this study were maintained under these conditions except where noted. All cages were inspected daily for birth of new litters. 18 EXPERIMENT 1: RETRIEVAL Because of the high mortality observed among scented pups during the pilot study, I hypothesized that altered odor of the treated pups had generated some change in the mother's behavior toward these young. To test this hypothesis a retrieval study was initiated. Materials and Methods Subjects The subjects were 12 naive (having had no prior experience with anise-scented pups), multiparous £3 eremicus females. Mothers with their newborn litters were, on the day after birth, transferred to a fresh cage and placed in a separate room adjacent to the colony room. This testing room.was maintained at the same temperature- light regime described above. Animals were kept in this room until the day of the retrieval test (day 6 after birth). Test Apparatus The cage housing mother and pups was identical to the breeding cage except for the presence of a 1" hole in one 19 20 end of the cage. The hole, until testing, was blocked with a rubber stopper. This cage was placed within a larger, freshly cleaned, clear plastic tub (19 1/2" x 15 1/2" x 8 1/4"). An opaque plastic sheet with a viewing slit (2" x 15") separated the observer from the subjects (Figure 1). Hon: LARGE CREE TUB EmT .____. ,7\, / \ PUP PUP OPAQLLE ScREEA/ >< OBSERVER Figure 1. Retrieval test apparatus. 21 Procedure All retrieval tests were conducted between 9 a.m. and 11 a.m. On day 6 after birth, all pups were removed from their mother and taken to the adjacent colony room. Simultaneously, the rubber stopper was removed from the nesting cage, allowing the mother free access to the large tub for the first time. Two pups were randomly selected from her litter and toe-clipped for identification. A drop of anise oil was placed on the back of one pup. The mother was allowed to explore the tub for at least 5 minutes from the time of pup removal. After 5 minutes, the observer returned to the testing room and waited behind the plastic sheet until the mother was observed to return to the home cage from the tub area. Once the mother was back in her home cage, the two pups were quickly placed in the tub at corners about 8" from the home cage exit hole. Pup position in a given corner was determined by coin toss. Measurement and Observations The time when pups were positioned in the tub was recorded, and the test was concluded when both pups had been retrieved to the home cage by the mother or when 15 minutes had elapsed. Two measurements were made of the mother's behavior: approach to a pup and retrieval of a pup. An approach was scored when the mother touched a pup with her nose or 22 forepaws. A retrieval was scored when a pup was carried through the hole and into the home cage. A mother was tested only once and returned, with her young, to the colony room at the end of the test session. Results All data was analyzed via a binomial table of cumula- tive probabilities which summed the probability of occur- rence of the events observed and all worse deviations from randomness (Beyer, 1966). In 12 trials all mothers approached and retrieved all pups presented. Anise-scenting did not affect which pup would be approaChed first. Scented pups were approached first four times and control pups were approached first eight times. This was not significantly different from random (P = 0.1208; n - 12). Anise-scenting did effect which pup would be retrieved first. Scented pups were retrieved first twice and control pups were retrieved first ten times during the 12 trials. This was significantly different from random (P = 0.0192; n = 12). Examination of the relationship between first approach and first retrieval revealed another difference in the mother's behavior toward scented and control pups. Control pups were approached first in 8 of twelve trials. In all cases, these pups were retrieved first (P = 0.0039; n = 8). 23 Scented pups were approached first four times, but retrieved first only twice (P = 0.6875; n = 4). Conclusions Anise scenting of pups generated two effects in this test situation. While scenting did not eliminate pup retrieval or affect which pup would be approached first, it significantly lowered the probability that a scented pup would be retrieved first. The difference in retrieval appeared to be due to the mother's response upon first approaching a pup. If a control pup was approached first, it was invariably retrieved first. If a scented pup was approached first, it had a reduced probability of being immediately retrieved. The behavioral alterations produced in this study do not explain the high mortality observed among anise- scented pups during the pilot study. However, they do illustrate that g, eremicus mothers do respond differently to pups with altered olfactory qualities. Furthermore, the direction of the reponse observed here is consonant with the hypothesis that altered olfactory cues from pups alter maternal care—taking behavior to the detriment of the pups. EXPERIMENT 2: ALTERED SCENT, PRIOR EXPERIENCE AND PUP AGE Introduction Several factors led to the design of the study presented below. Though the pilot study employed anise oil placement on the backs and ano-genital areas of young, the retrieval study demonstrated an alteration in the mother's behavior due to oil placement on the back only. Further- more, oil on the ano-genital area could be more easily absorbed through the pup's epidermis and possibly poison the pup. Therefore, it was decided to limit anise scenting to the backs of young. During the pilot study, several pups of anise-naive mothers were accidentally treated with anise late in the lactation period (between days 12 and 17). And though such pups were treated only once, they showed immediate weight loss. Death followed within 3 days in all cases. Evidence cited above indicated that stimuli from young have differ- ent effects upon the mother's behavior as pups age. Therefore, I included an experimental group that was treated with anise later in the lactation period. 24 25 Since a mother's prior experiences could possibly alter the effects of an olfactory manipulation, a group of anise-experienced mothers was incorporated in the design. Materials and Methogg Animals A11 mothers used were Fl offspring of wild-caught a. eremicus parents, maintained as described earlier. All had reared at least one prior litter to weaning age. Cages were inspected daily for new litters. As litters were born, anise-naive mothers and their young were randomly assigned to one of three experimental groups. On the day after birth (hereafter called day 1) male mates were removed from nest cages and litter size was reduced to 2 pups. Experimental Groups: Anise-naive Mothers 1. Weight Control (WC): N = 10 mothers. Pups weighed daily from day l onwards. 2. Extended Oil on Back (EXOB): N = 10 mothers. One pup per litter treated daily with a single drop of anise oil on the back. Both pups weighed daily from day l onwards. 3. Delayed Oil on Back (DOB): N = 10 mothers. One pup per litter treated a single time at day 9 with oil on the back. Both pups weighed daily from day 1 onwards. 26 To examine the effects of prior experience with anise- scented pups, mothers used in the EXOB group were placed in a fourth experimental group when they had given birth to a new litter. 4. Delaypd Oil on Back, Experienced (DOBE): N = 10 anise-experienced mothers. One pup per litter treated one time at day 9 with oil on the back. Both pups weighed daily from day l onward. Procedure On day 1, the sire and all pups were removed from the nest cage. Note was made of whether the pups were in or out of the nest and whether the pups were on or off of the mother's nipples. Pups were toe-clipped for identification and weighed. All but the two heaviest pups were killed. The heaviest pups were then simultaneously returned to their mother unless anise-scenting was appropriate for a given litter. If scenting was called for, it was applied as follows: A pup was chosen by coin toss and a single drop of anise oil (Magnus, Naybee and Reynard Inc., N.Y.) was applied with a rubber—bulbed glass pipette. The oil was placed on the pup's mid-dorsal line, between the shoulders. Both pups of a litter were then simultaneously returned to the next cage. Pups were not placed in the nest but were positioned in the cage corner furthest from the nest. The cage, now 27 containing both mother and young, was then returned to its shelf in the colony room. One hour later cages were inspected and note taken of the pup's positions as explained above. All observations were made daily between 8 a.m. and 11 a.m. Results The .05 probability level was set apriori as the criterion for existence of significant differences. Com- parisons of mean values of experimental and control animals within each experimental group were performed via paired, two tailed t-tests. Comparisons of means between experi- mental groups employed the Bonferoni t statistic which raised the alpha level required to reject the null hypothe- sis in accordance with the number of non-independent comparisons made (Kirk, 1968; Miller, 1966). Data concerning pup presence on or off the mother's nipple were analysed via x2 tests for day 10. Data concerning pup presence or absence from the nest area were discarded because "nest area" could not be accurately defined in many cases and because pup presence in or absence from the nest appeared correlated with pup attachment to the mother's nipple. Weight data were analyzed over two principle time segments: days 1-9 and 9-12. A x2 analysis was also performed, for reasons to be discussed later, concerning 28 numbers of pups losing or gaining weight over days 1-2 and days 9-10. Pup Weight Changes (see Figure 2 fOr outline of comparisons made) Analysis of pup weight changes was performed by first calculating the average daily gain (a.d.g.) for each pup for the time period of concern. The differences in a.d.g. of the two pups (experimental and control) in each litter were then summed over the ten litters in an exPerimental group. These sums were then divided by 10 to produce a mean difference in a.d.g. for each treatment group. To examine treatment effects within a treatment group, the following null hypothesis was tested: mean difference a.d.g. = 0 Rejection of the null hypothesis would indicate that treatment and control animals, within a treatment group, differed in their a.d.g. These same mean differences were used to make comparisons between experimental groups via the Bonferoni t-test (see Figure 3). To examine the effects of treatments on control animals within treatment groups, the controls were compared to the WC animals via the Bonferoni statistic. In this case, difference figures were not used; instead, the mean a.d.g. of controls within each treatment group were compared to the mean a.d.g. of controls in group WC. Treatment and 29 N = 10 Litters/treatment group; 2 pups/litter (experimental and control). EXOB: Daily treatment with anise over days 1-12. DOBE: Same mothers as in EXOB; anise treatment on day 9 only. DOB: Anise-naive mothers; anise treatment on day 9 only. WC: No anise treatment; pups handled same as all other control pups. EXoB EKPT DOBE DOB \uc Figure 2. Experimental design and comparisons. Comparison a) Comparison b) Comparison c) Intra-group comparisgns; experimental vs control pups. Ho: YA = 0. See Tables 1, 4. Inter—group comparisons of treatment group controls to WC. Ho: Ya.d.g. = Ya.d.g. See Tables 2, 6. Inter-group comparisons of mean difference values. Ho: YA = YA‘ See Tables 3, 5. 30 2C1 Yiz t A .Bonferoni = . .2 ‘6' CiZ/ri (ET? - 1)) i Figure 3. Bonferoni t-test. C1 = orthogonal polynomials F1 = ith treatment mean r1 = number of data points comprising the ith mean Yi2 = sums of squares of the ith treatment control a.d.g. data for all groups are presented, but not tested, in Figure 4. Intra-Group Comparisons: Days I-9 Within all treatment groups, none of the mean differ- ences were significantly different from zero (Table 1). These results were not unexpected for groups DOB and DOBE, as experimental and control pups were identically treated (weighed only) during this time period. However, EXOB experimental animals were scented daily, yet their a.d.g. was equivalent to that of controls. Inter-Group Comparisons to WC?—-Days 1:9— Controls within groups DOB, DOBE, and EXOB were compared to WC animals as described above (see Table 2). No significant differences were detected. a.d.g. treatment Days 1-9 DOB + DOBE + EXOB + .4162 i. .01 .3975 .2962 |+ .066 .083 |+ a.d.g. treatment Days 1-12 DOB - .170 i .413 DOBE - .276 i. .395 EXOB + .359 i .145 Figure Note: 4. 1. 31 a.d.g. Days 1-9 DOB + .407 |+ DOBE + .378 I+ EXOB + .356 [+- WC + .325 |+ a.d.g. Days 9-12 DOB + .269 |+ DOBE + .136 |+ EXOB + .306 |+ WC + .346 |+ Average daily gain data. control .01 .0685 .008 .08 control .200 .355 .0984 .1 Notice positive a.d.g. values for EXOB experi- mentals and negative a.d.g. values of DOB & DOBE experimentals over days 9-12. Tests of significance of intra-group control and treatment differences appear in Tables 1 and 4. Tests of significance of inter-group differences appear in Tables 2, 3, 5, and 6. 32 Table 1.--Paired t-tests: Within treatment group compari- sons of experimental and control pup mean differ- ences of a.d.g. days 1-9. Presented below are mean differences, standard errors, and t values. All tests are two-tailed at 9 df. Ho: YA = 0 DOB = 2.074 NS YA = -.011 :_.017 DOBE t = 1.5 NS RA = -.018 i .039 EXOB t — .979 NS RA = +.037 1 .012 t .05, 9 = 2.262 Table 2.--tBonferoni: Comparisons of mean a.d.g. of treatment group controls to WC animals days 1-9. Ho: YWC - Y1 Y we - .325 + .087 a.d.g. - Y DOB = .407 + .101 t = 2.474 NS E DOBE = .378 i .068 t = 1.59 NS Y EXOB = .356 1 .0916 t = .924 NS 33 her—F2 All possible comparisons between DOB, DOBE, and EXOB were made using the mean difference figures described above (see Table 3). No significant differences were observed, supporting the results obtained from intra-group comparisons. Table 3.--tBonferoni: Comparison of mean differences of a.d.g. between experimental groups days 1-9. DOB RA = -.011 i .017 DOBE RA = -.018 1 .039 EXOB RA = + .037 1 .012 Ho: YA1 - YAZ = 0 DOB vs DOBE t = .2272 NS DOB vs EXOB t = 1.4606 NS DOBE vs EXOB t = 1.687 NS tBonferoni .05, 3 comparisons, 26 df = 2.559 Iptra-GroupComparisons Days 9412 Testing the mean difference of group EXOB experimental and control pups revealed no significant difference (Table 4). Testing the mean difference of group DOB experimental and control pups revealed a significant difference 34 Table 4.--Paired t-tests: Within-treatment group com- parisons of experimental and control pup mean differences of a.d.g. days 9-12. Presented below are mean differences, standard errors and t values. All tests are 2-tailed at 9 df. Ho: YA = 0 DOB t = 3.250** YA = +.439 i .412 DOBE t = 4.68** SEA = +.591 i .403 EXOB t = .3214 NS TA = -.023 i .230 t .05, 9 df = 2.262 indicating that control pups had a significantly greater a.d.g. than eXperimentals (see also Figure 3). The same was true of DOBE controls and experimentals. Here, also, control pups have a significantly greater a.d.g. than experimentals. inter-Treatment Group Comparisons: Days -12 All possible comparisons between DOB, DOBE, and EXOB were made over days 9-12. No significant differences were revealed between groups DOB and DOBE (Table 5). Significant differences were revealed between groups EXOB and DOE and between groups EXOB and DOBE. In both DOB and DOBE, treatment and control animals had a greater difference than did treatment and controls in EXOB. 35 Table 5.--tBonferoni: Comparison of mean differences of a.d.g. between experimental groups, days 9-12. DOB RA = .439 i .412 DOBE YA = .591 i .403 EXOB YA = -.023 I. .230 Ho: §Al - §A2 = 0 DOB vs DOBE t = .959 NS DOB vs EXOB t = 2.93* DOBE vs EXOB t = 3.887** tBonferoni .05, 3 comparisons, 26 df = 2.559 Inter-Grou Comparisons to WC: Days 5-12_ Control pups within groups DOB, DOBE, and EXOB were compared to WC animals (Table 6). No significant differ- ences were observed between DOB control and WC or between EXOB controls and WC. DOBE controls were significantly different from WC animals; DOBE controls had a significantly lower a.d.g. than WC. Because of these results, another Bonferoni t-test was performed to test for a difference between DOB and DOBE. No significant difference was observed. Pup Presence on the Nipple: Day Analysis of data regarding pup presence on the nipple was performed via x2 using 2X2 contingency tables. Separate 36 Table 6:7'tBonferoni‘ (1) Comparisons of mean a.d.g. of treatment group controls to WC animals days 9-12, (2) comparison of DOB a.d.g. to DOBE a.d.g. 1. Ho: Y we = fi ia.d.g. wc = .346 i .1063 i? DOB = .269 1 .199 t = 1.009 115 R DOBE = .136 1.;355 t = 2.756* R EXOB = .306 :_.098 t = .562 NS tBonferoni .05, 40, 4 comparisons = 2.616 tBonferoni .05, 50, 4 comparisons = 2.591 2. Ho: 9 DOB = Y DOBE t = 1.8 NS analyses were performed for first and second observations on day 10 (see Tables 7 and 8). Intra-GroupaComparisons: 551 10 In tests of independence of rows and columns (to test whether nipple attachment was independent of treatment), EXOB treatment pups and control pups showed no significant differences in their tendency to be on the nipple. During the first observation, both were on the nipple more than they were off (see Table 7). During the second observation (occurring one hour after the first), both treatment and control EXOB pups showed a non-significant reduction in their tendency to be on the nipple as compared to the first 37 Table 7.--On and off the nipple intra-group x2 analysis day 10. First Observation EXPTL CnTL 2 X = EXPTL CnTL 2 X = EXPTL CnTL 2- X _ on off 9 l 8 2 .392 NS; N 20 on off 4 6 9 1 5.494*; N = 20 on off 3 7 6 4 1.818 NS, N = 20 on off 8 2 8 2 0.0 NS; N = 20 EXOB DOBE Second Observation EXPTL CnTL 2- X _ EXPTL CnTL EXPTL CnTL on off 6 4 6 4 0.0 NS; N = 20 on off 2 8 7 3 5.05*- = 20 on off 3 7 9 1 7.5**° = 20 on off 10 0 10 0 0.0 NS; N = 20 38 observation. There was no difference in the proportion of treatment and control pups being on or off the nipple. WC pups tended to be on the nipple; no significant differences were observed between controls on either first or second observations. Significant differences do appear upon examination of DOB treatment and control pups and DOBE treatment and control pups. In DOB, a significant difference in response occurred. Control pups were on the nipple more than treatment pups. This relationship held for both first and second observations. In DOBE, no significant difference existed between treatment and control pups for the first observation, though treated pups were off the nipple more than controls. In the second observation, treated pups were off the nipple significantly more than controls. Inter-Gropp Comparisons: fly It was of interest to make a number of inter—group comparisons (Table 8). EXOB experimental pups compared to WC pups showed no significant differences in their tendency to be on the nipple during the first observation. However, during the second observation, EXOB experimental pups showed a significantly reduced tendency to be on the nipple compared to WC pups. Comparison of DOB and EXOB experimental pups showed a significant difference during the first observation; EXOB Table 8.-—On and off the nipple inter-group x2 day 10. 39 analysis First Observation on off WC 16 4 EXPTL 9 l EXOB x2 = .480 NS; N = 30 on off DOB 4 6 EXOB 9 l x2 = 5.494*; N = 20 on off DOBE 3 7 EXOB 9 l 2 _ _ on off DOB 4 6 DOBE 3 7 2 x = .019 NS; N = 20 Experimental _DOB vs EXOB Secon EC vs EXPTL EXOB WC EXPTL 2 X = Experimental DOBE VS EXOB DOB EXOB 2- x _- Experimental DOBE EXOB 2 X = DOB vs DOBE DOB DOBE 2 x = d Observation on off 20 0 6 4 9.23**; N = 30 on off 2 8 6 4 3.33 NS; N = 20 on off 3 7 6 4 1.818 NS; N = 20 on off 2 8 3 7 .266 NS; N = 20 40 experimentals were on the nipple more than DOB experimentals. During the second observation, both experimentals showed a decreased tendency to be on the nipple such that there was no longer any significant difference between them. The same relationships during first and second observations hold for a comparison of EXOB and DOBE experimentals pups. A comparison of DOB and DOBE experimentals during first and second observations reveals no differences between them. Numbers of Animals Showin Weight Change: Days I-2, Days 9-10 Because confounding exists between length of treatment and day of treatment, it was necessary to make four inter- treatment group comparisons using unconfounded subsets of the data. While these comparisons will be discussed in detail later, please note that comparisons l and 2 below differ only in day of application of treatment, anise- experience being the same for all mothers. Comparisons 3 and 4 differ only in prior experience of the mother with anise, day of application of treatment being the same for all mothers (see Table 9). The data in Table 9 represent numbers of experimental animals displaying weight gain or weight loss over the time periods days 1-2 or days 9-10. Weight loss is defined as any weight change other than a positive one. 41 Table 9.--Numbers of pups showing weight loss--inter-group chi square analysis days 1-2 and 9-10. EXPTL EXPTL EXPTL EXPTL EXPTL EXPTL EXPTL EXPTL EXOB Days 1-2 DOB Days 9-10 EXOB Days 1-2 DOBE Days 9-10 EXOB Days 9-10 DOB Days 9-10 EXOB Days 9-10 DOBE Days 9-10 we WL 10 0 3 7 = 10.7923** = 20 we WL 10 0 4 6 2 = 8.571 ** = 20 we WL 10 0 3 7 2- - 10.7923** = 20 we WL 10 0 4 6 x2 = 8.571** 20 42 Comparing EXOB (day 1-2) to DOB (day 9-10), a significantly greater number of DOB animals lost weight. Comparing EXOB (day 1-2) to DOBE animals (day 9- 10), a significantly greater number of DOBE lost weight. Comparing EXOB (day 9-10) to DOB (day 9-10), a significantly greater number of DOB lost weight. Comparing EXOB (day 9-10) to DOBE (day 9—10), a significantly greater number of DOBE lost weight. l_ _l— i EXPERIMENT 3: PUP SUCKLING The results of the previous experiment show that DOB 13 and DOBE pups gained less weight than controls after application of anise and that decreased weight gain was coincidental with a lessened occurrence of experimental E pups on their mothers' nipples. Anise-scenting could hypothetically produce these results in two ways: 1. Scenting could produce decreased weight gain because mothers actively prevented scented pups from suckling. 2. Scenting could produce decreased weight gain by affecting the pups directly (irritating, poisoning or disorienting them) thereby preventing them from suckling. A third experiment was initiated to test these two hypotheses. An anesthetized mother could not actively prevent her young from suckling. Therefore, if hypothesis 1 is correct, there should be no difference in the ability of scented and unscented pups to attach themselves to their 43 44 anesthetized mother's nipples. If this hypothesis is rejected, one would accept hypothesis 2. Materials and Methods As mentioned earlier, the colony was inspected daily for the birth of new litters. As births occurred, twelve a. eremicus females and their litters were selected for the experiment. On day 9 females were removed from the home cage, weighed and injected, intraperitonially with Nembutal (Sodium Pento—Barbital; 3/4 gm/ml; Abbott Laboratories, North Chicago, Ill.) at a dosage of .01 ml per gram of body weight. They were then observed in a holding cage and repeatedly pushed over on their sides. When an injected animal could no longer right itself, it was returned to the nest area of the home cage. All pups were then removed and weighed. The two heaviest pups of each litter were toe-clipped for identifi- cation and one pup, selected by coin-toss, was scented with anise as described above. The two pups were then simul- taneously returned to the nest cage and placed beneath their quiescent mother's ano-genital area. Measurements At five minute intervals, starting from the moment pups were returned, mothers were gently lifted by their tails to determine if the pups were attached. Note was taken as to which pups had attached to the nipple and the time attachment was first observed. Tests were concluded 45 15 minutes after pups were first placed beneath their mother or after both pups were observed suckling, which ever occurred first. Results Pup_Ability_to Gra§p_ NippIe: Day 9 A x2 analysis of the ability of scented and control pups to grasp their mother's nipples reveals no significant differences (see Table 10). Table 10.--On and off the nipple anesthetized mothers scented vs unscented pups. scent cntl off 6 5 on 6 7 2 x = .1768 NS N = 24 DISCUSSION The results of experiment 2 indicate that olfactory information plays time-dependent and experience-dependent roles in maternal caretaking behavior of £3 aremicus. EXOB experimental and control pups showed equivalent a.d.g. through days 1-9 and days 9-12 as shown by the non- significant t-tests of experimental and control mean differences. Furthermore, there is no detectable influence of the treatment upon EXOB control pups since they and WC pups had an equivalent a.d.g. over days 1-9 and 9-12. Therefore, one can conclude that early, sustained, alter- ation of pup odor produces no effects in E. eremicus as measured by pup weight changes. Measure of pup nipple attachment revealed no differ- ence between EXOB experimental and control pup nipple clinging in first or second observations. Likewise, there was no difference between WC animals and EXOB experimentals in regard to nipple attachment on the first observation of day 10. Comparison of EXOB experimentals and WC on the second observation does reveal a significant difference; WC animals were on the nipple more than EXOB. This may be 46 47 interpreted as a statistically significant short-term effect of anise scenting, but its importance to pup maintainance must be considered as minimal for several reasons. First, the second observations were made only one hour after the first with scenting and weighing occurring during this interval. Second, the first observations, occurring about 23 hours after scenting on day 9, reveal no differences in nipple attachment between EXOB experimentals and controls or between EXOB experimentals and WC pups. Third, EXOB controls showed no differences when compared to EXOB experimentals on the second observation. Fourth, EXOB controls showed no differences in a.d.g. when compared to EXOB experimentals and when compared to WC pups. With regard to EXOB one may assume that the presence of two types of pup odor (anise and natural) in the nest had no detectable effect over days 1-9 and 9-12. In effect, mothers, if exposed early enough, learn to broaden their olfactory definition of a pup and to disregard such differences between pups, at least until day 12. These results and their interpretation will be discussed later in light of the findings of other investigators. In contrast to the null effects of extended daily oil treatment seen in group EXOB are the striking effects of delayed oil treatment seen in both DOB and DOBE treatment groups. A single drop of anise at day 9 was enough to cause weight loss and thereby significant mean differences 48 in a.d.g. over days 9-12 of experimental pups in both groups when compared to their controls. DOB and DOBE mean differences were both significantly different from that of EXOB. It should be remembered that DOE and DOBE pups had equivalent a.d.g. over days 1-9 as compared to their controls. Furthermore, DOB and DOBE controls had equivalent a.d.g. over days 1-9 when compared to WC. Group DOBE controls also displayed an uneXpected reaction to oil treatment of their litter-mates at day 9. DOBE control a.d.g. declined enough over days 9-12 to be significantly different from a.d.g. of WC pups. DOBE controls, though having lesser a.d.g., were not signifi- cantly different from DOB controls. DOBE experimentals declined even more than DOBE controls such that a significant mean difference occurred between them as stated above. Nipple attachment of DOB and DOBE pups appears corre- lated with weight changes. In DOB, experimental pups were "off" the nipple significantly more than controls during both the first and second observations. In DOBE, experi- mental pups were off the nipple more than controls, but a significant difference occurred only in the second observation. These results appear correlated with a.d.g. changes; DOBE controls gained significantly less weight than WC pups over days 9-12 and, as mentioned above, DOBE 49 experimentals gained significantly less weight than DOBE controls over days 9-12. In light of the results of experiment 3, this may be interpreted as a result of active rejection of DOB and DOBE experimentals by their mothers. Comparison of EXOB experimentals with DOB and DOBE experimentals with regard to their nipple attachment yields significant differences for the first observation. EXOB experimentals were on more than DOB and DOBE experimentals. The same comparison for the second observation produces no significant differences. Loss of significance between first and second observations may be regarded as due to a short-term rejection on the part of EXOB mothers as discussed above; in the case of DOB and DOBE mothers, rejection is a long-term process which produces significant mean differences in the a.d.g. of groups DOB and DOBE. As mentioned earlier, the pilot study produced a high degree of pup mortality. Such mortality was not seen in this study. Speculation as to the causes of the different results is impossible at this time, since the pilot study was uncontrolled as to litter size and as to day of anise treatment. The retrieval study performed on day 6 mothers and pups produced a preference in mothers for unscented pups. However, scented pups were all retrieved. It is, there- fore, possible that continuous anise-scenting starting at 50 day 6 would produce results similar to those obtained from group EXOB. Certainly an area for future investigation would lie in the determination of the exact time at which olfaction be- comes a crucial determinant of maternal care-taking behavior in E. eremicus. This study shows that day 9 olfactory changes in pups will produce effects. If olfactory cues are altered earlier, one can only speculate as to the result. One could argue, in examining the design of this study, that length of treatment has been confounded with time of treatment, and, therefore, results are impossible to interpret. Confounding is undeniable since EXOB pups were treated only once on day 9. However, analysis of uncon- founded subsets of the data indicates that both experience with anise and pup age affect results. I refer the reader to Table 9. Four x2 analyses are presented concerning pup weight loss between days 1-2 and 9—10. EXOB experimental pups received their first anise treatment on day 1 while DOB and DOBE experimental pups received their first anise treatment on day 9. All EXOB pups gained weight from day l to day 2. Significantly fewer DOB and DOBE pups gained weight from days 9-10. Since mothers in EXOB and DOB were anise-naive prior to treatment, one can conclude that time of treatment affected the results obtained in group DOB. DOBE mothers 51 were anise-naive insofar as their second litters were con- cerned and their litters were affected as much as DOB litters. If one compares EXOB experimental pups over days 9-10 with DOB and DOBE experimental pups, one again sees significant differences in numbers gaining weight. All EXOB pups gained weight over days 9-10. Significantly jfl fewer DOB and DOBE pups gained. This comparison demon- strates the effect of experience with anise prior to day 9, 1 since all pups were the same age and differed only by scent 9i condition prior to day 9. It is therefore logical to assume, though by no means closed to question, that continued anise treatment beyond day 9 in groups DOB and DOBE would not have reduced the mean difference a.d.g. between experimental and control pups or changed their a.d.g. from negative to positive. The results of this study tend both to support and to contradict the results of other investigations in this area. Growth and survival of EXOB animals indicates that olfaction is not as crucial to maternal behavior toward young pups as the work of Gandelman, et al. (1971, 1972), seems to show. In those studies bulbectomy produced cannibalism of young. The present study is in closer agreement with the theoretic of Beach and Jaynes (1956b) and Herrenkohl and Rosenberg (1972), who proposed a multisensory control 52 mechanism of maternal care-taking behavior. However, occurrences after day 9 scenting in groups DOB and DOBE conflict with this rationale as well. Nor does this work agree completely with the uni- multisensory hypothesis of Gandelman et a1. (1971). They proposed that olfaction plays a key role in the early days of lactation while other cues come into play later. Again, early alteration of olfactory cues produced no effect in this study while later alteration did produce effects. The results of this study indicate that some re- thinking is necessary regarding the role of olfaction in maternal behavior patterns. While more work needs to be done, it is reasonable to conclude now that maternal behavior patterns are not rigidly dependent upon mothers receiving "correct" olfactory cues. What is "correct" can be modified some time between day 1 and day 9 in g, eremicus. Nevertheless, novel "incorrect" cues, received after day 9, produce a decline in care—taking behavior as indicated by DOB results. Reappearance of a once familiar, "incorrect" olfactory cue may produce greater effects as indicated by reduced a.d.g. of DOBE controls as compared to WC pups over days 9-12. Why this may occur is inexplicable at this time. However, it should be pointed out that DOE and DOBE controls did not differ in a.d.g. over days 9-12 and, therefore, there is reason to doubt the existence of this phenomenon. SUMMARY Pup retrieval by g. eremicus mothers is signifi- cantly affected by alteration of pup odor with anise oil. Early (day l), sustained anise-scenting of pups does not alter maternal behavior. Late (day 9) single dose scenting of pups reduces maternal care-taking behavior as measured by pup weight changes and reduced occurrence of pup nipple attachment. Reduced occurrence of scented pup nipple attach- ment is due to maternal rejection as evidenced by equivalent ability of scented and control pups to attach to the nipples of their anesthetized mothers. 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