Intake and use of milk nutrients by rat pups suckled in small, medium, or large litters MARTA L. FIOROTTO, DOUGLAS G. BURRIN, MARGOT PEREZ, AND PETER J. REEDS United States Department of Agriculture/Agricultural Research Service, Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030 FIOROTTO, MARTA L., DOUGLAS G. BURRIN, MARGOT PEREZ, AND PETER J. REEDS. Intake and use of milk nutrients by rat pups suckled in small, medium, or large litters. Am. J. Physiol. 260 (Regulatory Integrative Comp. Physiol. 29): Rll04-Rlll3,1991.-We investigated the extent to which the altered weight gain of rat pups suckled in litters of varying sizes (4, 10, and 16 pups/litter) is attributable to differences in milk nutrient intake. Milk intake was estimated from the rate of dilution over 2 days of a dose of 3H20 administered at 4, 8, and 14 days of age after correction for the water and milk carbon deposited in body tissues over the measurement period. Protein and energy intakes were estimated from the volume of milk consumed by individual pups and the composition of milk from each dam. Significant effects of litter size on milk fat and protein concentration were observed. Weight gain was highly correlated with energy intake in pups suckled in litters of 4 and 10 but not 16. These findings were attributed to a higher energy expenditure of pups Cl0 days old suckled in litters of 16; specifically these pups had higher maintenance energy needs than pups suckled in litters of 4 and 10 and a higher energy cost of tissue synthesis. The latter was ascribed to an ability of immature pups to maximize the efficiency of protein utilization, thereby blunting the deleterious effects of a reduced nutrient intake on protein deposition. growth; milk composition; balance; protein balance
evaporative
water
losses; energy
STUDIES OF THE EFFECTS of altered nutrient intake on growth and development of the rat frequently use the model described by Widdowson and McCance (29), in which the number of pups suckled by a dam is altered; increasing litter size diminished the pups’ growth rate, whereas acceleration of growth was achieved by decreasing litter size. These studies implicitly assumed that alterations in growth rate are entirely attributable to the amount of milk available to each pup. The variations in growth rate induced by this technique, however, may not depend solely on the volume of milk consumed by the pups; alterations in milk composition may also modulate the differences in volume intake, and differences may occur in the efficiency of nutrient utilization (2). Evidence exists to indicate that maternal and pup behaviors are influenced by alterations in litter size, which in turn could affect the efficiency of nutrient use. Dams nursing small litters spend more time in the nest and grooming their pups (12), which may in itself stimulate growth independently of nutrient intake (28). The reverse may be true when litters are abnormally large
and dams spend less time in the nest. The dam also assists in the regulation of pup body temperature. Departures of the dam from the nest lead to an increase in thermoregulatory energy expenditure by the pups and hence less energy available for growth. Because of developmental changes in pup body composition and the maturation of their thermoregulatory mechanisms, this consequence of maternal behavior is likely to diminish during the postnatal 3rd wk. Energy expenditure may also be influenced by differences in the activity levels of pups of varied nutritional states. Lighter undernourished young rats are more active than heavier well-nourished animals (15). Activityinduced differences in energy expenditure would be measured as higher maintenance energy needs and in young rat pups would become evident toward the end of the 2nd wk of life as locomotor skills mature (1). We undertook the following study to determine the extent to which differences in nutrient intake, as opposed to differences in the efficiency of nutrient use, are responsible for differences in the weight gain of pups suckled in small, average, and large litters. The study concentrated on the first 16 days of life, during which milk is the only source of nutrients for rat pups. To accomplish these objectives, we estimated the volume and composition of milk consumed by the pups at 4-6, 8-10, and 14-16 days of age and their energy and protein balances during the study period. MATERIALS
AND
METHODS
Experimental design. We assigned pups to litters of 4 [small (S)], 10 [medium (M)], or 16 [large (L)]. Milk intake was estimated from the rate of dilution of a dose of 3Hz0 administered to the pups at 4, 8, and 14 days of age. Three litters of each size were studied at 4-6, B-10, or 14-16 days; each litter was studied only once. At the end of each study pups were killed, stomachs were emptied, and their total water, fat, and contents were determined. One litter of each size was also killed and analyzed at 4 days of age. At the end of a study, the dams were milked, and the milk was analyzed for water, fat, total and nonprotein N, and lactose. Dams whose pups were killed at 4 days of age were also milked. Animal care and experimentation were in accord with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” (Institute of Laboratory Animal Resources, National Research Council, DHHS Publ. 85-23)
R1104
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INTAKE
AND
USE
OF
MILK
and the protocol was approved by the Baylor College of Medicine Animal Protocol Review Committee. Animals. Timed-pregnant (2nd pregnancy) SpragueDawley (CRL:SD/VRL) dams (Charles River Laboratories, Wilmington, MA) were received at 15 days of gestation and housed individually in polycarbonate cages containing wood-chip bedding. Dams were fed a commercial rodent diet (MRH 22/5 Rodent Blox, Wayne Research Animal Diets, Chicago, IL) and water ad libiturn. A 12:12 h light-dark cycle was used in a room maintained at 22-24°C. Within 24 h of birth, pups were pooled and litters were reformed with 4, 10, or 16 pups/ litter, with it ensured that each litter contained equal numbers of males and females and that average pup weight was similar. Three days postpartum, and every 3rd day thereafter, pups were weighed. In addition, each pup was tattooed on its paws to ensure correct identification. To determine whether handling of the pups altered their growth, we compared the body weights of pups in this experiment with weights of pups from 26 litters (S, n = 10; M, n = 8; L, n = 8) of identical origin and treatment but on which only weight measurements were made. Estimation of milk consumption by tritiated water dilution measurements. The procedure used to estimate milk consumption was similar to that described by Coward et al. (7). On the 1st day of study (4,8, or 14 days of age), litters were removed from their dams, induced to urinate, and weighed. Four M, six L, and two S pups were injected intraperitoneally with 50 ~1 of 154 mM NaCl containing 2 &i 3H20 (Du Pont-New England Nuclear, Boston, MA). Two M, four L, and two S pups per litter were used as shams to determine recycling of label between pups and dams (3); they were injected with 50 ~1 of 154 mM NaCl. All pups were returned to their home cage. Approximately 5,24, and 48 h after injection, pups were induced to urinate, and a lo- to loo-p1 aliquot of urine was collected from injected and sham pups and expelled into vials containing scintillation fluid (3a70, Research Products International, Mt. Prospect, IL). Elapsed time from the point of injection was recorded. Specific gravity was measured by refractometry on a second aliquot of urine (Clinical Refractometer 57112021, Schuko, Japan). All pups were weighed before they were returned to their cages. Urine water content was estimated from its specific gravity. Together with the radioactivity content of the corresponding urine sample, urine water specific radioactivity (U,,) was calculated. To correct for recyling of label from dam to pups, U,, from sham pups within a litter was averaged at each time point and subtracted from the corresponding value for the labeled pups as described by Baverstock and Green (3). The degree of recycling, indicated by the U,, of sham relative to injected pups 48 h after injection, was age dependent (P < 0.001). It increased from 0.025 t 0.005 and 0.027 t 0.005% at 6 and 10 days of age, respectively, to 0.035 t 0.005% at 16 days of age. There was no effect of litter size on this value. Daily water intake (Win) of each pup was estimated from the slope (kd) of the line of a logarithmic plot of U,, against time and the total body water of that pup at the
NUTRIENTS
BY
RAT
RI105
PUPS
time of injection (V,) and at the end of the measurement period (V,) according to the equation W;,(g/day)
=
[(vb
-
Ve)/ln(Vb/Ve)l
x
kd
Milk intake is usually estimated from Win after subtraction of water input that arises from metabolic sources and correction for th .e water content of rat milk. Because of the pups’ rapid growth rate, however, we did not assume that metabolic water was derived from the complete oxidation of all milk solids. Rather, the amount of protein and fat gained by each pup during the measurement period was estimated from body composition measurements and used to adjust metabolic water input. We assumed that body protein and fat were derived from milk protein and fat. The minimum volume of milk required to provide the amounts of protein and fat that were deposited was determined (M,). The water yield (W,) of M,, i.e., its water content plusthe water produced by the complete oxidation of its carbohydrate content (0.55 g water/g carbohydrate ), was derived and subtracted from Win. The difference (Win - W,) was assumed to arise from the consumption of a quantity of milk (M,,) that had a total W, that was the sum of its water content (Wm) and the water derived from the complete oxidation of its fat, protein, and carbohydrate components producing 1.07, 0.41, and 0.55 g water/g substrate, respectively. Thus W;,was the sum of W,, W,, and water arising from the oxidation of M,,, and Mi, was (M, + M,,). The individual milk composition of a litter’s dam was used to estimate 8- to lo- and 14- to 16-day intakes; for the 4- to 6-day measurements, we assumed that the concentration of milk components changed linearly and thus used the means of the 4- and 6-day compositions. Body composition analyses. The empty bodies were lyophilized for 24 h, followed by desiccation to constant weight (3-4 days) at 60°C under vacuum (10 mTorr). Total body water was calculated from the difference between wet and dry body weights. The dry bodies were pulverized and redried to constant weight at 60°C under vacuum. Aliquots (1.5-3 g) of the powdered bodies were weighed into cellulose thimbles and placed in a Soxhlet apparatus, and fat was extracted by percolating with diethyl ether (~200 ml) for 24 h. The thimbles then were dried at 97°C for 24 h and reweighed. The extraction procedure was repeated until the absence of weight change indicated that all fat had been extracted. The fat content of the dried samples was calculated from the change in weight before and after extraction; from this value and the total body dry weight, total body fat was calculated. The N content of duplicate 25-mg fat-free dried samples was measured by the Kjeldahl technique (Kjeltec Auto Analyzer 1030, Tecator, Hoganas, Sweden); total body protein was assumed to equal total body N x 6.25. Fat, protein, and water accretion rates over each intake measurement period were calculated on an individual pup basis as follows. Regressions were performed between body weight and total body fat, protein, or water of individual pups. Three separate regressions were performed for each litter size, grouping together pups that were 4 and 6 (S, n = 10; M, n = 18; L, n = 27), 6 and 10
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Rl106
INTAKE
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OF
MILK
@ 4 = 12; M, n = 24; L, n = 36), or 10 and 16 days of we (S, n = 12; M, n = 24; L, n = 36). The slope of the regression line indicated the amount of fat, protein, or water gained per gram of weight gained between 4 and 6, between 6 and 10, and 10 and 16 days of age according to litter size. These values, multiplied by the weight gained by individual pups between 4 and 6, 8, and 10, and 14 and 16 days of age, were used to calculate both the composition of the weight gained over the 2day study periods and the composition of each pup at the beginning of the measurement period. Milk collection and analysis. Milk was collected -2 h after the pups had been rem .oved from the dams. Each dam was lightly anesthetized with ether, after which 0.5 IU oxytocin (Sigma Chemical, St. Louis, MO) was injected intraperitoneally and 0.5 IU intravenously. Two minutes later, milk collection was initiated by applying intermittent mild suction to each nipple through a polyethylene tube as each gland was stripped. Milk was drawn directly into a 5-ml Vacutainer tube. All glands were milked as completely as possible; a minimum of 4 ml of milk were collected from each dam over 15-20 min. Complete evacuation of the mammary glands was verified by the absence of any further milk letdown after a second intravenous dose of 1 IU oxytocin. Milk water content was determined from the weight lost on lyophilization of duplicate 0.25-ml milk samples; values for duplicates were on average within t0.5% of the mean value. Total lipid content was measured on duplicate 0.5ml samples by a modified Folch method (5); duplicate values were within t1.3% of the mean value. Total N was measured by Kjeldahl analysis on duplicate 0.25-ml samples of whole milk, and protein N was determined by Kjeldahl analysis of a trichloroacetic acid precipitate of duplicate 0.5-ml samples of whole milk; protein was assumed to be protein N x 6.38. Duplicate values were within t0.5% of the mean values. Lactose was measured in duplicate on a O&ml sample of skimmed milk sample by use of an automated enzymatic technique (Yellow Springs Instruments, Yellow Springs, OH); duplicate values were within t0.6% of the mean value. Interassay variability and accuracy for all analyses were determined by use of a control pooled human milk sample processed in duplicate with each assay. Assays were repeated if the values for the control varied from the mean value by >l SD (determined from a minimum of 25 replications). The mean interassay variability for fat, total and protein N, and lactose was t3 (n = II), 1 (n = lo), 1 (n = 14), and 1% (n = 5), respectively. Evaporative water Losses. In a separate experiment, estimates of net evaporative water losses (EWL) were determined in 11 additional litters (S, n = 4; M, n = 3; L, n = 4) by measuring the rate of weight loss of the individual litters over 3 h. Measurements were made at 6 (all litters), 10 (S, n = 1; M, n = 1; L, n = 2), and 16 days of age (S, n = 2; M, n = 1; L, n = 2). Each litter was studied twice. Pups were removed from their home cage, stimulated to urinate, and weighed at time 0. The pups were returned to a separate cage and placed in a huddle in a deep nest made of bedding material; an inverted cage filter cover of porous spun polyester was placed on the cage -3 cm above the huddle. After 1 h,
NUTRIENTS
BY
RAT
PUPS
each litter was removed from the cage, weighed, and returned to the nest; the pups were handled carefully to ensure that urination or defecation did not occur. The procedure was repeated after 2 and 3 h. Pups were observed over the 3 h, and pups that strayed from the nest were immediately returned to the huddle. CaZculations. Daily milk output was defined as the product of the average daily intake for a single pup and the total number of pups in the litter. Daily energy intake was estimated from the milk volume consumed and the dam’s milk composition. For the 4- to 6-day intake calculations, the mean of the 4- and 6-day milk compositions was used. The energy values used for fat and lactose are those for their heats of combustion, i.e., 9.25 and 3.95 kcal/g, respectively; 5.65 kcal/g was used for protein that was subsequently deposited (the heat of combustion of milk protein) by the pup and 4.4 kcal/g for protein that was oxidized. The term “corrected gross energy intake” (CGE) is used to define this estimate of energy intake. In pups that lost body fat during the m.easurement period, a value of 9.5 kcal/g of body fat lost was added to the milk-derived energy intake. Protein intake was estimated from the milk volume consumed by each pup and the protein concentration of the milk from that pup’s dam. The amount of energy gained as tissue by the pups during the study periods was estimated from assumed energy equivalents of 9.50 kcal/g fat and 5.65 kcal/g protein deposited. Energy loss was calculated as the differen .ce between energy intake and the energy gained; it therefore represents energy excreted in the feces and urine and energy lost as heat. When values for pup nutrient intake, gain, or loss are expressed per unit body size, the weight or composition of the pup at the midpoint of the intake measurements, i.e., 5, 9, or 15 days, was used. The rate of weight loss was assumed to be due to net EWL and was determined from the slope of a regression line fitted to the litter weights at 0, 1, 2, and 3 h. The formula of Diack (8) was used to calculate individual pup surface area. Statistical analyses. Because individual animals within a litter cannot be regarded as statistically independent, data from pups within a litter were averaged. The litter mean is used in the analyses with n equal to the number of litters. Effects of age and litter size (treatment) were tested by two-way analysis of variance (ANOVA). When the interaction between treatment and age was not significant, the main effects were tested directly from the twoway ANOVA. When the interaction was significant, a one-way ANOVA was performed on age at each treatment level or on treatment at each age level. If the F test was significant in either case, differences between any two means were determined by a t test with a Bonferroni correction for multiple comparisons. Because of the multistep statistical procedure, the relevant statistics have been omitted from Tables 1 and 2 and Figs. l-5 and are reported in the text. The effects of treatment and age on the relationships between protein intake and protein gain or between energy intake and energy gain were determined by analy-
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INTAKE
AND
USE
sis of covariance. Unless noted otherwise, applies to differences specified as significantly values are presented as means t SD.
OF
MILK
NUTRIENTS
BY
P c 0.05 different;
m,
A 85 --I
*-
'75-cn 65 --
250 i
i ~
i
2500--
200-1 \
\ CJ 2000--
..
Is,
150--
.2 1500--
lOO--
501
lOOO! 0
3
6
Days
12
15
18
Postpartum
(A), protein
0
I 3
: 6
Days
(B),
I 9
: 12
I 15
I 18
Postpartum
fat (C), and energy (D) concentrations in milk of rat dams at 4, 6, 10, and 16 days postpartum suckling small (4 pups), medium (10 pups), or large (16 pups) litters. Values are means & SD of 3 dams at each time point. SDS smaller than symbols are not shown. FIG.
1. Lactose
9
4 pups/litter
I
100 2 -0
2MiLh composition. Figure 1 shows milk composition at 4, 6, 10, and 16 days postpartum; on day 4 milk compositions were similar irrespective of litter size, and an average value was derived for each milk component. Twoway ANOVA using days 6, 10, and 16 identified no interaction between litter size and days postpartum for any of the measured milk components. Lactose concentration decreased between 4 and 6 days in all dams and then increased until 16 days (P < O.OOl), with no notable effect of litter size. Protein concentration also increased for all litters as lactation progressed (P < 0.001); after 4 days postpartum, milk from S dams always had a lower protein concentration than that from M or L dams (P < 0.04). At 4 days of age protein N constituted -70% of total N. Thereafter the proportion of total N that was protein N (data not shown) remained constant and was similar for all litters (90 t 3%). Milk fat concentration decreased markedly in all dams between 4 and 6 days. From 6 days postpartum, there was a significant effect of litter size on fat concentration (P < 0.001) but no additional effect of age. We observed, however, that milk fat concentration was negatively correlated with total daily milk volume (r = -0.79; P < 0.001). This relationship accounted for the changes observed between 4 and 6 days and the subsequent apparent litter size differences; i.e., litter size and days postpartum influenced milk fat concentration only insofar as these parameters affected milk volume. The differences in milk composition over time and between litters resulted in parallel
Rl107
PUPS
A
120
\
RESULTS
RAT
8o 60
"E
40
2
175
-0
150
\
125
0
100
;
75 50 25 0
4-6
8-1014-16 Days
Postpartum
FIG. 2. Daily milk volume (A), energy (B), and protein (C) yield of rat dams suckling small (4 pups), medium (10 pups), or large (16 pups) litters at 4-6, 8-10, and 14-16 days postpartum. Values are means & SD of 3 dams at each time point.
changes in the milk protein-to-energy ratio. The ratio increased among all litters over time from 0.18 t 0.01 at 6 days of age to 0.28 t 0.04 at 16 days. After 4 days postpartum, the milk protein-to-energy ratio of L dams was significantly higher than that of S dams, and that of M dams was intermediate. Milk volume output. The average daily milk volume, energy, and protein yield of dams suckling S, M, or L litters are shown in Fig. 2. Two-way ANOVA indicated a significant interaction between treatment and age for all three parameters. Daily milk volume increased over the first 16 days of lactation, and at any given time S dams produced significantly less milk than M dams (56 t 2%); there was no change in the proportional difference over time. L dams produced more milk than M dams over 4-6 and 8-10 days, but by 14-16 days of lactation, the difference was not statistically significant. In L dams, the significant increase in volume over 46 and 8-10 days was not associated with as marked an increase in energy, because milk energy density decreased concurrently (1,980 t 63 and 1,421 t 112 kcal/l at 5 and 10 days, respectively). Moreover, although the daily volume was higher for L dams than for M dams at 8-10 days (P < 0.05), their total energy output was not significant different. Due to the litter size effect on milk protein concentration and total volume, total milk protein output was significantly higher in L dams at all time points. It should be noted that the average of days 4 and 6 milk compositions was used in computing total milk energy and protein outputs for the 4- to 6-d period. Growth. The weight gain of pups in this study was not
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R1108
INTAKE
AND
USE
OF
MILK
significantly different from that of the reference litters. At 16 days of age the mean body weights of pups from the two populations were 43.7 t 3.0 and 41.9 t 2.6, 34.5 t 2 and 32.9 t 3.2, and 25.1 t 1.8 and 26.2 t 2.1 g for S, M, and L, respectively. Figure 3 shows the body weights and the daily weight gain calculated from the weight gain over the &day milk intake measurement period; data are shown at the midpoint of the &day interval. Litter size and age significantly influenced both body weight and weight gain, although a significant interaction (P < 0.001) indicated that the effect of litter size depended on the age of the pups. Although pup daily weight gain was highly correlated with the daily volume of milk consumed (all litters considered together), a significantly greater proportion of the variance in weight gain (85 vs. 70%) was accounted for when weight gain was correlated with energy intake. When the three groups were examined individually, no correlation between daily energy intake and weight gain could be identified for L pups. Body composition. Table 1 shows the mean concentration of fat and protein in pups at 5, 9, and 15 days postpartum and daily rates of fat and protein accretion over the 2-day intake measurements. All parameters were influenced by litter size and age, although a significant interaction (P < 0.001) indicated that the effect of litter size depended on the age of the pups and vice versa. The rate of fat accretion (g fat gained/day) was highly correlated with the rate of weight gain (g weight gained/ day) among all litters (r = 0.93; P < 0.001) and was highest in S pups throughout the study. Significant fat accretion occurred only in one litter of L pups between 8 and 10 days and in all L litters between 14 and 16 days. Indeed, no increase in body fat concentration of L pups was observed until 15 days of age. Because of the continued growth of lean body components and loss of fat by some pups between 4 and 6 and between 8 and 10 days, L pups were leaner at 9 days of age than at 5 or 15 days (P < 0.06). In S and M litters, protein constituted an increasingly greater proportion of the weight gained with age and reflected an absolute increase in the rate of protein gained per day. Protein accretion in L pups was the same as that of M pups at 5 days of age but did not increase substantially thereafter. Thus, between 8 and 10 days, protein accretion in L pups was significantly lower than O- -0,4 O-O,10
pups/litter pups/litter A---- A, 16 pups/lilter,‘1
A
G
4.0 l3.5
T /A+---Y
\ 3.0 0) -2.5 $ 2.0 0, 1.5 2 .o> 1.0 g 0
3
6 Age
9
(days)
12
0.5
0.0 15
18
t 1
0
.
I I.
3
.
1 I
6 Age
. .,
, :
9
:
12
:
:
15
:
18
(days)
FIG. 3. Body weights at 5, 9, and 15 days of age and weight gain at 4-6, 8-10, and 14-16 days of age of pups suckled in small (4 pups), medium (10 pups) and large (16 pups) litters. Values are means t SD of 3 litters at each time point. SDS smaller than symbols are not shown.
NUTRIENTS
BY
RAT
PUPS
1. Body concentration and accretion rates of fat and protein for pups raised in small, medium, or large litters TABLE
Litter
S 5 days (4-6
Size M
L
days)*
Fat g/kg
body
wt
g/day
Protein g/kg
body
wt
g/day
8Ok4 0.49t0.4
0.22rto.01
5223 0.02*0.01
115t2 0.24kO.01
116k2 0.20t0.01
114t0.4 0.20t0.01
9 days (B-10
65t7
days)*
Fat g/kg
body
wt
g/day
Protein g/kg
body
wt
g/day
91s
148&l 0.67t0.04
0.26t0.02
421k2 0.02t0.05
12353 0.46t0.02
132rt2 0.37kO.03
0.22t0.07
15 days (14-16
13325
days)*
Fat g/kg
body
wt
g/day
Protein g/kg g/day
body
wt
161tll
102tlO
0.63kO.05
0.35t0.02
61t8 0.14t0.01
135t6 0.57t0.05
145t2 0.40t0.02
153-+1 0.27kO.02
Values are means t SD; n = 3 litters per age per litter size. S, small (4 pups); M, medium (10 pups); L, large (16 pups). * Interval over which accretion rates (g/day) were estimated.
that for M pups. Energy and protein balances. To determine whether litter size influenced the efficiency with which the pups used dietary energy and protein for growth, daily energy and protein balances were computed; the data are summarized in Table 2. To facilitate interpretation of data among pups of varying size and body composition, data are expressed per gram body protein. Because of the differences in milk composition, only 87% of the variability in energy intake could be ascribed to volume intake. Energy intake per gram of body protein decreased significantly over time among all litters (P < 0.001); the largest decrease occurred between 4-6 and B10 days of age. Comparison among litter sizes indicated that the energy intake (per g body protein) of S pups was consistently higher than that for M and L pups, whereas there was only a trend for M pups to have a higher intake than L pups (P < 0.08). Energy gain per unit body protein decreased over time for S and M litters and up to 10 days of age in L litters. S pups deposited more energy than M pups at all time points. Despite the relatively small differences in energy intake, when scaled to “metabolic mass,” (i.e., body protein), L pups gained proportionally less energy than M pups at all time points. Hence the gross efficiency of energy gain (energy gain/energy intake) was significantly lower for L than for M pups at all time points (P < 0.05): 18 t 2 vs. 35 t 1, 19 t 13 vs. 40 t 3, and 26 t 2 vs. 32 -+ 3% at 4-6, B-10, and 14-16 days, respectively. The variation among L pups at B-10 days was relatively high (coefficient of variation 62%), because the efficiency of energy gain in two of the three litters was similar to that
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INTAKE
AND
USE
OF
MILK
2. Energy and protein balances for pups raised in small, medium, or large litters TABLE
Litter
S
M
5 days (4-6 Energy, kcal . g body protein-’ Intake 8.56t0.28 Gain 4.OlkO.17 Loss 4.54t0.23 Protein, Intake Gain Loss
go g body
Size
l
L
days)”
day-l
protein-l. 027+00?-' . -. 0.16-c-0.01 0.10t0.004
6.81t0.21 2.41t0.01 4.40t0.21
6.2320.25 1.12kO.06 5.08rtO.30
0.22kO.02 0.15t0.004 0.07t0.02
0.21t0.001 0.17kO.02 0.04t0.02
9 days (B-10 Energy, kcal . g body protein-’ day-’ Intake 5.89t0.55 Gain 2.67kO.30 Loss 3.22t0.43 Protein, go g body protein-’ 023+OOd3ay-l . -. Intake Gain 0.14-+0.02 0.09~0.01 Loss
days)*
l
3.91-to.33 1.57kO.04 2.35t0.30
3.89t0.35 0.70t0.40 3.19t0.70
0.22*0.002 0.13t0.003 0.09*0.001
0.24-t-0.01 0.11t0.03 0.13t0.03
l
15 days (14-16 Energy, kcal . g body protein-’ Intake 4.45kO.67 Gain 1.57t0.05 Loss 2.83t0.63 Protein, go g body protein-’ Intake 020+ooY1 . -. Gain 0.10t0.003 Loss 0.04~0.01 Values are means over which balances tions.
l
days)*
day-’ 3.51k0.36 1.12sto.12 2.38t0.30
2.91t0.31 0.76t0.03 2.15t0.28
0.18t0.003 0.08t0.01 0.10~0.01
0.17t0.01 0.07t0.003 0.10-1-0.01
l
& SD; n = 3 litters per age per litter size. * Interval were estimated. See Table 1 legend for abbrevia-
8 Xint = 2.36
;slope
= 0.30
3 ‘int
; slope
= 0.52
= 1.28
4.0
6.0
Energy (kcal
8.0
10.0
12.0
NUTRIENTS
RAT
R1109
PUPS
maintenance (zero gain) in these pups, was 1.28 kcal. g body protein-‘. day-l. The slope of the regression line, which represents the efficiency with which energy consumed in excess of maintenance was gained, was 0.52 t 0.03 kcal deposited/kcal CGE ingested. The regression line (r = 0.93) for the second population was significantly different (P < 0.001). This group comprised all L litters at 4-6 days of age and two of the three L litters at 8-10 days of age. The CGE needed for maintenance of these pups (2.36 kcal . g body protein-‘. day-‘) was significantly higher than for the other litters, and their efficiency of energy gain (0.30 t 0.07 kcal deposited/kcal consumed) was significantly lower. To facilitate comparison with other published data, the values for maintenance energy needs were also derived after standardization of energy intake and gain per body weight0.75; the corresponding values for the first and second populations of rats were 86 and 135 kcal/kg”*75, respectively. Presumably because of the marked differences in body composition, the relationship between intake and gain expressed per body weight0*75was not as close as when data were standardized per unit body protein (r = 0.78 and 0.92, respectively). As for energy gain, the gross efficiency of protein gain was similar for S and M pups between 4 and 6 and between 8 and 10 days of age (62 t 5%) and then decreased in both groups to 47 t 3% between 14 and 16 days of age. At 4-6 days of age, however, the gross efficiency with which L pups gained protein (83 t 8%) was significantly higher than that for S and M litters (P < 0.01). At 8-10 days and thereafter, efficiency among L pups had decreased and was not different from values for S and M pups. To determine whether these litter and age effects were attributable to changes in the protein needed for maintenance or in the efficiency with which the milk protein was used for growth, we examined the relationship between protein intake and protein retained in individual litters (Fig. 5). The three L litters at 4-6 days of age again formed a statistically different group. All remaining litters showed a continuous positive linear relationship between protein intake and gain (r = 0.91; P < 0.001). The slope of the line indicated that the efficiency of milk protein use was 92 t 9%. The protein required for maintenance (zero gain) was 0.085 gog body pro-
Intake
/ g protein
BY
0.30 /
/ d)
FIG. 4. Relationship between daily energy intake and energy gain at 4-6, 8-10, and 14-16 days of age. A, Large litters at 4-6 (n = 3) and 8-10 (n = 2) days; 0, all other litters. Symbols represent values for individual litters.
of litters at 4-6 days (efficiencies of 9 and 16%, respectively), whereas one litter showed an efficiency of 32%, similar to values at 14-16 days of age. These data are further evaluated in Fig. 4, where the relationship between energy intake and energy gain is shown. Two populations of pups can be discerned; a continuous relationship between energy intake and gain can be observed for all S and M litters and for four of nine L litters (r = 0.96; P < 0.001). The x-intercept of the regression line, which represents the CGE needed for
G .r \
0
0.25
,Xint=
0.085
/
; SlOpe=O.g3
t
g lg 0.20+ s% ‘a, ki 0.15 zm a,L ; 0.10 -
i
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
Protein Intake (g / 9 protein / d) FIG. 5. Relationship between daily protein intake and protein gain at 4-6, 8-10, and 14-16 days of age. A, Large litters measured at 4-6 days: 0, all other litters. Svmbols represent values for individual litters.
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RlllO
INTAKE
AND
USE
OF
MILK
tein-’ day-‘. With data for L litters at 4-6 days of age excluded, there was no effect of age or litter size on the slope of the regression line, but both factors influenced the line intercept to a small extent (
evaporative
water
losses; energy
STUDIES OF THE EFFECTS of altered nutrient intake on growth and development of the rat frequently use the model described by Widdowson and McCance (29), in which the number of pups suckled by a dam is altered; increasing litter size diminished the pups’ growth rate, whereas acceleration of growth was achieved by decreasing litter size. These studies implicitly assumed that alterations in growth rate are entirely attributable to the amount of milk available to each pup. The variations in growth rate induced by this technique, however, may not depend solely on the volume of milk consumed by the pups; alterations in milk composition may also modulate the differences in volume intake, and differences may occur in the efficiency of nutrient utilization (2). Evidence exists to indicate that maternal and pup behaviors are influenced by alterations in litter size, which in turn could affect the efficiency of nutrient use. Dams nursing small litters spend more time in the nest and grooming their pups (12), which may in itself stimulate growth independently of nutrient intake (28). The reverse may be true when litters are abnormally large
and dams spend less time in the nest. The dam also assists in the regulation of pup body temperature. Departures of the dam from the nest lead to an increase in thermoregulatory energy expenditure by the pups and hence less energy available for growth. Because of developmental changes in pup body composition and the maturation of their thermoregulatory mechanisms, this consequence of maternal behavior is likely to diminish during the postnatal 3rd wk. Energy expenditure may also be influenced by differences in the activity levels of pups of varied nutritional states. Lighter undernourished young rats are more active than heavier well-nourished animals (15). Activityinduced differences in energy expenditure would be measured as higher maintenance energy needs and in young rat pups would become evident toward the end of the 2nd wk of life as locomotor skills mature (1). We undertook the following study to determine the extent to which differences in nutrient intake, as opposed to differences in the efficiency of nutrient use, are responsible for differences in the weight gain of pups suckled in small, average, and large litters. The study concentrated on the first 16 days of life, during which milk is the only source of nutrients for rat pups. To accomplish these objectives, we estimated the volume and composition of milk consumed by the pups at 4-6, 8-10, and 14-16 days of age and their energy and protein balances during the study period. MATERIALS
AND
METHODS
Experimental design. We assigned pups to litters of 4 [small (S)], 10 [medium (M)], or 16 [large (L)]. Milk intake was estimated from the rate of dilution of a dose of 3Hz0 administered to the pups at 4, 8, and 14 days of age. Three litters of each size were studied at 4-6, B-10, or 14-16 days; each litter was studied only once. At the end of each study pups were killed, stomachs were emptied, and their total water, fat, and contents were determined. One litter of each size was also killed and analyzed at 4 days of age. At the end of a study, the dams were milked, and the milk was analyzed for water, fat, total and nonprotein N, and lactose. Dams whose pups were killed at 4 days of age were also milked. Animal care and experimentation were in accord with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” (Institute of Laboratory Animal Resources, National Research Council, DHHS Publ. 85-23)
R1104
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (133.006.082.173) on July 29, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
INTAKE
AND
USE
OF
MILK
and the protocol was approved by the Baylor College of Medicine Animal Protocol Review Committee. Animals. Timed-pregnant (2nd pregnancy) SpragueDawley (CRL:SD/VRL) dams (Charles River Laboratories, Wilmington, MA) were received at 15 days of gestation and housed individually in polycarbonate cages containing wood-chip bedding. Dams were fed a commercial rodent diet (MRH 22/5 Rodent Blox, Wayne Research Animal Diets, Chicago, IL) and water ad libiturn. A 12:12 h light-dark cycle was used in a room maintained at 22-24°C. Within 24 h of birth, pups were pooled and litters were reformed with 4, 10, or 16 pups/ litter, with it ensured that each litter contained equal numbers of males and females and that average pup weight was similar. Three days postpartum, and every 3rd day thereafter, pups were weighed. In addition, each pup was tattooed on its paws to ensure correct identification. To determine whether handling of the pups altered their growth, we compared the body weights of pups in this experiment with weights of pups from 26 litters (S, n = 10; M, n = 8; L, n = 8) of identical origin and treatment but on which only weight measurements were made. Estimation of milk consumption by tritiated water dilution measurements. The procedure used to estimate milk consumption was similar to that described by Coward et al. (7). On the 1st day of study (4,8, or 14 days of age), litters were removed from their dams, induced to urinate, and weighed. Four M, six L, and two S pups were injected intraperitoneally with 50 ~1 of 154 mM NaCl containing 2 &i 3H20 (Du Pont-New England Nuclear, Boston, MA). Two M, four L, and two S pups per litter were used as shams to determine recycling of label between pups and dams (3); they were injected with 50 ~1 of 154 mM NaCl. All pups were returned to their home cage. Approximately 5,24, and 48 h after injection, pups were induced to urinate, and a lo- to loo-p1 aliquot of urine was collected from injected and sham pups and expelled into vials containing scintillation fluid (3a70, Research Products International, Mt. Prospect, IL). Elapsed time from the point of injection was recorded. Specific gravity was measured by refractometry on a second aliquot of urine (Clinical Refractometer 57112021, Schuko, Japan). All pups were weighed before they were returned to their cages. Urine water content was estimated from its specific gravity. Together with the radioactivity content of the corresponding urine sample, urine water specific radioactivity (U,,) was calculated. To correct for recyling of label from dam to pups, U,, from sham pups within a litter was averaged at each time point and subtracted from the corresponding value for the labeled pups as described by Baverstock and Green (3). The degree of recycling, indicated by the U,, of sham relative to injected pups 48 h after injection, was age dependent (P < 0.001). It increased from 0.025 t 0.005 and 0.027 t 0.005% at 6 and 10 days of age, respectively, to 0.035 t 0.005% at 16 days of age. There was no effect of litter size on this value. Daily water intake (Win) of each pup was estimated from the slope (kd) of the line of a logarithmic plot of U,, against time and the total body water of that pup at the
NUTRIENTS
BY
RAT
RI105
PUPS
time of injection (V,) and at the end of the measurement period (V,) according to the equation W;,(g/day)
=
[(vb
-
Ve)/ln(Vb/Ve)l
x
kd
Milk intake is usually estimated from Win after subtraction of water input that arises from metabolic sources and correction for th .e water content of rat milk. Because of the pups’ rapid growth rate, however, we did not assume that metabolic water was derived from the complete oxidation of all milk solids. Rather, the amount of protein and fat gained by each pup during the measurement period was estimated from body composition measurements and used to adjust metabolic water input. We assumed that body protein and fat were derived from milk protein and fat. The minimum volume of milk required to provide the amounts of protein and fat that were deposited was determined (M,). The water yield (W,) of M,, i.e., its water content plusthe water produced by the complete oxidation of its carbohydrate content (0.55 g water/g carbohydrate ), was derived and subtracted from Win. The difference (Win - W,) was assumed to arise from the consumption of a quantity of milk (M,,) that had a total W, that was the sum of its water content (Wm) and the water derived from the complete oxidation of its fat, protein, and carbohydrate components producing 1.07, 0.41, and 0.55 g water/g substrate, respectively. Thus W;,was the sum of W,, W,, and water arising from the oxidation of M,,, and Mi, was (M, + M,,). The individual milk composition of a litter’s dam was used to estimate 8- to lo- and 14- to 16-day intakes; for the 4- to 6-day measurements, we assumed that the concentration of milk components changed linearly and thus used the means of the 4- and 6-day compositions. Body composition analyses. The empty bodies were lyophilized for 24 h, followed by desiccation to constant weight (3-4 days) at 60°C under vacuum (10 mTorr). Total body water was calculated from the difference between wet and dry body weights. The dry bodies were pulverized and redried to constant weight at 60°C under vacuum. Aliquots (1.5-3 g) of the powdered bodies were weighed into cellulose thimbles and placed in a Soxhlet apparatus, and fat was extracted by percolating with diethyl ether (~200 ml) for 24 h. The thimbles then were dried at 97°C for 24 h and reweighed. The extraction procedure was repeated until the absence of weight change indicated that all fat had been extracted. The fat content of the dried samples was calculated from the change in weight before and after extraction; from this value and the total body dry weight, total body fat was calculated. The N content of duplicate 25-mg fat-free dried samples was measured by the Kjeldahl technique (Kjeltec Auto Analyzer 1030, Tecator, Hoganas, Sweden); total body protein was assumed to equal total body N x 6.25. Fat, protein, and water accretion rates over each intake measurement period were calculated on an individual pup basis as follows. Regressions were performed between body weight and total body fat, protein, or water of individual pups. Three separate regressions were performed for each litter size, grouping together pups that were 4 and 6 (S, n = 10; M, n = 18; L, n = 27), 6 and 10
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Rl106
INTAKE
AND
USE
OF
MILK
@ 4 = 12; M, n = 24; L, n = 36), or 10 and 16 days of we (S, n = 12; M, n = 24; L, n = 36). The slope of the regression line indicated the amount of fat, protein, or water gained per gram of weight gained between 4 and 6, between 6 and 10, and 10 and 16 days of age according to litter size. These values, multiplied by the weight gained by individual pups between 4 and 6, 8, and 10, and 14 and 16 days of age, were used to calculate both the composition of the weight gained over the 2day study periods and the composition of each pup at the beginning of the measurement period. Milk collection and analysis. Milk was collected -2 h after the pups had been rem .oved from the dams. Each dam was lightly anesthetized with ether, after which 0.5 IU oxytocin (Sigma Chemical, St. Louis, MO) was injected intraperitoneally and 0.5 IU intravenously. Two minutes later, milk collection was initiated by applying intermittent mild suction to each nipple through a polyethylene tube as each gland was stripped. Milk was drawn directly into a 5-ml Vacutainer tube. All glands were milked as completely as possible; a minimum of 4 ml of milk were collected from each dam over 15-20 min. Complete evacuation of the mammary glands was verified by the absence of any further milk letdown after a second intravenous dose of 1 IU oxytocin. Milk water content was determined from the weight lost on lyophilization of duplicate 0.25-ml milk samples; values for duplicates were on average within t0.5% of the mean value. Total lipid content was measured on duplicate 0.5ml samples by a modified Folch method (5); duplicate values were within t1.3% of the mean value. Total N was measured by Kjeldahl analysis on duplicate 0.25-ml samples of whole milk, and protein N was determined by Kjeldahl analysis of a trichloroacetic acid precipitate of duplicate 0.5-ml samples of whole milk; protein was assumed to be protein N x 6.38. Duplicate values were within t0.5% of the mean values. Lactose was measured in duplicate on a O&ml sample of skimmed milk sample by use of an automated enzymatic technique (Yellow Springs Instruments, Yellow Springs, OH); duplicate values were within t0.6% of the mean value. Interassay variability and accuracy for all analyses were determined by use of a control pooled human milk sample processed in duplicate with each assay. Assays were repeated if the values for the control varied from the mean value by >l SD (determined from a minimum of 25 replications). The mean interassay variability for fat, total and protein N, and lactose was t3 (n = II), 1 (n = lo), 1 (n = 14), and 1% (n = 5), respectively. Evaporative water Losses. In a separate experiment, estimates of net evaporative water losses (EWL) were determined in 11 additional litters (S, n = 4; M, n = 3; L, n = 4) by measuring the rate of weight loss of the individual litters over 3 h. Measurements were made at 6 (all litters), 10 (S, n = 1; M, n = 1; L, n = 2), and 16 days of age (S, n = 2; M, n = 1; L, n = 2). Each litter was studied twice. Pups were removed from their home cage, stimulated to urinate, and weighed at time 0. The pups were returned to a separate cage and placed in a huddle in a deep nest made of bedding material; an inverted cage filter cover of porous spun polyester was placed on the cage -3 cm above the huddle. After 1 h,
NUTRIENTS
BY
RAT
PUPS
each litter was removed from the cage, weighed, and returned to the nest; the pups were handled carefully to ensure that urination or defecation did not occur. The procedure was repeated after 2 and 3 h. Pups were observed over the 3 h, and pups that strayed from the nest were immediately returned to the huddle. CaZculations. Daily milk output was defined as the product of the average daily intake for a single pup and the total number of pups in the litter. Daily energy intake was estimated from the milk volume consumed and the dam’s milk composition. For the 4- to 6-day intake calculations, the mean of the 4- and 6-day milk compositions was used. The energy values used for fat and lactose are those for their heats of combustion, i.e., 9.25 and 3.95 kcal/g, respectively; 5.65 kcal/g was used for protein that was subsequently deposited (the heat of combustion of milk protein) by the pup and 4.4 kcal/g for protein that was oxidized. The term “corrected gross energy intake” (CGE) is used to define this estimate of energy intake. In pups that lost body fat during the m.easurement period, a value of 9.5 kcal/g of body fat lost was added to the milk-derived energy intake. Protein intake was estimated from the milk volume consumed by each pup and the protein concentration of the milk from that pup’s dam. The amount of energy gained as tissue by the pups during the study periods was estimated from assumed energy equivalents of 9.50 kcal/g fat and 5.65 kcal/g protein deposited. Energy loss was calculated as the differen .ce between energy intake and the energy gained; it therefore represents energy excreted in the feces and urine and energy lost as heat. When values for pup nutrient intake, gain, or loss are expressed per unit body size, the weight or composition of the pup at the midpoint of the intake measurements, i.e., 5, 9, or 15 days, was used. The rate of weight loss was assumed to be due to net EWL and was determined from the slope of a regression line fitted to the litter weights at 0, 1, 2, and 3 h. The formula of Diack (8) was used to calculate individual pup surface area. Statistical analyses. Because individual animals within a litter cannot be regarded as statistically independent, data from pups within a litter were averaged. The litter mean is used in the analyses with n equal to the number of litters. Effects of age and litter size (treatment) were tested by two-way analysis of variance (ANOVA). When the interaction between treatment and age was not significant, the main effects were tested directly from the twoway ANOVA. When the interaction was significant, a one-way ANOVA was performed on age at each treatment level or on treatment at each age level. If the F test was significant in either case, differences between any two means were determined by a t test with a Bonferroni correction for multiple comparisons. Because of the multistep statistical procedure, the relevant statistics have been omitted from Tables 1 and 2 and Figs. l-5 and are reported in the text. The effects of treatment and age on the relationships between protein intake and protein gain or between energy intake and energy gain were determined by analy-
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INTAKE
AND
USE
sis of covariance. Unless noted otherwise, applies to differences specified as significantly values are presented as means t SD.
OF
MILK
NUTRIENTS
BY
P c 0.05 different;
m,
A 85 --I
*-
'75-cn 65 --
250 i
i ~
i
2500--
200-1 \
\ CJ 2000--
..
Is,
150--
.2 1500--
lOO--
501
lOOO! 0
3
6
Days
12
15
18
Postpartum
(A), protein
0
I 3
: 6
Days
(B),
I 9
: 12
I 15
I 18
Postpartum
fat (C), and energy (D) concentrations in milk of rat dams at 4, 6, 10, and 16 days postpartum suckling small (4 pups), medium (10 pups), or large (16 pups) litters. Values are means & SD of 3 dams at each time point. SDS smaller than symbols are not shown. FIG.
1. Lactose
9
4 pups/litter
I
100 2 -0
2MiLh composition. Figure 1 shows milk composition at 4, 6, 10, and 16 days postpartum; on day 4 milk compositions were similar irrespective of litter size, and an average value was derived for each milk component. Twoway ANOVA using days 6, 10, and 16 identified no interaction between litter size and days postpartum for any of the measured milk components. Lactose concentration decreased between 4 and 6 days in all dams and then increased until 16 days (P < O.OOl), with no notable effect of litter size. Protein concentration also increased for all litters as lactation progressed (P < 0.001); after 4 days postpartum, milk from S dams always had a lower protein concentration than that from M or L dams (P < 0.04). At 4 days of age protein N constituted -70% of total N. Thereafter the proportion of total N that was protein N (data not shown) remained constant and was similar for all litters (90 t 3%). Milk fat concentration decreased markedly in all dams between 4 and 6 days. From 6 days postpartum, there was a significant effect of litter size on fat concentration (P < 0.001) but no additional effect of age. We observed, however, that milk fat concentration was negatively correlated with total daily milk volume (r = -0.79; P < 0.001). This relationship accounted for the changes observed between 4 and 6 days and the subsequent apparent litter size differences; i.e., litter size and days postpartum influenced milk fat concentration only insofar as these parameters affected milk volume. The differences in milk composition over time and between litters resulted in parallel
Rl107
PUPS
A
120
\
RESULTS
RAT
8o 60
"E
40
2
175
-0
150
\
125
0
100
;
75 50 25 0
4-6
8-1014-16 Days
Postpartum
FIG. 2. Daily milk volume (A), energy (B), and protein (C) yield of rat dams suckling small (4 pups), medium (10 pups), or large (16 pups) litters at 4-6, 8-10, and 14-16 days postpartum. Values are means & SD of 3 dams at each time point.
changes in the milk protein-to-energy ratio. The ratio increased among all litters over time from 0.18 t 0.01 at 6 days of age to 0.28 t 0.04 at 16 days. After 4 days postpartum, the milk protein-to-energy ratio of L dams was significantly higher than that of S dams, and that of M dams was intermediate. Milk volume output. The average daily milk volume, energy, and protein yield of dams suckling S, M, or L litters are shown in Fig. 2. Two-way ANOVA indicated a significant interaction between treatment and age for all three parameters. Daily milk volume increased over the first 16 days of lactation, and at any given time S dams produced significantly less milk than M dams (56 t 2%); there was no change in the proportional difference over time. L dams produced more milk than M dams over 4-6 and 8-10 days, but by 14-16 days of lactation, the difference was not statistically significant. In L dams, the significant increase in volume over 46 and 8-10 days was not associated with as marked an increase in energy, because milk energy density decreased concurrently (1,980 t 63 and 1,421 t 112 kcal/l at 5 and 10 days, respectively). Moreover, although the daily volume was higher for L dams than for M dams at 8-10 days (P < 0.05), their total energy output was not significant different. Due to the litter size effect on milk protein concentration and total volume, total milk protein output was significantly higher in L dams at all time points. It should be noted that the average of days 4 and 6 milk compositions was used in computing total milk energy and protein outputs for the 4- to 6-d period. Growth. The weight gain of pups in this study was not
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R1108
INTAKE
AND
USE
OF
MILK
significantly different from that of the reference litters. At 16 days of age the mean body weights of pups from the two populations were 43.7 t 3.0 and 41.9 t 2.6, 34.5 t 2 and 32.9 t 3.2, and 25.1 t 1.8 and 26.2 t 2.1 g for S, M, and L, respectively. Figure 3 shows the body weights and the daily weight gain calculated from the weight gain over the &day milk intake measurement period; data are shown at the midpoint of the &day interval. Litter size and age significantly influenced both body weight and weight gain, although a significant interaction (P < 0.001) indicated that the effect of litter size depended on the age of the pups. Although pup daily weight gain was highly correlated with the daily volume of milk consumed (all litters considered together), a significantly greater proportion of the variance in weight gain (85 vs. 70%) was accounted for when weight gain was correlated with energy intake. When the three groups were examined individually, no correlation between daily energy intake and weight gain could be identified for L pups. Body composition. Table 1 shows the mean concentration of fat and protein in pups at 5, 9, and 15 days postpartum and daily rates of fat and protein accretion over the 2-day intake measurements. All parameters were influenced by litter size and age, although a significant interaction (P < 0.001) indicated that the effect of litter size depended on the age of the pups and vice versa. The rate of fat accretion (g fat gained/day) was highly correlated with the rate of weight gain (g weight gained/ day) among all litters (r = 0.93; P < 0.001) and was highest in S pups throughout the study. Significant fat accretion occurred only in one litter of L pups between 8 and 10 days and in all L litters between 14 and 16 days. Indeed, no increase in body fat concentration of L pups was observed until 15 days of age. Because of the continued growth of lean body components and loss of fat by some pups between 4 and 6 and between 8 and 10 days, L pups were leaner at 9 days of age than at 5 or 15 days (P < 0.06). In S and M litters, protein constituted an increasingly greater proportion of the weight gained with age and reflected an absolute increase in the rate of protein gained per day. Protein accretion in L pups was the same as that of M pups at 5 days of age but did not increase substantially thereafter. Thus, between 8 and 10 days, protein accretion in L pups was significantly lower than O- -0,4 O-O,10
pups/litter pups/litter A---- A, 16 pups/lilter,‘1
A
G
4.0 l3.5
T /A+---Y
\ 3.0 0) -2.5 $ 2.0 0, 1.5 2 .o> 1.0 g 0
3
6 Age
9
(days)
12
0.5
0.0 15
18
t 1
0
.
I I.
3
.
1 I
6 Age
. .,
, :
9
:
12
:
:
15
:
18
(days)
FIG. 3. Body weights at 5, 9, and 15 days of age and weight gain at 4-6, 8-10, and 14-16 days of age of pups suckled in small (4 pups), medium (10 pups) and large (16 pups) litters. Values are means t SD of 3 litters at each time point. SDS smaller than symbols are not shown.
NUTRIENTS
BY
RAT
PUPS
1. Body concentration and accretion rates of fat and protein for pups raised in small, medium, or large litters TABLE
Litter
S 5 days (4-6
Size M
L
days)*
Fat g/kg
body
wt
g/day
Protein g/kg
body
wt
g/day
8Ok4 0.49t0.4
0.22rto.01
5223 0.02*0.01
115t2 0.24kO.01
116k2 0.20t0.01
114t0.4 0.20t0.01
9 days (B-10
65t7
days)*
Fat g/kg
body
wt
g/day
Protein g/kg
body
wt
g/day
91s
148&l 0.67t0.04
0.26t0.02
421k2 0.02t0.05
12353 0.46t0.02
132rt2 0.37kO.03
0.22t0.07
15 days (14-16
13325
days)*
Fat g/kg
body
wt
g/day
Protein g/kg g/day
body
wt
161tll
102tlO
0.63kO.05
0.35t0.02
61t8 0.14t0.01
135t6 0.57t0.05
145t2 0.40t0.02
153-+1 0.27kO.02
Values are means t SD; n = 3 litters per age per litter size. S, small (4 pups); M, medium (10 pups); L, large (16 pups). * Interval over which accretion rates (g/day) were estimated.
that for M pups. Energy and protein balances. To determine whether litter size influenced the efficiency with which the pups used dietary energy and protein for growth, daily energy and protein balances were computed; the data are summarized in Table 2. To facilitate interpretation of data among pups of varying size and body composition, data are expressed per gram body protein. Because of the differences in milk composition, only 87% of the variability in energy intake could be ascribed to volume intake. Energy intake per gram of body protein decreased significantly over time among all litters (P < 0.001); the largest decrease occurred between 4-6 and B10 days of age. Comparison among litter sizes indicated that the energy intake (per g body protein) of S pups was consistently higher than that for M and L pups, whereas there was only a trend for M pups to have a higher intake than L pups (P < 0.08). Energy gain per unit body protein decreased over time for S and M litters and up to 10 days of age in L litters. S pups deposited more energy than M pups at all time points. Despite the relatively small differences in energy intake, when scaled to “metabolic mass,” (i.e., body protein), L pups gained proportionally less energy than M pups at all time points. Hence the gross efficiency of energy gain (energy gain/energy intake) was significantly lower for L than for M pups at all time points (P < 0.05): 18 t 2 vs. 35 t 1, 19 t 13 vs. 40 t 3, and 26 t 2 vs. 32 -+ 3% at 4-6, B-10, and 14-16 days, respectively. The variation among L pups at B-10 days was relatively high (coefficient of variation 62%), because the efficiency of energy gain in two of the three litters was similar to that
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INTAKE
AND
USE
OF
MILK
2. Energy and protein balances for pups raised in small, medium, or large litters TABLE
Litter
S
M
5 days (4-6 Energy, kcal . g body protein-’ Intake 8.56t0.28 Gain 4.OlkO.17 Loss 4.54t0.23 Protein, Intake Gain Loss
go g body
Size
l
L
days)”
day-l
protein-l. 027+00?-' . -. 0.16-c-0.01 0.10t0.004
6.81t0.21 2.41t0.01 4.40t0.21
6.2320.25 1.12kO.06 5.08rtO.30
0.22kO.02 0.15t0.004 0.07t0.02
0.21t0.001 0.17kO.02 0.04t0.02
9 days (B-10 Energy, kcal . g body protein-’ day-’ Intake 5.89t0.55 Gain 2.67kO.30 Loss 3.22t0.43 Protein, go g body protein-’ 023+OOd3ay-l . -. Intake Gain 0.14-+0.02 0.09~0.01 Loss
days)*
l
3.91-to.33 1.57kO.04 2.35t0.30
3.89t0.35 0.70t0.40 3.19t0.70
0.22*0.002 0.13t0.003 0.09*0.001
0.24-t-0.01 0.11t0.03 0.13t0.03
l
15 days (14-16 Energy, kcal . g body protein-’ Intake 4.45kO.67 Gain 1.57t0.05 Loss 2.83t0.63 Protein, go g body protein-’ Intake 020+ooY1 . -. Gain 0.10t0.003 Loss 0.04~0.01 Values are means over which balances tions.
l
days)*
day-’ 3.51k0.36 1.12sto.12 2.38t0.30
2.91t0.31 0.76t0.03 2.15t0.28
0.18t0.003 0.08t0.01 0.10~0.01
0.17t0.01 0.07t0.003 0.10-1-0.01
l
& SD; n = 3 litters per age per litter size. * Interval were estimated. See Table 1 legend for abbrevia-
8 Xint = 2.36
;slope
= 0.30
3 ‘int
; slope
= 0.52
= 1.28
4.0
6.0
Energy (kcal
8.0
10.0
12.0
NUTRIENTS
RAT
R1109
PUPS
maintenance (zero gain) in these pups, was 1.28 kcal. g body protein-‘. day-l. The slope of the regression line, which represents the efficiency with which energy consumed in excess of maintenance was gained, was 0.52 t 0.03 kcal deposited/kcal CGE ingested. The regression line (r = 0.93) for the second population was significantly different (P < 0.001). This group comprised all L litters at 4-6 days of age and two of the three L litters at 8-10 days of age. The CGE needed for maintenance of these pups (2.36 kcal . g body protein-‘. day-‘) was significantly higher than for the other litters, and their efficiency of energy gain (0.30 t 0.07 kcal deposited/kcal consumed) was significantly lower. To facilitate comparison with other published data, the values for maintenance energy needs were also derived after standardization of energy intake and gain per body weight0.75; the corresponding values for the first and second populations of rats were 86 and 135 kcal/kg”*75, respectively. Presumably because of the marked differences in body composition, the relationship between intake and gain expressed per body weight0*75was not as close as when data were standardized per unit body protein (r = 0.78 and 0.92, respectively). As for energy gain, the gross efficiency of protein gain was similar for S and M pups between 4 and 6 and between 8 and 10 days of age (62 t 5%) and then decreased in both groups to 47 t 3% between 14 and 16 days of age. At 4-6 days of age, however, the gross efficiency with which L pups gained protein (83 t 8%) was significantly higher than that for S and M litters (P < 0.01). At 8-10 days and thereafter, efficiency among L pups had decreased and was not different from values for S and M pups. To determine whether these litter and age effects were attributable to changes in the protein needed for maintenance or in the efficiency with which the milk protein was used for growth, we examined the relationship between protein intake and protein retained in individual litters (Fig. 5). The three L litters at 4-6 days of age again formed a statistically different group. All remaining litters showed a continuous positive linear relationship between protein intake and gain (r = 0.91; P < 0.001). The slope of the line indicated that the efficiency of milk protein use was 92 t 9%. The protein required for maintenance (zero gain) was 0.085 gog body pro-
Intake
/ g protein
BY
0.30 /
/ d)
FIG. 4. Relationship between daily energy intake and energy gain at 4-6, 8-10, and 14-16 days of age. A, Large litters at 4-6 (n = 3) and 8-10 (n = 2) days; 0, all other litters. Symbols represent values for individual litters.
of litters at 4-6 days (efficiencies of 9 and 16%, respectively), whereas one litter showed an efficiency of 32%, similar to values at 14-16 days of age. These data are further evaluated in Fig. 4, where the relationship between energy intake and energy gain is shown. Two populations of pups can be discerned; a continuous relationship between energy intake and gain can be observed for all S and M litters and for four of nine L litters (r = 0.96; P < 0.001). The x-intercept of the regression line, which represents the CGE needed for
G .r \
0
0.25
,Xint=
0.085
/
; SlOpe=O.g3
t
g lg 0.20+ s% ‘a, ki 0.15 zm a,L ; 0.10 -
i
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
Protein Intake (g / 9 protein / d) FIG. 5. Relationship between daily protein intake and protein gain at 4-6, 8-10, and 14-16 days of age. A, Large litters measured at 4-6 days: 0, all other litters. Svmbols represent values for individual litters.
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RlllO
INTAKE
AND
USE
OF
MILK
tein-’ day-‘. With data for L litters at 4-6 days of age excluded, there was no effect of age or litter size on the slope of the regression line, but both factors influenced the line intercept to a small extent (
