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The Journal of Nutrition Vol. 127 No. 3 March 1997, pp. 458-462
Copyright ©1997 by the American Society for Nutritional Sciences

Maternal Lipid Intake During Pregnancy and Lactation Alters Milk Composition and Production and Litter Growth in Rats1,2

Martha Del Prado, Guadalupe Delgado, and Salvador Villalpando

Unidad de Investigación en Nutrición, Instituto Mexicano del Seguro Social, Apartado Postal 7-1069, México, DF 06700

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED

ABSTRACT

The relationship between dietary fat content and milk composition, production and litter growth was studied in rats fed during pregnancy and lactation purified diets of equal energy density containing 2.5 or 20 g fat/100 g diet. A subsample of rats (HL-EP group) fed the high lipid (HL) diet but pair-fed on an energy basis with the low lipid (LL) diet group was also studied in a separate experiment. Food intake, dam body weight and litter weight were recorded daily. Rats were milked on d 14 of lactation. Milk lipid, lactose and protein concentration and milk production were measured. Lactating rats fed the HL diet had significantly higher energy intakes (P < 0.01) and milk production (P < 0.05) than rats fed the LL diet. Milk lipid concentration and daily milk volume and lipid production were significantly higher in the HL group. The HL-EP dams had significantly higher milk lipid, protein and lactose concentrations (P < 0.05) and tended to have higher daily lipid and energy outputs (P = 0.08) than LL rats. Birth weights of pups were similar among groups, but from d 6 on, the pups from the HL and HL-EP groups were significantly heavier (P < 0.05) than pups from the LL group. This investigation presents evidence that the milk fat concentration and the daily output of fat, protein and lactose of lactating rats are altered by dietary fat manipulations, which in turn affect growth of the litter.

Key words: lactation, dietary fat, milk composition, rats, litter growth.

INTRODUCTION

The fatty acids of milk triacylglycerols are derived from de novo synthesis within the mammary gland from lipids of dietary origin or lipids mobilized from adipose tissue (Williamson and Da Costa 1993). Variations in the fatty acid composition of the maternal diet during lactation alter the fatty acid composition of milk (Brandorff 1980, Grigor and Warren 1980, Harzer et al. 1984, Insull et al. 1959, Mellies et al. 1979). Increment changes in the proportion of fat in the diets of rats during lactation have been shown to increase (Grigor and Warren 1980) or decrease (Beare et al. 1961) the milk lipid concentration. Others have found no effect (Burnol et al. 1987, Farid et al. 1978, Green et al. 1981).

Feeding lactating rats high fat diets has been reported to result in diminished mammary gland lipogenesis and in reduced growth rates and increased mortality of the pups (Agius et al. 1980, Rolls et al. 1980). Other studies have reported that despite reduced mammary gland lipogenesis, pups of dams fed high fat diets grew better than did controls (Grigor and Warren 1980).

These contradictory results might be due to differences in the experimental designs. Some studies have provided the experimental diets during short periods after parturition (4 d) while others did so throughout pregnancy and lactation. The proportion of fat in the high fat diets differed among the studies, ranging between 10 and 60 g fat/100 g diet.

Other variables also may confound the results, such as the marked diurnal changes in the rate of mammary gland lipogenesis in lactating rats fed a nonpurified diet. The peak rate occurs at night and falls dramatically during the daylight hours, coinciding with the periods of lowest food intake (Williamson et al. 1984). The food intake pattern of rats can be modified by altering the composition of the diet (Grigor and Thompson 1987). This, in time, might have some effects on the circadian variations in the lipid concentration and composition of milk. None of the studies mentioned above was designed to account for circadian changes in the composition of milk.

The specific aims of this study were to measure the milk fat concentration and output and the growth performance of the litters from rat dams fed freely diets with either a low or a high concentration of fat. Such a design allows investigation of whether milk fat concentrations or output might be further increased by dietary modifications, and whether the magnitude of potential modifications may be reflected in the growth performance of the litter. Specific efforts were made to control for known confounders such as total energy intake of the dams and circadian variations in milk fat concentration.

The primary hypothesis was that the intake of a high fat diet during pregnancy and lactation would increase milk lipid concentration and litter growth.

MATERIALS AND METHODS

Animals. Female Sprague-Dawley rats (Charles River, Wilmington, MA) were randomly assigned to group cages housed in a temperature-controlled room (22 ± 2°C) with a 12-h light:dark cycle (lights on from 0700 h). From weaning to 12 wk-of-age rats were fed a pelleted nonpurified diet (Purina, Guadalajara, México) containing (per 100 g diet) 28 g protein, 61 g carbohydrate and 2.5 g fat. Rats were maintained and handled in accordance with the guidelines for experimental animals of the NIH and Secretaria de Salubridad of México. The rats were adapted to a pelleted purified diet containing the same nutrient composition as the pelleted commercial diet for 2 wk before mating. Body weight and food intake were recorded every other day during this period.

Animals weighing 220-280 g at 14 wk-of-age were mated. The day on which sperm was identified in vaginal smears was designated d 1 of pregnancy. Pregnant females were housed individually. On the day of parturition, designated d 1 of lactation, litters were weighed and adjusted to 8 pups per dam. No gender differentiation was done. Dams were weighed daily from d 1 of pregnancy, and both dams and litters were weighed daily throughout lactation.

Experimental diets. After mating, rats were randomly assigned to one of two purified diets containing 2.5 g fat/100 g diet (LL) or 20 g fat/100 g diet (HL). Purified diets had the same energy density and supplied 14.64 kJ of digestible energy per gram of dry diet. Compositions are shown in Table 1. Lipid contributed 42% of total energy to the high fat diet and 3% to the low fat diet. Casein contributed 30% of total energy to both diets. The diets were pelleted and stored at 4°C until use. Rats had free access to diet and water at all times. Daily food intake was assessed by weighing the food before and 24 h after it was made available to the rats. To monitor differences in the diurnal pattern of maternal food intake associated with different concentrations of lipid in the diets, food intake was assessed at 4-h intervals throughout the day in a subsample (HL, n = 20; LL, n = 22) during d 12 of lactation. Digestibility of macronutrients was assessed as the relative difference between daily intake and 24-h excretion in feces by the following formula:
Digestibility = [(Dietary intake
− Fecal excretion)/Dietary intake]⋅100

Table 1. Composition of experimental diets1

[View Table]

In the initial experiment dams fed the HL diet had a significantly higher energy intake than did LL dams. To control for the higher energy intake of the HL group, a control group composed of a subsample of HL rats (n = 12), was pair-fed the mean daily energy intake recorded for the LL group (group HL-EP). The diurnal pattern of maternal food intake was not assessed in the HL-EP group.

Within each dietary group, dams were assigned randomly to a milk yield (n = 10) or to a milk composition subgroup (n = 36 of HL and LL groups, and n = 12 of HL-EP group).

Milk collection and analysis. Milk samples were collected between d 12 and 14 postpartum from 36 rats of each dietary group. To control for circadian variations in milk composition, the rats were randomly assigned to one of six subgroups (n = 6) milked at one of the following time points: 0700, 1100, 1500, 1900, 2300 or 0300 h. Rats were milked on one occasion because serial milking affects milk composition (Keen et al. 1980a). The dams were separated from litters for a period of 4 h before milking; longer periods of separation were avoided as this can affect milk composition (Keen et al. 1980b). Milk was expressed manually from all teats in rats after intraperitoneal injection of oxytocin (4 UI) under pentobarbital anesthesia (35 mg/kg body weight). Milk samples were frozen at -20°C until further analysis.

Milk lipid concentration was measured gravimetrically after chloroform-methanol extraction by a modified Folch method (Jensen et al. 1985). Protein concentration was determined by the method of Lowry (1951) with bovine serum albumin as standard. Milk lactose concentration was estimated by measuring glucose after the hydrolysis of lactose with beta -galactosidase (Trinder 1969).

Milk production. Milk production was measured on d 14 of lactation in a subsample of 10 rats drafted from the original pool of rats from each dietary group. Milk yield was determined by the tritiated water method described by Godbole et al. (1981), modified by Warman and Rasmussen (1983).

Statistical analysis. The Minitab statistical software program (release 10X, Minitab Inc., State College, PA) was used for analysis of variance for comparisons between HL and LL dietary groups. Analysis of variance and covariance with repeated measures were used to asses the differences between pre and post parturition time points for maternal body weight, energy and food intakes. Sheffé's test was used as a post hoc test (Harrison, 1972). The alpha level was set at P < 0.05. The differences in mean pup body weight, number of delivered pups, energy, fat, protein, lactose concentrations and milk production between HL and LL groups were analyzed by Student's t test for independent samples.
Fig. 1. Daily energy intake of rat dams fed a high lipid (HL) diet or a low lipid (LL) diet. Values are means ± SD, n = 36 animals per group. The high lipid diet (HL) contained 20% and the low lipid diet (LL) 2.5% corn oil on a dry weight basis. Asterisks (*) indicate significant differences (P < 0.05) between groups.
[View Larger Version of this Image (22K GIF file)]

Table 2. Maternal body weight during pregnancy and lactation of rats fed a low lipid (LL) diet, a high lipid (HL) diet or paid-fed the HL diet to the energy intake of the LL group (HL-EP)1,2,3

[View Table]

RESULTS

Maternal energy intake. The daily energy intake was similar in HL and LL groups and varied very little longitudinally during pregnancy. After parturition energy intake increased progressively and significantly (P < 0.01) in both groups. The energy intake of the HL group was significantly higher (P < 0.01) than that of LL group from d 8 of lactation on (Fig. 1). The sum of energy consumed from d 1 to d 12 was significantly higher in the HL than in the LL group (P < 0.05).

The circadian pattern of food intake was comparable in the HL and LL groups. The period of maximal food intake occurred between 1900 and 2300 h, corresponding to 20-30% of the total daily food intake. Digestibility of lipid and protein measured during lactation in a subgroup of 6 rats from each dietary group did not differ (lipid: 93.5 ± 2.3% in LL, 96.8 ± 0.7% in HL; protein: 94.7 ± 0.8% in LL, 96.7 ± 0.4% in HL).

Maternal body weight. The mean body weight of dams increased progressively, and no differences were observed between groups during pregnancy (Table 2). Despite the greater food intake of the HL rats, no differences in maternal body weight during lactation were noted between LL and HL groups, both remaining essentially stable (Table 2). The rats of the HL-EP group showed a progressive loss of body weight after parturition, totaling an average of 34 g at d 12 of lactation.

Pup body weight. There were no differences in pup birth weights (7.49 ± 1.0 and 6.98 ±0.5) or in the number of pups per litter (11 ± 3 and 11 ± 2) between LL and HL groups. The pups of HL mothers gained weight more rapidly, and from d 6 had significantly higher body weights than LL pups (P < 0.01) (Table 3). The mean body weight of HL pups exceeded by about 17% that of LL pups by d 12 of lactation.

Table 3. Body weights of pups of dams fed a high lipid (HL) diet, a low lipid (LL) diet or paid-fed the HL diet to the energy intake of the LL group (HL-EP)1,2,3,4

[View Table]

Milk composition and 24-h nutrient production. The concentration of lactose, protein and lipid in milk from both dietary groups did not show any circadian variation. Accordingly, individual values of milk lactose, protein and lipid concentrations in the cross-sectional study were pooled from all sampling schedules and are presented as 24-h milk composition in Table 4. Milk lipid concentration was significantly higher (P < 0.001) from the HL than from LL rats. There were no differences in the protein and lactose concentrations of milk from HL and LL groups (Table 4). Daily milk volume and production of lipid, protein, lactose and total energy were significantly greater (P < 0.01) in the HL than in the LL group (Table 5).

Table 4. Lipid, protein and lactose concentration of milk from rats fed a low lipid (LL) diet, a high lipid (HL) diet or pair-fed the HL diet to the energy intake of the LL group (HL-EP)1,2,3,4

[View Table]

Table 5. Milk volume and 24-h production of lipid, protein and lactose in rats fed a low lipid (LL) diet, a high lipid (HL) diet or pair-fed the HL diet to the energy intake of the LL group (HL-EP)1,2,3

[View Table]

Paired energy intake group. The pups of the HL pair-fed group grew similarly (P > 0.2) to the pups of HL dams fed freely (Table 3). The daily milk volume of HL-EP dams was similar to that of LL group and significantly lower than that of the HL rats (P < 0.01) (Table 5). The daily milk lipid and energy output tended (P = 0.09) to be higher in the HL-EP group than in the LL group. The milk protein and lactose output were comparable to that in HL rats and significantly higher (P < 0.05) than LL rats.

DISCUSSION

We present evidence that a high fat diet fed to Sprague-Dawley rats during pregnancy and lactation increases daily milk volume, milk lipid concentration and daily lipid production. The daily output of protein and lactose, but not their concentration in milk, was increased as well. These changes in milk were associated with faster growth of pups during lactation.

The milk fat responses to high lipid diet must have at least two separate components. The first is represented by the increase of daily milk volume, which is mostly driven by demand from the pups (Russel 1980). The second is the increase of milk fat concentration, which must be controlled by mechanisms in the maternal compartment. The data presented here suggest a critical role of dietary fat in modifying the composition of milk.

The faster growth observed in the HL pups is consistent with the larger milk volume and energy output measured in the HL dams, but does not explain which factor initiated the accelerated growth of HL pups. The body weight and the carcass composition of HL and LL pups were similar at birth (lipid: 1.03 ± 0.22% in HL, 1.03 ± 0.28% in LL; protein: 9.34 ± 0.35% in HL, 9.75 ± 0.25% in LL). Their body weight remained comparable up to d 6 postpartum. One plausible hypothesis might consider a higher level of energy in the milk from the early days of lactation due to its higher content of fat. However, we did not measure the milk fat concentration at this stage.

Energy intake of dams has been correlated with the growth rate of their pups (Rolls et al. 1984). Even when the energy intake of the HL dams was restricted to that of LL rats, the growth of the HL-EP pups was still comparable to those of freely fed HL dams. This faster growth may be associated with the apparently higher milk energy and protein output of the energy-paired dams compared to the LL group.

Improvement in the growth of pups from rats fed a high fat diet has not been a constant finding among the studies published. Experiments in lactating rats fed a cafeteria diet showed a depressed growth rate of the litter relative to that of controls fed a nonpurified commercial diet only (Rolls et al.1984). Grigor and Warren (1980) fed lactating rats a purified diet containing coconut oil (200 g/kg diet, 490 g lauric acid/kg oil) for 4 d. That diet resulted in a weight gain rate in the pups similar to that of pups from dams fed a nonpurified commercial diet. In contrast, pups from rats fed a purified diet containing peanut oil (a mixture of oleic and linoleic acids) had a growth rate greater than control rats fed a nonpurified commercial low lipid diet. The conflicting results among these experiments might be due to differences in experimental design. Some investigators added fat (vegetable oil or lard) to nonpurified diets (Green et al. 1981) or used cafeteria diets (Rolls et al. 1984). These approaches dilute the proportion of dietary protein and other nutrients, which in time may affect the nutritional status and milk production of the dams. In the experiments reported here, dams were fed a purified experimental diet with a higher concentration of fat but similar energy density and protein concentration to the purified control diet. Therefore, an appropriate intake of protein and protein/energy ratio were provided to both groups. Addition of cellulose as a bulking agent to the HL diet assured an equal content of nutients and energy density per gram of dry diet and a similar nutrient/energy ratio, except for lipid and carbohydrates. Theoretically, a larger intake of cellulose might impair the digestibility of nutrients, but no differences were found in actual digestibilities of fat and protein between HL and LL rats.

The HL-EP dams lost body weight from parturition to d 12 of lactation, while the mean weight of their freely eating counterparts remained essentially stable. Such a loss is indicative of negative energy balance explained by the combination of limited energy intake and high energy loss to milk production (energy exported to milk was similar to HL rats). The differences in body weight among these groups are attributable more to differences in the content of gastrointestinal tract, since we did not find differences in the weight of carcasses among the groups (data not shown).

The differences in the growth of the LL and HL pups may have been related to differences in milk lipid composition. A high fat diet changes the fatty acid composition of rat milk by increasing the long-chain fatty acid content at the expense of medium-chain fatty acids (Brandorff 1980). The medium-chain fatty acids released from dietary triacylglycerols are not reesterified in intestinal cells and are transported directly, via the portal circulation, to the liver for either partial oxidation to ketone bodies or complete oxidation to carbon dioxide (Bach and Babayab 1982). Consequently, dietary medium-chain fatty acids are more readily utilized for energy and less effectively incorporated into adipose tissue triacylglycerols than long-chain fatty acids (Bray et al. 1980, Geliebter et al. 1983).

Demand of the pups seems not to be the only driving force for milk volume and fat output. Both decreased when the energy intake of the dams fed the HL diet was restricted to the energy intake of LL dams, while the growth of their pups was still comparable. Milk fat concentration was higher in the HL-EP group than in the LL group, despite their having the same energy intake. This suggests that the differences in milk fat concentration reported here are attributable more to the dietary fat intake of the dams than to total energy intake.

The daily food and energy intake of the freely eating HL group was comparable to that of LL group throughout pregnancy and during the first days of lactation. This evidence suggests that the hyperphagia noted in the later stage of lactation is not related to increased palatability of the HL diet and is not the explanation for enhanced energy intake of the free-eating HL dams.

In summary, this investigation presents evidence that the milk fat concentration and the daily output of fat, protein and lactose of rats are altered by dietary fat concentration. These in time affect the growth of pups. Precise identification of the mechanisms by which dietary fat controls milk composition and production requires further research.

FOOTNOTES

1   Research was supported by a grant (3320-M9308) from the Consejo Nacional de Ciencia y Tecnología (CONACYT) México.
2   The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

Manuscript received 14 December 1995. Initial reviews completed 16 February 1996. Revision accepted 24 September 1996.

LITERATURE CITED

0022-3166/97 $3.00 ©1997 American Society for Nutritional Sciences



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