Order Number 9307780

The comparative reproductive energetics of the gray short-tailed o p o ssu m{Monodelphxs domestica) and the golden hamster (Mesocricetus auratus)

Hsu, Jy-Minna, Ph.D.

The Ohio State University, 1992

U MI 300 N. Zeeb Rd. Ann Arbor, MI 48106 THE COMPARATIVE REPRODUCTIVE ENERGETICS OF

THE GRAY SHORT-TAILED OPOSSUM (Monodelphis domestica)

AND

THE GOLDEN HAMSTER (Mesocricetus aiiratus)

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Jy-M inna Hsu, B.A., M.S. * * * * * The Ohio State University 1992

Dissertation Committee: Approver by

D. W. Garton T. C. Grubb, Jr. J. D. H arder S. I. Lustick Advisor D epartm ent of Zoology To My Parents

ii ACKNOWLEDGMENTS

I am most grateful to my advisor, John. D. Harder, for his unwavering support and guidance throughout this work and for his endless discussion and editing of manuscripts. I sincerely thank the other members of my committee: Drs. David W. Garton, Thomas C. Grubb, Jr., and Shelly I.

Lustick, for their interest in my work and thoughtful criticisms of this dissertation. I am grateful to the following people for their assistance in bomb calorimetry: Jackie Pondy, Amy Cremean, Bobbie Walker, Beth

Kennedy, Amy Lavender and Mark Prior. I benefitted from the advice and assistance of M. J. Stonerook and G. Durka. Drs. S. I. Lustick, P. Pappas

and D. W. Garton kindly provided equipments and the Department of

Zoology provided financial support throughout this study. I thank D. W.

Garton and F. Ruland for valuable statistical advice. The Academic

Computer Center, The Ohio State University provided funding and

computer time for data analysis. I am especially indebted to my parents;

without their efforts and support this work would never have been

completed. I also appreciate the help of my sisters in fulfilling my

responsibilities as a mother and daughter. Finally, I thank Drs. Shelly I.

Lustick and Agoramoorthy Govindasamy for their support and

encouragement throughout this work. VITA

February 23, 1961 ...... Bom- Gaushyong, Taiwan, Republic of C hina

1 9 8 2...... B. A., National Taiwan University, Taipei, Taiwan

1984 ...... M. S., National Taiwan University

1984 - 1992 ...... Graduate Teaching or Research Associate, Department of Zoology, The Ohio State University, Columbus, Ohio

1988...... M. S., The Ohio State University, Columbus, Ohio

PUBLICATIONS

Hsu, M., J. D. Harder and S. I. Lustick. 1988. Seasonal energetics of opossums (Didelnhis virginiana) in Ohio. Comparative Biochemistry and Physiology 90A: 441-443.

Hsu, M. and M. J. Humpert. 1988. Use of artificial nest cavities along Ohio interstate highways by bluebirds (Sialia sislis) and mice (Peromvscus sp.). Ohio Journal of Science8 8: 151-154.

FIELDS OF STUDY

Major Field: Zoology

Studies in Physiological Ecology and Mammalian Reproduction. TABLE OF CONTENTS

ACKNOWLEDGMENTS...... ii

VITA ...... iv

LIST OF TABLES...... vii

LIST OF FIGURES ...... ix

ABSTRACT...... xii

CHAPTER PAGE

I Introduction...... 1

II Effects of Environmental Temperature on Metabolic Rates During Gestation and Lactation in Golden Hamsters (Mesocricetus auratus)...... 16

A b stra c t ...... 16 Introduction...... 17 Materials and methods ...... 20 R e su lts...... 26 D iscu ssio n...... 38

v CHAPTER PAGE

III Effect of Body Temperature on the Metabolic Rate of the Gray Short-tailed Opossum (Monodelnhis domestical during Gestation and Lactation ...... 45

A b stra c t ...... 45 Introduction...... 46 Materials and methods ...... 51 R e su lts...... 59 D isc u ssio n...... 70

IV Comparative Reproductive Energetics of Golden Hamsters and Gray Short-tailed Opossums...... 78

A b stra c t ...... 78 Intro d u ctio n ...... 79 Materials and Methods ...... 82 R e su lts...... 91 D isc u ssio n...... 103 V Conclusions...... 112

LITERATURE CITED 118 LIST OF TABLES

TABLES PAGE

Table 2.1. Significance levels from ANCOVA for oxygen consumption (RMR, log ml 02 hr'1), thermal conductance (C, Kcal “C 1 hr'1) and rectal temperature (Tb, °C) and from ANOVA for respiratory quotient (RQ) in 16 RA-30, 21 NR-30, 20 RA-24 and 16 NR-24 female golden hamsters...... 32

Table 3.1 Average whole-animal resting metabolic rates (RMR, ml 0 2 h r'1), body mass (g) and body tem perature (Tb, °C) and percentage of Kleiber-predicted heat production (BMR) of Reproductively Active (RA) females and non- reproductive (NR) female opossums. Data for mother plus young measured together are presented in the second row below each designated day of lactation. . 60

Table 3.2 F values and significance levels from the ANCOVA evaluation of the treatment effects on RMR (ml 02 hr'1), heat production (HP, Kcal h r1) and Tb (°C) of 20 reproductively active (RA) and 19 non-reproductive (NR) opossums at 30 °C. Sample sizes for each stage were 18-20, except n = 7 on day 30 of lactation 63 Table 4.1 The body composition of reproductively active (RA) and non-reproductive (NR) golden hamsters used in this study. Values are means + SEM...... 94

Table 4.2 The body composition of 20 reproductively active (RA) and 19 non-reproductive (NR) female opossums. All units of mass are in grams...... 98

Table 4.3 Comparisons of absolute energy (Kcal) assimilated and used from conception to weaning for reproduction of gray short-tailed opossums and golden hamsters. . 100

Table 4.4 Comparisons of energy (Kcal) used for respiration from conception to weaning based on measurements of metabolic rates and energy stored in young of gray short-tailed opossums and golden hamsters...... 104

Table 4.5 Comparisons of reproductive parameters of golden hamsters and gray-short tailed opossums...... 107

Table 4.6 Comparisons of reproductive energetics (Kcal) of marsupials and eutherians in relation to basal metabolic rate, and the length of gestation (G) and from conception to weaning (C-W)...... 110

Table 4.7 Comparisons of reproductive energetics on a daily basis (Kcal day'1) of marsupials and eutherians...... I l l LIST OF FIGURES

FIGURES PAGE

Fig. 1.1 Experimental design of the comparative reproductive energetics of golden hamsters (Mesocricetus auratus) and gray short-tailed opossums (Monodelphis domestica). . . 13

Fig. 2.1 Average body mass (g animal'1) of reproductively active (RA) female golden hamsters maintained at 24 °C (n=20) or 30 °C (n=16) and of non-reproductive (NR) females maintained over the same experimental period at 24 °C (n=16) or 30 °C (n=21). Data are plotted relative to the day of birth (day 0)...... 27

Fig. 2.2 Adjusted mean RMR (resting metabolic rate, ml 0 2 h r'1, + 1 SEM) of RA golden hamsters at 24 °C and 30 °C. Adjusted means were from ANCOVA models adjusted to the average body mass 115.3 g and rectal temperature 37.5 °C at 24 °C and 111.9 g and 36.6 °C at 30 °C. The average of adjusted mean RMR of NR females is presented as dashed or dotted lines for females maintained at 24 °C and 30 °C, respectively...... 29

Fig. 2.3 A) Wet mass (mg) of interscapular BAT of golden hamsters. Data of pregnant and lactating dams maintained at 24 °C (filled diamond) were redrawn from Wade et at. (1986). Unshaded triangle, NR-24 females; unshaded circle, RA-24 dams; shaded circle, RA-30 dams. B) Average Reproductive Increment of Metabolism (RIM, m l 0 2 h r1) of RA-24 and RA-30 dams. RIM was calculated at each stage by subtracting non-reproductive metabolism estimated from the equation generated for NR females with identical body mass and Tb with each reproductive stage from observed RMR of each RA dam ...... 3 5

Fig. 2.4 Adjusted mean Tb (+ 1 SEM) of golden hamsters. Adjusted means were from ANCOVA models adjusted to the average body mass 115.3 g and RMR 221.6 ml Oa h '1 a t 24 °C and 111.9 g and 134.7 ml Oa h '1 a t 30 °C. Symbols are the same as those used in Fig. 2.2...... 36

Fig. 3.1 Average respiratory quotient (RQ,± 1 SEM) of opossums during the reproductive cycle compared to that of non- reproductive females during an equivalent 80-day period. 64

Fig. 3.2 Average proportion of Kleiber-predicted BMR (heat production) of RA (n = 20) and NR (n = 19) opossums. Lactating heat production of dams were converted from estimated RMR. Heat production of litters was calculated from measured RMR of litters and estimated RMR of litters if data were not measured ...... 65

Fig. 3.3 Adjusted means of reproductive increment of metabolism (RIM, m l 0 2 h r'1, + 1 SEM) of RA opossums. RIM was calculated at each stage by subtracting RMR estimated from the equation generated for NR females with identical body mass and Tb with each reproductive stage from observed RMR of each RA dam. Adjusted means were from ANCOVA models adjusted to the average rectal temperature 33.63 °C ...... 68

x Fig. 4.1 Average (+ SEM) whole-animal Assimilated Energy (Kcal day'1 animal'1) of reproductively active (RA) golden hamsters during gestation and lactation maintained at 24 °C (n=2 0 ) or 30 °C (n=16) and non-reproductive (NR) females maintained over the same experimental period at 24 °C (n=16) or 30 °C (n=21). Data are plotted relative to the day of birth (day 0). The symbols of RA-24 and RA-30 females were overlapping on day2 2 ...... 92

Fig. 4.2 Average (+ SEM) whole-animal Assimilated Energy (Kcal day'1 anim al1) of reproductively active (RA) gray short­ tailed opossums during gestation and lactation maintained at 30 °C (n=20) and non-reproductive (NR) females maintained over the same experimental period (n=19). Data are plotted relative to the day of birth (day 0). ... 96

xi ABSTRACT

Eutherians and marsupials reflect the evolution of alternative strategies for viviparity and lactation. The consequences of thism a m m a lia n dichotomy were examined through comparison of the reproductive energetics, from conception to weaning (C-W), of golden hamsters (Mesocricetus auratus) and gray short-tailed opossums (Monodelphis domestica). The resting metabolic rate (RMR, with body mass and body temperature as covariates) was elevated (P < 0.05) during gestation in reproductively active hamsters maintained in thermoneutrality (30 °C) but not in opossums at thermoneutrality or hamsters maintained at 24 °C. Adjusted mean RMR (at

30 °C) was elevated (P < 0.05) in lactating dams of both species with highest levels reached on day 7 (164.5 + 5.0 ml Oa hr *1 animal*1) of the 21-day lactation period in hamsters and on day 40 (79.4 + 8.9 ml 20 hr'1 animal'1) of the 56-day lactation period in opossums. Body temperature in opossums increased from 32.1 + 0 .2 °C at diestrus to 35.0 + 0.3 °C during lactation and, thus, most of the increase in RMR in opossums was associated with increased expenditures for maintenance and was not closely correlated with reproductive output. Total energy assimilated from food, from C-W, (hamsters: 1647.5 +

60.6 Kcal, and opossums: 1261.3 + 28.0 Kcal) was positively correlated with litter size and mass per young in both species. Total energy assimilated was higher in hamsters than in opossums during gestation (P < 0.001), but not during lactation or from C-W (P > 0.05). The Incremental Cost of

Reproduction from C-W (adjusted for body mass) of RA-30 hamsters was not significantly different from that in RA-30 opossums. Efficiency of offspring production was higher in hamsters than in opossums and, in both species, it was higher during lactation than in gestation. However, on a per young and on a per young per day basis, the cost of reproduction was consistently higher in hamsters than in opossums.

The observed interspecific differences were due largely to a lower

BMR and a longer period of lactation in opossums. Thus, the marsupial mode of reproduction, as seen in opossums, yields young at a lower cost but at the expense of a longer reproductive period than is the case for eutherians, particularly the golden hamster. CHAPTER I

Introduction

The Marsupial - Eutherian Dichotomy

Fundamental differences in reproductive adaptations and strategies separate the Infraclasses Metatheria (marsupials) and Eutheria (all other viviparous mammals). Marsupial reproduction is characterized by a very brief period of gestation (range 12-46 days), followed by birth of embryonic neonates which develop during an extended period of lactation. Gestation accounts for an average of 12% of the time from conception to weaning (C-W interval) in marsupials (n = 32 species) compared to 56% of C-W in eutherians (n = 132 species). Therefore, the period of lactation in marsupials averages about 40% longer than that in similar-sized eutherians

(Hayssen, Lacy and Parker 1985). The interval from conception to weaning is also relatively long for marsupials, almost 1.5 times that of similar-sized eutherians (Braithwaite and Lee 1979; Lee and Cockbum 1985; Thompson

1987).

Maternal investment in marsupial gestation, in terms of mass and energy content of neonates, is much lower than has been recorded among

1 similar-sized eutherians (Hay8sen, Lacy, and Parker 1985). Neonatal marsupials are small with whole body weights ranging from0 .0 1 to 1 g

(Russell 1982a). Marsupial litters at birth do not exceed 0.2% of maternal body mass (Russell 1982a), while litters of some small insectivores and rodents weigh well over 50% of their mother's mass at birth (Lee and

Cockbum 1985). However, these altricial marsupial offspring exhibit a mosaic of developmental states. For example the olfactory system of the neonate is precociously functional and the muscles and claws of forelimbs are well-developed. At the same time, the hindlimbs are represented only by condensed mesenchyme (Sharman 1973).

Lactation, which is the dominant mode of energy transfer to the developing marsupial young, proceeds in two phases: the teat attachment phase and the nest phase (Eisenberg 1988; Harder 1992; fig. 1.1). Following birth young must crawl unassisted from the opening of the urogenital sinus to the mammary area (within the pouch in some marsupials). The neonate must then locate an unoccupied teat and take it into its well-developed oral cavity wherein it becomes firmly affixed. The teat is not voluntarily released during the teat attachment phase. Maternal care in marsupials is strongly influenced by presence or absence of the pouch, which in term is related to adult body size and litter size (Russell 1982a). Pouchless marsupials are usually less than2 0 0 g in body mass and often have large

(>3) litters. The teat attachment phase of pouchless species is shorter than that of species with a well-developed pouch (one-forth to one-third versus two thirds of the lactation period, respectively, Harder 1992). By the time young are left in the nest, the total litter mass of small marsupials can reach more than 50 % of maternal body mass, even through each young weighs only 1.0-1.1 g in Ningaui sp. and Planieale maculatus (Russell

1982a). By comparison, the total litter mass of large, pouched species may reach 20 % of maternal body mass by the time that young are no longer carried by the mother. The burden of carrying young is much greater for small arboreal animals or active predators than for terrestrial herbivores

(Russell 1982a). The pouchless condition might reflect some advantages for small-sized marsupials associated with leaving young in the nest in an early stage while the mother forages (Harder 1992).

The pouchless condition is prevalent among New World marsupials occurs in 64 of 78 species (Harder 1992), particularly among species of

Marmosa and Monodelphis of family Didelphidae (Kirsch 1977).

Didelphidae is the primitive marsupial family from which all other marsupials apparently descended (Clemens, 1968). Thus the pouch is considered a derived condition (Tyndale-Biscoe and Renfree 1987).

Evolution of the pouch and reduced the dependence on a nest might have play an important role in the adaptive radiation of marsupials (Harder

1992). The marsupial pouch also provides protection and thermal 4 regulation for the neonates; they are maintained at a relatively constant temperature (Gemmell, Cepon, and Barnes 1987).

The marsupial strategy for viviparity and postnatal care of young has been viewed as an inferior, less efficient process than the eutherian approach (Lillegraven 1975), which relies more heavily on in utero development and transfer of nutrients across thin placental membranes from the maternal to the fetal blood system. Tyndale-Biscoe (1973) concluded that the choriovitelline placenta of marsupials is less efficient than the chorio-allantoic placenta of eutherians. However, growth and maintenance of the eutherian placenta consumes energy in addition to that required by the young (Silver and Steven 1975) and, thus, differences in energetic efficiencies between the two groups might be small (Fleming,

Harder, and Wukie 1981). Comparisons of growth rates and energetics between marsupials and eutherians are hindered by the difficulty of determining comparable stages in growth of the young (Russell 1982a). The developmental stage at the attainment of homeothermy in a young marsupial has been suggested approximately equivalent to the developmental stage at birth in many eutherians (Russell 1982a).

length of gestation and growth rates are correlated with basal metabolic rate (BMR) in eutherians. Species with higher BMR have higher growth rates and shorter gestation periods (McNab 1980 and 1986). On the

other hand, this relationship does not appear to hold for marsupials. BMRs (or standard metabolic rates) of marsupials are about 30% lower (MacMillen and Nelson 1969; Dawson and Hulbert 1970; McNab 1978) than the Kleiber- predicted BMR for eutherians (heat production in Kcal day'1 = 70 x (body mass, Kg)0,76, Kleiber 1961). Low BMR might contribute to lower rates of development in metatherians (Morton et al. 1982) and consequently, lower energy demands during reproduction. This maybe allow marsupials to reproduce during low food availability.

Low (1978) postulated that because marsupials shift to lactation relatively early in the development of offspring, compared to eutherians, they might expend more energy per unit mass of offspring than eutherians.

Morton et al. (1982) suggested that marsupials might invest greater total

energy during the course of the entire reproductive cycle (C-W) but may

require less energy per unit time than ecologically similar eutherians.

Unfortunately, little quantitative data on maternal energetic investment in reproduction is available for objective evaluation of these interesting

hypotheses. The study of Thompson and Nicoll (1986) is one of few

designed to evaluate reproductive energetics of marsupials and eutherians.

Comparisons of reproductive energetics across studies and species are

extremely difficult to make because variation in body temperature (Tb) and

ambient temperature (Ta) outside thermoneutrality can strongly influence

food assimilation and resting metabolic rates (RMR) of endotherms (see

review by Whittow 1971). Thus, different methodologies and experimental 6 conditions often frustrate attempts at interspecific comparisons of RMR, maternal Tb, and mass from conception to weaning.

Physiological Changes during the Reproductive Cvcle

When Ta is below the zone of thermoneutrality in homeotherms, additional energy is required for thermoregulation, including shivering and non­ shivering thermogenesis. Nonshivering thermogenesis has a major effect on differences in total thermogenic capacity in cold tolerance (Heldmaier,

Steinlechner, and Rafael 1982). Brown adipose tissue (BAT), in both hibemators and non-hibemators, is the dominant site of nonshivering thermogenesis (Foster and Frydman 1978). Cold acclimation generally leads to the changes in thermogenic capacity that are evident in the bioenergetic properties of mitochondria of BAT (Nicholls 1979; Hamilton et al. 1986).

Most measurements of RMR have been made at normal room temperature (21-25 °C), which is below the zone of thermoneutrality for many small mammals. Mover, Hellwing, and Ar (1988) argued that the true metabolic cost of gestation is different when measurements are made below rather than within thermoneutrality. Thermogenesis which is included in measurement RMR may be assumed to remain unchanged between reproductive and non-reproductive conditions. However, thermogenesis by BAT in vitro is suppressed during pregnancy and 7 lactation in laboratory mice and rats (Trayhurn 1985) and hamsters (Wade,

Jennings, and Trayhurn 1986; Schneider and Wade 1987). A reduction in the capacity for non-shivering thermogenesis during lactation also occurs in vivo (Isler, Trayhurn, and Lunn 1984). Suppression of non-shivering thermogenesis apparently reflects increased metabolic heat production from fetal growth and milk synthesis, thus reducing requirements for thermoregulatory heat production (Trayhurn, Douglas, and McGuckin 1982;

Trayhurn 1985). Relatively few studies have employed measurements of

RMR within the zone of thermoneutrality in order to diminish the influence of uncontrolled thermogenesis (Fleming, Harder, and Wukie 1981; Nicoll and Thompson 1987; Mover, Ar, and Hellwing 1989). In addition BAT has been identified in only one marsupial, the Bennett’s Wallaby (Loudon,

Rothwell, and Stock 1985). The gray short-tailed opossum and other marsupials appear to lack of BAT (Rowlatt, Mrosovsky, and English 1971;

Dawson and Olson 1988). Therefore, measurements of energy used for reproduction should be made on animals maintained in their zone of thermoneutrality in order to minimize any possible energy allocated from reproduction to thermogenesis.

Interpretation of oxygen consumption values for pregnant dams is complicated not only by difficulty in separating fetal metabolism from that of the mother, but also by changing total body mass of mother and her fetuses. Body mass of small eutherians can increase 30-50% above pre- 8 mating body mass during late gestation (Mattingly and McClure 1982) and, in at least one study of laboratory mice the increase was 320% (Studier,

1979). Total whole-animal oxygen consumption of small terrestrial eutherians usually peaks just before parturition. However, when whole- animal oxygen consumption of pregnant dams is divided directly by increasing body mass to obtain mass-specific metabolic rate, resulting estimates show no increase or even a reduction compared to non-pregnant animals (e.g., Studier 1979; Innes and Millar 1981).

Average body temperatures (Tb) of marsupials under standard conditions (resting and within thermoneutrality) are generally in the range of 34-36 °C (Dawson and Wolfers 1978; McNab 1978), 2-3 °C lower than the normal range for eutherians (Dawson and Hulbert 1970). The lower level Tb is one of the major correlates of low BMR in marsupials (Dawson and

Hulbert 1970). Small-sized marsupials also have daily Tb of 3-5 °C fluctuations depending on their activity and ambient temperatures

(Kleinknecht, Erkert and Nelson 1985). Increases in RMR during lactation

(Thompson and Nicoll 1986) might parallel elevated Tb.

Although the use of ratios to adjust biological data that vary allometrically with body size has been criticized (Tanner 1949; Atchley and

Anderson 1978), such scaling is commonly employed in physiology. In cases

of isometry the ratio (Y / X) does not vary with independent variable (X)

such as body size (Y = bX), but the ratio does vary with body size in cases of allometry (Y = a + bX, a><0, i. e., a non-linear relationship between X and

Y). Instead of using ratios to adjust physiological variables (e. g., direct division by body mass, analysis of covariance (ANCOVA) can be used to control for the effects of differences in body mass between treatment groups.

ANCOVA is based on regression and analysis of variance (Fisher 1932).

ANCOVA can improve the precision (reducing the coefficients of variation) and the validity of the conclusions through removing confounding effects of body size (Cochran 1957; Packard and Boardman 1988). In this study covariance analysis is employed for comparison of RMRs independent of body mass and Tb changes.

Reproductive Energetics and Reproductive Effort

Studies of reproductive energetics should integrate measurements of food intake and assimilation, energy stored or used from maternal body reserves, and changes of RMR to estimate energy allocated to reproduction (Mattingly and McClure 1982; Oswald and McClure 1990). Although RMR is a useful parameter for comparative energetics, it does not, in of itself, provide a

satisfactory basis for calculating the total cost of reproduction or for

estimating the allocation of energy to maintenance, growth and

reproduction. Studier (1979) argued that food intake was the most accurate

estimate of the bioenergetic requirements of reproductively active mice. 10 In addition to substantial hyperphagia and suppression of metabolic energy expenditure during pregnancy, rats and mice can store energy in white adipose tissue, to be mobilized later during lactation (Naismith,

Richardson, and Pritchard 1982). In golden hamsters and Djungarian hamsters, 40-50% of maternal lipid stores were lost during pregnancy

(Wade, Jennings, and Trayhurn 1986; Schneider and Wade 1987).

Therefore, estimation of total energy used during reproduction should also include the examination of maternal body composition and comparison of

energetics between non-reproductive controls and females with young

successfully weaned.

Millar (1977) proposed that comparison of reproductive energetics

among mammalian species be based on an algorithm, reproductive effort

(Re), which is equivalent to the amount of energy required by a female to

support a litter from conception to weaning. Re = (N Ww° 76 ) / M° 76 where

N is litter size, Ww is individual young mass at weaning, and M is maternal body mass before mating. According to this approach, the principal underlying parameters which determine energetic investment in mammals

are maternal body mass and litter size (Millar 1977).

Because growth rate and size at weaning of mammals are fairly

constant, and are related to 0.69-0.73 power of adult body mass, Millar

(1977) concluded that variation in litter size and time to weaning reflect

adaptions to environmental constraints. Furthermore, the postnatal 11 energetic cost of individual offspring is negatively correlated with litter size

(Glazier 1985a). Therefore, litter size appears to play a dominant role in determining energy requirements of mammalian reproduction.

The major goal of this study was a thorough examination of the reproductive energetics of the gray short-tailed opossum (Monodelphis domestica) and the golden hamster (Mesocricetus auratus). The data obtained permit a meaningful comparison of a small didelphid marsupial with a typical rodent relative to the energetic costs, energy used from conception to weaning, and efficiency of offspring production.

Natural History of the Study Species

Gray short-tailed opossums and golden hamsters were chosen for this study because they have similar adult body masses and litter sizes and because both breed readily under laboratory conditions. Maternal investment in reproduction is high in both species, as evidenced in the ratios of litter size or litter mass to maternal body mass (Russell 1982a; Martin and

MacLamon 1985).

The gray short-tailed opossum is pouchless and widely distributed in

the Chaco, Cerrado and Caatinga biomes of south eastern Brazil. It is

found in the remnant Atlantic rainforest and in every major habitat type of

Caatinga, including low and high thorn scrub, disturbed areas and high 12 granitic outcroppings (Streilein 1982). This species feeds on invertebrates, especially insects, small vertebrates, and fruits.

Sexual maturity of female opossums can occur at four months of age, depending on the exposure to male pheromone in male-deposited feces, urine, and scent marks (Stonerook and Harder 1992). Adult female body mass varies from 60 to 100 g. Litter size ranges from 3 to 14, with an average of 7; some females can rear four litters in a year (Fadem et al.

1982). The duration of gestation is 14-15 days and young are weaned at 56 days of age (Fadem et al. 1982; Harder, Stonerook, and Pondy in press).

Neonates migrate to exposed (pouchless) teats on the posterior venter immediately after birth and remain attached continuously for about 14 days

(Fadem et al. 1982). Young are weaned approximately 56 days; thus the total duration from conception to weaning is 70 days (fig. 1.1).

Although golden hamsters are abundant in cultivated fields in

Aleppo, Syria, where wheat is their major diet (Murphy 1971), relatively little is known of their natural history. However, numerous laboratory investigations have shown hamsters to be generally representative of small­ sized eutherians with altricial young. An average litter of 10 is bom after a gestation period 16 days; lactation lasts21 days (fig. 1.1), and, thus, the interval from conception to weaning is only 37 days (Murphy 1971).

Hamsters exhibit high post-natal growth rates and females reach sexual maturity at 28 days of age (Selle 1945). Seasonal anestrus occurs in golden hamsters (Mesocricetus auratus) and gray short-tailed opossums opossums short-tailed domestica). gray and (Monodelphis of auratus) energetics (Mesocricetus reproductive hamsters comparative golden the of design Experimental 1.1 Fig. Opossums Hamsters A A B B E AA A A □ T" A RMR measurement RMRmeasurement A e meaning I Non-reproductiue Non-reproductiue control 38 days acclimation days 38 A * A A A A A Al A A A A ___ 10 A /////////A V 20 n Days 30 ------

40 r~ LACTATION GESTHTION et phase nest phase 50 teat attachm ent ent attachm teat I 60 T Al 70 Al 13 14 reproductively active hamsters following their exposure to short photoperiod

(Seegel and Goldman 1975).

Organization and Objectives of Dissertation

The major goal of this study was a thorough and systematic comparison of the reproductive energetics of opossums and golden hamsters. The following questions were tested: 1) Do gestation and lactation impose similar maternal metabolic costs in both species? 2) Do reproductively active opossums and hamsters have the similar physiological responses

(changes in RMR, heat production, Tb, thermal conductance) compared to non-reproductive conspecies? 3) Do opossums and hamsters compensate for the energy demand of reproduction in the similar ways (such as increasing assimilation efficiency or increasing total assimilated energy)? 4) Do they

(the two species) have the same reproductive efficiency and offspring production efficiency? 5) Do they allocate similar proportions of energy into reproduction, maintenance and growth from conception to weaning (C-W)?

6) Do they assimilate and use the same amount of energy during C-W? On a per young or on a per young per day basis, do they use the same amount of energy? 7) Do they have the same incremental energetic costs (per young or per young per day) during C-W? Resting metabolic rates and assimilated energy were compared between reproductively active (RA) and non- reproductive (NR) female (controls) opossums and hamsters maintained 15 within their zone of thermoneutrality (30 °G). In addition, effects of ambient temperature on reproductive energetics was evaluated in hamsters.

This study was designed to properly compare the reproductive energetics of a representative marsupial with that of a eutherian by controlling or adjusting for confounding effects such as thermoregulation and changes in body mass and body temperature during the reproductive cycle.

Chapters II and in of this dissertation were prepared as manuscripts for publication. Chapter II explores the influence of ambient temperature on changes of metabolic rates of golden hamsters during gestation and lactation. Temperature and reproductive effects were compared among RA-

30 and NR-30 (maintained at 30 °C), and RA-24 and NR-24 females

(maintained at 24 °C). Chapter III examines the relationship of body temperature to RMR of opossums during gestation and lactation. In

Chapter IV, the total cost of reproduction (energy per offspring) and the allocation of energy intake to maintenance, growth, and reproduction from conception to weaning in hamsters and opossums are compared.

Data obtained from these experiments provide a quantitative basis for comparing the total cost and efficiency of reproduction in a representative didelphid marsupial and an eutherian, the golden hamster, and, thereby, contribute to our understanding of the two major reproductive strategies employed by viviparous mammals. CHAPTER H

Effects of Environmental Temperature on Metabolic Rates

During Gestation and Lactation in Golden Hamsters

(Mesocricetus auratus)

A bstract

We determined the influence of ambient temperature on energetic costs of reproduction by measuring resting metabolic rates (RMR), rectal temperature (Tb) and respiratory quotient (RQ) at six reproductive stages

(pre-mating, mid- and late gestation, early, mid- and late lactation) of

Reproductively Active (RA) golden hamsters maintained at 24 °C (RA-24) and 30 °C (RA-30), using non-reproductive (NR) females as controls. RA and NR hamsters consumed more oxygen at 24 °C than at 30 °C (within the zone of thermoneutrality), but only NR females had a lower RQ at the lower

Ta. Gestation caused a significant (P < 0.05) increase in the adjusted RMR of RA-30 dams (compared to NR-30 control), but this effect of gestation was not detected in RA-24 dams. Lactating hamsters had higher RMR than NR females at both Tas. In addition, RA-30 dams had lower RQ during gestation, and higher Tb during lactation. These results indicate that

16 17 increased metabolic costs of gestation are undetectable in golden hamsters maintained below thermoneutrality and that RMR is highest midway through the period of lactation at both T.’s.

Introduction

The mammalian (therian) mode of reproduction is characterized by a unique combination of viviparity and lactation. From conception to weaning, the changing needs of developing offspring are inseparably linked to the physiological status of the mother. The demands of gestation are quite different from those of lactation and, thus, the allocation of maternal energetic resources to maintenance and reproduction during each of the reproductive stages has received considerable attention (Glazier 1985a;

McNab 1986; Thompson and Nicoll 1986; Gittleman and Thompson 1988).

Reproductive costs and efficiency can be evaluated through measures of food consumption or maternal metabolic rate relative to litter size or the mass of young produced. Basal metabolic rate (BMR), the minimum energetic requirement for maintenance, is measured as oxygen consumption under

"standard" conditions, i.e., the animal is non-reproductive, at rest, post-

absorptive, and maintained within the its zone of thermoneutrality. Resting

metabolic rate (RMR) is measured when the animal is at rest, but other

conditions such as the ambient temperature and reproductive status are not 18 standard but defined by the study. Although estimation of maternal food assimilation provides information on total energy expenditures during the reproductive cycle, measurement of RMR provides an attractive approach to the estimation of the energetic costs of reproduction because the confounding effects of physical activity are eliminated (Gittleman and

Thompson 1988).

Interspecific comparisons have revealed a positive correlation between

BMR and reproductive rate (McNab 1986; Hennemann 1983; Derting 1989).

In small eutherians (10 - 1000 g body mass), increased BMR is associated with short gestation, reduced time from conception to weaning, and increased post-natal growth rates (McNab 1986). These associations between reproductive parameters and BMR make the study of maternal increment of RMR particularly meaningful as a direct measure of reproductive costs.

Estimates of the true metabolic cost of reproduction are difficult to obtain because variation in body temperature (Tb) and ambient temperature

(Ta) outside thermoneutrality can strongly influence the RMR of endotherms

(reviewed by Whittow 1971; Mover, Hellwing, and Ar 1988). Measurements of RMR made below thermoneutrality include the metabolic costs of thermogenesis. Unfortunately, relatively few studies have measured RMR within the zone of thermoneutrality in order to control for thermogenic effects (Fleming, Harder, and Wukie 1981; Thompson and Nicoll 1986; 19 Mover, Ar, and Hellwing 1989). Additional complications arise in reproductive studies, because non-shivering thermogenesis by brown adipose tissue (BAT) is suppressed during pregnancy and lactation, at least in small eutherians (Isler, Trayhum, and Lunn 1984; Trayhum 1985; Wade,

Jennings, and Trayhum 1986; Schneider and Wade 1987).

Interpretation of changes in RMR during pregnancy is also complicated by the changing body mass of dams, which in small eutherians can increase by 30-50% during gestation (Mattingly and McClure 1982).

Therefore, proper controls (sexually mature, non-reproductive females) and adjustments for changes in body mass during gestation and lactation need to be included in analysis of metabolism during the reproductive cycle.

Total oxygen consumption usually reaches highest levels just before parturition in small mammals. However, because maternal body mass also increases during gestation, estimates of simple mass-specific RMR often show no change or even a reduction in RMR compared to that of non­ pregnant animals (e.g., Studier 1979; Innes and Millar 1981). Although the use of ratios to adjust biological data that vary allometrically with body size has been criticized (Tanner 1949; Atchley and Anderson 1978), such scaling is commonly employed in physiology. Analysis of covariance (ANCOVA) should be employed instead of simple ratios to improve the validity and precision of the conclusions (Packard and Boardman 1988). 20 The purpose of this study was to determine the effect of ambient temperature on the metabolic cost of reproduction by comparing maternal

RMR independent of changes in body mass and body temperature. The

Syrian golden hamster (Mesocricetus auratus) was chosen for this study because it breeds readily in captivity and is representative of small-sized eutherians with altricial young.

Materials and methods

Experimental Design and Animal Care

Nulliparous golden hamsters (50 - 150 days of age) were obtained from a breeding colony in the Department of Zoology at The Ohio State University.

Sexually mature females were maintained singly in 50x40x30 cm plastic or steel cages supplied with pine shavings for bedding and strips of paper towel for nesting material. Water and food (Purina Rodent Chow) were provided ad libitum. Photoperiod was constant at 14L:10D, with lights on at 0600 hrs Eastern Standard Time. Hamsters were acclimated for at least one month at one of two ambient temperatures: 24 or 30 + 0.5 °C. Adolph and Lawrow (1951) and our preliminary studies indicate that the zone of thermoneutrality for golden hamsters is 28-35 °C. Females acclimated at each Ta (24 or 30 °C) were randomly assigned to either a reproductively active (RA-24 or RA-30) or to a non-reproductive group (NR-24 and NR-30). 21 Estrous cycles were monitored and females in the RA group were paired with individual males prior to the expected night of estrus and ovulation. Golden hamsters have an invariant four-day estrous cycle and the appearance of a conspicuous, sticky vaginal discharge indicates the occurrence of estrus and ovulation the night before (Selle 1945). The cages of pregnant dams were examined daily (after day 14 of gestation) for the presence of neonates (day 0 of lactation). Young were weaned at 21 days of lactation. Body mass of each female with and without her litter was

measured at least every other day from the day prior to mating and

conception to the day after weaning of young. Litter size in hamsters varies

widely (3-14), in part because dams frequently cannibalize a portion of their

litters during the first 5 days postpartum (Fleming 1978; Labov et al. 1986).

Therefore, no attempt was made to adjust litter size. However, because

litter size is positively correlated with energy demands (Fleming 1978;

Glazier 1985a), a litter size of five was chosen as the lower limit for

inclusion of dams in the data analysis.

Measurement of Resting Metabolic Rate

Resting metabolic rates (RMR) of hamsters were initially measured at 30 °C

for 21 NR-30 females and 16 RA-30 females, and at 24 °C for 16 NR-24

females and 20 RA-24 females. Measurements of oxygen consumption were

made at 7-10 day intervals, which included 6 stages in the reproductive 22 cycle of HA dams: 1) prior to estrus, i. e., diestrus of the estrous cycle; 2) early gestation, 7 days after conception; 3) late gestation, 13*15 days after conception and 1-3 days before parturition; 4) early lactation (day 7); 5) mid- lactation (day 14); and 6) at the time of weaning (day 21). RMR of lactating dams was measured first with dam and young occupying the metabolic chamber and, then with the mother alone. Total RMR of the whole litter was estimated from the difference between these two measurements. Food was not withheld from females prior to measurement of RMR. The natural feeding conditions were simulated to avoid potential interference with milk production (Brody 1945; Borer et al. 1979). Therefore, in this study, measures of RMR reflect combined energy costs of BMR and some diet- induced thermogenesis (specific dynamic action) for all females, reproductive metabolism for gestation or lactation (in RA females), and thermoregulation in RA-24 and NR-24 females.

All measurements of RMR were made during 0800-1700 hrs, EST, a time when hamsters are normally inactive with procedures described previously (Hsu, Harder, and Lustick 1988). Each female or lactating dam with young was placed in a plexiglass metabolic chamber (volume approximately 9000 cm3) with air flow set at 370 cm3/min. Carbon dioxide

(C02) production was calculated from the percentage of0 8 in the exiting air

stream before and after C0 2 was absorbed by ascarite. The minimum RMR

(ml 0 2 h'1) was calculated from the lowest oxygen consumption after animals 23 were at rest or sleeping and RMR had reached a steady state for 15-20 minutes. Each trial normally ran for 150-180 min. Rectal temperature (Tb) for each dam or female was recorded alter each trail with a thermistor probe. Each animal was then weighed to the nearest 0.1 gram.

Calculations of 02 consumption were made according to Depocas and Hart

(1957) after C0 2 was absorbed. The respiratory quotient (RQ) was calculated as C0 2 produced/ 0 2 consumed and was used to convert oxygen consumption into heat production (Brody 1945). The relationship between heat production and oxygen consumption is Kcal/ml2 0= 3.815 + 1.232 RQ

(King and Famer 1961). Thermal conductance (Kcal “C '1 animal'1) was calculated as heat production/(Tb - 30).

Post-weaning (one day after) RA and NR females were euthanized with C 0 2 and weighed to the nearest 0.1 g. Interscapular BAT, an index of thermogenic capacity, was carefully separated from parametrial white adipose tissue and weighed, then combined with the remaining carcass.

Dry body mass was determined by drying carcasses to constant weight at 70

+ 5 °C.

Absolute values for RMR, heat production, and thermal conductance were initially derived without adjustment for variation among females in body mass or Tb and are referred to as whole-animal RMR, whole-animal heat production and whole-animal thermal conductance. 24 Statistical Analysis

All statistical analyses were made using Statistical Analysis System software (SAS Institute Inc. 1985). Although litter sizes were not manipulated or controlled, data from females with litter size less than 5 at the time of weaning were excluded from the analyses. However, analyses that included all RA females gave similar results. The final data analyses were based on the following sample sizes: 20 RA-24, 16 RA-30 and 16 NR-24 and 21 NA-30 females. The non-parametric Wilcoxon test was used to compare means of body mass and percent water content between RA and

NR females within temperature treatments and difference in litter sizes and

average weaning mass of young per litter between RA-24 and RA-30 dams.

All mean values through the text are presented + 1 standard error of the

mean (SEM). The effects of temperature and reproduction on interscapular

BAT were tested using ANCOVA, with dry body mass as the covariate to

control for the effect of differences in body mass.

In small eutherians, metabolic rate is positively correlated with both

body mass and body temperature. Therefore, a repeated-measures (or

nested) experimental design within ANCOVA was used to analyze

dependent variables (RMR, heat production, Tb and thermal conductance)

during the reproductive stages of RA dams, or sequential measurements of

NR females through the equivalent period. These dependent variables and

their covariates (in parentheses) were: RMR (body mass and Tb), heat 25 production (body mass and Tb), thermal conductance (body mass), and Tb

(body mass and RMR). Adjusted means of these dependent variables

obtained from ANCOVA were used for within and between treatment

comparisons. A similar experimental design with analysis of variance

(ANOVA) was used to analyze RQ. Significance of main effects

(temperature and reproductive status) were tested using mean square for

individual females within temperature or within reproductive treatment,

respectively, as the error term. If individual variation within treatment

was not significant, the treatment main effects were then tested using mean

square for error. Reproductive periods were divided into gestation and

lactation to further examine changes of dependent variables. Analyses

using log-transformed RMR and body mass resulted in slightly better

explanatory power (higher R2, or coefficient of determination) than analyses

using untransformed data. Therefore, data sets were analyzed after log-

transformation of metabolic rate and body mass. Homogeneity of slopes

across treatments were tested for each reproductive stage, and the

appropriate ANCOVA model (i.e. common or separate slope model) was

selected before performing all statistical tests for treatment effects, and

calculating adjusted means of dependent variables.

The effects of temperature, litter size and sex of young on the

weaning mass of young were tested using ANOVA. Temperature effect was

tested using mean square for dams within temperature, litter size effect 26 using mean square for dams within litter size, and sex of young effect using mean square for sex interaction with dams within litter size. Relationships between maternal physical conditions and production (litter size or litter mass) were tested using multiple regression, with litter size or litter mass as the dependent variable and body mass and RMR of RA-30 dams measured before mating as independent variables. Tests were performed on data collected on days 7, 14 and 21 of lactation.

The effects of temperature, developmental stage (days 7,14 and 21 of lactation) on the estimated RMR of litters were tested using ANCOVA, with total litter mass as the covariate. A similar experimental design with

ANOVA was used to analyze RQ of the dam and her young.

R esults

Changes in Maternal Body Mass and Interscapular BAT

Patterns of change in body mass of RA-24 and RA-30 females were similar

(fig. 2.1). Body mass of RA-24 females increased an average of 37.5 % (41.3

+ 1.8 g), compared to a 40.4% (40.3 + 1.3 g) in RA-30 females. Loss of weight after parturition was also similar in RA-24 and RA-30 dams, 20.8% and 19.3%, respectively. However, body mass declined more in RA-24 (15.5

+ 1.0 g) than in RA-30 (6.4 + 1.4 g) dams during the 21-day lactation period

(P < 0.001). The body mass of NR-24 females increased 19.1 + 1.4 g while 27

o- RA-24 CJ11 5 0 - •- RA-30 A- NR—24 / CO NR—30 6 $ CO k i / <

> - 100 Q O mating birth weaning CD J, GESTATION | LACTATION

-1 6 •7 0 14 21 DAYS

Fig. 2.1 Average body mass (g anim al1) of reproductively active (RA) female golden hamsters maintained at 24 °C (n=20) or 30 °C (n=16) and of non- reproductive (NR) females maintained over the same experimental period at

24 °C (n=16) or 30 °C (ri=21). Data are plotted relative to the day of birth

(day 0 ). 28 that of NR-30 females increased 15.7 + 1.8 g over a 38-day time period (P <

0 .0 0 1 ), equivalent to the time required for mating gestation and lactation to weaning in RA females.

Reproduction resulted in a significant reduction of interscapular BAT of dams at weaning compared to NR females (F168=10.4, P < 0.002).

Females maintained in thermoneutrality had less interscapular BAT than females maintained at 24 °C (F16a=18.9, PcO.OOl). Because water content of RA females was significantly higher (PcO.OOl) than in NR females (24 °C:

72.7 + 0.5 % versus 6 8 .8 + 0.7 %, z=-3.52; 30 °C: 71.0 + 0.2 % versus 68.0 +

0.5 %, z=4.52), dry body mass was used as the covariate for ANCOVA of interscapular BAT. The average wet mass of interscapular BAT (adjusted to the average dry body mass of 35.07 g) was 116.5 + 8 .6 mg in post- weaning RA-30 females, 143.7 + 7.8 mg in NR-30, 143.5 + 8 .6 mg in RA-24, and 184.0 ± 9.2 mg in NR-24 females.

Effects of Ambient Temperature on RMR

After removing the effects of differences in body mass and Tb, average RMR throughout the experimental period was higher for animals maintained at

24°C than at 30°C. Average RMR of NR-24 females was 51.7% higher than that in NR-30 females (Fii36 = 110.9, P < 0.001), while this difference was

45.8% in RA females (F 134 = 212.7, P < 0.001; fig. 2.2). Significant differences of RMR were also found among different reproductive stages 29

o RA-24 250*- RA-30

2 4 °C -C 200 4- CM O 150 + fc 3 0 ° C rv bg 100 + a : mating birth weaning + GESTATION J, LACTATION + 50 —*— I------1------T ------1------1------T“ — 16 - 7 0 7 14 21 DAYS

Fig. 2.2 Ac^uBted mean RMR (resting metabolic rate, ml 0 2 h r 1, ± 1 SEM) of RA golden hamsters at 24 °C and 30 °C. Adjusted meanB were from

ANCOVA models adjusted to the average body mass 115.3 g and rectal temperature 37.5 °C at 24 “C and 111.9 g and 36.6 °C at 30 °C. The average of adjusted mean RMR of NR females is presented as dashed or dotted lines for females maintained at 24 °C and 30 °C, respectively. 30 (Fg.ioi - 15.3, P < 0.001) and for the interaction of temperature and stage of

RA females (Fb i161 = 2.34, P < 0.05). These differences indicated that patterns of increased RMR were not the same for RA-30 and RA-24 females

(fig. 2.2). In contrast, RMR of NR females did not change over equivalent time periods (P > 0.96), nor was there a significant interaction between sequential measurements and temperature (P > 0.67). Therefore, the average of adjusted mean RMRs recorded over the 38-day experimental period for NR females was represented as a single baseline for each temperature treatment (fig. 2.2). Because whole-animal heat production

(Kcal h r 1 animal'1) is derived from RMR values and because the results of statistical analyses for RMR and heat production were similar, only results from RMR analyses are reported here.

The increase in thermal conductance below thermoneutrality was similar to the increase in RMR. The adjusted mean thermal conductance of

NR-24 females was 130.93 Kcal “C 1 anim al1 compared to 93.98 Kcal °C1 animal'1 in NR-30 females (at average body mass 113.7 + 1.2 g).

Average RQ was lower in animals maintained at 24 °C than in those maintained within thermoneutrality. Average RQ was 0.75 + 0.02 for NR-

24 females compared to 0.83 + 0.01 for NR-30 females (Fi132= 17.25, P

<0.001) but average RQ was 0.77 + 0.01 for RA-24 dams, similar to 0.79 +

0.01 for RA-30 dams (F1150 = 3.77, P > 0.54). Ambient temperature did not have a significant effect on Tb within each RA or NR group. Rather, RMR 31 and individual variation within temperature treatment accounted for a significant amount of the variability of Tb (P < 0.01).

Effects of Gestation and Lactation

The average whole-animal RMR of RA-24 dams increased 27.9 + 3.3% from premating levels to late gestation, similar to the 28.3 + 1.6 % increase seen in RA-30 dams during the same interval (fig. 2.2). Average whole-animal

RMR through lactation increased 33.7 + 3.7 % from premating levels in RA-

24 dams and 40.4 + 2.7 % in RA-30 dams. Because RMR changes were associated with body mass and Tb changes through gestation and lactation, the later two variables were standardized (used as covariates) for comparisons of RMR of RA and NR females.

An energetic cost of gestation, identified as a significant elevation in

RMR of RA over NR females, was detected in RA-30, but not RA-24 females

(table 2.1). Individuals within treatment and Tb accounted for most of the variability of RMR at 24 °C. When body mass and Tb effects were held constant, RMR of RA-24 females during mid- and late gestation were not different from premating RMR’s (P > 0.05). By contrast, RMR of RA-30 females during late gestation was higher than the pre-mating RMR and the

RMR of NR-30 females (P < 0.05, fig. 2.2).

RMR of lactating RA-24 and RA-30 females was higher (P < 0.05) than that of NR females maintained at the same temperature (table 2 .1). Table 2.1. Significance levels from ANCOVA for oxygen consumption (RMR, log ml 02 h r 1), therm al conductance (C, Kcal "C1 h r 1) and rectal temperature (Tb, °C) and from ANOVA for respiratory quotient (RQ) in 16

RA-30, 21 NR-30, 20 RA-24 and 16 NR-24 female golden hamsters.

Gestation Lactation

24 °C 30 "C 24 °C 30 °C Source RMR C Tb RQ RMR C Tb RQ RMR C Tb RMR C Tb

Treatment effects

R.E. ** ** ** *> ** ** • ID(R.E.) * * ** ** ** ** Stage * ** R.E. x stage ** *

Covariates

Log(mass, g) - Tb ** - - * - * -- - log(RMR) - ** - - * - * -

R2 .85 .81 .77.60 .88 .81 .51 .66 .77 .64 .77 .87 .73 .76

** : P<0.01, * : P<0.05, - : not included in the model. R. E. : abbreviation for reproductive effects, i. e. RA females vs. NR females; significance of R. E. is tested by mean square from ID(R. E.) if ID(R. E.) had a significant effect on dependant variables. ID : abbreviation for individual animals. Stage : gestation measurements made at before mating, mid- and late gestation, while lactation data sets included early, mid- and late (weaning) lactation. 33 The adjusted mean RMR was highest during early lactation, but was not significantly different from rates at mid-lactation at either temperature treatment. The adjusted mean RMRs of RA-30 females remained significantly higher than baseline (RMR of NR-30 females) at all stages of lactation (P < 0.05). However, the adjusted mean RMR of RA-24 females declined at weaning and was not significantly different from the baseline value (fig. 2 .2 ).

To compare the cost of reproduction without confounding effects of thermogenesis between RA-24 and RA-30 dams, the whole-animal RMR of each RA dam was partitioned into the non-reproductive portion

(maintenance) and reproductive increment of metabolism (RIM) for maintaining body mass and Tb as a function of reproductive stage. First, a regression equation for estimation of the RMR of the non-reproductive condition was derived from whole-animal RMR (ml 0 2 h r1) of NR-24 and

NR-30 females with body mass and Tb as independent variables.

(1) a t 24°C, log10(RMR) = -0.2547 + 0.57 x log 10 (mass, g)

+ 0.0369 x Tb (°C), (r 2 = 0.40, P < 0.001)

(2) at 30°C, RMR = -210.0309 + 0.5738 x (mass, g) + 7.3807 x Tb,

(r2 = 0.47, P < 0.001).

Next, the non-reproductive portion of RMR of RA dams was estimated from the regression equation using the observed body mass and Tb of RA dams.

The reproductive increment of metabolism was then obtained as the 34 difference between the observed whole-animal RMR and the estimated non- reproductive metabolism of RA females. A RIM of 0 indicated no increase in RMR over the non-reproductive condition. RIM waB analyzed through

ANOVA because mass and Tb had no residual effect on RIM. The effects of temperature were tested using mean square for individuals within temperature as the error term when individuals within temperature had a significant effect on RIM.

Mean values of RIM were similar for RA-24 and RA-30 dams (P >

0 .11), but stage, stage and temperature interaction and individual variation had significant effects on RIM (P < 0.02). RIMs of both groups were close to

0 during early gestation and then increased to highest levels in early lactation (fig. 2.3B). Significant differences between average RIMs of RA-24 and RA-30 dams was found at early and mid-lactation.

Thermal conductance in RA-30 dams was significantly higher than

NR-30 females throughout gestation and lactation (table 2.1). However, no significant difference in thermal conductance was found between RA-24 and

NR-24 females (table 2.1). The highest thermal conductance was at day 7 of lactation in both RA-30 (108.6 Kcal "C1 animal'1) and RA-24 dams (161.3

Kcal “C'1 animal'1).

When body mass and BMR effects were held constant, Tb of lactating

RA-30 females was higher than that of NR-30 females (table 2.1 and fig.

2.4). The adjusted mean Tb of RA-30 dams increased from 36.6 + 0.1 °C at 35

!< m 2 0 0 -

-16 -7

^ 6 0 - - _ l o UJ 0Q o R A -24 R A -30 i r

o o 2 0 0 d h - Q_ Z LU IaJ o r 2 o ■ UJ ££ mating birth weaning a \l/ GESTATION \ LACTATION | -1 6 - 7 14 21 DAYS

Fig. 2.3 A) Wet mass (mg) of interscapular BAT of golden hamsters. Data of pregnant and lactating dams maintained at 24 °C (filled diamond) were redrawn from Wade et at. (1986). Unshaded triangle, NR-24 females; unshaded circle, RA-24 dams; shaded circle, RA-30 dams. B) Average Reproductive Increment of Metabolism (RIM, ml Oa hr'1) of RA- 24 and RA-30 dams. RIM was calculated at each stage by subtracting non- reproductive metabolism estimated from the equation generated for NR females with identical body mass and Tb with each reproductive stage from observed RMR of each RA dam. 36

o - r O - R A -24 O O • R A -30 m 3 8 < 24°C c r LU Q_ i 3 7 - 30 C mating birth weaning O 1 GESTATION | LACTATION LU 36 4 m 16 - 7 0 7 14 21 DAYS

Fig. 2.4 Adjusted mean Tb (+ 1 SEM) of golden hamsters. Adjusted means

were from ANCOVA models adjusted to the average body mass 115.3 g and

RMR 221.6 ml 0 2 h 1 at 24 °C and 111.9 g and 134.7 ml Oa h 1 a t 30 °C.

Symbols are the same as those used in Fig. 2.2. 37 mid-gestation to 37.2 + 0.1 °C at day 7 of lactation, higher than at any other stage (P < 0.05), and then decreased to 36.7 + 0.1 °C at weaning. The adjusted mean Tbs during pregnancy and lactation of RA-24 dams were not different from those of NR-24 females (P > 0.05). Apparently, individual variation and RMR accounted for the most variability of Th at ambient temperature below thermoneutrality (table2 .1).

Pregnant RA-30 dams had lower average RQ than NR-30 females

(F 133 =10.2, P < 0.01, table 2.1). However, no significant difference in RQ was found between RA-24 and NR-24 females (P > 0.73); individual patterns accounted for variability of RQ at 24 °C. No significant difference in RQ was found between lactating dams and NR females.

Growth and RMR of Young

Weaning mass of individual young was affected by individual dam within litter size (P < 0.001; 24 °C: F 13135 = 34.0; 30 °C: F 11>94 = 8.1), but not by the sex of young (P > 0.87), litter size (P > 0.49) or Ta (P > 0.85). The RMR and body maBS of RA-30 dams measured before conception did not explain a

significant amount of variation in litter size, or total litter mass on days 7,

14 or 21 of lactation. The average mass of young from RA-24 dams at

weaning was 36.9 + 1.4 g litter'1, very similar to 37.2 + 1.0 g litter'1 for RA-

30 dams (P > 0.83). The average litter size at weaning was 7.8 + 0.4 for

RA-24 dams, not significantly different than 6.9 + 0.4 for RA-30 dams (P > 38

0.22) The overall average was 7.4 + 0.3 (n = 36), a reduction of about 1.6 pups from day 1 of lactation. Although sex ratios (percent males) within litters ranged from 25% to 80%, overall average sex ratio was 48% (n = 36).

Ambient temperature and total litter mass had significant effects on the estimated RMR of litters (P < 0.05). The average total litter mass nearly doubled every seven days during lactation. Because RMR of litters was positively correlated with litter mass (r = 0.968, P < 0.001, n = 108), litter size was not the main effect on RMR of litters (P > 0.05) when litter mass was also included in the analysis. Stages of lactation and Ta had

significant effects (P < 0.001) on RQ of the dam and her young. The

average RQ of RA-24 dams and litter together was 0.82 + 0.01, less than

observed for RA-30 dams and young (0.89 ± 0.01, P < 0.001). Higher RQ of

dams and litter together was found at weaning than day 7 and day 14 of

lactation (P < 0.05).

D iscussion

The effect of gestation on RMR, RQ, and thermal conductance was detected

in RA-30 but not in RA-24 dams. The fact that gestational effects on RMR

were seen in females maintained in thermoneutrality but not in those held

at 24 °C clearly indicate that the energy requirements of gestation are not

simply additive to thermogenic costs of maintaining body temperature below 39 thermoneutrality. The reasons for this are not entirely dear but probably involve a complex interaction of thermogenic factors and changes in body size during gestation.

Thermogenic caparity and the amount of interscapular BAT in hamsters rise progressively with decreasing T. from 30° to 13 °C (Trayhum et al. 1983). However, the thermogenic capacity of BAT also declines during gestation and lactation (Trayhum 1985; Wade, Jennings, and Trayhum

1986). Nonshivering thermogenic activity also declines with increasing litter size (Isler, Trayhum, and Lunn 1984). The difference of thermogenic caparity between RA-24 dams and NR-24 females illustrates the difficulty of measuring gestational coBts when RMR comparisons are made below thermoneutrality, as in Mattingly and McClure (1982).

Although hamsters gain weight rapidly during gestation, they actually lose some lipid content and carcass energy during late pregnancy

(Schneider and Wade 1987; Wade, Jennings, and Trayhum 1986). This is consistent with the observation that pregnant RA-30 had a lower RQ of pregnant RA-30 dams than NR-30 females, indicating a greater rate of fat catabolism. This might also explain the increase in thermal conductance in

RA-30 dams, if thermal conductance increases as a result of lower body fat.

However, mass-specific minimum thermal conductance measured below thermoneutrality decreases with increasing body size (Herreid and Kessel

1967), and after removing the effect of differences in body size, no 40 significant difference of thermal conductance was found between RA-24 and

NR-24 females. These observations reflect a distinct conservation of energy in pregnant RA-24 dams. Maternal physiological changes, possibly coupled with increased heat from active reproductive organs and growing fetuses, might contribute to meeting the thermogenic demands of thermoregulation.

The highest estimates of RIM were made on day 7 of lactation, indicating that milk production might be higher during early lactation rather than late lactation. These observations are consistent with life history and development patterns in golden hamsters. Hamster pups start ingesting caecotrophe beginning on day 5 of lactation (Dieterlen 1959).

Caecotrophe is a semisolid digestive excreta emanating from the maternal caecum, which is rich in bacteria and nutrients (Leon 1974). Pups start to eat solid food on day10 of lactation (Daly 1976). Suckling and milk production apparently reduced after 10 days of lactation as indicated by nipple shifting behavior; all pups shift nipples at 15 days of age (Hall and

Rosenblatt 1979). Thus it would appear that milk is the major food source only during the first10 days of lactation; thereafter the proportion of solid food in the diet increases dramatically towards weaning.

The greater increase of RMR during lactation in RA-24 dams relative to RA-30 dams was probably caused by a combination of higher milk production and thermoregulatory demands. Because the growth rate of RA-

24 pups was similar to that of pups raised at 30 °C, the RA-24 dams would 41 have required more energy compensate for the greater heat loss at 24 °C.

Hamster pups lack physiological mechanisms for thermoregulation before 10 days of age but they can regulate body temperature within 2 °C of nest temperature at 14 days (Leonard 1982). In addition, maternal thermoregulatory costs could increase following loss of mass involved with parturition and fat mobilization during gestation (Wade, Jennings, and

Trayhum 1986). However, the mass of interscapular BAT in RA-24 dams was similar to that in NR-30 females, indicating low thermogenic capacity of RA-24 dams as NR-30 females maintained within thermoneutrality. At the same time, thermogenesis associated with milk synthesis in RA-24 dams might have contributed heat for thermoregulation and allowed RA-24 dams to maintain thermal conductance similar to that in NR-24 females.

Although the cost of lactation was higher in RA-24 dams than in RA-

30 dams, the proportion of energy allocated to reproduction is relatively small compared to total metabolic energy expenditures, which include thermoregulation. Average whole-animal RMR increased 34% during lactation in RA-24 dams and 40% in RA-30 dams, a relatively small difference. The incremental cost of reproduction (gestation and lactation combined) from RIM was not significantly different between RA-24 and RA-

30 dams.

Adjusted mean RMR declined sharply near the time of weaning in

RA-24 dams. This probably reflected reduced milk synthesis and weaning 42 of the young. Similar average weaning masses of the young of RA-30 and

RA-24 dams indicate that dams and young compensate for thermoregulatory costs through increased milk production and food intake as in golden- mantled ground squirrels, Spermophilus saturatus (Kenagy, Stevenson, and

Masman 1989). Low Ta can actually stimulate hyperphagia (Whittow 1971), and tissue growth and milk synthesis are higher than in warm environments (Brody 1945).

The ad libitum food availability used in this experiment probably simulated natural conditions. Reproducing golden hamsters do not increase food intake until the time of lactation, but significant food hoarding begins on day 12 of pregnancy (Fleming 1978). In contrast, fats accumulated in pregnant rats through hyperphagia are used later in lactation (Naismith,

Richardson, and Pritchard 1982). Loss of maternal lipid reserves in hamsters (Schneider and Wade 1987; Wade, Jennings, and Trayhum 1986) suggests that the energy demands of lactation cannot be met solely by food hoarding or increased food intake. Also, environmental factors might limit maintenance water balance during lactation. Because golden hamsters are native to desert environments (Murphy 1971) and pups begin consumption of solids at an early age (Daly 1976), an adequate water supply is crucial for growth and survival of young.

The litter size and growth rates of nursing young observed in this study are comparable to those reported in previous studies with ad libitum 43 feeding (Fleming 1978; Labov et al. 1986). However, the weaning mass of young in our study was not affected by litter size. Fleming (1978) found that the average weight of young hamsters in litters of 5-6 to be less than that for young in litters of 1-4, and Glazier (1985a) reported that growth of young is inversely related to litter size in Peromvscus.

Weaning mass and growth rate of hamster young in our study were not correlated with maternal RMR and body mass before conception. This is consistent with the observation that intraspecific variation in BMR is not correlated with the production of young (Derting and McClure 1989; Hayes,

Garland and Dohm 1992). However, energy expenditure in both lactation and postnatal growth rates is correlated with BMR of five species of

Peromvscus (Glazier 1985b). In addition to transferring energy, nutrition and water to neonates, maternal care such as helping pups in urination and defecation is also critical for young survival. Therefore, individual variation of dams plays a significant role in many physiological and reproductive processes, and also provides the fundamental basis for natural selection.

This study indicates that maternal energy requirements are most pronounced during lactation, particularly in ambient temperatures below thermoneutrality. Lactation is therefore a critical period for determining reproductive success for small eutherians, such as the golden hamster.

Altricial hamster pups must ingest maternal caecotrophe followed by solid food at an early age in order to reduce milk dependence. In contrast, 44 energy conservation during gestation at Ta below thermoneutrality is achieved by increasing body mass and decreased BAT thermogenesis.

Additional energy saving of dams can be achieved by allocating pre-existing expenditures into reproduction, such as decreasing activities and changing behavior (Richards 1966). Therefore, changes in ambient temperature aifect reproductive costs and may play an important role in the evolution of increased body size with prolonged gestation in eutherian mammals.

However, ecological components and environmental limitations such as food and water availability, in space and time also influence physiological and reproductive strategies. CHAPTER i n

Effect of Body Temperature on the Metabolic Rate of

the Gray Short-tailed Opossum (Monodelnhis domestica)

during Gestation and Lactation.

A bstract

Resting metabolic rates (RMR), body temperature (Tb) and respiratory

quotient (RQ) of reproductively active (RA) opossums maintained at 30 °C

were compared with non-reproductive (NR) control females. Average body

mass, Tb as well as whole-animal RMR increased in RA dams throughout

gestation and lactation. However, when the effects of the differences of

body mass and Tb were removed, RMR and heat production of RA dams

during gestation were similar (P > 0.05) to those of NR females, but during

lactation were higher (P < 0.05) than those of NR females. Adjusted mean

RMR was highest at day 40 of lactation, significantly higher than at

diestrus, estrus, or at the time of weaning. Day 40 of lactation was also the

only stage in which the adjusted mean reproductive increment of

metabolism (RIM) differed from zero. Adjusted means of Tb and thermal

conductance were similar between RA and NR females. Litter size and

45 46 litter mass after day 40 of lactation were inversely related to the maternal

RMR at diestrus (BMR). Although opossums increase energy expenditures during reproduction, most of this increase was associated with higher maintenance metabolism and not closely correlated with gestation and lactation.

Introduction

Mammals are in general homeothermic; i.e., able to maintain relatively

constant body temperature (Tb), independent of environmental conditions.

However, circadian rhythms in Tb and metabolic rates have been recognized

in mammals (reviewed by Aschoff et al. 1974), with highest levels being

recorded during the active or waking phase and lowest levels during the

inactive or sleeping phase (Heller and Glotzbach 1977). Even greater

variation in metabolic rates have been recorded in some mammalian taxa

during torpidity and hibernation (reviewed by Lyman et al. 1982). Of

particular interest are differences in Tb and metabolic rates between

marsupials and eutherians (MacMillen and Nelson 1969; Dawson and

Hulbert 1970; McNab 1978), two infraclasses (Metatheria and Eutheria)

which are more commonly recognized for differences in their modes of

reproduction. 47 Body temperatures of marsupials under standard conditions (resting and within thermoneutrality) are typically between 33-35 °G (Dawson and

Wolfers 1978; McNab 1978), lower than that of eutherians (36-38 °C). Tb in marsupials varies not only with the circadian cycle (Kleinknecht, Erkert, and Nelson 1985) but also with ambient temperature and physical activity

(e.g. Morrison and McNab 1962; Wallis 1976; Geiser 1986). The lower

critical temperature of thermoneutrality is not well defined in some species, because Tb is maintained only 2-3 °C above ambient temperatures, as seen in pigmy possums, Cercaertus nanus (Bartholomew and Hudson 1962).

Basal metabolic rates (BMR) of eutherians generally follow the three-

fourths power of body mass relationship (heat production in Kcal day1 = 70

x (body mass, Kg)0 75, Kleiber 1961). This relationship is based on data from

eutherians, which have Tbs ranging 36-38 °C and correspondingly high

BMRs. McNab (1978, 1980) demonstrated that differences in food habits

among eutherians are correlated with the degree of departure from the

Kleiber-predicted BMR and has suggested that these differences represent

ecological constraints. BMRs of marsupials are about 30% lower than the

Kleiber-predicted BMR for eutherians (MacMillen and Nelson 1969; Dawson

and Hulbert 1970; McNab 1978) but interspecific variation in BMRs does

not appear to be related to diet (McNab 1980). Low BMRs of marsupials

are partially related to low Tb (Dawson and Hulbert 1970). 48 Although the energetic costs of reproduction have been studied in a variety of mammalian species, few studies have fully accounted for the effect of variable Tb on resting metabolic rate (RMR). Interspecific comparisons of reproductive energetics are difficult to make because variation in Tbs and ambient temperatures below the lower critical temperature of thermoneutrality strongly influence RMR of animals.

Information on changes in Tb during the reproductive cycle (conception to weaning) and on the effect of such changes on RMR is scarce. This might reflect the fact that biologists have not generally recognized the existence of wide daily or seasonal variation in Tb (3-5 °C) in many mammals.

Mammals with BMR lower than predicted from Kleiber’s body-mass relationship show large increases in average maternal RMR during reproduction (Thompson and Nicoll 1986). Such increases in RMR during gestation and lactation may parallel elevation in Tb, or they might be influenced by other physiological changes associated with reproduction. To date, energetic studies have not partitioned the effects of changing RMR as a result of elevated Tb associated with reproduction or vice versa.

In addition to having lower BMRs and labile Tbs, marsupials exhibit a mode of reproduction that differs markedly from that of eutherians.

Gestation, which is very brief in marsupials relative to lactation, is followed by birth of embryonic neonates which develop during an extended period of lactation. Neonates of marsupials are very small; litter mass at birth 49 averages 0.09% (32 species) of maternal body mass, much less than 15.5%

(132 species) for eutherians (Hayssen, Lacy, and Parker 1985).

Lactation, which is for marsupials the dominant mode of energy transfer to the developing young, proceeds in two phases: the teat attachment phase and the nest phase. Upon locating a teat, the neonate takes the teat into its well-developed oral cavity wherein it becomes firmly affixed and it is not voluntarily released during the teat attachment phase.

This stage is roughly equivalent to the latter part of intrauterine gestation of eutherians (Sharman 1970). Neonates are ectothermic at birth and thermoregulatory abilities are correlated with the development of pelage and thyroid function (Setchell 1974). In species with a well-developed pouch, a constant temperature in the pouch serves to regulate Tb of young

(Setchell 1974). Thermal protection is reduced in pouchless species or in those with open pouches (Geiser, Matwiejczyk, and Baudinette 1986).

Maternal investment of marsupials is strongly influenced by presence or absence of the pouch, which in term is related to adult body size and litter size (Russell 1982a). Most New World marsupials are pouchless (64 out of 78 species). Pouchless marsupials are often small (< 200 g) but with large litter sizes (>3). The teat attachment phase of pouchless species is shorter than that of species with a well-developed pouch (one third versus two thirds of the lactation period, respectively Harder 1992). However, the ratio of litter mass to maternal mass in small, pouchless marsupials is high, 50 even early in lactation. Therefore, the pouchless condition might reflect advantages for small-sized marsupials associated with leaving young in the nest in an early stage while the mother forages (Harder 1992).

The major objectives of this study were to assess 1) the interaction of

Tb and metabolism during diestrus, estrus, gestation, and lactation and 2) the relationships between RMR and the production of young (litter size and litter mass) in a small didelphid marsupial. To provide insight into the relationship between increased RMR and T„,I examined metabolic patterns during the reproductive cycle of a small(100 g) pouchless opossum native to

Brazil, the gray short-tailed opossum (Monodelphis domestica. Marsupialia:

Didelphidae) maintained within thermoneutrality (30 °C). BMR of gray

short-tailed opossums (hereinafter opossums) is 55-64% of the Kleiber-

predicted value (Thompson and Nicoll 1986; Dawson and Olson 1988).

Average Tb of this species within thermoneutrality is 32.6 °C (Dawson and

Olson 1988), which is typical of neotropical marsupials (McNab 1978). Both

RMR and Tb of opossums increase during lactation (Thompson and Nicoll

1986) and I hypothesized that elevation of RMR in reproducing opossums is

associated with litter production (size and mass) and is independent of

changes in Tb. If increases in RMR during reproduction is correlated with

Tb and can be explained by temperature effects and is independent of litter

production, then an alternative explanation is that the proportion of RMR 51 invested in reproduction is relatively small compared to increased maintenance costs during the reproductive cycle.

Materials and methods

Experimental design and animal care

Forty-four female opossums, aged 4-18 months, were obtained from a breeding colony maintained in the Department of Zoology, at The Ohio State

University, Columbus, Ohio. They were randomly assigned to the

Reproductively Active (RA) group or the Non-Reproductive (NR) control

group. Opossums were housed singly in opaque polycarbonate rat cages

with pine shavings as bedding. Strips of paper towels and square plastic

containers were provided for nesting. Ambient temperature within the

colony was maintained at 30 +0.5 °C which was within thermoneutrality for

opossums (Dawson and Olson 1988). Females were acclimated at least one

month before the start of the experiment. Photoperiod was maintained at

14L:10D, with lights on at 0600 hrs, EST. Water and food (Fox

Reproduction Food, Milk Specialties Product Inc., New Holstein, WI) were

provided ad lihitum.

Estrus and ovulation in opossums are induced by male stimuli

(Fadem 1987). Estrus, characterized as comified epithelial cells in

urogenital smears (Fadem and Rayve 1985), was induced in RA females by 52 their exposure to male pheromone by cage switching for 5-7 days (Fadem

1987). Upon expression of urogenital estrus, each female was paired with her cage-switch male until copulation was confirmed through time-lapse videorecording or until leukocyte infiltration appeared in urogenital smears

(Baggott, Davis-Butler, and Moore 1987). Pregnant dams were examined daily toward the end of gestation (14-15 days, Fadem et al. 1982) for the presence of newborn. The first day that neonates were observed was designated day 0 of lactation. The teat attachment phase of lactation is about 14 days (Kraus and Fadem 1987), but young are occasionally left in the nest as early as 12 days old (personal observation). Body mass of each dam or dam with her litter was measured at least every other day. Young were weaned on day 56 of lactation.

All females were nulliparous except two which had litters 7 months before this study. The potentially confounding effects of unusually large dams or extremely small litter sizes were controlled by excluding from the analysis dams larger than 105 g (n = 1) or with litter sizes of less than six

(n =4). Final data analyses were based on data from 20 RA dams and 19

NR females. The average age of NR (8.2 + 0.8 months) and RA females (7.6

+ 0.7 months) and body mass (table 2) of these two groups were similar at the beginning of this study (Wilcoxon test, P > 0.05). Measurement of metabolic rate

Resting metabolic rate (RMR) was measured (at 30 + 0.1 °C) at 10 stages of the reproductive cycle of RA females. These included: 1) diestrus, prior to pheromonal induction of estrus; 2) estrus, 0-3 days before copulation; 3) early gestation, days 5-7 after copulation; 4) late gestation, days 12-14 of gestation (1-3 days prior to parturition). Stages 5-9 were days 10, 20, 30,

40, and 50 of lactation and stage 10 was the day of weaning (day 56).

RMRs of lactating dams were first measured with their young, then the

RMR of the litter alone was measured when time permitted and the separation of young and dams became possible, i.e., young were firmly attached to teats at day 10 of lactation. Measurements of whole litter RMR were based on sample sizes of 2, 9, 19, 19, and 17 litters for stages 6-10, respectively. I was particularly concerned about the disturbing the relationship between dams and young at stages 6 and 7. In addition, some litters at stage 8-10 did not remain in a resting state when separated from their mothers. Therefore, whole Utter RMR for stages 5-10 was estimated through least square regression analysis with age and litter mass as independent variables. The RMR of lactating females alone was estimated by substraction of the estimated RMR of Utters from the measured RMR of the mother with her Utter.

The RMR of NR females was measured seven times at 7-14-day intervals over a period of 80 days (fig.1 ), equivalent to the time required for 54 induction of estruB, mating, gestation and lactation to weaning in RA females. Occurrence of estrus is very low in isolated female opossums

(Fadem 1987) and urogenital smears confirmed that all NR females were in diestrus at the time of each RMR measurement.

In order to simulate natural condition and to avoid the potential interference with milk production (Brody 1945), food was not withheld from animals prior to measurement of RMR. Therefore, measures of RMR reflect combined energy costs of BMR and diet-induced thermogenesis, and reproductive metabolism for gestation or lactation (in RA dams). The equipment, procedures, and calculations of oxygen consumption, C02 production and respiratory quotient (RQ) was described Chapter II. Rectal temperature (Tb) for each NR or RA dam was recorded with a thermistor probe after each trial. Because it was critical that these measurements reflect the actual Tb of the animal at the time of 02 consumption measurement, Tb measurements were recorded only if they could be made immediately (within 3 minutes after the metabolic chamber was removed from water bath) or before signs of disturbance occurred in the animals.

Each animal was then weighed to the nearest 0.1 grams. Post-weaning RA and NR females were euthanized with C0 2 and weighed to the nearest 0.1 g. Dry body mass was determined by drying carcasses to constant weight at

70 + 5 °C. 55

Heat production (Kcal hr'1 anim al1) was calculated by multiplying

RMR and energy equivalent of oxygen conversion factor for RQ (Brody

1945). Each lactating dam and her litter were assumed to have the same value of RQ as was measured in dam and litter together. Thermal conductance (Kcal h r 1 a n im a l1 °C) was obtained by dividing heat production by (Tb - 30). The temperature coefficient (Q10) was derived from the correlation coefficient of log (RMR / body mass) or log (heat production / body mass) related to Th. This derivation was based on the relationship: log Rj -log Ra = log Q10 * (Tbl - Tb2) / 10. The percentage of Kleiber-predicted was calculated as heat production (Kcal day'1) divided by 70 x (body mass,

Kg)0 76. Therefore, it is mass-specific heat production presented as the percentage of Kleiber-predicted BMR for eutherians.

Absolute values for RMR, heat production, and thermal conductance were initially derived without adjustment for variation among females in body mass or Tb and are referred to as whole-animal RMR, whole-animal heat production and whole-animal thermal conductance.

Statistical Analysis

All statistical analyses were conducted using Statistical Analysis System software (SAS Institute Inc. 1985). The non-parametric Wilcoxon test was uBed to compare the average of age, body mass and percent water content of 56 RA and NR females. All mean values are presented + 1 standard error of

the mean (SEM).

RMR was correlated (P < 0.001) with body mass and Tb for RA dams

(n = 119; body mass: r = 0.362; Tb: r = 0.695) and for NR females (n = 133; body mass: r = 0.343; Tb: r = 0.347). Therefore, a repeated-measures (or

nested) experimental design within analysis of covariance (ANCOVA) was

used to analyze dependent variables to control for the effects of differences

in RMR, body mass or Tb during the reproductive stages of dams, or

sequential measurements of NR females through the equivalent time period.

These dependent variables and their covariates Oisted in parentheses)

included: RMR (mass and Tb), heat production (mass and Tb), Tb (mass and

RMR), and thermal conductance (mass). Adjusted means of these

dependent variables obtained from ANCOVA were used for within and

between treatment comparisons. A similar experimental design with

analysis of variance (ANOVA) was used to analyze RQ. Significance of

reproductive status (RA or NR) on dependent variables were tested using

mean square for individual females within reproductive treatment as the

error term. If individual variation within treatment was not significant,

then treatment effects were tested using mean square for error.

Differences between RA and NR females were initially tested for the

entire reproductive cycle (conception to weaning except days10 and 20 of

lactation). Reproductive periods were subsequently divided into gestation 57 and lactation to further examine changes of dependent variables. Because Tb data were not obtained from females on days10 and 20 of lactation, another

ANCOVA model was used with body mass as the covariate for testing BMR and heat production between RA and NR females.

Analyses using log-transformed RMR and body mass resulted in better explanatoiy power (higher R2, or coefficient of determination) than

analyses based on untransformed data. Therefore, data sets were analyzed

after log-transformation of metabolic rate and body mass. Homogeneity of

slopes across treatments were tested for each reproductive stage, and the

appropriate ANCOVA model (i.e. common or separate slope model) was

selected before performing all statistical tests for treatment effects, and

calculating adjusted mean of dependent variables.

To further estimate the cost of reproduction, the whole-animal RMR

of each RA dam was partitioned into the non-reproductive portion

(maintenance) and reproductive increment of metabolism (RIM) for

maintaining body mass and Tb as a function of reproductive stage. To do

this, regression equations for estimation of the RMR (ml 2 0hr'1) and heat

production (cal hr _1) of the non-reproductive condition were derived from

NR females with body mass and Tb as independent variables.

log10 (RMR) = -1.631 + 0.89 x log10(m ass, g) + 0.05087 x Tb (°C), (rf = 0.48,

PcO.001), and 58

log10 (heat production) = -0.93677 + 0.87153 x log10(mass, g) + 0.05114 x Tb (°C), (if = 0.47, PcO.OOl), respectively.

Next, the non-reproductive portion of RMR of RA dams was estimated from the regression equation using the observed body mass and Tb of RA dams.

The reproductive increment of metabolism was the difference between the observed whole-animal RMR or heat production and the estimated non- reproductive metabolism. A RIM of 0 indicated no increase in RMR over the non-reproductive condition. Differences in RIM of reproductive stages were tested using ANCOVA, with Tb as the covariate as Tb still had significant residual effects on Tb-corrected dependent variables.

The effect of litter size on dependent variables (RMR, heat production, Tb, RIM) was tested among RA dams using ANCOVA using the statistical model described above. Litter sizes were classified as small (6 and 7), medium (8 and 9) and large (10 and above). The effect of litter size was tested using dams within litter size as the error term. The effects of litter size and sex of young on the weaning mass of young were tested using

ANOVA. Effects of litter size on weaning mass of young was tested using mean square for dams within litter size. The effect of sex of young was tested using mean square for interaction of sex of young with dams within litter size.

Relationships between maternal physical conditions and production

(litter size or litter mass) were tested using multiple regression, with litter 59 size or litter mass as the dependent variable and body mass and RMR of dams in diestrus as independent variables. Similar tests were also preformed with body mass and RMR of dams during lactation as independent variables. Relationships between litter size and litter mass,

RMR, and Tb were also tested using multiple regression, with RMR or Tb as the dependent variable and the rest as independent variables. Tests were performed on data from days 30, 40, 50, or 56 of lactation. The effects of developmental stage of young (days 30, 40, 50 and 56 of lactation) and individual litters on the measured RMR of litters were tested using

ANCOVA, with total litter mass as the covariate.

R esu lts

Changes in Body Mass

Although the initial average body masses of RA and NR females were

nearly identical (P > 0.05, table 3.1), RA dams gained more weight than NR

females during the 80-day study period. The average final (one day after

weaning) body mass of RA dams was 87.2 + 2.0 g while that of NR females

was 70.7 + 2.4 g. However, no significant difference (P > 0.05) was found in

the dry mass of carcasses between RA and NR groups. The difference

between RA and NR females in fresh body mass was due to higher (P <

0.001) water content in RA dams (71.4 + 0.5 %) compared to that (65.7 + 60

Table 3.1 Average whole-animal resting metabolic rates (RMR, ml 0 2 h r 1), body mass (g) and body temperature (Tb, °C) and percentage of Kleiber- predicted heat production (BMR) of Reproductively Active (RA) females and non-reproductive (NR) female opossums. Data for mother plus young measured together are presented in the second row below each designated day of lactation.

% of Kleiber Reproductive RMR Body mass Tu predicted BMR Stage______mean s.e. mean s.e. mean s.e. mean s.e. RA fem ales Diestrus 50.0 3.1 64.1 2.4 32.1 0.2 63.4 3.5 E strus 51.9 2.0 70.7 2.4 32.4 0.2 61.2 2.1 G estation mid- 69.5 1.6 71.1 1.8 33.5 0.2 81.7 1.8 late 74.4 1.9 77.3 2.0 33.5 0.3 82.8 1.8 Lactation day 10 60.6 3.1 75.5 2.0 67.9 3.0 65.8 3.1 80.7 2.0 69.7 2.6 day 20 62.4 3.5 78.2 7.6 6 8 .2 3.4 81.6 3.3 97.8 1.6 74.9 2.5 day 30 72.7 4.2 80.8 1.9 34.0 0 .2 77.7 5.0 104.1 4.4 121.4 2.3 81.6 3.8 day 40 96.3 10.3 80.5 1.7 34.1 0.2 105.3 11.6 153.6 11.6 148.9 3.3 1 02.8 7.1 day 50 121.3 14.2 82.3 1.6 34.8 0.3 142.6 17.5 243.0 17.8 184.6 4.1 138.2 9.4 weaning 127.1 18.2 83.3 1.7 35.0 0.3 136.3 19.3 (day 56) 309.1 21.8 230.0 5.3 148.2 8.7

NR females trial 1 47.7 2.3 63.7 2.8 32.8 0.3 61.5 3.3 trial 2 45.1 2.5 65.2 2.9 32.6 0.2 56.5 2.2 Trial 3 41.9 3.4 6 6 .0 3.0 32.2 0.2 50.9 2.8 Trial 4 41.9 1.6 66.7 2 .6 32.1 0.2 51.0 1.9 T rial 5 42.7 2.6 67.7 2.8 32.1 0.3 51.5 3.0 Trial 6 42.9 3.3 68 .1 2.7 32.5 0.2 51.2 3.8 Trial 7 42.6 2 .6 6 8 .6 2 .6 32.9 0.2 50.5 3.6 61 0.6%) of NR females. The weight gain in NR females was not correlated with their RMR nor initial body mass (P > 0.05).

Metabolic Rate during Diestrus and Estrus

The adjusted mean RMR of RA dams at diestrus was not significantly different (P > 0.20) from that of NR females at the beginning of the study.

The percentages of Kleiber-predicted value of RA dams at diestrus was also similar to that of NR females at the beginning of the study (table 3.1).

However, the percentage of Kleiber-predicted value decreased in NR females during repeated respiratory measurement. The Q10 calculated from RMR of

RA and NR females at diestrus was 3.16, and similar to the Q 10 calculated from heat production (3.07).

Variation in Tb explained a significant amount of variability of RMR and heat production over time (P < 0.001), and heat production of NR females was constant among sequential measurements and individual animals. However, adjusted mean RMR varied through time without

obvious patterns (Fg 100 = 3.51, P <0.01).

The average body mass of RA dams increased over 5-7 day period

from 64.1 + 2.3 g at diestrus to 70.7 + 2.4 g at estrus (P < 0.04). However,

average RQ, adjusted mean T* and adjusted mean RMR remained constant

from diestrus to estrus (P > 0.05). Variation of Tb was correlated with RMR 62 between diestrus and estrus (P < 0.03). In addition, Tb was influenced by individual variation (Fig 17 = 4.93, P < 0.001).

Effects of Tv and Reproduction on Metabolic Rate

Average whole-animal RMR of RA dams was 40 + 7% higher in gestation and 77 + 13 % higher in lactation than in diestrus. Because variation of whole-animal RMR and whole-animal heat production associated with body mass and Tb changes through gestation and lactation, the later two variables were standardized (used as covariates) for comparisons of RMR and heat production of RA and NR females.

RMR and Tb were highly correlated (table 3.2) during gestation

(partial R2 = 0.025) and lactation (partial R 2 = 0.068). RMR and heat production (corrected for mass and Tb) of pregnant RA dams were similar to those of NR females (table 3.2). Average RQ of pregnant RA dams was also similar to that of NR females (fig. 3.1). Because Tb data were not obtained from females on days10 and 20 of lactation, only body mass was taken into account in RMR comparisons and no significant difference of adjusted mean

RMR was found between RA dams and NR females. Average heat production of RA dams expressed as proportion of Kleiber-predicted (table

3.1 & fig. 3.2) at days 10 and 20 of lactation were within 10% range of that at diestrus. Because similar results were obtained for analysis of RMR and 63 Table 3.2 F values and significance levels from the ANCOVA evaluation of the treatment effects on RMR (ml 02 hr'1), heat production (HP, Kcal hr'1) and Tb (°C) of 20 reproductively active (RA) and 19 non-reproductive (NR) opossums at 30 °C. Sample sizes for each stage were 18-20, except n = 7 on day 30 of lactation.

Gestation Lactation Source df* RMR HP Tb d f RMR H P Tb

Treatment effects

R. E .1 1 1 7.89b 8.91b ID2(R. E.) 37 1.82c 1.93b 3.24“ 37 3.00“ 2.78“ Stage3 2 9.34“ 8.34“ 3 4.17b R. E. x Stage 2 6.15b 5.25b 4.25* 3

Covariates

Log(mass)4 3(1) 8.72“ 8.30“ 1

Tb 1 4.92c 8.41b - 1 7.58b 6.55* -

Log(RMR) 1 - - 4.38* 1 -- 46.59“

Tb x T b --- 1 9.5 lb 8.36b - E rror 6 6(6 8) 91(92) Total 112 138

S? 0.87 0.88 0.76 0.91 0.91 0.81

* : df of Tb in the parenthesis if different from RMR; - : not in the model; a: PcO.OOl; b: P<0.01;c: P<0.05.; blank not significant. 1 : abbreviation for reproductive effect, i. e. RA and NR females; 2: abbreviation for individual animals. 3 : gestation measurements made at estrus, mid- and late gestation, lactation measurements made at day 30, 40, 50, 56 (weaning) of lactation. 4 : this covariate of RMR and heat in gestation was log(mass( stage)) with df=3. Fig. 3.1 Average respiratory quotient (RQ, (RQ, quotient respiratory Average 3.1 Fig. the reproductive cycle compared to th a t of non-reproductive females during during females non-reproductive of t a th to compared cycle reproductive the an equivalent 80-day period. 80-day equivalent an Respiratory Quotient 0 1 .9 0 0 0.7- 0.5 . . 8 6 - - tn birth ating m 0 4 1 - ^estotior^/ ^estotior^/ RA females NRfemales DAYS 428 14 ±

1 SEM) of opossums during during opossums of SEM) Lactation 42 weaning 56 64 65

I Q) 1.6 • -----• RA females _Q o-----o NR females *o)Ct:i.4-! A A + young — a young alone QQ1.2 M— ° - o , 0 O) 1 0 o oo.aJ I p 0 ) O x°.6H Q - (U O 0.4-1 mating birth weaning Q. |>estatior^ Lactation i -14 0 14 28 42 56 DAYS

Fig. 3.2 Average proportion of Kleiber-predicted BMR (heat production) of

RA (n = 20) and NR (n = 19) opossums. Lactating heat production of dams

were converted from estimated RMR. Heat production of litters was

calculated from measured RMR of litters and estimated RMR of litters if

data were not measured. 66 heat production, only results from analysis of RMR will be presented hereafter.

In contrast to the situation during gestation, RMR and RQ of lactating RA dams were higher than those of NR females (table 3.2 and fig.

3.1). However, the average RQs of RA dams were not higher than those of

NR females at any stage except weaning. The square of Tb also had a significant effect on RMR during lactation (table 3.2), indicating a non­ linear relationship between Tb and RMR.

RMR of RA dams varied significantly among reproductive stages,

dams, and Tb within stage, after the effects of differences in body mass and

Tb were removed. Adjusted mean RMR was highest at day 40 of lactation

which was the only stage that adjusted mean RMR was significantly higher

than that recorded in females in diestrus, estrus and at weaning. Adjusted

mean RMRs recorded during gestation were not different from those

measured throughout lactation. From diestrus until day 40 of lactation,10 Q

calculated from RMR of RA dams was 2.42, but Q 10 increased to 6.45 when

data from days 50 and 56 of lactation were included.

Adjusted mean Tb’s of RA and NR females were similar through

gestation (P > 0.70) and lactation (P> 0.68). Adjusted mean Tb of RA dams

increased from estrus through gestation and peaked at weaning. Adjusted

mean Tb s on lactation days 40, 50 and 56 were higher (P < 0.05) than those

of diestrus and estrus, but only at weaning was the adjusted mean Tb 67 higher than that at gestation. However, the adjusted mean Tb at weaning was not higher than all adjusted mean Tb’s of NR females.

Thermal conductance was similar between RA and NR females (P >

0.14), but was affected by individual within reproductive treatment (P <

0 .001), and the interaction between body mass within reproductive treatment and stage (P <0.04). Average whole-animal thermal conductance was 107.32 + 5.78 (cal hr'1 °C animal'1, n = 39, at an average body mass of

71.81 g).

Individual variation within RA dams accounted for a significant amount of variance of RMR (P < 0.02), heat production (P < 0.03) and Tb (P

< 0.001). About 19-25% of this individual variation was associated with litter size but the influence of fitter size was not significant (P > 0.14).

RIM changed between reproductive stages, with litter size, and with

Tb within stage (P < 0.05), but only RIM calculated from heat production was affected by dams within litter size (P < 0.04). Adjusted means of RIM increased from gestation to lactation and peaked at day 40 of lactation which was the only stage different from zero (P < 0.05, fig. 3.3).

Growth and RMR of Young

The average growth rate of young (g day1 litter1, n = 20) increased throughout lactation: 0.048 + 0.002 (day 0-10), 0.164 + 0.007 (day 10-20),

0.330 + 0.013 (day 30-40), 0.411 + 0.023 (day 40-50) and peaked at weaning 68

0 ) > o £ D « 4 0 ~ o — o o )- _Q S ' ° ct:(D -*_j o)2o

c w o d) c 0) E "D d) mating birth weaning 0) L_ -4—' ^estatior^ Lactation CO o 4 D — -14 0 14 28 42 56 ~ o < DAYS

Fig. 3.3 Adjusted means of reproductive increment of metabolism (RIM, ml

0 2 h r \ ± 1 SEM) of RA opossums. RIM was calculated at each stage by subtracting RMR estimated from the equation generated for NR females with identical body mass and Tb with each reproductive stage from observed

RMR of each RA dam. Adjusted means were from ANCOVA models adjusted to the average rectal temperature 33.63 °C. 0.863 + 0.034 (day 50-56). Litter size at weaning was affected by body mass

(Fx 17 = 5.16, P < 0.037) and RMR of dams measured in diestrus (Fj 17 = 6.97,

P < 0.018). Litter size was positively correlated with the body mass (g) of dams but inversely correlated with dam RMR (ml 0 2 hr'1 animal1) at diestrus (litter size = 7.1518 - 0.0656 x RMR + 0.0745 x mass). Total litter mass at lactation days 40, 50 and 56 were inversely related to RMR of dams at diestrus (P < 0.003). No significant correlation occurred between production of young (litter size and litter mass) and maternal Tb, or between production of young and maternal RMR during days 30-56 of lactation.

Weaning mass of individual young was affected by dam within litter size (F13 jag = 13.7, P < 0.001) but was not affected by sex of young (Fx 19 =

3.93, P > 0.06) or litter size (Fg 13 = 1.67, P > 0.2). However, weaning mass of individual young decreased in linear fashion as litter size increased from

6 to 12 (Fj 13 = 5.82, P < 0.032). The highest average weaning mass was

19.0 + 0.7 g (n = 2) at litter size of6 , while the lowest was 12.9 + 0.5 g at litter size of 11 (n = 1). The average weaning mass of young was 17.2 + 0.5 g litter1, at an average litter size 8.65 + 0.36 pups. Although sex ratios (no. females /no. males) within litters ranged from 1:6 to 7:2, the overall average sex ratio was 1.25:1, not significantly different from1:1 (t = -0.57, P > 0.57).

Neither age nor mass of dam was correlated with litter size or sex ratio, or between sex ratio and litter size. 70 RMR of litters was affected by total litter mass (P < 0.05) but not by age of pups and individual litters. Adjusted mean RMR of litters decreased during lactation but did not differ significantly among day 30, 40, 50 and 56 of lactation. Litter size was not the main effect of RMR of litters (P > 0.20) when litter mass was also included in the analysis. Therefore, only litter mass was included in the regression model (RMR (ml 0 2 h'1 litter'1):: (total litter mass)10014 ) to estimate total RMR of litters when actual data were missing. The average RQ of litters was 0.68 + 0.02 litter'1, and varied among litters (P < 0.03) but not as a function of the age of pups (P > 0.2).

D iscu ssio n

This study clearly indicates that energetic demands of reproduction in the gray short-tailed opossum are low until late lactation. Although whole-

animal RMR increased during gestation and lactation, significant

reproductive effects were only detected at day 40 of lactation, e.g., higher

adjusted mean RMR than RMR at diestrus, and RIM > 0. Litter size had a

significant effect on RIM but not on RMR and Tb and, thus RIM appeared to

be a particularly useful parameter for evaluating reproductive costs

independent of concurrent changing body mass and Tb.

Milk production in opossums is probably highest around day 40 of

lactation, rather than near the time of weaning. Although average whole- 71 animal RMRs of dams peaked on days 50-56 of lactation rather than at day

40, most of the elevated maternal RMR late in lactation was explained by increased Tb. Opossum young depend on milk as the sole source of nourishment through 28-35 days postpartum; they start to eat solid food after 40 days of age and can be weaned and maintained exclusively on a solid diet at day 50 of lactation (Fadem et al. 1982). Therefore, the metabolic costs of milk synthesis probably were highest around day 40 of lactation and declined thereafter, as indicated by RIM.

The average mass-specific RMRs of RA and NR females (n = 39) at diestrus was 87 % of the BMR predicted for marsupials by Dawson and

Hulbert (1970) and 93 % of that predicted by MacMillen and Nelson (1969).

The average mass-specific heat production of RA opossums at diestrus was

63.4 % of Kleiber-predicted value and nearly identical to the BMR of opossums reported by Thompson and Nicoll (1986) and close to the BMR reported by Dawson and Olson (1988, 64% and 55%, respectively). Thus, the RMR of diestrus females in our study appears to be equivalent to the

BMR of these animals.

Although adjusted mean Tb of RA dams increased (P < 0.05) from estrus to mid-gestation and was highest at weaning, the adjusted mean Tbs of RA dams were similar to those of NR females (P > 0.05). The higher Tb of RA dams was a consequence of a higher RMR, because thermal conductance of RA dams was similar to that of NR females. In addition, 72 variation of Tb was higher between the RA and NR groups than within the

RA group. The higher water content (and specific heat) of RA dams might have reduced diurnal variation in Tb compared to NR females.

Average Tb of RA and NR females at diestrus was lower than Tb of marsupials recorded by Dawson and Hulbert (1970) but similar to previous reports of Monodelnhis domestica (Kraus and Fadem 1987; Dawson and

Olson 1988) and another neotropical marsupial, M. brevicaudata (McNab

1978). McNab (1978) indicated that didelphids tend to have lower Tb than dasyurids (family of carnivorous, Australian marsupials) at small body masses (< 200 g). Metabolism is reduced and energy is conserved with reduction of Tb to near ambient temperature (Dawson and Hulbert 1970;

Walker and Berger 1980). Dawson and Wolfers (1978) observed that a low body temperature (33.5 °C) in Antechinus and Planigale. even within thermoneutrality. Even small reductions in Tb(1 or 2 °C in sleep or 5-20 °C in shallow torpor) can result in detectable decreases in metabolism.

Patterns of slow wave sleep (SWS) and shallow torpor are temporally continuous and isomorphic and apparently have evolved as an adaption for energy conservation (Walker, Garber, and Berger 1979). Cold and food deprivation frequently induce torpor in small mammals (e.g. Wallis 1976 ;

Walker, Garber, and Berger 1979; Geiser 1986), but torpor (Tb = 27-30.5 °C) may occur spontaneously in mice in the presence of food at T, of 20 °C

(Hudson and Scott 1979). Monodelnhis brevicaudata becomes torpid (Tb = 73 27 °C) following food deprivation at 24 °C (McNab 1978). Daily torpor or prolonged torpor in marsupials might beu n d e r precise control and hence similar to hibernation in placental mammals (Geiser 1987). Lower Tb during sleep or shallow torpor may be the reason that BMR of opossums in this study was about 14-28 % lower than BMR predicted for Australian marsupials by Dawson and Hulbert (1970).

The Q10 values of 6-7 observed in our females at days 50-56 of lactation are considerably greater than the Q10 of 2-3 that characterizes biological reactions influenced by temperature in general. This implies physiological or behavioral changes of lactating dams on day 50-56 of lactation beyond simple temperature effects on biological reactions. Values of Q10 greater than 3 also occurred for diestrus NR females (Tb range 30.5-

35.0 °C). Large Q10’s at high Tb’s and small Q10*s at low Tb’s have been observed in hibemators passing from normothermia to torpor (Hudson and

Scott 1979; Geiser 1988). Metabolism may be inhibited during entry into torpor, above and beyond effects of reduced Tb. A Q 10 of 7.6 of torpid white mice occurred as Tb declined from 36.0 to 32.0 °C, but Q 10 was only 2.1 as Tb declined from 29.5 to 28 °C (Hudson and Scott 1979).

Reduction in the duration and frequency of hypothermia would be advantageous for milk production and growth of young opossums. The effects of hypothermia on reproduction include decreased fetal development, prolongation of gestation and decreased post-natal growth rates. For 74 example, the length of gestation is extended in heterothermic bats because fetal development ceases during diurnal torpor (Racey and Speakman 1987).

Conversely reducing the frequency of torpor during lactation increases growth rates of young bats (Audet and Fenton 1988). In this study, variations in energy expenditure (RMR) and growth rates of young opossums among litters during late lactation were greater than early lactation. Individual variability in Tb is positively associated with energy expenditure for maintenance in non-reproductive animals (Lynch, Vogt, and

Smith 1978). The average unadjusted Tb of RA opossums continued to increase during gestation and lactation and was at a higher level than during diestrus. This might permit increased milk production, and growth of young opossums. Because there was no significant correlation between

RMR during lactation and production (litter size and total litter mass), partitioning RMR into RIM and maintenance (a higher Tb) might not be consistent from conception to weaning in opossums.

Litter size of opossums was positively associated with maternal body mass as in another didelphid marsupial, Caluromvs philander (Julien-

Laferriere and Atramentowicz 1990). This suggests that maternal nutritional status influences litter size, starting with ovulation rate. Litter size of Virginia opossums in New York State was positively correlated with maternal hind leg fat index, which reflects fat reserves following energetic demands of winter (Hosseler and Harder in press). 75 Although the average weaning mass of young in this study (17.2 +

0.5) was smaller than previously reported (22.5 ± 3.8 g; Cothran, Aivaliotis, and Vandeberg 1985), the average litter size in our study was more than twice as large as in their study (8.65 versus 3.79). This suggests a possible trade-offs between size of litter and weaning mass of young as in Caluromvs philander (Atramentowicz 1992).

Marsupials appear to differ from eutherians relative to allocation of energy into maintenance and reproduction. McNab (1980, 1987) has demonstration that BMR among eutherians is positively associated with high rates of reproduction, but there is little or no evidence from interspecific comparisons among marsupials that BMR affects the length of gestation, post-natal development or fecundity.

Derting and McClure (1989) argued that energy assimilation is limited by physiological constraints. When BMR was experimentally elevated (T4 implants) in cotton rats provided with unlimited food, BMR was associated with higher ingestion rate, faster growth, and earlier sexual development, but high BMR was associated with negative growth when food was restricted (Derting 1989). In another study, intraspecific variation in

BMR of seven populations of cotton rats (range 39.6 - 52.3 KJ day *) was not correlated with post-natal growth, reproduction, or rates of assimilation

of energy (Derting and McClure 1989). Because all cotton rats assimilated

similar amounts of energy irrespective of differences in BMR, they further 76 suggested that production rates were inversely related to BMR within a species.

Physiological restrictions, such as limitation on energy assimilation and milk production, might force opossums to choose between reproduction and maintenance. Litter mass of opossums during late lactation (days 40-

56) was inversely correlated with maternal RMR at diestrus (BMR). This suggests that when the growth and energetic demands of young were high, opossums with high maintenance cost (as high BMR) allocated less energy to reproduction. This also indicates that sustained maximum metabolic demands accompanying maximum food assimilation capacity occurred in opossums during day 40 and 56 of lactation.

This study supports the hypothesis that mammals with low BMR can compensate and increase RMR during reproduction (Thompson and

Nicoll 1986). However, most of the increased RMR observed during reproduction was correlated with higher Tb, and was not correlated with litter size or litter mass. Elevations in Tb and RMR occurred more frequently during the reproductive cycles of RA females than during diestrus in NR females, but these elevations did not occur in every RA dam throughout the reproductive period. Incremental cost of reproduction beyond maintaining a higher Tb from conception to weaning remained low until late during lactation. Therefore, the increased energy expenditures are associated with higher maintenance metabolism, and cannot be directly 77 interpreted as absolute reproductive costs. Additional studies are needed to evaluate the benefits and trade-offs of maintaining normothermic Tb during reproduction instead of using torpor as a method of energy conservation. CHAPTER IV

Comparative Reproductive energetics of golden hamsters

and gray short-tailed opossums.

A b stract

The reproductive energetics of golden hamsters (Mesocricetus auratus) and gray short-tailed opossums (Monodelphis domestical were compared from conception to weaning (C-W). The resting metabolic rate (RMR) measured by respiratory, and energy assimilation from food in reproductively active

(RA) hamsters and opossums when analyzed in reference to those measurements in non-reproductive (NR) females provided estimates of energy allocated to maintenance, activity, and reproduction.

RA opossums and RA-30 hamsters (both maintained at 30 °C) both stored some energy in body reserves during the reproductive cycle (17.7 versus 5.2 Kcal, respectively). However, an average of 26.6 Kcal was used from body reserves of RA-24 hamsters (maintained at 24 °C) during reproduction. Total energy assimilated from food, from C-W, (hamsters:

1647.5 + 60.6 Kcal, and opossums: 1261.3 + 28.0 Kcal) was positively

78 79 correlated with litter size and mass per young in both species. Total energy assimilated was higher in hamsters than in opossums during gestation

(P < 0.001), but not during lactation or from C-W (P > 0.05). The

Incremental Cost of Reproduction from C-W (adjusted for body mass) of RA-

30 hamsters was not significantly different from that in RA-30 opossums.

Efficiency of offspring production was higher in hamsters than in opossums and, in both species, it was higher during lactation than in gestation.

However, on a per young and on a per young per day basis, the cost of reproduction was consistently higher in hamsters than in opossums.

The observed interspecific differences were due largely to a lower

BMR and a longer period of lactation in opossums. Thus, the marsupial mode of reproduction, as seen in opossums, yields young at a lower cost but at the expense of a longer reproductive period than is the case for eutherians, particularly the golden hamster.

Introduction

Although resting metabolic rate (RMR) is a useful parameter for comparative energetics, the total cost of reproduction estimated from RMR may be similar or lower than estimated energy assimilated from food. In addition, energy can be further stored or mobilized from maternal body reserves during reproduction (Naismith, Richardson, and Pritchard 1982; 80 Wade, Jennings, and Trayhum 1986). Studier (1979) argued that food intake is the most accurate measure of the bioenergetic requirements of reproductively active mice. Thus, total energy used from conception to weaning (C-W) should combine assimilated energy with energy balance of maternal body stores. In this study the total cost of reproduction was evaluated through energy assimilation from C-W and through comparisons of changes of RMR from previous chapters.

The major objective of this chapter is to compare the gray short-tailed opossum (Monodelnhis domestica) and the golden hamster (Mesocricetus auratus) relative to the cost of reproduction (energy per offspring) and allocation of assimilated energy to maintenance, growth, activity and reproduction. Resting metabolic rates and assimilated energy were compared between reproductively active (RA) and non-reproductive (NR) female (controls) opossums and hamsters maintained within their zone of thermoneutrality (30 °C). In addition, effects of ambient temperature on assimilated energy and incremental cost of reproduction were evaluated in hamsters maintained at 24 °C (RA-24 and NR-24).

The following questions were tested:

1) Do gestation and lactation impose similar maternal metabolic costs

(compared to non-reproductive conspedfics) in both hamsters and

opossums? Does assimilated energy increase in gestation and/or

lactation in both spedes? 81 2) Do opossums and hamsters compensate for the energy demand of

reproduction in the similar ways (such as increasing assimilation

efficiency or increasing total assimilated energy)?

3) Do they assimilate and use the same amount of energy during gestation,

lactation and from C-W?

4) Do they allocate similar proportions of energy into reproduction,

maintenance and growth from C-W?

5) On a per young or on a per young per day basis, do they use the same

amount of energy from C-W?

6) Do they have the same incremental energetic costs (per young or per

young per day) from C-W?

7) Do they have the same reproductive efficiency (produce number of

offsprings per unit energy used from C-W) and offspring production

efficiency (incremental energy transfer to young)?

8) When the effects of differences of maternal body mass, litter size and

average mass per young were removed, do they use the same amount

of energy and have the same incremental energetic costs? How about

on a per day basis?

Data obtained from these experiments provide a quantitative basis

for comparing the total cost and efficiency of reproduction in marsupials and

eutherians and thereby contribute to our understanding of the two major

reproductive strategies employed by viviparous mammals. Additional 82 background information and rational for this comparison is presented in

C hapter I.

M aterials and Methods

Experimental animals

Golden hamsters and gray short-tailed opossums were obtained from breeding colonies maintained by the Department of Zoology, The Ohio State

University. Details of experimental designs, breeding procedures and colony maintenance were described in Chapters II and III. Body mass and unconsumed food of each Non-Reproductive (NR) female and Reproductively

Active (RA) dam and her litter were measured at least every other day throughout the reproductive cycle (from conception to weaning, C-W) or the equivalent period of time for NR controls.

Hamsters hoard food and the amount of food hoarded is associated with reproductive success (Labov et al. 1986). Hamster pups start ingesting caecotrophe beginning on day 5 of the 21-day period of lactation (Dieterlen

1959). Caecotrophe is a semisolid digestive excreta, emanating from the maternal caecum, which is rich in bacteria and nutrients (Leon 1974).

Hamster pups begin to eat some solid food on day 10 of lactation (Daly

1976) and opossum pups start to eat solid food after day 40 of lactation

(Fadem et al. 1982). Thus, in order to simulate natural conditions and to 83 provide solid food for nursery young, preweighed pelleted food (usually 200-

300 g) was placed in container on the floor of each cage in order to provide food in excess for mother and young of both species. Uneaten food was retrieved and weighed every one or two days. Food consumption and body mass of weaned young were measured for a 24-hour period after they were separated from dams.

Although litter sizes were not manipulated or controlled, data from dams with litters of less than five at the time of weaning were excluded in the analysis. The final data analysis was based on the following sample sizes: 20 RA and 19 NR opossums, 16 RA-30 and 21 NR-30 hamsters

(maintained at 30 °C, within thermoneutrality for both opossums and hamsters) and 20 RA-24 and 16 NR-24 hamsters (maintained at 24 °C).

Energy Intake and Assimilation

Dry food consumption and energy equivalence were calculated on the basis of a 9.4% water content and 4.215 Kcal g'1 in Purina Rodent Chow for hamsters and a 6.3% water content and 4.534 Kcal g 1 in Fox Reproduction

Food (Milk Specialties Product Inc., New Holstein, WI) for opossums.

Samples of food and feces were dried to a constant weight at 65-70 °C to determine water content. Energy content of dehydrated food and feces was estimated by bomb calorimetry (Phillipson Co.). Samples were ground to uniform consistency in a micromill (Thomas Scientific Co.) and portions (10- 84 20 mg) of the ground mixtures were pelleted, weighed and burned in a microbomb calorimeter calibrated with dry benzoic acid standards.

Changes of food digestibility in hamsters were calculated through 48- h series digestion trials that measured the mass of all food supplied to and the mass of feces collected from the cages of female hamsters at six intervals: before pairing, days 7-9, and 13-15 of gestation, days 5-7, 13-15, and 19-21 of lactation or for equivalent time intervals for NR females.

Complete longitudinal digestion trials were conducted in four female opossums, at diestrus and after pairing (gestation) until weaning of young.

Feces and uneaten food were collected at 1-day and at 6-day intervals in these four dams. For all other opossums, 48-h digestion trial were conducted during diestrus. Energy loss through urine is generally low in mammals (Grodzinski and Wunder 1975) and was assumed to be about 2% of total energy ingested in this study. It was considered a constant in calculations and was not measured directly. Digestive efficiency

(absorption) was calculated as:

(total energy in food intake - energy lost in feces) total energy in food intake

Thus, the assimilated energy is equal to the total energy in food intake times digestive efficiency minus 2 % for urine energy loss.

Because both opossum and hamster young eat increasing amounts of

solid food during the latter stages of lactation, it was difficult to accurately 85 estimate the proportion of energy assimilated from solid food by young.

Therefore, all assimilated energy during lactation of a breeding unit (dam and young) was treated as assimilated energy of the RA dam.

Changes in digestive efficiency were tested among different stages (7-

10 days interval during gestation and lactation) of reproductive cycle within individuals using analysis of covariance (ANCOVA). Energy in feces was the dependant variable, and mass of feces or energy in food intake was the covariate. Energy in feces (corrected for mass) was similar among NR hamsters, pregnant RA dams, lactating RA dams and young at mid- and late lactation. However, energy in feces of lactating dams and young was lower at day 5 of lactation than at other times. Therefore, the average digestive efficiency of 0.781 (± 0.004) was used to estimate food absorbed by hamsters except that 0.743 was used for lactating hamsters and young during day 1-5 of lactation.

Energy in feces (corrected for mass) of four RA opossums did not change from gestation to day 35 of lactation but it was lower (P < 0.05) than at diestrus. Digestive efficiency was not significantly lower in 1-day than in

6-day digestive trials. Individual variation was also not significant.

Therefore, the average digestive efficiency in diestrus was 0.83 + 0.01, and the average digestive efficiency during gestation until day 35 of lactation was 0.80 ± 0.01. From day 35 of lactation to weaning, the average digestive efficiency of lactating opossums and young was 0.76 + 0.01. 8 6 Energy Content and Composition of Carcasses

Carcass composition was measured by a modification of the method of

Leshner, Li twin, and Squibb (1972). NR females, post-weaning dams and two young per litter were euthanized and weighed to determine live body mass. The interscapular brown adipose tissue (BAT) of female hamsters was carefully separated from parametrial white adipose tissue, weighed, and returned to the carcasses. Dehydrated carcasses were ground to uniform consistency in a Thomas-Wiley laboratory mill (Thomas Scientific

Co.). Preweighed samples (0.3-0.5 g) of carcass mixtures also were extracted to a constant weight with petroleum ether to determine lipid content. The mass of the remaining tissue represented the lipid-free dry mass. Protein content of each lipid-free dry mass was assayed by modified

Lowry method using bovine serum albumin as a standard (Markwell et al.

1978). Energy content of carcasses was also determined by bomb calorimetry.

Initial whole-animal energy content was estimated by multiplying initial dry body mass by equivalent energy value of individual animals as determined at the end of the experiment. Initial dry body mass of NR females was calculated by assuming that percent water content did not

change through this experiment in NR females and percent water content

was assumed to be the same in RA and NR animals at the beginning of the

experiment. 87 Calculations for Cost of Reproduction

Available Energy, i.e., total energy used from C-W, was calculated as the sum of daily assimilated energy (Assimilated Energy) plus energy derived from body reserves and minus any energy accumulated in maternal body stores. Incremental Cost of Reproduction was calculated as Available

Energy of RA dams minus the average energy used by NR control females maintained at the same T„.

Absolute values for Assimilated Energy, Available Energy,

Incremental Cost of Reproduction were initially derived without adjustment for variation among females in body mass and are referred to as whole- animal Assimilated Energy, whole-animal Available Energy, whole-animal

Incremental Cost of Reproduction.

Efficiency of Offspring Production

Efficiency of offspring production at weaning was calculated as the ratio of total energy stored in young mass at birth and weaning to the incremental cost of reproduction at birth and weaning, respectively. Because young consumed directly some of the solid food delivered to the cages during the last 28% (opossums) or 50% (hamsters) of the lactation, it was essential to estimate the efficiency of energy transfer from mother to young at birth

(efficiency during gestation), 50% of lactation, lactation, and from C-W.

Energy content of young during different reproductive stages was estimated 88 from the percent water content and energy equivalent of dry mass. The percent water content of opossum newborn was 93.6% (n = 2) and 85.2 % in a 6-day-old hamster young (personal observation). Because percent water content of mammalian carcasses is higher in newborn and decreases near weaning (Kenagy, Stevenson, and Masman 1989), percent water content of young opossums and hamsters was assumed to be 93.6 % at birth, 85% at

50% of lactation. For purposes of calculation the energy content of offspring per unit of dry mass was assumed to be constant from birth to that measured at weaning which was 5.69 + 0.14 Kcal g'1 dry (n = 20) in hamsters and 5.80 + 0.27 Kcal g'1 dry (n =13) in opossums.

Statistical Analysis

All statistical analyses were made using Statistical Analysis System software (SAS Institute Inc. 1985). The non-parametric Wilcoxon test was used to compare means of litter size, energy assimilation, energy stored in young, percentage increment of RMR, reproductive efforts and efficiency of offspring production between opossums and RA-30 hamsters and between

RA-24 and RA-30 hamsters. All mean values through the text are presented ± 1 standard error of the mean (SEM).

In mammals, food consumption and energy assimilated per day is associated with body mass. Therefore, a repeated-measures (or nested) experimental design for each species within ANCOVA was used to analyze 89 maternal assimilated energy per day during gestation or lactation by using maternal body mass (wet) as a covariate. Significance of temperature and reproductive status on food assimilation during gestation or lactation were tested using mean square for individual females within temperature or within reproductive treatment, respectively, as the error term. If individual variation within treatment was not significant, the main effects on the dependent variable were tested using mean square for error. These models also include day, and interaction of day and treatment groups. Energy

assimilated by RA dams alone was calculated in days of gestation from pre­ mating and every other day of gestation until parturition (8 times in opossums and 9 times in hamsters), and in days of lactation (every other days, 20 times in opossums and 4 times in hamsters).

Untransformed data were used in all analyses. Homogeneity of

slopes across treatments were tested for each reproductive stage, and the

appropriate ANCOVA model (i.e. common or separate slope model) was

selected before performing statistical tests for treatment effects and

calculating adjusted mean of dependent variables.

Relationships between young production, maternal physical

conditions and energy assimilation were tested using multiple regression,

with whole-animal Assimilated Energy as dependent variable, and litter

size, average mass per young (of a single litter), body mass and RMR of

dams measured before mating as independent variables. Relationships 90 between body mass, RMR (independent variables) and whole-animal

Assimilated Energy (dependent variables) were also tested using multiple regression. The effects of temperature and/or reproduction on lipid, protein and energy content of carcasses of hamsters and opossums were tested using ANCOVA, with dry body mass as the covariate.

The effect of species (RA opossums and RA-30 hamsters) or T, (RA-30 and RA-24 hamsters) on Assimilated Energy, Available Energy (total, per young or per young per day) and Incremental Cost of Reproduction (total per young or per young per day) was also tested using ANCOVA with body mass before mating as the covariate. Adjusted means of these dependent variables obtained from ANCOVA were used for within and between treatment comparisons. Litter size and average mass per young were also included in the ANCOVA. The effects of species or T. on total and average daily Assimilated Energy, total and average daily Available Energy, and total and average daily Incremental Cost of Reproduction were tested by removing the differences of maternal body mass, litter size and average mass per young. 91 R esu lts

Energy Assimilation bv Hamsters

Average whole-animal Assimilated Energy of RA hamsters increased above that in NR females maintained at same Ta during lactation but not during gestation (fig. 4.1). Daily assimilated energy (corrected for body mass) of

RA hamsters decreased toward parturition, but was not significantly lower than that of NR females (P > 0.05). In addition, energy assimilation during gestation was influenced by day, body mass and individual animals within treatment groups (P < 0.001). In contrast, from day 1-7 of lactation, daily assimilated energy (corrected for body mass) of RA hamsters was higher than that of NR females maintained at same T, (P < 0.001).

Assimilated Energy of RA hamsters and young from C-W was positively correlated with litter size (P < 0.001), average mass per young (P

< 0.004), and maternal body mass (P < 0.02), but not with RMR before mating (P > 0.64). Assimilated Energy of NR-30 hamsters during the same interval of time was positively correlated with initial body mass (P < 0.001), and growth (P < 0.01), but Assimilated Energy of NR-24 hamsters was only correlated with initial body mass (P < 0.001). In both cases, initial RMR was inversely correlated with total Assimilated Energy, but not significantly

(P > 0.62 and 0.25, respectively). 92

O O 200 o RA—24 • R A -3 0 Q A- a NR—24 LU 100 A- a NR—30 r 5 50 40 CO 0 0 30 < 20 • a - = A ' y~ o mating birthK!nfk weaning QC yjf GESTATION LACTATION LU 10 - k - Z - 1 6 - 7 14 21 LU DAYS

Fig. 4.1 Average (+ SEM) whole-animal Assimilated Energy (Kcal day'1 animal *) of reproductively active (RA) golden hamsters during gestation and lactation maintained at 24 °C (n=20) or 30 “C (n=16) and non-reproductive

(NR) females maintained over the same experimental period at 24 "C (n=16) or 30 °C (n=21). Data are plotted relative to the day of birth (day 0). The symbols of RA-24 and RA-30 females were overlapping on day 22. 93 Changes of Body Composition nf Hamsters

An average of 26.6 + 8.7 Kcal was depleted from the body reserves of RA-24 hamsters during reproduction while RA-30 dams gained an average of 5.2 +

6.5 Kcal. During the same interval of time, NR-24 females gained 38.8 +

7.4 Kcal and NR-30 females gained 34.7 + 5.2 Kcal. However, energy content (corrected for dry body mass) of RA hamsters was similar to NR females (P > 0.28); but energy content of females maintained at 24 °C was higher (P < 0.05) than females maintained at 24 °C (204.15 + 5.09 versus

189.75 + 5.02 Kcal, respectively). Energy contents of offspring raised at two

Tas were similar, with a combined average of 1.54 + 0.05 Kcal g'1 wet (n =

20) or 5.69 + 0.14 Kcal g 1 diy and a water content of 73.0% ± 0.3 (n = 52).

The amount of protein, but not lipid, was significantly correlated with dry body mass of RA dams. Protein and lipid content were both correlated with dry body mass of NR-30 females (r = 0.552, P < 0.01 for protein and r

= 0.480, P < 0.05 for lipid) but only the amount of lipid was correlated with dry body mass of NR-24 females (r = 0.613, P < 0.011). Therefore, dry body mass was used as the covariate in the ANCOVA analysis of reproductive effects on energy, protein and lipid content.

The lipid content of ham sters declined during reproduction (C-W), especially at low Ta, but the remaining lipid content after weaning was still higher in RA-24 than in RA-30 dams (P < 0.05, table 4.1). Hamsters maintained at 24 °C accumulated a significantly greater amount of lipid 94 Table 4.1 The body composition of reproductively active (RA) and non- reproductive (NR) golden hamsters used in this study. Values are means

+ SEM.

RA-30 NR-30 RA-24 NR-24 variable m ean s.e. m ean s.e. m ean s.e. m ean s.e.

Age(month) 3.3 0.1 4.3 0.4 3.6 0.1 4.0 0.1

Initial wet mass(g) 95.3 2.8 100.8 3.9 106.0 2.7 106.7 2.9 Final w et mass(g) 111.6 2.9 120.2 2.9 110.9 2.5 126.5 2.9

Dry mass(g) 32.3 0.8 38.4 1.0 30.3 0.9 39.5 1.2

W ater(%) 71.1 0.2 68.0 0.5 72.7 0.5 68.8 0.7

Protein(g)1 16.0 0.6 14.2 0.5 14.8 0.6 11.3 0.6

Lipid(g)1 5.1 0.6 8.5 0.6 6.9 0.6 13.6 0.7

Interscapular BAT1 112.8 8.6 148.1 7.8 137.2 8.6 189.9 9.2

Energy1 189.5 7.9 191.4 7.2 189.5 7.9 216.1 8.4

Sample size 16 21 20 16

1: protein(g), lipid(g), interscapular BAT (interscapular brown adipose tissue, mg) and energy content (Kcal) were adjusted to the average dry body mass,

35.07 g + 0.67 (from ANCOVA analysis). 95 than those maintained at 30 °C. Therefore, the difference in the amount of lipid between RA-24 and NR-24 females was much greater than between

RA-30 and NR-30 females. In addition, the relationship of dry body mass and lipid content changed between RA and NR groups.

Post-weaning RA hamsters had proportionally higher protein content of dry carcass mass than NR females (P < 0.001, table 4.1). As a consequence of lower amount of lipid, protein content was higher in females maintained at 30 °C than maintained at 24 °C (P < 0.001),

Energy Assimilation bv Onnssuma

The average whole-animal Assimilated Energy of pregnant or lactating RA

opossums was higher than that of NR females (P < 0.01). Average daily

assimilated energy of RA dams and young continued to increase through

lactation (fig. 4.2). Daily energy assimilated (corrected for body mass) by

RA opossums during gestation was not higher than that of NR females (P >

0.08) but it was significantly higher during lactation (P < 0.001). Adjusted

mean daily energy assimilated by RA opossums was higher than that of NR

females after day 32 of lactation (P < 0.05). Energy assimilation was

further influenced by body mass and individual animals within treatment

groups (P < 0.001) as well as by day, and day interaction with treatment

groups (P < 0.05). 96

O O 200 RA-24 RA-30 O' Q A- J 9 100 •A NR—24 LU A- ■A NR-30 / 50 o '. 40 CO u i 30 < 20 A - f r c , Kir4h uionnl o mating 9T birth weaning (H GESTATION LACTATION LU 10 ± -* * z : -1 6 - 7 0 14 21 LU DAYS

Fig. 4.2 Average (+ SEM) whole-animal Assimilated Energy (Kcal day'1 anim al1) of reproductively active (RA) gray short-tailed opossums during

gestation and lactation maintained at 30 °C (n=20) and non-reproductive

(NR) females maintained over the same experimental period (n=19). Data

are plotted relative to the day of birth (day 0). 97 Assimilated Energy of RA opossums from C-W was positively correlated with litter size (P < 0.001) and average mass per young (P < 0.03).

Assimilated Energy was negatively correlated with RMR in diestrus (P <

0.05), only when litter size and mass were not included in the regression analysis. Assimilated Energy of NR females during the same interval of time was positively correlated with initial body mass (P < 0.04) and RMR at diestrus (P < 0.02).

Changes of Body Composition of Qnosfliims

Although RA dams gained more mass through reproduction than NR females (table 4.2), the dry mass of RA and NR opossums were similar (P >

0.85) and both put a similar amount of energy into body reserves (17.7 + 6.6 versus 13.0 + 3.9 Kcal, respectively). Therefore, total energy content was similar between RA and NR females (P >0.71) with an average of 6.07 +

0.16 Kcal g'1 dry (n = 39). Energy content of offspring ages 57 days old was

1.44 + 0.08 Kcal g'1 wet (n = 13) or 5.80 + 0.27 Kcal g 1 dry and water

content was 75.2 % ± 0.3 (n = 30). The average lipid content of RA dams was lower than that of NR dams (P <0.001, table 4.2), but they have a

similar amount of protein (P > 0.09). 98 Table 4.2 The body composition of 20 reproductively active (RA) and 19

non-reproductive (NR) female opossums. All units of mass are in grams.

V ariable RA OnnsanmB NR Opossums

Age (month) 10.30 0.65 10.92 0.83

Initial wet mass 65.18 2.33 64.79 2.87

Final wet mass 87.20 2.02 70.68 2.40

D ry m ass 24.94 0.71 24.46 1.17

W ater(%) 71.39 0.52 65.65 0.65

Protein1 12.09 0.52 10.78 0.54

Lipid1 4.74 0.39 7.61 0.40

Energy1 150.61 5.37 147.81 5.51

1 : Protein(g), lipid (g) and energy content (Kcal) were adjusted to the

average dry body mass, 24.70 g± 0.67 (from ANCOVA analysis). 99 Comparisonof Hamsters and OnnsanmR

1) Assimilated and Available Energy from Conception to Weaning

Average whole-animal Assimilated Energy was higher in hamsters than in opossums throughout gestation and lactation (table 4.3). After the effects of differences in body mass were removed, RA-30 hamsters assimilated more energy during gestation than did RA opossums (P < 0.001) but during lactation the difference was not significant (P > 0.35). Although more energy was stored in body reserves in RA opossums than in RA-30 hamsters

(P <0.05), Assimilated Energy and Available Energy of RA-30 hamsters

(corrected for body mass) from C-W were not significantly higher than those of RA opossums (P > 0.051 and 0.07, respectively).

The Assimilated Energy of RA-24 hamsters was higher than that of

RA-30 hamsters during gestation (P < 0.04), but not during lactation (P >

0.16). Although energy was mobilized from body reserves in RA-24 hamsters, Available Energy of RA-24 hamsters (corrected for mass) from C-

W was not significantly higher than that of RA-30 hamsters.

2) Cost of Reproduction

On a per young and on a per young per day basis, Available Energy and

Incremental Cost of Reproduction from C-W in RA-30 hamsters were consistently higher than those of opossums (table 4.3). However, 43%

Incremental Cost of Reproduction of RA-30 hamsters was concentrated in the last 25% of the time duration from C-W. The proportion of Incremental 100 Table 4.3 Comparisons of absolute energy (Kcal) assimilated and used from conception to weaning for reproduction of gray short-tailed opossums and golden hamsters.

Opossums H am sters RA-30 RA-24 V ariables m ean s.e. P 1 m ean s.e. P2 m ean s.e.

Litter size 8.7 0.4 b 6.9 0.4 NS 7.8 0.4

Assimilated Enererv

G estation 162.0 5.7 a 288.3 8.8 b 330.7 7.8 Lactation 1099.4 25.2 a* 1359.2 56.1 NS 1535.4 62.1 Total 1261.3 28.0 a* 1647.5 60.6 NS 1866.1 65.5

Available Enerev3

Total 1243.7 27.8 a* 1642.3 62.1 c* 1892.7 66.7 stored in young 211.7 7.1 a 395.7 21.5 NS 438.3 25.6 maternal RMR 630.3 30.1 NS 669.0 11.6 a 1040.9 24.0 % maternal RMR 50.6 2.0 41.5 1.5 a 56.2 2.2 cost/young 147.4 5.4 a 245.4 10.6 NS 251.6 11.4 cost/young/day 2.1 0.1a 6.6 0.3 NS 6.8 0.3

NR females

energy used 592.6 26.2 655.3 16.7 b 745.8 18.2 % use for RMR 58.6 2.3 81.8 1.2 a 100.5 3.5

Cost for Reproduction4

Incremental 651.0 27.8 a* 987.0 62.1 NS 1146.9 66.7 increment/young 76.4 3.2 a 145.4 7.6 NS 150.3 7.8 increment/young/day 1.1 <.0 a 3.9 0.2 NS 4.1 0.2

a, P < 0,001; b, P < 0.01; c, P < 0.05, and NS based on Wilcoxon test. *, P > 0.05 based on ANCOVA with maternal body mass as the covariate. 1: compare between opossum and hamsters maintained at 30 °C. 2: compare within hamsters. 3: energy assimilated minus energy stored in body reservoirs. *: available energy minus energy used for maintenance and activity of NR females. 101 Cost of Reproduction at 50% and 75% of the time duration from C-W (0.45 ±

0.02 and 0.85 + 0.03, respectively) in opossums were significantly higher (P

< 0.001) than those in RA-30 hamsters (0.27 + 0.01 and 0.57 ± 0.02, respectively). After the effect of differences of maternal body mass was removed, Incremental Cost of Reproduction of RA-30 hamsters was not

significantly higher than that of RA opossums (P > 0.29, table 4.3).

After the effects of differences in maternal body mass, litter size and

average mass per young were removed, Assimilated Energy, Available

Energy from C-W in RA opossums were similar to those in RA-30 hamsters

(P > 0.22), but Incremental Cost of Reproduction from C-W in opossums was

higher (P < 0.047). However, on a per day basis, Available Energy and

Incremental Cost of Reproduction from C-W (corrected for maternal body

mass, litter size and average mass per young) in RA-30 hamsters were

consistently higher than those in RA opossums (P < 0.02).

3) Efficiency of Offspring Production

a. comparisons between gestation and lactation

In both species, percent energy transferred from mother to young

was higher (P < 0.001) during lactation than during gestation. Efficiencies

of offspring production in opossums were 0.004 + 0.0003 during gestation

and 0.23 + 0.005 during lactation. For RA-30 hamsters, these values were

0.05 + 0.003 and 0.31 + 0.007, respectively. In both species, efficiencies of

offspring production at half way through lactation (opossums 0.02 + 0.0005 102 and hamsters 0.26 + 0.007) were much higher than those during gestation

(P < 0.001). b. comparisons between species

Average efficiency of offspring production was consistently lower in opossums than in hamsters (0.04 + 0.002 versus 0.08 + 0.003 at 50% C-W and 0.41 + 0.01 versus 0.33 + 0.01 at weaning).

Comparisons withinH am sters

Available Energy and Incremental Cost of Reproduction were similar in RA-

24 and RA-30 hamsters on a per young basis. Although whole-animal

Available Energy of RA-30 hamsters at 50% and 75% C-W were significantly lower than those of RA-24 hamsters, hamsters maintained at the two temperatures used similar proportions of Available Energy during reproduction. No difference of efficiency of offspring production was found between RA-30 and RA-24 hamsters (P > 0.78).

Reproductive Energetics from Respiratory

Cost of reproduction based on energy used for respiration from C-W was estimated from daily whole-animal RMR (Chapter II and III) multiplied by the corresponding duration (days), then averaged to represent the average energy used per day during gestation or lactation. Mean-cost of gestation or of lactation was compared to pre-mating BMR (Kcal day1). Total Energy 103 was calculated as the total energy used for RMR and plus energy stored in young. Increment of RMR was Total Energy minus (pre-mating energy used per day x C-W).

Daily energy used for RMR of RA-30 hamsters was lower than that of RA-24 hamsters, but the RMR incremental cost per young was similar between these two groups (table 4.4). Total whole-animal energy used for maternal RMR from C-W was similar between RA opossums and RA-30 hamsters (table 4.3). However, on a daily basis, whole-animal and adjusted mean energy used for RMR of RA opossums were consistently lower than those of RA-30 hamsters from C-W (table 4.3).

D iscu ssion

Strategies of Accommodating Maternal Energy Requirements during

Reproduction

Both hamsters and opossums increased energy assimilation by increased food consumption but neither increased energy assimilation through a reduction in fecal losses. In fact, assimilation efficiency decreased during reproduction parallel with increase food consumption in both species.

Mobilization of body reserves by hamsters during reproduction would provide additional energy needed for the very high fetal and neonatal 104 Table 4.4 Comparisons of energy (Kcal) used for respiration from conception to weaning based on measurements of metabolic rates and energy stored in young of gray short-tailed opossums and golden hamsters.

Opossums H am sters RA-30 RA-24 Variables mean s.e. P1 m ean s.e. P2 m ean s.e.

Pre-m ating % of Kleiber 63.97 3.51 a 115.67 2.61 a 170.75 2.65 predicted BMR Kcal/day 5.64 0.36 a 13.41 0.28 a 21.54 0.42 Gestation (duration) 14 16 % of Kleiber 77.29 1.36 a 118.58 2.09 a 179.26 3.35 mean-cost3 1.40 0.07 NS 1.28 0.02 NS 1.28 0.03 Kcal/day 7.48 0.18 a 17.20 0.36 a 27.44 0.65 Lactation (duration) 56 21 dam s % of Kleiber 90.32 5.22 a 137.11 3.08 a 209.03 4.34 m ean cost 1.77 0.13 c 1.40 0.03 NS 1.34 0.04 Kcal/day 9.39 0.52 a 18.76 0.32 a 28.66 0.70 dams and young m ean-cost 3.52 0.25 a 4.96 0.18 b 4.08 0.13 Kcal/day 18.40 0.73 a 66.31 2.51 a 87.48 2.87

Total energy* Kcal 1135.21 41.16 a 1667.68 55.66 a 2276.15 60.60 use/young 132.87 3.53 a 249.19 9.59 b 303.42 13.05

Increm ent5 Kcal 740.13 52.19 a 1171.41 52.12 b 1479.10 57.88 use/young 84.64 4.22 a 173.24 5.56 N S 195.36 8.16

% Kleiber increase6 G estation 27.61 5.82 b 2.90 1.83 NS 5.60 2.86 Lactation 51.16 12.27 NS 18.91 2.56 NS 23.16 3.53

a, P < 0.001; b, P < 0.01; c, P < 0.05 (based on Wilcoxon test). 1: compare between opossum and hamsters maintained at 30 °C.

2: compare within hamsters. 3: over premating RMR.

4: RMR of mother and young plus energy stored in young

6: total energy minus (RMR x duration).

6: average of proportion of Kleiber-predicted BMR through gestation lactation divided by initial proportion of Kleiber-predicted BMR. 106 growth rates seen in this species. Hoarding of food during late lactation

(Fleming 1978) would provide additional energy needed during lactation.

Thus, pregnant hamsters do not increase energy assimilation through increasing food consumption but rather they are apparently able to provide the energy needed for growing fetus by reducing activities and mobilizing fat from body reserves during pregnancy.

Comparison of Reproductive Parameters in Hamsters and O n n san m a

Although the time from conception to weaning in opossums is almost as twice long as that in hamsters, the estimated life time production of offspring is similar (table 4.5). For each litter, the reproductive effort

(Millar 1977) of opossums is 3.01 + 0.11, similar to 3.41 + 0.18 of RA-30 hamsters (P > 0.56). If a female hamster produced litters (average size of

6.5) continuously from puberty to death she would produce a total of 58.5 during her lifetime, which is similar to a value of 56 for a female opossum with 7 litters and litter size of 8. Although little is known of golden hamsters in the wild, a close related species, the Turkish hamsters

(Mesocricetus brandti) is known to be a seasonal breeder and to hibernate during winter. Under these conditions, wild Turkish hamsters produce a maximum of two litters a year (Lyman and O'Brien 1977). No female

Turkish produced more than 3 litters per season in the laboratory (Lyman 107 Table 4.5 Comparisons of reproductive parameters of golden hamsters and gray-short tailed opossums.

Hamster Opossums

M aturity 28 days 4-5 months breeding seasonal (wild) year round

4 litters/year (lab)

Rep success decrease after d after 2 years old

5 litters up to 28 m in females

up to 39 m in males

Rep period 400-500 days (lab) litter/life 9 7 litter size 6.5 8

offspring/life 58.5 56 108 and O’Brien 1977). Also, under laboratory condition length of time in hibernation was positively associated with longevity (Lyman et. al. 1981).

Thus, it appears that hamsters experience a trade-off between the number of breeding events and length of life.

Comparisons of Reproductive Energetics

Assimilated Energy and Available Energy from C-W (corrected for maternal body mass, litter size and average mass per young) in RA opossums were

similar to those in RA-30 hamsters. Although the Incremental Cost of

Reproduction from C-W (corrected for maternal body mass, litter size and

average mass per young) in RA opossums were higher than in RA-30

hamsters, it was lower on a daily basis in opossums. This might be due to

the fact that the weaning mass of a hamster is more than twice that of

opossums at weaning (37 versus 17 g). In addition, partial increased

metabolism in opossums was associated maintaining higher body

temperature during reproduction (Chapter III). Therefore, when this

increased metabolism associated with maintenance was counted as

incremental cost of reproduction, it reduced the efficiency of offspring

production in opossums.

The efficiency of offspring production is higher in hamsters than in

opossums. However, from an evolutionary perspective, the number of young

weaned is more important than mass per young at weaning, as long as the 109 weaning mass of young is beyond the critical point for independence and survival.

On a per young basis, the reproductive efficiency of the gray short­ tailed opossum is higher than hamsters (Table 4.6). Incremental cost from

C-W per young of opossums in this study were dose to those of S. hispidus. but much less than hamsters and other eutherians (table 4.6). However, on a daily basis, incremental cost from C-W per young of S. hispidus was about twice of that in M. domestdca. even that weaning mass per young was

similar in both species (table 4.7). The observed interspecific differences were due largely to a lower BMR and a longer period of lactation in

opossums. Thus, the marsupial mode of reproduction, as seen in opossums,

yields young at a lower cost but at the expense of a longer reproductive

period than is the case for eutherians, particularly the golden hamster. 110 Table 4.6 Comparisons of reproductive energetics (Kcal) of marsupials and eutherians in relation to basal metabolic rate, and the length of gestation

(G) and from conception to weaning (C-W).

litter % duration Total Cost Incremental Species mass size KP* 6 C-W total young7' total young'1 Reference

MonodelDhis 70 8.7 64 14 70 1244 147 651 76 this study1 domestica 20% 1135 133 740 85 this study3

82 8 64 1333 167 853 107 Thompson & Nicoll 1986 Eleohantulus 84 1 82 57 72 872 872 226226 same as above rufescens 79% Elephant shrews

EchinoDS 141 5 28 60 83 1054 211 681 136 same as above relfairi 72% Tenrec

Mesocricetus 95 6.9 116 16 37 1642 245 987 145 this study1 auratus 43% 1668 249 1171 173 this study3

Siermodon 126 4.9 95 26 38 1776 362 490 100 Randolph et al. hispidus 68% 1977 Cotton rats 134 4.6 1804 392 477 104 Mattingly & McClurel982 175 6.9 2125 308 687 100 same as above

*: percentage of Kleiber-predicted BMR.

1: from Available Energy and Incremental cost of Reproduction.

2: from RMR measurements and energy stored in young. I l l Table 4.7 Comparisons of reproductive energetics on a daily basis (Kcal day'1) of marsupials and eutherians.

litter Cost dav'1 Increment dav'1 mass at birth Species m ass size total young-1total young"1 to weaning (g)

Monodelnhis 70 8.7 17.8 2.1 9.3 l . l 1 0.1 - 17.2 domestica 16.2 1.9 10.6 1.22

82 8 19.0 2.4 12.2 1.5

Elenhantulus 84 1 12.1 12.1 3.2 3.2 rufescens shrews

EchinoDS 141 5 12.7 2.5 8.2 1.6 relfairi Tenrec I-1

Mesocricetus 95 6.9 44.4 6.6 26.7 CO CO 2.8 - 37.2 auratus 45.1 6.7 31.6 W

Siermodon 126 4.9 46.7 9.5 12.9 2.6 6.8 - 16.8 hisoidus Cotton rats 134 4.6 47.5 10.3 12.6 2.7 6.7 - 18.2

175 6.9 55.9 8.1 18.1 2.6 6.8 - 21.7

*: from Available Energy and Incremental cost of Reproduction.

2: from RMR measurements and energy stored in young. CHAPTER V

Conclusions

1. Resting metabolic rates (RMR), heat production, and thermal

conductance of both Reproductively Active (RA) and Non-reproductive

(NR) hamsters increased at ambient temperature (TJ below

thermoneutrality (Chapter II). Adjusted mean Tbs were not affected by

Ta within each RA or NR group because RMR and individual variation

accounted for a significant amount of the variability of Tb.

2. Energetic requirements of reproduction in hamsters are not simply

additive to the thermogenic costs of maintaining Tb below

thermoneutrality (Chapter II). Gestation was not related to significant

changes in RMR, RQ, and thermal conductance in RA-24 hamsters as

it was in RA-30 hamsters. This reflects a distinct energy conservation

for pregnant RA-24 hamsters. Maternal physiological changes,

possibly coupled with increased heat from active reproductive organs

and growing fetuses contribute heat for thermoregulation. The greater

increase of RMR during lactation in RA-24 relative to RA-30 hamsters

112 may have been caused by a combination of higher milk synthesis and increased thermoregulatory demands of nursing young.

Effects of reproduction on energetics are more readily detected without the confounding effect of thermoregulation (Chapter II). RA-30 hamsters had higher adjusted mean RMR and lower RQ during gestation, and higher adjusted mean Tb during lactation than those of

NR-30 hamsters.

Higher energy demands occurred during lactation than in gestation in both species (Chapter II, III and IV). Adjusted mean RMR of hamsters at day 7 of lactation was higher than other reproductive stages, corresponding with peak milk dependence by young (Chapter II). The reproductive increment of metabolism (RIM) of RA-30 hamsters was similar to that of RA-24 hamsters. Average RIM of hamsters significantly differed from zero during late-gestation and lactation.

Average whole-animal RMR, Tb as well as body mass increased in RA opossums throughout gestation and lactation (Chapter III). Most of this increased RMR in opossums was associated with higher T„, which can be explained by the temperature effects on physiological processes and reactions as a higher maintenance cost.

The maternal energetic costs of reproduction may be defined as a higher adjusted mean RMR of RA dams compared to that of NR females and a RIM significantly greater than 0. This is not the 114 amount of energy directly used by reproductive tissues during

reproduction. This indicates that the metabolism of reproductive

organs and the growth of fetuses (gestation) or metabolism of milk

synthesis Oactation) impose higher RMR than other tissues after the

effects of differences of body mass and Tb are removed by ANCOVA.

7. Energetic costs of reproduction of opossums was small except late

during lactation (Chapter III). Adjusted mean RMR of opossums was

highest at day 40 of lactation, significantly higher than at diestrus,

estrus, or at the time of weaning. After the effects of differences of Tb

in reproductive stages were removed, day 40 of lactation was also the

only stage in which the RIM differed from zero.

8. Energy assimilated from food from conception to weaning (C-W) in

both species was positively correlated with maternal body mass, litter

size and mass per young. But litter size and litter mass of opossums

were inversely related to the maternal RMR in diestrus. These

relationships were not observed in hamsters. Daily Assimilated

Energy (corrected for maternal body mass) for both species increased

during lactation but not during gestation (Chapter IV).

9. Whole-animal and adjusted mean Assimilated Energy were higher in

RA-24 hamsters than in RA-30 hamsters during gestation. Otherwise,

patterns of energetic costs per young and efficiencies of offspring

production obtained from energy assimilation and maternal energy balance were similar between RA-24 and RA-30 hamsters. Total

Whole-animal Assimilated Energy of RA hamsters did not increase

during gestation, which indicated that energy might be allocated from

decreased physical activities into reproduction (Chapter IV).

10. In general, respiratory measurements provided a similar estimation of

reproductive costs as energy assimilation in animals maintained within

their thermoneutrality (Chapter IV). The whole-animal Available

Energy and the whole-animal Incremental Cost of Reproduction might

not reflect accurately the energy used for reproduction from C-W at T.

of 24 °C. Temperature gradient (Tb - T.) of RA-24 hamsters in the

colony may be reduced by insulation within bedding.

11. Average whole-animal Assimilated Energy was higher in hamsters

than in opossums throughout gestation and lactation. However, total

energy used at 50% and 75% of C-W was similar between opossums

and RA-30 hamsters (P > 0.05). The greatest energy demands

reproduction in hamsters occurs in the last 25 % of C-W, which is

associated with onset direct food consumption by hamster young. This

may partially explain the greater efficiency of offspring production in

hamsters compared to opossums (Chapter IV).

12. Efficiency of offspring production in both species was higher during

lactation than gestation (Chapter IV), which may be related to short

lengths of gestation relative to lactation, and to direct consumption of 1 1 6 food by young. For small eutkerians living below thermoneutrality,

gestation imposes much lower energetic costs than lactation, but

energy conservation and allocation can occur.

13. After the effects of the differences in body mass were removed through

ANCOVA, RA-30 hamsters showed higher energy assimilation than RA

opossums during gestation (P < 0.001), but not during lactation (P >

0.35) or from C-W (P > 0.05). These differences were related to lower

BMR and a longer period of lactation in opossums (Chapter IV).

However, Available Energy and Incremental Cost of Reproduction

(corrected for mass) of RA-30 hamsters from C-W were not

significantly different from those of RA opossums.

14. On a per young and on a per young per day basis, Available Energy,

and Incremental Cost of Reproduction from C-W were consistently

higher in RA-30 hamsters than in RA opossums.

15. On a per day basis, after the effects of differences in maternal body

mass, litter size and average young mass were removed, Available

Energy, and Incremental Cost of Reproduction from C-W were

consistently higher in RA-30 hamsters than those in RA opossums.

16. After the effects of differences in maternal body mass, fitter size and

average young mass were removed, Assimilated Energy from C-W in

RA opossums was not significantly higher than in RA-30 hamsters

(1429.2 + 64.3 versus 1319.6 + 60.2 Kcal, respectively, P > 0.34), but 117 the Incremental Cost of Reproduction from C-W was higher in RA

opossums than in RA-30 hamsters (876 + 54.6 versus 678.9 ± 51.2

Kcal, respectively, P < 0.047). This may due to the fact that opossums

reduce the frequency of heterothermy as energy conservation, and

increase the costs of maintaining a higher body temperature during

gestation and late lactation (Chapter III).

17. Although the efficiency of offspring production was lower in opossums

than in RA-30 hamsters, the two species had similar reproductive

efforts. The observed interspecific differences were due largely to a

lower BMR and a longer period of lactation in opossums. Thus, the

marsupial mode of reproduction, as seen in opossums, yields young at

a lower cost but at the expense of a longer reproductive period than is

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