Energetics of lactation in domestic dog (Canis familiaris) breeds of two sizes

Comparative Biochemistry and Physiology Part A 125 (2000) 197 – 210

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Energetics of lactation in domestic dog (Canis familiaris) breeds of two sizes Michael Scantlebury a,b,*, Richard Butterwick b , John R. Speakman a a

Department of Zoology, Aberdeen Centre for Energy Regulation and Obesity, Uni6ersity of Aberdeen, Aberdeen, AB24 2TZ, UK b Waltham Centre for Pet Nutrition, Waltham-on-the-Wolds, Melton Mowbray, Leicestershire, LE14 4RT, UK Received 31 May 1999; received in revised form 15 October 1999; accepted 4 November 1999

Abstract The energetics of lactation was measured in two breeds of domestic dog during peak lactation. Labrador Retrievers (30 kg) had larger litter sizes than Miniature Schnauzers (6 kg). During the 7-day experimental period, Labrador pups increased more in mass than Schnauzer pups, both absolutely and relatively. Consequently, the energy demands of the litter, relative to maternal metabolism, were higher in Labradors than Schnauzers. Milk composition and gross efficiency of milk production were not significantly different between breeds and the costs of lactation were fuelled by increases in food intake. Metabolisable energy intake was higher than predicted in Labradors, but lower than predicted in Schnauzers. These patterns differ from interspecific expectations, which would predict larger animals to reproduce more slowly, have smaller litter sizes, and invest less energy in reproduction. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Domestic dog; Lactation; Energetics; Deuterium; Isotope; Water turnover; Allometry; Body composition; Milk composition

1. Introduction The functional significance of differences in body size across mammals has received considerable attention in the literature (Kleiber et al., 1961; Bonner et al., 1965; Linzell et al., 1972; Harvey et al., 1985; Kozlowski et al., 1997). In the context of reproduction, larger animals generally exhibit reduced reproductive rates (Bonner et al., 1965; Krzyzanowski et al., 1975; Clutton-Brock et al., 1983), increased lengths of gestation, increased weaning times and take longer to reach maturity * Corresponding author. Tel.: + 44-1224-272879; fax: + 441224-272396. E-mail address: [email protected] (M. Scantlebury)

(Sacher et al., 1959; Millar, 1977; Western, 1979; Sacher and Staffeldt, 1999). Other variables, such as food intake, milk production and offspring growth rate tend to be greater in smaller mammals when expressed relative to adult mass (Kleiber et al., 1961; Oftedal and Gittleman, 1989; Calder, 1996). As relatively less milk is produced in larger animals, less food is required to produce the milk, as a proportion of adult body mass. The effects of body mass on reproductive energetics are normally compared between groups of higher taxa (Millar, 1983; Millar and Zammuto, 1983; Harvey et al., 1985) because intraspecific variation in body mass is small compared with interspecific differences (Harvey et al., 1989). However, the effects of mass in these comparisons are confounded by phylogenetic and ecological

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covariation. An intraspecific study, however, would have the advantage of being phylogenetically distinct (Felsenstein et al., 1985) and independent of ecology. It would be particularly worthwhile if a species could be found with a large variation in body mass. The domestic dog Canis familiaris has the largest range in adult body mass of any mammalian species (Burger and Johnson, 1991). This variation provided an opportunity to examine the differences in reproductive energetics between animals whose principal difference was body mass. Although some energy additional to the maintenance level is required during gestation, lactation is the most energetically demanding period of mammalian reproduction (Hanwell et al., 1977; Millar, 1979; Oftedal and Iverson, 1987; Gittleman et al., 1988; Reilly et al., 1996). This period of maximal energy intake is likely to be a key determinant of life history as it defines the amount of resources available to produce offspring (Sadleir, 1984; Weiner, 1987). Consequently, observations were made during peak lactation in domestic dogs (Paragon et al., 1993). The aims of this study were to obtain quantitative information on the mass, water and energy transfer between mothers and offspring during lactation in dog breeds of different masses, and investigate whether the intraspecific relationships with body mass were consistent with (interspecific) allometric predictions. In particular, we aimed to measure the following variables: litter size, body mass change in adults and offspring, food intake and faecal production, apparent digestibility of food, water kinetics, milk intake, milk composition, body composition of adults and offspring and the gross efficiency of lactation.

2. Materials and methods

2.1. Animals Twelve Labrador Retrievers (‘Labradors’) and six Miniature Schnauzers (‘Schnauzers’) plus litters (totalling 77 and 21 pups, respectively) were used in this study. Experiments took place over periods of 7 days between 24 and 30 days post partum that spanned the period of peak lactation for both breeds. Animals were bred and reared at the Waltham Centre for Pet Nutrition (WCPN), Melton Mowbray, UK.

2.2. Diets and housing Adults were fed a commercial diet (Waltham® Formula® Expert Growth) from the onset of gestation until the offspring were weaned. The diet consisted of 26.9% crude protein, 14.4% fat, 44.8% nitrogen free extract, 7.4% water and 6.5% ash. The metabolisable energy content of the diet was determined by complete faeces and urine collections and was 15.6 kJ/g (WCPN unpublished data). Food and water were supplied in deep stainless steel bowls that were too high for pups to drink or eat from. Food intake was determined by weighing (Sartorius F150 balance 90.1 g) the food bowl at 08:00 h each day. Mothers were housed with their litters in individual pens with constant access to outside runs and fresh water (Loveridge, 1994). Food intake and faeces collections were for 7 days. Urinary losses were calculated by subtraction of the metabolisable energy intake and apparent faecal losses from the gross energy intake.

2.3. Analytical methods Samples of food, faeces and milk were analysed for crude protein (nitrogen× 6.25 for food and faeces, nitrogen ×6.38 for milk) (McDonald et al., 1981) by the combustion method (DUMAS, LECO FP428 analyser), fat by acid hydrolysis and extraction with petroleum ether (Soxhelet, Gerhardt system), water by drying to constant weight at 105°C, and ash by baking at 550°C to constant weight. The gross energy contents of food and faeces were determined by adiabatic bomb calorimetry (Parr 1271, Auto Calorimeter). Milk energy density was derived from the proximate composition and was estimated using the equation: Energy (kJ/g) = [(fat(g)× 9.11+ protein(g)× 5.86 + sugar(g)× 3.95)/100] × 4.186/100

(1)

(Oftedal, 1984a,b; Perrin, 1958).

2.4. Body composition analysis Body composition was determined by dual energy X-ray absorptiometry (DXA) (Hologic QDR 1000/W Densitometer, Hologic, Inc. Waltham MA) (Mazess et al., 1990; Jebb et al., 1997).

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Adults and pups were DXA scanned twice, once at the beginning and once at the end of the experimental period. Offspring and adults were scanned using the infant whole body and adult whole body programmes, respectively (Brunton et al., 1996; Picaud et al., 1996), providing measurements of bone mineral density (BMD, g/cm2), bone mineral content (BMC, g), lean tissue (g) and fat (g). Water content (g) was calculated as lean tissue ×0.73 (Pace et al., 1945) in adults and lean tissue×0.80 in offspring (Sawicka-Kapusta, 1974). Individual scans took approx. 10 min during which the animals were anaesthetised (0.1 ml/kg Vetalar, 0.1 ml/kg Rompun).

2.5. Milk collection Milk samples were collected at the same time each day to minimise any effect of circadian variation in milk composition (Oftedal, 1984a). Three milk samples were collected from each female at 1500 on days 24, 28 and 30 post partum. Pups were removed from their mothers for an hour prior to milking. Milk was collected by manual expression immediately after an injection of oxytocin (Intra-muscular, 10 IU for Labradors and 5 IU for Schnauzers). All nipples were milked and all glands were emptied as completely as possible producing 40–80 ml of milk per sample (Oftedal, 1984a). Milk samples were stored in rubber-sealed glass containers at − 80°C until analysis. Milking took approx. 20 min after which the pups were returned to their mother.

2.6. Drinking water Drinking water of adults was measured from the drop in the water level of a header tank supplying the water bowl, accurate to 0.1 l. This provided an upper limit of the amount of free water drunk as it included any water that was spilt. Evaporation was discounted as a possible source of error because previous validation studies had shown evaporative water losses from the tank and drinking bowl were less than 100 ml/ week (mean 80 ml/week, S.D. 10.1, n = 4). Pups had no access to water and only consumed milk.

2.7. E6aporati6e water loss Adults were placed in whole body gas analysis (WBGA) chambers [internal dimensions were

199

1.5× 1× 1 m for Labradors and 1× 0.75× 0.75 metres for Schnauzers] for 1 h at some point during the experimental week. Animals were habituated to these chambers prior to entry. The chambers had been purpose built for analysis of dietary gasses such as methane and hydrogen sulphide (Papasouliotis et al., 1993), and consisted of an airtight transparent glass door with plywood sides to which an open circuit airflow was supplied. The air inlet was cooled (20°C) and the humidity controlled (40–50% RH) prior to entering the chamber by a water trap. Airflow was adjusted so that increases in temperature and humidity were approximately the same for each animal. Three internal thermohygrographs were fitted at various locations inside the chambers and in the outflow. One hour was sufficient for temperature and humidity to equilibrate at flow rates of approx. 100 l/min. Evaporative water loss of the animals was measured by the difference in water contained in the air of the outflow with and without the animal inside the chamber. Water loss was calculated using information on atmospheric humidity, temperature and pressure (Webb et al., 1995) from standard Smithsonian tables (Weast et al., 1964).

2.8. Total body water turno6er Deuterated water (99.9% D2O, MSD Isotopes Inc) was used to determine body water space (Nd) and water turnover rates (rH2O) in adults and offspring (Nagy et al., 1980). Doses, administered IV, were 7.0 ml for adult Labradors (n= 12), 2.5 ml for adult Schnauzers (n= 6) (approx. 0.1 g/kg) 1.0 ml for Labrador pups (n= 36) and 0.9 ml for Schnauzer pups (n=10) (approx. 0.5g/kg). Six of the 12 Labrador litters were used for maternal body water measurements (litter sizes 5, 7, 7, 6, 7, 5) and six were used to measure offspring (litter sizes 6, 6, 6, 7, 8, 7). Similarly, four Schnauzer litters were used for maternal body water measurements (litter sizes 2, 3, 2, 4) and three litters for measurements of offspring (litter sizes 4, 4, 6). The calculations assume that all substances entering the animal are labelled at background levels and there is no recycling or re-entry of deuterium (Lifson et al., 1966; Nagy, 1980; Oftedal, 1984a; Oftedal et al., 1993b). Adults and offspring were labelled in separate experiments and only half the offspring of any litter were labelled so that the effects of isotope recycling could be corrected for.

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We have previously shown that recycling of isotopes is unlikely to cause significant errors in maternal DEE calculations, but may cause an underestimate of pup water turnover by up to 10.9% (Scantlebury et al., 2000). We took seven blood samples per mother and seven samples per pup (1 per day). Blood samples were vacuum distilled into Pasteur pipettes (Nagy, 1983). Hydrogen, obtained by reacting the resulting distillate with LiAlH4 (Speakman, 1997; Ward et al., 2000), was used for the determination of 2H:1H ratio using a mass spectrometer (Optima, Micromass IRMS, Manchester, UK). Water turnover was calculated using a dedicated computer package, which took into account the effects of small deviations from 24 h for the sampling intervals and the effects of mass changes on body water pool size over the experimental period. Total body water efflux (Lifson et al., 1966) in adults and pups was calculated according to the equation: rH2O=kdNd × F

(2)

where F is the fractionation factor of the isotope and determined by: F = [pE× (0.941)+ (1 − pE) × (1.0)]

(3)

where pE is the proportion of evaporation of the isotope. Zero pE would imply a fractionation factor of 1.00, whereas 100% pE would imply a value of 0.941 (Speakman, 1997). There is uncertainty about the magnitude of in vivo fractiona-

tion in humans and animals (Schoeller et al., 1986; Speakman, 1997). However, based on our measurements of evaporative water loss in dogs (Figs. 1 and 2), a value of 25% pE was used. Eq. (2) reduces to: rH2O= kdNd[(0.941+3)×0.25].

(4)

2.9. Calculation of milk intake from deuterium elimination in labelled pups Milk intake (MI) (g/day) per offspring was calculated as: MI=100×(TWI+ 1.07FD + 0.42PrD) /(%WM + 1.07%FM + 0.42%PrM + 0.58%SM)

(5)

(Oftedal et al., 1987; Oftedal and Iverson, 1987; Perrin, 1958) (Abbreviations according to Oftedal et al., 1993a) in which total water intake (TWI) = (rH2O+ G); where rH2O is the daily water turnover calculated by deuterium elimination and G is the daily increase in body water as a result of offspring growth. FD and PrD are fat and protein deposition (g/d) and%WM,%FM,%PrM and %SM are percentages of water, fat, protein and total sugar in the ingested milk respectively. Milk composition data for each female was averaged over the time period. Rates of fat and protein deposition in pups were measured by changes in the DXA estimates of body composition. Gross efficiency of lactation was calculated as: Efficiency= EMILK/[(EFIL + EM)− EFIP]

(6)

in which EFIL was the net energy intake during lactation, EM was the energy obtained from mass loss during lactation, EFIP was the net energy intake post lactation and EMILK is the milk energy during lactation (English et al., 1985).

2.10. Statistical methods

Fig. 1. Bone mineral content (BMC) plotted against body mass in 10 non-lactating Labradors (closed symbols) and 10 lactating Labradors (open symbols). There was a significant decrease in BMC in the lactating individuals.

Means are displayed with standard deviations. Paired t-tests were used to analyse changes over the experimental period in the same individuals for body mass, BMC, lean and fat tissue. Two sample t-tests were used to examine differences between breeds in food intake, faecal output, apparent absorption and dry matter digestibility. Analysis of covariance (ANCOVA) was used to test for differences between pups; body mass and litter size were entered as co-variates with breed as

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a categorical factor. One pup per litter was selected at random for inclusion in the analysis to prevent pseudoreplication. Statistical analyses were performed using Minitab 5.2 software (Ryan et al., 1985).

3. Results

3.1. Animals Adult Labradors were significantly heavier than Schnauzers (2-sample t16 =22.85, P B 0.001) (Table 1). Within a breed, there was no significant change in mean body mass over the 7 day period (paired t11 = − 2.21, P =0.054, for Labradors and t6 = 1.58, P =0.19 for Schnauzers). Labradors had significantly larger litter sizes than Schnauzers (2-sample t16 =4.83, P = 0.0013) (Table 1). Litter size varied in Labradors from four to nine pups and in Schnauzers from two to six pups. We created divisions within breeds of ‘large’ and ‘small’ litter sizes corresponding to four to six and seven to nine pups in Labradors and one to three and four to six pups in Schnauzers. There was no significant difference in body mass for either breed between females with large and small litter sizes (2-sample t10 =0.77, P= 0.47 for Labradors and t4 =0.82, P =0.47 for Schnauzers). Labrador pups were heavier than Schnauzer pups at 24 days (2-sample t66 =18.28, P B0.001) and pups of both breeds increased significantly in body mass from 24 – 30-days-old (paired t50 = 19.86, PB0.001 in Labradors and t16 =6.13, PB 0.001 in Schnauzers) (Table 2). These increases were significantly different between breeds when the effect of body mass was not taken into account (two-sample t66 =10.71, P B0.001, data pooled across all pups), but mass change was not significantly correlated with pup body mass (ANCOVA: F1,9 = 1.40, P =0.267), breed (F1,9 =0.18, P= 0.679) or litter size (F1,9 =1.07, P =0.327) when mass was included as a co-variate.

3.2. Food intake Although Labradors consumed more food than Schnauzers (2-sample t16 =7.11, P B0.001) (Table 1), there was no difference in food intake between breeds when body mass was included as

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a co-variate (ANCOVA: F1,15 = 3.87, P= 0.068). The gross energy intakes of Labradors and Schnauzers during lactation (27367 kJ/day and 6505 kJ/day, respectively) (Table 1) were greater than that measured during the period pre-breeding (7430 (S.D. 873) kJ/day and 2506 (S.D. 336) kJ/day, respectively). Labradors with larger litters consumed significantly more food than those with smaller litters (1074 g/day, S.D. 461 for 4–6 pups, 1611 g/day, S.D. 264, for 7–9 pups, 2-sample t11 = 2.56, P =0.038). There was no significant difference in food intake between the Schnauzers that had small and large litters.

3.3. Faecal production Labradors produced more faeces than Schnauzers (2-sample t16 = 7.65, P B0.001) (Table 1) and the dry matter had a greater energy content (2–sample t16 = 3.27, P=0.004) (Table 3). Mean faecal energy losses represented 6.1 and 5.4% of the gross energy intake of Labradors and Schnauzers respectively. Labradors with large litter sizes produced more faeces than those with small litter sizes (mean 244 g/day, S.D. 65 for large littered individuals and mean 159 g/day S.D. 23 for small littered individuals, 2-sample t11 = 3.01 P=0.024).

3.4. Apparent dry matter digestibility There was no significant difference in apparent dry matter digestibility between breeds (2-sample t16 = 0.66, P= 0.54) or within breeds between individuals with large and small litter sizes (2-sample t10 = 1.13, P= 0.30 in Labradors and t4 = 0.43, P= 0.71 in Schnauzers) (Table 1).

3.5. Adult body composition Although Labradors contained significantly more BMC, lean and fat tissue than Schnauzers because they were larger, there was no significant difference in percent BMC, lean tissue or fat between Labradors and Schnauzers (Table 3). Equally, there were no significant differences in body composition for either breed between individuals with large and small litter sizes. The only significant change in body composition over the experimental period, was a decrease in fat content of the Labradors (paired t11 = − 2.42, P= 0.039) (Table 3). However, BMC in the lactating moth-

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Table 1 Mean and standard deviations (S.D.) of energy balance taken for adult dogs Energy balance

Body mass (kg) Litter size Chg (body mass) (kg) Food intake (g/day) Gross energy intake (kJ/day) Metabolised energy intake (kJ/day) Faeces produced (g/day) Faecal energy output (kJ/day) Urine energy output (kJ/day) Apparent digestibility Milk produced (g/day) Milk energy output (kJ/day) % Lactation efficiency

Labradors (n = 12)

Schnauzers (n =6)

Mean

S.D.

Mean

S.D.

Pa

30.26 6.42 −0.525 1439* 27367 22448 202* 3437* 1485 0.81 1303 7715 63

3.30 0.90 0.751 241 4170 3760 65 1097 474 0.10 815 4346

6.47 3.57 0.111 345 6505 5382 45 739 394 0.82 389 2304 61

0.11 1.40 0.074 150 2828 2340 21 340 184 0.08 212 1132

B0.001 0.00 0.88 B0.001 B0.001 B0.001 B0.001 B0.001 B0.001 0.88 0.0024 0.0024

a P values denote a significant difference between Labradors and Schnauzers. * Denotes a significant difference between large and small litter sizes. Data for body mass (kg), litter size, change in body mass Chg (body mass) (kg), food intake (g/day), gross energy intake (kJ/day), metabolised energy intake (kJ/day), faeces produced (g/day), faecal energy output (kJ/day), urine energy output (kJ/day), apparent dry matter digestibility, milk produced (g/day), milk energy output (kJ/day), lactation efficiency (%).

Fig. 2. Flow diagram showing mass, water and energy transfer in a lactating Labrador and offspring. Arrow sizes are proportional to flow rates. Unknown quantities are denoted by dotted arrows.

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Table 2 Mean and standard deviations (S.D.) of kinetic measurements taken for adult dogs Kinetic measurements

Drinking water (l/day) Evaporated water (l/day) k(d) (per h) N(d) (l) N (DXA) (l) Total water turnover (l/day) % Body water

Labradors (n= 12)

Pa

Schnauzers (n =6)

Mean

S.D.

Mean

S.D.

5.32 1.11 0.01193 18.80 18.0 5.40 62

1.54 0.40 0.00306 2.59 1.1 2.80 6

1.26 0.38 0.00783 3.69 3.4 0.68 57

0.26 0.21 0.00101 0.74 0.7 0.18 2

B0.001 0.0017 0.022 B0.001 B0.001 0.00

a

P values denote a significant difference between Labradors and Schnauzers. Data for drinking water (l/day), evaporated water (l/day), kd (deuterium elimination rate, h−1), Nd (body water space estimated by deuterium dilution, litres), N(DXA) (body water space estimated by DXA, litres), total water turnover estimated by deuterium elimination (l/day) % body water calculated by deuterium dilution.

ers was significantly lower than that measured in non-breeding individuals (unpublished data, ANCOVA: F1,17 =38.11, P B 0.001, with body mass as a co-variate, Fig. 1).

dors than Schnauzers (2-sample t16 = 4.24, P= 0.0017) (Table 2). These differences were not significant, when mass was entered as a co-variate (ANCOVA: F1,10 = 0.37, P= 0.557). There was no difference in evaporative water loss between indi-

3.6. Pup body composition There was no significant difference in percent BMC between Labrador and Schnauzer pups. However, Labrador pups contained relatively less fat and more lean tissue than Schnauzer pups (Tables 4 and 5). There were no differences in body composition between individuals of either breed that came from large and small litter sizes. All pups increased significantly in BMC and lean tissue during the experimental period, although only the Labrador pups increased significantly in fat (Table 5).

3.7. Water kinetics and milk intake 3.7.1. Drinking water Adult Labradors drank more water than Schnauzers (2-sample t16 =8.54, P B0.001) (Table 2). However, these differences were not different when mass was entered as a co-variate (ANCOVA: F1,16 =1.80, P =0.198). There were no differences in the amount of water used between mothers that raised large and small litters (t11 = 1.62, P= 0.16 for Labradors and t6 =0.46, P = 0.69 for Schnauzers). 3.8. E6aporati6e water loss Evaporative water loss was greater in Labra-

Fig. 3. Flow diagram showing mass, water and energy transfer

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Table 3 Mean and S.D. of body composition taken for adult dogs. Body composition

% BMC % LEAN % FAT BMC (g) LEAN (g) FAT (g) Chg (BMC) (g) Chg (Lean) (g) Chg (Fat) (g)

Labradors (n = 12)

Pa

Schnauzers (n = 6)

Mean

S.D.

Mean

S.D.

2 79 19 652 22474 5335 0.27 −50 −224**

0 4 4 80 1516 1460 12.5 716 293

2 82 16 129 4191 999 3.98 −202 −16

0.3 3 2.8 24.6 1141 439 6.73 63 63

0.27 0.076 0.082 B0.001 B0.001 B0.001 0.47 0.68 0.057

a

P values denote a significant difference between Labradors and Schnauzers. ** Denotes a significant change over time (24–30 days post partum). Data for % BMC (percent bone mineral content), % LEAN (percent lean tissue), % FAT (percent fat tissue), BMC (bone mineral content, g), LEAN (lean tissue content, g), FAT (fat tissue content, g), Chg (BMC) (change in bone mineral content from 24 to 30 days of lactation), Chg (LEAN) (change in lean tissue content), Chg (FAT) (change in fat tissue content). Table 4 Mean and S.D. of faeces composition taken for adult dogs Faeces composition

% Water % Ash % Crude protein % Crude fat % Nitrogen free extract Dry matter energy (kJ/g)

Labradors (n= 12)

Pa

Schnauzers (n =6)

Mean

S.D.

Mean

S.D.

72.7 6.0 9.6 1.7 10.1 17.0

2.1 1.1 1.3 0.3 1.2 0.7

66.2 8.2 10.7 2.0 12.9 16.2

1.4 1.0 0.8 0.1 0.5 0.5

B0.001 0.0002 0.026 0.001 B0.001 0.004

a P values denote a significant difference between Labradors and Schnauzers. Data for percent water, percent ash, percent crude protein, percent crude fat, percent nitrogen free extract, dry matter energy (kJ/g).

viduals that raised large litters and small litters (2-sample t11 = 0.23. P =0.83, data for Labradors only).

3.9. Total body water turno6er In all cases the log converted enrichments of hydrogen above background decreased linearly and were correlated with time over the experimental period. The r 2 values averaged 99.5% in both breeds. Adult Labradors had larger deuterium dilution spaces (Nd) than Schnauzers (2-sample t16 = 13.47, P B 0.001), greater deuterium elimination rates (kd) (2-sample t16 =3.05, P=0.022) and higher water turnover rates (rH2O) (2-sample t16 =5.26, P =0.0033) (Table 2). Independent body water spaces 2

measured by DXA in Labradors and Schnauzers (Table 2) were 4 and 7%, respectively less than deuterium dilution spaces. These differences are consistent with isotope based calculations of body water producing overestimates of between 5 and 10%, because of additional isotope pools (Speakman, 1997). There were no significant differences in either Nd, kd or rH2O between individuals of either breed that reared large litters and small litters. Deuterium dilution spaces in Labrador pups were greater than Schnauzer pups (2-sample t66 = 9.55, PB0.001) (Table 5). In contrast to adults, deuterium elimination rates were not significantly different between breeds (2-sample t66 = 0.11, P=0.91). Water turnover rates were greater in Labrador than Schnauzer pups (2-sample t66 = 5.99, PB 0.001). DXA estimates

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Table 5 Mean and S.D. of measurements taken for offspring Labradors (n= 51)

Schnauzers (n =17)

P*

Mean

S.D.

Mean

S.D.

2350 241** 15060

520 87 2300

860 69** 310

160 45 810

B0.001 B0.001 B0.001

2 84 14 46 1971 345 6.89** 221** 15.3**

0 2 2 13 405 117 4 91 30

2 80 18 13 674 166 3.03** 54.2** 12

0 4 4 4 109 59 4 91 26

0.067 0.0001 B0.001 B0.001 B0.001 B0.001 0.0015 B0.001 B0.001

0.005379 1710 1665 195 203 1201 73

0.00095 319 327 47 127 752 9

0.005613 660 561 84 109 645 77

0.00211 106 95 20 60 352 6

0.91 B0.001 B0.001 B0.001 0.0024 B0.001

a

Body mass (g) Chg (body mass) (g) Total litter mass (g) Body compositionb %BMC %LEAN %FAT BMC (g) LEAN (g) FAT (g) Chg (BMC) (g) Chg (LEAN) (g) Chg (FAT) (g) Water kinetics and energy balancec k(d) (per h) N(d) (ml) N (DXA) (ml) Water turnover (ml/day) Milk consumption (g/day) Milk energy intake (kJ/day) % Body water a

Body mass (kg), change in body mass Chg (body mass) (g), total litter mass (g). Body composition; % BMC (percent bone mineral content), % LEAN (percent lean tissue), % FAT (percent fat tissue), BMC (bone mineral, g), LEAN (lean tissue content, g), FAT (fat tissue content, g), Chg (BMC) (change in bone mineral content from 24 to 30 days of lactation), Chg (LEAN) (change in lean tissue content), Chg (FAT) (change in fat tissue content). c Water kinetics and energy balance: kd (deuterium elimination rate, h−1), Nd (body water space estimated by deuterium dilution, ml), N(DXA) (body water space estimated by DXA, ml), water turnover calculated by isotope elimination (ml/day), milk consumption (g/day), milk energy intake (kJ/day), % body water calculated by deuterium dilution. * P values denote a significant difference between Labradors and Schnauzers. ** Denotes a significant change over time during peak lactation (24–30 days post partum). b

of body water space in Labrador and Schnauzer pups were 4 and 10%, respectively less than the deuterium dilution spaces.

protein, 8.11 (S.D. 1.91)% fat and 4.86 (S.D.1.42)% sugar. Milk energy averaged 5.92 (S.D. 0.60) kJ gross per gram.

3.10. Milk intake, milk composition and milk energy output

4. Discussion

Milk intake was greater in Labrador than Schnauzer pups (2-sample t66 =3.70, P =0.0024) (Table 5). There were no differences in kd, Nd or milk intake between those individuals that came from large or small litters. In addition, there were no differences in milk composition between breeds, within breeds with different litter sizes, or between day 24 and day 30 of lactation within individuals. Milk contained on average 21.2 (S.D. 1.33)% dry matter, 8.23 (S.D. 0.66)% crude

4.1. Animals There is a positive relationship between litter size and body mass across dog breeds (Robinson et al., 1973), in contrast to the general mammalian pattern (Blueweiss et al., 1978). However, gestation length (Krzyzanowski et al., 1975), oestrus cycle length, weaning times (Evans and White, 1988) and age at first breeding are remarkably uniform across dog breeds. This is surprising

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given the convincing allometric patterns for these traits across mammals of similar size ranges, with larger animals generally reproducing more slowly and investing less overall resources into reproduction (Hanwell et al., 1977; Calder, 1996). Hence, one might expect the (intraspecific) patterns of energy allocation observed across dogs of different masses to differ from expected (interspecific) allometric variation. Figs. 2 and 3 show the mass, water and energy transfer in lactating Labradors and Schnauzers, respectively (24 – 28 days post partum).

4.2. Food intake Metabolisable energy intakes (MEI) during peak lactation were similar to the maximum predicted rates of energy assimilation. MEI’s of Labradors were 29% higher than the Weiner (1989) prediction and 13% higher than the Kirkwood (1983) prediction. In contrast, Schnauzers were 14% lower than the Weiner prediction and 18% lower than the Kirkwood prediction (Fig. 4). Hence, Labradors appeared to be working harder during peak lactation than Schnauzers, independently of their mass. These increases are consistent with Labradors having the larger litters. The fact that differences in food intake between large and small litters were only observed in the Labradors may reflect the larger litter sizes of Labradors. The extra cost of lactation was met by increases in food intake of 300 – 400% in Labradors and 200–300% in Schnauzers, in agreement with other studies on small mammals (Lonnerdal et al., 1981; Lochmiller et al., 1982; Glazier, 1985; Hammond et al., 1992. In contrast, when food supply is intermittent, or when lactation is necessarily brief, some large carnivores such as species of pinniped and bear cover some or all of the costs of lactation by utilising body reserves (e.g. Arnould and Ramsay, 1994; Lydersen et al., 1996, 1997; Oftedal, 1993; Oftedal et al., 1993a). This is likely to depend on the size of the mother, with relatively larger mothers being able to contribute more resources or fast for longer periods (Bowen et al., 1992; Fedak et al., 1996).

4.3. Body mass and body composition There was no change in mass for adults of either breed during peak lactation. This is consistent with previous studies in which the costs of

lactation are met by increases in food intake (Meyer et al., 1984; Oftedal, 1984a; Kienzle et al., 1985). Nevertheless, there was a significant decrease in the body fat of the Labradors. The loss in the larger breed is consistent with the idea that larger mothers may be better able to utilise body reserves during lactation (Bowen et al., 1992; Fedak et al., 1996), and it is tempting to regard this fat loss as a possible limit to energy assimilation. However, the large amount of variability in the data and the relatively minor loss of fat (0.74% reduction in body fat of the Labradors over the 7 day interval) indicate this loss is unlikely to be biologically significant. Although there was no loss of BMC during the week of peak lactation, lactating mothers had significantly less BMC than non-breeding individuals (Fig. 1). Maternal BMC was therefore likely to have been used to support offspring skeletal growth (Meyer et al., 1984; Fukuda et al., 1993). In contrast to adults, pups of both breeds increased in body mass during the experimental period. Lean tissue growth accounted for 90% of the mass increase in Labradors and 80% in Schnauzers (Table 5, Figs. 2 and 3). By comparison, offspring of some species of pinniped, with a necessarily short duration of lactation, are able to grow rapidly and 50–80% of the increase in mass is fat (Meyer et al., 1984; Oftedal et al., 1993b; Lydersen et al., 1995, 1996, 1997).

Fig. 4. Log-converted metabolisable energy intake (MEI) against log-converted body mass (kg) for 12 Labrador Retrievers (closed symbols) and five Miniature Schnauzers (open symbols). Predicted relationships are given as lines. The solid line is the Kirkwood (1983) prediction and the dotted line is the Weiner (1989) prediction. Labradors had higher and Schnauzers lower MEI’s than predicted.

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Fig. 5. Metabolic mass ratio (LMM/MMM) plotted against log-converted body mass (kg) for 12 Labrador Retrievers (closed symbols) and six Miniature Schnauzers (open symbols). The higher ratio in Labradors compared to Schnauzers indicates a higher demand of the litter relative to maternal metabolism.

4.4. Milk composition Milk production estimates in the current study were similar to the 1690 g/milk per day in a German Shepherd (66) and 1060 g/milk per day in Beagles at day 26 of lactation (Oftedal, 1984a). Although milk composition varies greatly between species (Jenness et al., 1970; Linzell et al., 1972), there appears to be little difference between dog breeds (Russe, 1961). Milk composition changes with period of lactation (Thomee, 1978; Lonnerdal et al., 1981; Oftedal et al., 1993a; Lydersen et al., 1995), which teat and how completely a gland is emptied (Oftedal, 1984b). In the current study, milk composition was similar to that measured in beagles at a corresponding stage of lactation (Oftedal, 1984a). We found no difference in milk composition between Labradors and Schnauzers measured during the same period of lactation or any evidence of more dilute milk being produced by mothers of larger litters (e.g. in cotton rats, Sigmodon hispidus) (Rogowitz et al., 1995). Therefore, milk composition is unlikely to be a source of disparity in lactation energetics between these two breeds.

4.5. Milk energy output In mammals, the magnitude of milk energy output roughly parallels body mass, with larger individuals producing more energy per day in

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milk (Oftedal, 1984b). Typically canids have high milk energy outputs, of which the domestic dog has one of the highest (Oftedal and Gittleman, 1989). Communal rearing of offspring might have allowed the evolution of large litter sizes which subsequently induced high milk energy outputs (Moehlem, 1989; Oftedal and Gittleman, 1989). These milk energy demands might be expected to differ, following selection for variations in body mass, with resulting litter size changes. Expression of milk energy output relative to maternal metabolic mass (MMM) [(M0.75): M= maternal body mass, 0.75 is the allometric exponent] is one way of correcting for size-related differences in energy yield (Oftedal, 1984b). Relative energy outputs calculated in this way were 598 kJ/kg0.75 in Labradors and 567 kJ/kg0.75 in Schnauzers. These values are similar, but Labradors maintained slightly higher milk energy outputs independent of their mass. Milk energy output can also be expressed in terms of litter metabolic mass (LMM) (allometric exponent is 0.83), which provides an estimate of the relative energy demands of the litter (Gittleman et al., 1987; Oftedal and Gittleman, 1989). Energy output per LMM was 591 kJ/kg0.83 in Labradors and 731 kJ/kg0.83 in Schnauzers. The ratio LMM/MMM indicates the demands of the litter relative to maternal metabolism. It explains approx. 97% of the variance in maternal energy production per kg0.75 and is valid for species that vary greatly in body mass (Oftedal, 1984b). LMM/MMM, was 1.01 in Labradors and 0.78 in Schnauzers, which indicates Labradors have a higher demand of the litter relative to maternal metabolism than Schnauzers (Fig. 5). In summary, the pattern of energy allocation during reproduction in Labradors and Schnauzers was different from the predicted interspecific pattern, which predicts larger animals to reproduce more slowly, have smaller litter sizes and invest less energy in reproduction. In the current observations, although milk composition and gross efficiency of milk production were not significantly different between breeds, Labradors had a relatively higher milk energy output than the Schnauzers and invested a greater amount of resources in reproduction. The costs of lactation were met by increases in metabolisable energy intake, which was higher than predicted in Labradors and lower than predicted in Schnauzers.

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Acknowledgements The work was funded by a grant from Waltham Centre for Pet Nutrition to the University of Aberdeen. We are grateful for the help and support of all those who helped in this project. At WCPN these included Ivan Burger, Derek Booles, Tom McCappin, Amanda Hawthorne, Waring Hynds, David Poore, Matthew Gilham, Pat Nugent and David Lawson. We would like to thank Sally Ward for valuable comments on earlier drafts of the ms and Peter Thomson for the mass spectrometry. Calculations of water turnover were made using a dedicated program; Lemen & Speakman, 1997 http://www.natureware.com/ double.htm.

References Arnould, J.P.Y., Ramsay, M.A., 1994. Milk-production and milk consumption in polar bears during the ice-free period in Western Hudson-Bay. Can. J. Zool. 72 (8), 1365–1370. Blueweiss, L., Fox, H., Kudzma, V., Nakashima, D., Peters, R., Sams, S., 1978. Relationships between body size and some life history parameters. Ecologia 37, 257–272. Bonner, J.T., 1965. Size and Cycle: An Essay on the Structure of Biology. Princeton University Press, Princeton, NJ. Bowen, W.D., Oftedal, O.T., Boness, D.J., 1992. Mass and energy-transfer during lactation in a small phocid, the Harbor seal (Phoca-6itulina). Physiol. Zool. 65 (4), 844–866. Brunton, J.A., Saigal, S., Wintrop, A.L., Atkinson, S.A., 1996. Body composition by doubly labelled water (DLW) and dual energy X-ray absorptiometry (DXA) in tiny premature infants (PI). FASEB. J. 10 (3), 1328. Burger, I.H., Johnson, J.V., 1991. Dogs large and small. The allometry of energy requirements within a single species. J. Nutr. 121, S18–S21. Calder, W.A., 1996. Size, Function and Life History. 2nd edition. Dover Publications, Mineola, NY, p. 1996. Clutton-Brock, T.H., Harvey, P.H., 1983. The Functional Significance of Variation of Body Size Among Mammals. In: Eisenberg, J.F., Kleinman, D.G., (Eds.), Advances in the study of Mammalian Behaviour. Special publication American Society of Mammalogists 7, 632–663. English, R.M., 1985. Breast milk production and energy exchange in human lactation. Br. J. Nutr. 53 (3), 459–466.

Evans, JM, White, K., 1988. A Complete Guide to Understanding and Caring for Bitches. Henston. Publ. Fedak, M.A., Arnbom, T., Boyd, I.L., 1996. The relation between the size of the elephant seal mothers, the growth of their pups, and the use of maternal energy, fat and protein during lactation. Physiol. Zool. 69 (4), 887 – 911. Felsenstein, J., 1985. Phylogenies and the comparative method. Am. Naturalist 125 (1), 1 – 15. Fukuda, S., Iida, H., 1993. Histomorphometric changes in iliac trabecular bone during pregnancy and lactation in beagle dogs. J. Vet. Med. Sci. 55 (4), 565 – 569. Gittleman, J.L., Oftedal, O.T., 1987. Comparative growth and lactation in carnivores. Symposium Zool. Soc. Lond. 57, 41 – 77. Gittleman, J.L., Thompson, S.D., 1988. Energy allocation in mammalian reproduction. Am. Zool. 28, 863 – 875. Glazier, D.S., 1985. Energetics of litter size in 5 species of Peromyscus with generalisations for other mammals. J. Mammal 66 (4), 629 – 642. Hammond, K.A., Diamond, J., 1992. An experimental test for a ceiling on sustained metabolic rate in lactating mice. Physiol. Zool. 65 (5), 952 – 977. Hanwell, A., Peaker, M., 1977. Physiological effects of lactation on the mother. Symp. Zool. Soc. Lond. 41, 297 – 312. Harvey, P.H., Clutton-Brock, T.H., 1985. Life history variation in primates. Evolution 39 (3), 559 – 581. Harvey, P.H., Promislov, D.E.L., Read, A.F., 1989. Causes and correlates of life history differences among mammals. In: Foley, R., Standen, V. (Eds.), Comparative Sociobiology. Blackwell, Oxford, pp. 305 – 318. Jebb, S.A., 1997. Measurement of soft tissue composition by dual energy X-ray absorptiometry. Br. J. Nut. 77 (2), 151 – 163. Jenness, R., Sloan, R.E., 1970. The comparison of milks of various species: a review. Dairy Sci. Abstr. 32, 599 – 612. Kienzle, E., Meyer, H., Dammers, C., Lohrie, H., 1985. Milk intake, weight development, milk digestibility and energy and nutrient retention in suckling puppies. In: Meyer, H., (Ed.). Investigations on nutrient requirements in breeding bitches and suckling pups. Advances in Animal Physiology and Animal Nutrition. Supplements to Journal of Animal Physiology and Animal Nutrition. Verlag Paul Parey, Hamburg und Berlin, pp. 26 – 50. Kirkwood, J.K., 1983. A limit to metabolisable energy intake in mammals and birds. Comp. Biochem. Physiol. 75A, 1 – 3. Kleiber, M., 1961. The Fire of Life. An Introduction to Animal Energetics. Wiley, New York.

M. Scantlebury et al. / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 197–210

Kozlowski, J., Weiner, J., 1997. Interspecific allometries are by-products of body size optimisation. Am. Naturalist 149 (2), 352–380. Krzyzanowski, J., Malinowski, E., Studnicki, W., 1975. Badania nad czasem trania ciazy u suk niektorych ras psow hodowanych w kraju. Medycyna Weterynaryjna 31 (6), 373–374. Lifson, N., McClintock, R., 1966. Theory of the turnover rate of body water for measuring energy and material balance. J. Theor. Biol. 12, 46–74. Linzell, J.L., 1972. Milk yield, energy loss in milk, and mammary gland weight in different species. Dairy Sci. Abstr. 34 (5), 351–360. Lochmiller, R.L., Whelan, J.B., Kirkpatrick, R.L., 1982. Energetic cost of lactation in Microtus pinetorum. J. Mammalogy 63 (3), 475–481. Lonnerdal, B., Keen, C.L., Hurley, L.S., Fisher, G.L., 1981. Developmental changes in the composition of Beagle dog milk. Am. J. Vet. Res. 42 (4), 662–666. Loveridge, G., 1994. Provision of environmentally enriched housing for dogs. Anim. Technol. 5 (1), 1–19. Lydersen, C., Hammill, M.O., Kovacs, K.M., 1995. Milk intake, growth and energy-consumption in pups of ice-breeding grey seals (Halichoerus-grypus) from the Gulf of St-Lawrence, Canada. J. Comp. Physiol. B-Biochem. Systematic Environ. Physiol. 164 (8), 585–592. Lydersen, C., Kovacs, K.M., Hammill, M.O., Gjertz, I., 1996. Energy intake and utilisation by nursing bearded seal (Erignathus barbatus) pups from Svalbard, Norway. J. Comp. Physiol. B-Biochem. Systematic Environ. Physiol. 166 (7), 405–411. Lydersen, C., Kovacs, K.M., Hammill, M.O., 1997. Energetics during nursing and early postweaning fasting in hooded seal (Crstophora cristata) pups from the Gulf of St Lawrence, Canada. J. Comp. Physiol. B-Biochem. Systematic Environ. Physiol. 167 (2), 81–88. Mazess, R.B., Barden, H.S., Bisek, J.P., Hanson, J., 1990. Dual energy X-ray absorptiometry for total body and regional bone mineral and soft tissue c. Am. J. Clin. Nutr. 51 (6), 1106–1112. McDonald, P., Edwards, R.A., Greenhalgh, J.F.D., 1981. Animal Nutrition, 3rd Edition. Longman, New York. Meyer, H., 1984. Mineral metabolism and requirements in bitches and suckling pups. In: Anderson, R.S. (Ed.), Nutrition and Behaviour in Dogs and Cats. Pergamon Press, Oxford, pp. 13–24. Millar, J.S., 1977. Adaptive features in mammalian reproduction. Evolution 31, 370–386. Millar, J.S., 1979. Energetics of lactation Peromyscus maniculatus. Can. J. Zool. 57, 1015–1019. Millar, J.S., 1983. Prepartum reproductive characteristics of eutherian mammals. Evolution 35 (6), 1149– 1163.

209

Millar, J.S., Zammuto, R.M., 1983. Life histories of mammals — an analysis of life tables. Ecology 64 (4), 631 – 635. Moehlem, P.D., 1989. Intraspecific variation in Canid social systems. In: Gittleman, J.L. (Ed.), Carnivore Behaviour Ecology and Evolution. Chapman and Hall, London, pp. 143 – 163. Nagy, K.A., 1980. CO2 production in animals: an analysis of potential errors in the doubly labelled water technique. Am. J. Physiol. 238, R466 – R473. Nagy, K.A., Costa, D.P., 1980. Water flux in animals: analysis of potential errors in the tritiated water method. Am. J. Physiol. 238, 454 – 465. Nagy, K.A., 1983. The doubly labelled water (3HH18O) method: a guide to its use. UCLA Publication No. 12-1417. University of Californin, Los Angeles, CA 90024. Oftedal, O.T., Inverson, S.J., 1987. Hydorgen isotope methodology for the measurement of milk intake and 20th energetics of growth in suckling young. In: Huntly, A.C., (Ed.), Marine Mammal Energetics. Society for marine mammalaogy, special publications, 1, 67 – 96. Oftedal, O.T., Iverson, S.J., Boness, D.J., 1987. Milk and energy intakes of suckling California sea lions Zalophus californianus pups in relation to growth and predicted maintenance requirements. Physiol. Zool. 60 (8), 560 – 575. Oftedal, O.T., Gittleman, J.L., 1989. Patterns of energy output during reproduction in Carnivores. In: Gittleman, J.L. (Ed.), Carnivore Behaviour, Ecology and Evolution. Chapman and Hall, London and Cornell University Press, Ithca, NY, pp. 355 – 378. Oftedal, O.T., 1993. The adaptation of milk secretion to the constraints of fasting in bears, seals and baleen whales. J. Dairy Sci. 76 (10), 3234 – 3246. Oftedal, O.T., Alt, G.L., Widdowson, E.M., Jakubasz, M.R., 1993a. Nutrition and growth of suckling black bears (Ursus americanus) during their mother’s winter fast. Br. J. Nutr. 70, 407 – 419. Oftedal, O.T., Bowen, W.D., Boness, D.J., 1993b. Energy-transfer by lactating hooded seals and nutrient deposition in their pups during the 4 days from birth to weaning. Physiol. Zool. 66 (3), 412 – 436. Oftedal, O.T., 1984a. Lactation in the dog: milk composition and intake by puppies. J. Nutr. 114, 803 – 812. Oftedal, O.T., 1984b. Milk composition, milk yield and energy output at peak lactation: a comparative review. Symposium. Zool. Soc. Lond. 51, 33 – 85. Pace, N., Rathbun, E.N., 1945. Studies on body composition. III. The body water and chemically combined nitrogen content in relation to fat content. J. Biol. Chem. 168, 685 – 691. Papasouliotis, K., Muir, P., Gruffydd-Jones, T.J., Cripps, P.J., Blaxter, A.C., 1993. The effect of short-

210

M. Scantlebury et al. / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 197–210

term dietary fibre administration on orocecal transittime in dogs. Diabetologia 36 (3), 207–211. Paragon, B.M., Grandjean, D., 1993. Conduite du rationnement de la chienne en periode de reproduction. Recueil. Med. Vet. 169 (4), 223–230. Perrin, D.R., 1958. The calorific value of milk of different species. J. Dairy Res. 25, 215–220. Picaud, J.C., Rigo, J., Nyamugabo, K., Milet, J., Senterre, J., 1996. Evaluation of dual energy X-ray absorptiometry for body composition assessment in piglets and term human neonates. Am. J. Clin. Nutr. 63 (2), 157–163. Reilly, J.J., Fedak, M.A., Thomas, D.H., Coward, W.A.A., Anderson, S.S., 1996. Water balance and the energetics of lactation in grey seals (halichoerus grypus) as studied by isotopically labelled water methods. J. Zool. 238 (1), 157–165. Robinson, R., 1973. Relationship between litter size and weight of dam in the dog. Vet. Record 92, 221–223. Rogowitz, G.L., McClure, P.A., 1995. Energy export and offspring growth during lactation in Cotton rats (Sigmodon hispidus). Funct. Ecol. 9, 143–150. Russe, I., 1961. Die Laktation der Hundin Zentraibl. Veterinaehrmed 8, 252–281. Ryan, B.F., Joiner, B.L., Ryan, T.A., 1985. Minitab Handbook, 2nd Edition. PWS-Kent, Boston, MS. Sacher, G.A., 1959. Relation of lifespan to brain weight and body weight in mammals. Ciba. Found Coll. 5, 115–141. Sacher, G.A., Staffeldt, E.F., 1999. Relation of gestation time to brain weight for placental mammals: implications for the theory of vertebrate growth. American Naturalist 74;108:593–615. Sadleir, R.M.F.S., 1984. Ecological consequences of lactation. Acta. Zool. Fennica 171, 179–182.

.

Sawicka-Kapusta, K., 1974. Changes in the gross body composition and energy value of bank voles during their postnatal development. Acta Theriol. 19 (3), 27 – 54. Scantlebury, M., Burger, I., Speakman, J.R., 2000. Isotope recycling in lactating dogs Canis familiaris. Am. J. Physiol. (in press). Schoeller, D.A., Leitch, C.A., Brown, C., 1986. Doubly labelled water method: in vivo oxygen and hydrogen isotope fractionation. Am. J. Physiol. 251, R1137 – R1143. Speakman, J.R., 1997. Doubly Labelled Water, Theory and Practice. Chapmann and Hall, London. Thomee, A., 1978. Composition, digestibility and tolerance of bitch milk and mixed food for puppies, taking into particular consideration the fat component. Doctor of Veterinary Medicine Dissertation. Tierarzliche Hochsuhule, Hannover. Publ. Ward, S., Scantlebury, M., Thomson, P., Speakman, J.R., 2000. Preparation of Hydrogen for mass spectrometric analysis using LiAlH4. Rapid communications in Mass Spectrometry (in press). Weast, R.C., Selby, S.M., Hodgman, C.D., 1964. Handbook of Chemistry and Physics. 45th edition. The Chemical Rubber Company, OH. Publ., 1964. Webb, P.I., Speakman, J.R., Racey, P.A., 1995. Evaporative water loss in two sympatric species of vespertilionid bat, Plecotus auritus and Myotis daubentoni: relation to foraging mode and implications for roost site selection. J. Zool. London 235, 269 – 278. Weiner, J., 1987. Maximum energy assimilation rates in the Djungarian hamster (Phodopus sungorus). Oecologia 72 (2), 297 – 302. Weiner, J., 1989. Metabolic constraints to mammalian energy budgets. Acta Theriol. 34 (1), 3 – 35. Western, D., 1979. Size, life history and ecology in mammals. African J. Ecol. 17, 185 – 204.