The lipid composition of milk from mice fed high or low fat diets

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1 Laboratory Animals (1992) 26, The lipid composition of milk from mice fed high or low fat diets JERALD SILVERMAN I, DOUGLAS W. STONE2 & JEAN D. POWERS3 IDepartment of Veterinary Preventive Medicine, The Ohio State University, College of Veterinary Medicine, 1900 Coffey Road, Columbus, OH 43210; 2University Laboratory Animal Resources, The Ohio State University, 1960 Kenny Road, Columbus, OH and 3Departments of Statistics and Veterinary Clinical Sciences, 1958 Neil A venue, The Ohio State University, Columbus, OH 43210, USA Summary Total fatty acids and the proportions of methyl esters of individual fatty acids were measured in mouse milk. Pregnant mice were fed either a high fat (OF) diet or a low fat (LF) diet from 14 days of gestation. After parturition, each dam was milked once a day for a period of 18 days. The mean total fatty acid concentration over the entire study period was 110 mg/g of milk (approximately 11,70/0 fat as triglyceride) for both dietary treatment groups. During days 2 to 6 postpartum, the mean total fatty acid concentration for dams fed OF diet was lower than for the LF group. Although the concentration of total fatty acids of mouse milk was not affected by the level of dietary fat fed to the dam, several variations in the proportions of individual fatty acids were observed. Keywords: Mice; Milk; Lipids; Fatty acids As part of a project investigating the mammary cancer promoting effect of high levels of dietary fat fed early in the life of a mouse (Silverman et 01., 1989), it became necessary to determine if the level of fat fed to a dam was related to her milk fat level and the proportion of individual fatty acids in her milk. In this study, the high fat (HF) diet was fat (as lard) and was chosen to simulate a typical western human diet. Correspondence to: Dr Silverman. Received 22 May /991; accepted29 October 1991 The low fat (LF) diet was 5% lard. Other dietary ingredients were formulated to provide for equivalent amounts of nutrients. The pelleted diets were provided ad libitum from 14 days of gestation. Mouse milk was collected from the dams over an 18day period postpartum. Total fatty acids and individual fatty acids were measured using gasliquid chromatography. Materials and methods Animals and husbandry Fourteendaypregnant Crl:CDl(ICR)BR mice, free of common pathogenic murine viruses and bacteria (as certified by the breeder), were received from Charles River Breeding Laboratories (Portage, MI, USA). On arrival, mice were housed individually in 29 X 19 X 13 cm polypropylene cages with ground corncob bedding. Clean cages and bedding were provided twice each week. Food and water were provided ad libitum. Room temperature was maintained at 22 ± 1 C and relative humidity was maintained at 50± 15%. The light cycle was 12:l2light:dark, with 1215 air changes per hour. The mice (dams n = 14) were identified as A to N; upon arrival, they were randomly placed into one of two groups and fed either a HF or LF diet. Diets Pelleted diets were obtained from a commercial source (BioServ Inc, Frenchtown, NJ, USA) and the stated contents (Table 1) of fat, carbohydrate, protein, crude fibre, moisture and ash were confirmed by an independent laboratory.

2 128 Silverman, Stone & Powers Table 1. Percentage composition of high and low fat diets Ingredient Low fat High fat Casein, vitamin free '50 dlmethionine 0,30 0,35 Cornstarch Dextrose 13,00 8,30 Alphacel 5,00 5'90 Lard 5,00 23'52 Mineral mix, AIN76 3'50 4 II Vitamin mix, AIN '18 Choline bitartrate Metabolizable energy (Kcal/g) 3, 88 (tow fat) and 4, 73 (high fat). Mice fed these two diets have been shown to consume them in an equicaloric manner (Silverman & Powers, 1991), and they were formulated to allow equivalent consumption of protein, fibre, vitamins and minerals for both diets. Lard was the only fat source in both diets. Diets were kept refrigerated and were used within 90 days of milking. Both groups of mice were fed the diets for an average of 7 days prior to parturition and continued on the same diet throughout the study period. Food intake was not measured. Milking After birth, the pups were kept with their dams. No attempt was made to cull litters to an equal number of pups. Dams were milked even if all or part of a litter did not survive. Approximately 15min before milking, the dams received O 2 USP units of oxytocin subcutaneously to stimulate milk collection, and all milking was performed at approximately 1600 h once daily for a maximum of 18 days postpartum. Milking was accomplished by a mechanical suction apparatus similar to that described by Haberman (1974) and continued for each mouse until there was no further milk yield from any nipple (approximately 5 min/mouse). Milk was collected in 5 ml glass culture tubes with Teflon screw caps and the tubes were weighed before and after milking. Milk yields ranged from 24,6212,0 mg/sample and the samples were stored at 20 DC prior to analysis. Analytical procedure for fatty acids in milk The milk samples were centrifuged at rpm for 30 min at 410 DC to cause the milk fat to form a solid layer on the top. The methodology was a modification of the procedure of Sukhija and Palmquist (1988), wherein the milk fat was methylated and hexane was substituted for benzene. Fatty acids were identified by comparison of their retention times with that of the internal standard (nonadecanoic acid) and reported retention times of known methyl esters. Total fatty acid concen.trations (mg/g of milk) were calculated using the peak areas obtained by gasliquid chromato.~raphy. The percentage concentrations of individual fatty acid methyl esters were calculated relative to the total fatty acid concentrations. Only those fatty acids that have been found to be most common in cow's milk are reported. In some instances, the total fatty acid concentrations wen: expressed as triglycerides in order to compare our data with other published data. To estimatl~the total fat as triglyceride, the concentrations (If total fatty acids were divided by a factor of C' 94. Statistical evalul1tion Two postpartum timeperiods were chosen (days 26 and days 2 18) for statistical comparisons as sparse literature was available for the first period. All data were compared using a twotailed Student's ttest. Results Thirtyfive samples were obtained and the results are shown in Table 2. All data are presented as there are limited literature reports for fatty acid analyses of m)use milk and none present individual animd data collected over time. There was an inadequate amount of milk on day I postpartum in most cases, and analyses began on day 2 or 3. Although all mice were milked daily, an insufficient volume was obtained for analysis in many cases, particularly in the group fed the LF diet. For these n:asons, no data are presented for some days.

3 Mouse milk lipids 129 Table 2. Total fatty acids (mg/g of milk) and proportions (weight 0/0) of methyl esters of major fatty acids in mouse milk Days Total post Number fatty Proportion Mouse partum of pups acids /0:0 /2:0 /4:0 /6:0 /6:1 18:0 /8:/ /8:2 /8:3 High fat diet A ' , ,8 9,9 1 4 A '1 6,9 9, ,3 3,8 36'1 8'5 1 3 A ,5 8,0 8, '4 5,9 35,9 9'0' A '5 8'7 9, ,7 5,7 37,0 10,9 " B ,8 4,9 20'5 4, , B ,7 5, ,9 3,3 44, " B '5 6,3 7, ' C ,4 3'6 6, , ' C ,3 4, '5 4,6 3, ,6 " C ,9 6, ,0 3,4 44,9 11'3 ' , ,0 2' ' ,6 6,0 7, , ,5 5,7 6, '5 3,4 43,5 10,4" ,4 7, , ,4 10'3 ' E ,0 5, , ,0 10,7 1 8 E , , ,6 9,4 1 1 E ,7 8, ' '0 8,9 1 2 E ,5 5'5 7, ' F , '6 4, , ' F '4 4,7 6, ,6 3'6 41'3 io'4 ' G ,0 7, ,6 4,9 40,8 10'1 1 0 G , , Low fat diet H '0 6' , ,0 9,6 1 4 H , , , '1 I ,8 6' ,6 3,0 33,8 7,4' J ,6 8, , ,4 1 0 J 6 1\ , ,3 3'5 28'3 4'9 1 0 K ,0 8,8 30,0 6,5 3,8 36,5 6,7 ' L , '3 5, '4 6'5 \ 0 L , , ,7 \ 2 M , '1 5'3 2' ,3 1 2 M , , ,5 4,8 O' 8 M ,4 9,7 27'3 5'6 3,8 30,8 6,3 1 2 N ,5 9,6 25'5 3'3 3'4 39'0 9'3 \'1 N 'Trace. When data for all samples in each group were When data were combined, there was no combined, there was no significant difference significant correlation between the number of (P> O 05) in the total fatty acid concentration nursing pups and the total fatty acid conof milk from mice fed HF versus LF diets. The centration with either the HF or LF diets mean total fatty acid values (mg/g ± SEM) were (r= 0,4 and 0'2, respectively). 110 ± 13 for those fed the HF diet (N = 22) and During days 26 postpartum, there was a 110 ± 11 SEM for the LF diet (N = 13). Figure 1 slight difference between groups in total fatty shows the total fatty acid concentration for each acid concentration: the values for the group fed day of analysis and mean values are presented the HF diet were lower than the group fed the LF where more than one sample was analysed. diet (93' 1 mg/ g ± 14 9 SEM versus mg/ g ±

4 130 Silverman, Stone & Powers 300 ;? 250 "e ~ 200.s. '" ~ 150 ~u. 100 Jil {!. 50. ~ ~ E.. " ~ Days Postpartum IIl!I High Fa' f I. Low Fat " ~ ;:.~ 5. " j I I I I Fig. 1. Mean total fatty acid concentration of milk from Crl:CDl mice fed a high fat (23'5070 lard) or low fat (5% lard) isocaloric diet from 14 days of gestation. 10'5 SEM; P=0 05). There was no significant correlation between the number of nursing pups and the total fatty acid concentration with either diet for this period. The relative weight percentages of individual fatty acid methyl esters are shown in Table 2. When examined without reference to the number of days postpartum, significant differences (P<0'05) were found between mice fed HF and LF diets for all fatty acids except 10:0 (caproic acid) and 12:0 (lauric acid). For the period 26 days postpartum, significant differences between the two dietary groups were observed for all of the measured fatty acids except the 10:0, 18:0, and 18:3 fractions. During the two periods studied, there was no significant correlation between number of nursing pups and the relative proportion of individual fatty acids from dams fed HF or LF diets. Discussion There were no statistically significant differences in the average total fatty acid concentration in the milk of mice fed HF or LF diets over the total timeperiod studied, although a small difference was observed between these two groups when milk from days 26 postpartum was examined. There is insufficient information in the carcinogenesis literature to categorically state that such a difference for total fatty acids would or would not make a significant difference in enhancing the development of rodent mammary cancers. When total falty acids were converted to total fat as triglycerides, the mean total milk fat was 11.7% over the course of the entire study for mice fed both HF and LF diets, which is similar to previously reported studies (Barnett & Dickson, 1984; Nagasawa et al., 1989). Mean milk fat concentrations \'ary from ' 4OJoin other published studie; (Smith et al., 1969; Baverstock et al., 1976; Knight et al., 1986). It is difficult to interpret differences in published studies on total milk triglyceride concentrations due to several factors which include dietary composition (particularly isoc:aloric versus fat added), dietary consumption, lactation status, strainspecific variation (Baflli~tt & Dickson, 1984; Nagasawa et al., 1989; Knight et al., 1986; Baverstock et al., 1976), milking methods (Keen et al., 1981; Barnett & Dicksl)n, 1984; Knight et al., 1986) and parity status (Nagasawa et al., 1989). These milk fat percentages are of interest to experimental oncologists as it has been suggested that dietary fat may enhance mouse and rat mammary carcinogenesis, when fed at approximately 1617OJo of the diet (Silverstone & Tannenbaum, 1950; Carroll & KhoJ', 1971; Beth et al., 1987). Milk fat concentrations are reported to change following parturition in other species with higher values observed in the first few days postpartum (Keen et al., 19m; Wheatley et al., 1981). Knight et al. (1986) have reported that on a standard diet (i.e. approximately 5OJofat by weight), mouse milk fat increased from day 2 to day 10 of lactation, then plateaued through to day 17. The present study has demonstrated the same pattern in that total fatty acids from HF fed dams peaked at 11 days postpartum and then decreased to day 18 postpartum (Fig. 1). Although the total fatty acid concentration of milk tended to increase until approximately day 11 postpartum, there were some individual animals where this did not occur (e.g. mice A, E and M). Our data show a wide variation in the total fatty acid concentration between animals, and in the same animal over a period of time; this variation ill the fat content of milk may have been caused by several factors associated with sample collection procedures.

5 Mouse milk lipids 131 In contrast to the limited differences seen in total fatty acids from the milk of mice fed HF or LF diets, several differences for individual fatty acids were observed. The mean proportion of 18: I was higher in mice fed the HF diet compared to the LF diet (40' 5 versus 34' 90/0; P<O'OOI), and the 18:2 fraction was also higher in mice fed the HF diet as compared to the LF diet (10'3% versus 6'4%; P<O'OOI); a similar pattern was observed for the Day 26 postpartum period. These findings are similar to those of Smith et al. (1969) who hypothesized that mice fed a HF diet absorb a larger proportion of long chain fatty acids from dietary fat. When all data are considered, the proportion of medium chain fatty acids (10:0, 12:0) in the milk did not differ between the HF and LF groups, which is in accordance with the findings of Smith et al. (1969) who found that the synthesis of these fatty acids by the mammary gland is unaffected by dietary manipulations. We did not find that 12:0 proportions differed significantly (P<0'05) between HF and LF groups when days 26 postpartum were analysed. A more precise understanding of the role of diet in influencing the lipid composition of mouse milk and subsequent carcinogenesis will necessitate feeding diets with differing fatty acid composition and concentration to mammary tumour virus positive mice and then following the course of tumour development. Acknowledgments This study was supported in part by grant 84B02 from the American Institute for Cancer Research. The authors thank Dr Donald Palmquist for his excellent advice and support and Dr PS Sukhija for the quantitation and analysis of milk fatty acids. References Barnett S & Dickson R (1984) Milk production and consumption and growth of young or wild mice after ten generations in a cold environment. Journal of Physiology 346, Baverstock P, Spencer L & Pollard C (1976) Water balance of small lactating rodents. II. Concentration and composition of milk of females on ad libitum and restricted water intakes. Comparative Biochemistry and Physiology 53A,4752 Beth M, Berger M, Aksoy M & Schmahl 0 (1987) Comparison between the effects of dietary fat level and of calorie intake on methylnitrosoureainduced mammary carcinogenesis in female SO rats. International Journal of Cancer 39, Carroll K & Khor H (1971)Effects of level and type of dietary fat on incidence of mammary tumors induced in female SpragueDawley rats by 7,12dimethylbenz(a)anthracene. Lipids 6, Haberman B (1974) Mechanical milk collection from mice for bittner virus isolation. Laboratory Animal Science 24, Keen C, Lonnerdal B, Clegg M & Hurley L (1981) Developmental changes in composition of rat milk: Trace elements, minerals, protein, carbohydrate and fat. Journal of Nutrition 111, Knight C, Maltz E & Docherty A (1986) Milk yield and composition in mice: Effects of litter size and lactation number. Comparative Biochemistry and Physiology 84A, Nagasawa H, Naito T & Kataoka K (t989) Relationship between milk composition and pup's growth in mice. Proceedings of the Society for Experimental Biology and Medicine 191, 7881 Silverman J, Powers J, Stromberg P & Kent S (1989) Effects on C3H mouse mammary cancer of changing from a high fat to a low fat diet before, at, or after puberty. Cancer Research 49, Silverman J & Powers J (1991) Body weights of C3H/HeN mice fed semipurified or commercial diets of different fat content. Nutrition and Cancer 15, Silverstone H & Tannenbaum A (1950) The effect of the proportion of dietary fat on the rate of formation of mammary carcinoma in mice. Cancer Research 10, Smith S, Gagne H, Pite]ka 0 & Abraham S (1969) The effect of dietary fat on lipogenesisin mammary gland and liverfrom lactating and virgin mice. Biochemical Journal 115, Sukhija P & Palmquist 0 (1988) Rapid method for determination of total fatty acid content and composition of feedstuffs and feces. Journal of Agricultural and Food Chemistry 36, Wheatley B, Green 1 & Green M (1981) Effect of dietary fat and cholesterol on milk composition, milk intake and cholesterol metabolism in the rabbit. Journal of Nutrition 111,