Camp. Biochem.
Physiol.
Vol.98A,
No. 314, pp. 535-542,
0300-9629/91
1991
$3.00 + 0.00
0 1991Pergamon Press plc
Printed in Great Britain
MILK SECRETION IN THE RAT: PROGRESSIVE CHANGES IN MILK COMPOSITION DURING LACTATION AND WEANING AND THE EFFECT OF DIET KEVINR. NICHoLAs*t and PETERE. HARTMANN Department of Biochemistry, University of Western Australia, Nedlands, W.A. 6009, Australia; and *Division of Wildlife and Ecology, CSIRO, PO Box 84, Lyneham, ACT. 2602, Australia, Telephone: (06) 242-1600, Fax; (06) 241-3343 (Received 26 June 1990) Abstract-l. Progressive changes in the composition of milk from rats has been studied from day 0 to 20 of lactation and for 3 days following separation of the dams and pups at day 20 post partum. 2. The changes in concentration of Na, K and lactose suggested that secretion both prepartum and following weaning occurred by a paracellular mechanism whereas a transcellular pathway existed during established lactation. 3. The concentration of total protein and casein increased gradually throughout lactation. In contrast, the concentration of serum albumin increased and transferrin decreased markedly during early lactation. The fat content of milk declined 3-fold within 5 days of birth but the concentration of Ca, Mg and inorganic P increased. The concentration of each of these milk constituents remained constant during established lactation. 4. Following weaning the pronounced decline in lactose, K and inorganic P was negatively correlated with an increase in all other milk constituents except fat. 5. Rats fed a low energy diet produced milk with a lower fat content but with an unaltered concentration of protein and carbohydrate. The growth rate of these litters was similar for the first 5 days of lactation when compared to litters from dams fed a high energy diet. The growth rate of litters thereafter and following weaning was greater for rats fed a high energy diet.
Hartmann et al., 1985). In view of the intensive use of the rat as a model for the study of lactation it is surprising that there is a lack of comprehensive analysis of the relative changes in the concentration of milk constituents throughout the lactation cycle (Luckey et ul., 1954; Chalk and Bailey, 1979; Keen et al., 1981; Godbole et al., 1981). This may reflect the difficulty in obtaining samples since. the volume of milk required for comprehensive analysis precludes the possibility of intensive sequential sampling and often results in analysis of milk at only a few time points. Indeed, Keen et al. (1980) have provided evidence to show that sequential sampling leads to erroneous values for the concentration of Zn in milk. This paper describes the progressive changes in the major milk constituents in rats during the first 20 days of lactation and for 3 days following the cessation of suckling at 20 days post partum. These changes are discussed in terms of what is known about the mechanisms of milk secretion in other species. In addition, the effects of energy content of the diet on milk composition and growth rate of the young before and after weaning is assessed.
INTRODUCTION The study of progressive changes in milk composition during initiation, maintenance and cessation of lactation can provide useful information on mammary gland metabolism and the process of secretion. For example, the differences in the relationships between the concentration of Na, K and lactose in milk during the initiation and cessation of lactation compared to established lactation suggests that different mechanisms of secretion operate during these stages of the lactation cycle (see Linzell and Peaker, 197 1a; Peaker, 1975, 1983). Additional factors also can contribute to changes in milk composition. For example, it is now clear that variation in the amount of food and quality of the diet can have profound influences on the composition of the major milk constituents and the rate at which milk is produced (Grigor and Warren, 1980; Grigor and Gain, 1983; Sampson and Jansen, 1984; Hartmann et al., 1985; Grigor et al., 1987; Grigor and Thompson, 1987; Geursen et al., 1987). The majority of studies on changes in milk composition have been limited to large domestic and laboratory animals such as the cow, goat, rabbit and guinea pig, and to the woman (Cowie, 1969; Peaker and Taylor, 1975; Peaker et al., 1975; Kulski and Hartmann, 1981; Peaker, 1983; Prosser et al., 1984;
MATERIALS
tAddress all corresnondence . Nicholas. _.._..-.-_, ___... R. ______r--------- to: Dr Kevin - ------- -_. Division of Wildlife and Ecology, CSIRO, PO Box 84, Lyneham ACT 2602, Australia.
AND METHODS
Materials Lactase (grade I 1, from Sacchuromyces fiugilis), peroxidase (type lj from_ __-.___-_____,, horseradish). ______ nlucose oxidase (tvne 11. \-,r- --, from Aspergillis niger) and tributyrin (glyceryl tributyrate, grade 1, approx. 99%) were obtained from the Sigma
535
KEVINR. NICHOLASand
536
Table 1. Comoosition of animal diets Fat Carbohydrate Protein Fibre Ca P Ash Moisture Metabolizable energy (kJ/a)
Diet A
Diet B
4.0 43.0 ii.0 5.0 1.3 0.9 5.0 9.0
1.5 56.3 i9.0 2.0 0.6 0.6 6.25 9.0
11.93
15.02
Analysis of the diets was provided by the companies from which the food was purchased. Metabolizable energy was calculated (Davidson er al., 1975)from the fat, carbohydrate and protein content of each diet.
Chemical Co. (St. Louis, MO). Oxytocin (Pitocin) was purchased from Park-Davis and Co. (Sydney, Australia). Animals
Rats were from the Wistar albino strain of Rarru.r norwegicus. Milk samples were expressed at O-5, 8, 10, 15, and 20 days of lactation and at days 1, 2 and 3 after separation of the pups and dam at day 20. During lactation the dam was separated from the litter for 0.5-l .Ohr, administered 0.1 iu of oxytocin (SC) and a milk sample collected within 5min under light ether anaesthesia. Milk was collected at day 0 within 2 hr of parturition. Dams which had been weaned also received 0.1 iu of oxytocin before milking. No rat was milked on more than one occasion. Whenever possible each milk sample was analysed for all parameters studied. The influence of diet on milk composition was studied in rats fed the 2 diets shown in Table 1. Diet A (Westfeeds Ptv Ltd, Perth, Western Australia) was formulated for rats. Diet B (W. H. Milne and Co Pty Ltd, Perth, Western Australia) was formulated as a creep ration for piglets. This latter diet was chosen as it represented the only other suitable commercial diet available which appeared to meet the nutritional requirements of the rat. Rats were maintained on each diet during mating, pregnancy and for the first 20 days of lactation. Litters weaned at day 20 continued with the same diet as their respective dams. The number and weight of pups in each litter was recorded. In addition, the food consumption (g food consumed/kg body wt/24 hr) of adult male rats fed each diet was assessed during a 7 day trial.
PETER
E. HARTMANN
water, dissolved by adjusting the pH to 8.0 and the phosphorus content determined as above. Protein content of the pellet was estimated by the method of Lowry et al. (1951) using crystalline lyophilized bovine serum albumin as standard, The concentration of casein in milk was calculated from the determination of protein phosphorus and assuming the phosphorus content of casein was 1.61 + 0.15% (mean f SD, n = 7). Inorganic phosphorus was measured in the protein free supematant and the method was similar to that for protein phosphorus except that the digestion procedure was omitted and all tubes (including standards) were extracted with one volume of ether before the absorbance was measured. (5) Serrun albumin and transferrin. Milk samples were diluted in 0.9% (w/v) NaCl and the concentration of serum albumin and transferrin determined by single radial immunodiffusion (Mancini er al., 1965). The serum albumin, transferrin and the corresponding antisera were kindly provided by Professor E. H. Morgan (Department of Physiology, University of Western Australia). (6) Zen confenr. Sodium and potassium were determined by flame photometry and calcium and magnesium by atomic absorption spectrophotometry. (7) Milk energy content. The metabolizable energy was calculated (Davidson et al., 1975) from the fat, carbohydrate and protein content of the milk. RESULTS (1) Changes in milk composition during the first 20 days of lactation Lactose, potassium and sodium. The concentration
of lactose remained relatively constant at approximately 73 mM during the first 5 days of lactation and then increased to a maximum value of 126 mM at 15 days (Fig. 1). There was a significant negative correlation (r = -0.42, P < 0.02, n = 30) between the increase in potassium and decline in sodium concentration during the first 5 days of lactation.
Milk analyses
(1) Lactose. Milk samples were diluted in water and assayed enzymatically as described previously (Nicholas et al., 1981). (2) Fa;. Milk fat was estimated as total esterified fatty acid by the method of Stem and Shapiro (1953). A standard curve of tributyrin was included with each set of detenninations. Milk fat (triacylglycerols) was calculated by assuming the average molecular weight of fatty acids in rat milk to be 245 (Glass et al., 1967). (3) Protein. Total protein nitrogen was determined by a micro-Kjeldahl method using the distillation apparatus designed by Leurquin and Delville (1950). Milk protein was calculated as N x 6.38. No correction was applied for non-protein nitrogen. (4) Casein and inorganic phosphorus. Casein was estimated as protein phosphorus by the method of Zilversmit and Davis (1950). The phosphorus content of casein was estimated in a total of seven milk samples collected at 5, 10, 15 and 20 days of lactation. Milk was centrifuged at 3000g for 10 min to remove fat and cell debris. The supematant was centrifuged at 45,000g for 2 hr at 4°C and the casein pellet retained and washed in water. The pellet was suspended in
o0
5
10
15
2c
tdays) Fig. 1. Changes in the concentration of lactose, sodium and potassium in milk during the first 20 days of lactation. The concentration of Na and K (flame photometry) and lactose was determined in each milk sample. Each value represents the mean + SEM for four to seven observations. TIME AFTER PARTURITION
537
Milk secretion in the rat
10 -
I
L 0
0
5
10
2( 1
15
TIME AFTER PARTURITION (days1 3 rhcanaee in the mnrentrst;nn V. nf tntal .Fio .e. 1. _.L..‘.b” ..1 .I._ ~“I.-_...L...L”.. .“I... nrntpin p”.-*.., carpin ‘Y”_.l.,
serum albumin and transferrin in milk during the first 20 days of lactation. The concentration of total protein was measured by the Kjeldahl method. Casein was measured as protein phosphorus by the method of Zilversmit and Davis (1950) and by assuming the phosphorus content of casein was 1.61%.Transferrin and serum albumin were estimated by single radial immunodiffusion as described by Mancini er ol. (1965).
or 5
10
TIME AFTER PARTURITION
15
20
(days)
Fig. 3. Changes in the concentration of fat in milk during the first 20 days of lactation. Fat was estimated as total esteritied fatty acid using the method of Stem and Shapiro (1953). Milk fat (triglyceride) was calculated by assuming the average molecular weight of fatty acids in rat milk to be 245 (Glass er al., 1967). Each point represents the mean f SEM for four to eight observations.
5 10 15 TIME AFTER PARTURITION(days1
2(
Fig. 4. Changes in the concentration of calcium, magnesium and inorganic phosphorus in milk during the first 20 days of lactation. The concentration of Ca and Mg was determined by atomic absorption spectrophotometry in each milk sample. The concentration of inorganic phosphorus was measured by the method of Zilversmit and Davis (1950). Each point represents the mean + SEM for four to
Thereafter the concentration of both sodium and potassium decreased gradually until day 20 of lactation. During this time there was a significant negative correlation (P < 0.01) between the concentration of lactose and potassium (r = -0.54, n = 22) and lactose and sodium (r = -0.57, n = 22). Total protein, casein, serum albumin and transferrin. A slight decrease (approx. 2 g/100 ml) in total protein from 8 to 6 g/ 100 ml during the first 2 days after birth was followed by a gradual increase to constant levels of 7-9g/lOOml for the remainder of lactation (Fig. 2). Casein concentration increased gradually from 2.5 g/100 ml at birth to a maximum of 4 g/100 ml at day 20 of lactation. In contrast, the changes in the concentration of transferrin and serum albumin were more pronounced. Serum albumin increased 2-fold in the first 2 days after birth but remained constant for the remaining 18 days. On the other hand, the concentration of transferrin decreased l-fold during the first day of lactation, remained constant until day IO and then increased to reach a maximum concentration on day 20. Fat. The concentration of fat (triacylglycerols) in milk declined 3-fold during the first 5 days post pat-turn and then remained constant for the remaining 15 days (Fig. 3). Calcium, magnesium and inorganic phosphorus. The level of Ca increased abruptly from 25 mM to 50 mM in the 24 hr after birth but thereafter more gradually to a maximum (70 mM) at mid-lactation (Fig. 4). The pattern of changes in concentration of inorganic P was similar to that for Ca. An initial increase from about 20 to 100 mM within the first 2 days of lactation was followed by relatively constant levels for the remainder of lactation. The concentration of Mg increased from approximately 17 to 25 mM
KEVIN
538
R.
NICHOLAS
and
PETER
E. HARTMANN
80 60 40 20
i
125 1
y/h4 Inorganic
P
P4
:
P- --__
--__
Ca
4
Fat
-
I
15
I
I
201
1
c
(
2 3
1
15
LACTATlONMlEANlNG
.
201
1
1
I
2 3
(days)
Fig. 5. Progressive changes in milk composition for 3 days after the cessation of suckling at day 20 of lactation. The dam and litter were separated at day 20 of lactation and milk samples collected on the subsequent 3 days. Each value represents the mean + SEM for four or five observations. during the first 5 days of lactation but thereafter did not change significantly. (2) Changes in milk composition after the cessation of suckling at day 20 of lactation The change in concentration of milk constituents during the cessation of lactation is shown in Fig. 5. Lactose, potassium and sodium. The concentration of lactose declined abruptly from 117 mM at day 20 of lactation to 15 mM 3 days after separation of the dam and the litter. In the corresponding time interval the concentration of K decreased 4-fold whereas the concentration of Na increased 2-fold. There were significant (P < 0.001) negative correlations between the concentration of lactose and Na (r = -0.87, n = 17) and K and Na (r = -0.82, n = 17) and a significant (P < 0.001) positive correlation between the concentration of lactose and K (r = 0.8 1, n = 17). Total protein, casein, transferrin and serum albumin. The concentration of total protein declined from 7.8 to 6.7 g/lOOml after the pups and dam had been separated for 1 day but thereafter the concentration increased markedly to 13 g/lOOml at day 3. The casein content of milk did not change significantly in the first 24 hr but doubled from 4.5 to 9.0 g/100 ml during the following two days. In contrast, the concentration of transferrin and serum albumin in-
creased 43% and 20% respectively in the first 24 hr after weaning and the concentration of both proteins increased further on each of the next 2 days. There were significant (r < -0.72, P < 0.001, n = 17) negative correlations between the concentration of lactose and the concentration of total protein, casein, serum albumin and transferrin. Fat. The concentration of fat in milk collected after the cessation of suckling did not change significantly from the concentration (11 g/l00 ml) at day 20 of lactation. There was no significant correlation between the concentration of fat and lactose. Calcium, magnesium and inorganic phosphorus. The concentration of Ca in milk increased abruptly from 60 mM to 90 mM during the first 2 days after the removal of the pups but remained constant for the next 24 hr. On the other hand, the concentration of inorganic P declined from 120 mM to 75 mM after 24 hr and remained relatively constant for the next 2 days. Magnesium concentration increased only slightly from 9.5 mM at day 20 of lactation to 11.2 mM after separation of the dam and litter for 2 days. (3) Assessment of diet Milk composition. The effects of diets A and B on the concentration of the major milk constituents during lactation is shown in Table 2. Whereas there
Milk secretion in the rat
539
Table 2. Effect of diet on milk comwsition
Fat
LXt0Se
Diet
A
B
Protein
A
B
A
B
Lactation (Day) 0 5 IO I5 20
2.56 k 0.1 I (3) 2.47 + 0.1 I (5) 3.21kO.29 (5) 4.31kO.21 (4) 4.04+ 0.48(4)
2.22 f 0.09 (5) 23.19 + 3.2 (5) 2.79 f 0.15 (5) l7.65 + 2.1(5) 3.51kO.16 (5) l8.% + 1.8(5) 4.30kO.18 (4) *IO.93f 1.7(8) 4.41f 0.27(5) 11.06a2.9 (4)
28.9 f 0.61 (5) 14.59f 0.48(5) 16.69k 1.46(5) 18.56kO.38(4) 13.10~0.71(5)
7.75 f 0.82 (5) 6.95f 0.42(5) 8.21kO.40 (5) 9.19kO.85 (4) 8.2820.28 (4)
8.18 20.35 (5) 7.002 0.18(5) 8.18kO.17 (5) 9.30f 0.41(4) 9.57f 0.32(5)
Rats were maintained on each diet during mating, pregnancy and lactation. Each vale nzpresents mean + SEM (g/100 ml) with the number of milk samples analysed shown in parentheses. The valuea obtained were analysed by a one-way analysis of variance. l, indicates values were significantly dilTercnt at the 5% level.
was no prolonged effect of diet on either protein or carbohydrate, milk from rats fed diet B had an elevated level of fat at days 5, IO and I5 of lactation. Body weight of pups during lactation and after weaning. The change in the mean body weight of pups
which suckled from dams fed each of the diets A and B is shown for the first 20 days of lactation in Fig. 6a. The mean body weight increased from approximately 5.5 g at birth to 9.0 g at day 5 of lactation in both experimental groups. However, from day 5 to day 20 of lactation the rate of gain in body weight was greater in the pups from dams fed diet B. The increase in body weight after the pups were removed from the dam at day 20 of lactation is seen in Fig. 6b. The weaned pups were maintained on the same diet as their respective dams. The body weight of rats fed diet B increased 3.5-fold after 23 days whereas the body weight of animals fed diet A increased 3.1 fold. Energy content. The energy content of milk expressed from rats fed diet A and B was 606 and 718 mJ/lOO ml, respectively, at day 10 and 523 and 810 mJ/lOO ml, respectively, at day 20 of lactation. Food consumption.The main food consumption (g food consumed/kg body weight/24 hr) in two groups of 3 adult rats, one group fed diet A and the other group fed diet B for a 7 day trial was 91.5 + 5.4 and 55.3 + 3. I (mean + SEM) respectively. This food consumption represents 1091.6 kJ and 830.6 kJ of metabolizable energy for rats consuming diet A and diet B respectively.
The transition from colostrum to milk is characterized by a number of compositional changes in the mammary secretion. The increase in the concentration of K and lactose and decline in the concentration of Na in the mammary secretion from 12 hr before (Nicholas and Hartmann, 1981) to 48 hr after parturition in the rat is consistent with the change from colostrum to milk over this period observed in other species (Peaker, 1975). The 2-fold increase in concentration of serum albumin in milk within 2 days of parturition in the rat is similar to that reported by Geursen and Grigor (1987) and is in contrast to the decline observed during the transition from colostrum to milk in the
(a)
30 -
20-
DlSCUS!SlON
Recent studies have stressed that the dose of oxytocin administered and the time interval between removal of the young and collection of the milk sample must be carefully controlled in order to obtain a physiologically represenative sample. Several studies in the past have reported separating the dam and pups for 4-12 hr and the administration of 0.3-2.5 units of oxytocin prior to collection of milk samples. Oxytocin administration to goats (Linzell and Peaker, 1971b) and rabbits (Linzell et al., 1975) has been shown to evoke a rapid change (less than 60 min) in milk composition and the response was accentuated following administration of increased levels of the hormone. It should be emphasized that in the present study rats were separated from the pups for OS-l.0 hr and were administered only 0.1 iu of oxytocin 5 min before a milk sample was collected, ensuring a sample accurately reflecting the physiological status of the mammary gland.
I1 2o 0
I
t
I
I
20 TIME AFTER PARTURITtONIWEANINQ(days) 5
10
15
Fig. 6. The effect of diet on the rate of gain in body weight of pups during the first 20 days of lactation and after weaning. (a) Lactation. Each value represents the mean f SEM for 3-7 litters. The litter size was 10.5 f 0.3 (50) and 9.8 f 0.4 (47) (mean f SEM) from rats fed diet A (0-e) and diet B (A---A) respectively. (b) Weating. Litters were separated from the dams at day 20 of lactation and the pups divided into groups of 10 animals. Each group continued to feed from the diet previously fed to their respective dams. The values from rats fed diet A (@-a) and diet B (A---A) represent the mean f SEM for three groups of rats and the individual values from two groups of rats respectively.
540
KEVINR. NICHOLAS and
cow (Schanbacher and Smith, 1975) and woman (Kulski and Hartmann, 1981). In these species the post partum decline in serum albumin concentration in milk is negatively correlated to the change in the concentration of lactose and probably results from the dilution by newly synthesized milk. The mRNA for serum albumin has not been detected in the rat mammary gland (Geursen and Grigor, 1987) and consequently the concentration of serum albumin in milk can be used as an index of diffusion from the blood, reflecting the integrity of the permeability barrier between the extracellular fluid and the milk. Therefore, the increase in both the synthesis of lactose and the accumulation of serum albumin in the mammary secretion in early lactation suggests that a specific mechanism for the transport of serum albumin from the extracellular fluid to the milk may become operational in the rat after parturition. In contrast to serum albumin the concentration of transferrin in milk declined after parturition and remained low until day 10, after which it increased to day 20. These changes are consistent with those reported by Grigor et al. (1988) and Jorden and Morgan (1967). Studies in vitro have shown that rat mammary tissue will incorporate 14C-leucine into a protein which will precipitate with an antiserum specific for serum transferrin (Grigor et al., 1988; Jorden and Morgan, 1969). The respective contributions of transferrin to the milk from the mammary gland and the serum throughout lactation remains to be determined. However, Grigor et al. (1988) have observed low levels of accumulation of transferrin mRNA in the mammary gland early in lactation with a significant increase late in lactation. During established lactation in the rat (day 5 to 20) the increase in lactose concentration is negatively correlated with the concentration of both Na and K. A similar result has been supported for the goat (Linzell and Peaker, 197lb), cow (Rook and Wheelock, 1967) and woman (Hartmann and Prosser, 1982) and is consistent with the transfer of lactose and ions between the extracellular fluid and the milk by a transcellular pathway (Linzell and Peaker, 197la; Peaker, 1975, 1983). The concentration of casein in milk increased gradually from approximately 30% of the total protein at term to approximately 50% at day 20 of lactation. Whereas the concentration of casein (3-4g/lOOml) was lower than reported by Cox and Meuller (1937) Luckey et al. (1954) and Dymsza et al. (1964), the concentration of total protein (7-9 g/l00 ml) was similar to the range reported by Luckey et al. (1954), Dymsza et al. (1964), Chalk and Bailey (1979) Godbole et al. (1981) and Keen et al. (1981). The decline in the concentration of fat in early lactation in the rat is in agreement with the report of Luckey et al. (1954) and Godbole et al. (1981) and similar to that observed during early lactation in other Rodentia (Cowie, 1969; Peaker et al., 1975). In contrast, the studies of Chalk and Bailey (1979) showed an increase in the concentration of milk fat during the first 10 days of lactation. However, they also reported that if rats progressed through two additional, successive lactations, the concentration of milk triglyceride did not change. Keen et al. (1981)
PETERE. HARTMANN reported that the concentration of fat in milk did not change significantly throughout lactation. The ratio of the concentration of Ca, Mg and inorganic phosphorus and that for lactose in the milk from rats is in agreement with the reciprocal relationship which exists between lactose and the concentration of salts in other species. For example, species such as women and cows with a high concentration of lactose in the milk have less salt than milks lower in lactose such as in the guinea pig and the rabbit (Peaker et al., 1975; Davies et al., 1983). Thus, a general reciprocal relationship between lactose and salt concentration helps maintain the total osmotic power of milk close to that of blood. The cessation of suckling on day 20 of lactation in the rat preceded an abrupt decline in the concentration of lactose and K together with an increase in concentration of Na in the milk. Similarly, milk expressed from rats at term had an elevated concentration of Na and a decreased concentration of K suggesting that secretion at both these times occurred by the paracellular pathway. This relationship is also observed during involution in the cow (Wheelock et al., 1967) and woman (Hartmann and Kulski, 1978) and is believed to result from changes in the zonulae occludentes which connect neighbouring secretory cells. Consequently, lactose and K pass out of, and Na and Cl into the extracellular fluid through leaky ‘tight’ junctions according to their concentration gradients (see Peaker, 1975, 1976, 1983). Further examples of this paracellular secretion have been reported in late pregnancy in the goat (Linzell and Peaker, 1974) during lactation in the rabbit (Peaker and Taylor, 1975) and the latter stages of lactation in the guinea pig (Peaker et al., 1975). The rapid decline in the concentration of lactose in the milk during the first 3 days after the cessation of suckling was accompanied by an increase in concentration of casein and the whey proteins. This is consistent with the removal of lactose, the major osmole in milk, to the extracellular fluid by the paracellular pathway. The inadequacies by the low energy diet A is reflected in the elevated food consumption of male rats during a 7-day trial and the diminished weight gain of pups after weaning. On the other hand the protein and carbohydrate content of milk from lactating rats fed each of the diets remained essentially unchanged. This is consistent with studies in other species (see Davies et al., 1983) showing that changes in diet, including the energy content, does not have significant effects on protein and carbohydrate content of milk. The major difference between milks in the present study was the concentration of fat which was largely responsible for the variation in energy content. Whereas the fat content of the diet may influence the fatty acid composition of the milk, the effect of energy content of the diet on milk composition is less clear (Davies et al., 1983). Milk yield, which can be estimated indirectly in the rat by measuring the rate of gain in the body weight of the pups (see Cowie and Tindal, 1971), did not differ significantly for the first 5 days of lactation in rats fed each of the diets. However the body weight of pups from dams fed diet B was higher than pups from dams fed diet A at day 20 of lactation. In the
Milk secretion in the rat
latter stages of lactation the young begin to sup plement the mother’s milk with solid food which most likely would contribute to the differences observed in pup weight. These results emphasize the importance of choosing the correct diet with a suitable quality control for animal experimentation. An earlier report has further emphasized this fact showing that rats fed diet A demonstrated an increasing concentration of glucose in the mammary gland just prior to lactogenesis (Nicholas and Hartmann, 1981). However, the concentration of mammary glucose in rats fed diet B remained low throughout the peripartum period. Furthermore, the choice of animal diet is important from an economic point of view since rats consumed twice as much of diet A (a low energy diet) when compared to the other diet. Acknowledgement-This
project was supported by a grant from the National Health and Medical Research Council.
REFERENCES
Chalk P. A. and Bailey E. (1979) Changes in the yield, and carbohydrate, lipid and protein content of milk during lactation in the rat. J. Develop. Physiol. 1, 61-79. Cowie A. T. (1969) Variations in the yield and composition of the milk during lactation in the rabbit and the galactopoietic effect of prolactin. J. Endocr. 44, 437-450. Cowie A. T. and Tindal J. S. (Eds) (1971) The Physiology of Lactation. Edward Arnold Ltd., London. Cox W. M. and Meuller A. J. (1937) The composition of milk from stock rats and an apparatus for milking small laboratory animals. J. Nutr. 13, 249-261. Davidson S., Passmore R. and Brock J. R. (Eds) (1975) Human Nutrition and Dietetics (6th edition), p. IS. Churchill Livingston, London. Davies D. T., Holt C. and Christie W. W. (1983) The composition of milk. In Biochemistry of Lacfarion (Edited by Mepham T. M.), pp. 71-l 17. Elsevier, Amsterdam. Dymsxa N. A., Czajka D. M. and Miller S. A. (1964) Influence of artificial diet on weight gain and body composition of the neonatal rat. J. NW. 84, 100-106. Geursen A., Came A. and Grigor M. R. (1987) Protein synthesis in isolated mammary acini: effect of maternal diet. J. Nulr. 117, 769-775. Geurson A. and Grigor M. R. (1987) Serum albumin secretion in rat milk. J. Physiol. 391, 419-427. Glass R. L., Troolin H. A. and Jenness R. (1967) Comparative biochemical studies of milks-IV. Constituent -fatty acids of milk fats. Come. Biochem. Phvsiol. 22. 415-425. Godbole V. Y., Grundleger M. L., Pasquine T. A. and Thenen S. W. (1981) Composition of rat milk from day 5 to 20 of lactation and milk intake of lean and preobese Zucker pups. J. Nurr. 111, 480-487. Grigor M. R., Allen J. E., Carington J. M., Came A., Geursen A., Young D., Thompson M. P., Coleman R. A. and Haynes E. B. (I 987) Effect of dietary protein and food restriction on milk production and composition, maternal tissues and enzymes in lactating rats. J. Nurr. 117, 1247-1258. Grigor M. R., Carne A.. Geursen A. and Flint D. J. (1988) Effect ofextended lactation and diet on transferrin concentrations in rat milk. J. Nutr. 118, 669674. Grigor M. R. and Gain K. R. (1983) The effect of starvation and refeeding on lipogenic enzymes in the mammary gland and livers of lactating rats. Biochem. J. 216, 515-518.
Grigor M. R. and Thompson M. P. (1987) Diurnal regulation of milk lipid production and milk secretion in the
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rat: effect ofdietary protein and energy restriction. J. Nufr. 117, 748-753. Grigor M. R. and Warren S. M. (1980) Dietary regulation of mammary lipogenesis in lactating rats. Biochem. J. 188, 61-65. Hartmann P. E. and Kulski J. K. (1978) Changes in the composition of the mammary secretion of women after abrupt termination of breast feeding. J. Physiol. (Land) 275, I-II. Hartrnann P. E. and Prosser C. G. (1982) Acute changes in the composition of milk during the ovulatory menstrual cycle in lactating women. J. Ph&iol. (bnd) 324, 21-u). Hartmann P. E.. Rattiaen S.. Saint L. and Suruivana 0. (1985) Variation i the .yield and composition of human milk. Oxford Reviews of Reproductive Biology 7, 118-167. Jorden S. M. and Morgan E. H. (1967) Albumin, transferrin and y-globulin metabolism during lactation in the rat. Q. J. Exp. Physiol. 52, 422429. Jorden S. and Morgan E. H. (l%9) Plasma protein synthesis in tissue slices from pregnant and lactating rats. Biochim. Biophys. Acta 174, 373-379.
Keen C. L., Lonnerdal B., Clegg M. and Hurley L. S. (198 I) Developmental changes in composition of rat milk: trace elements, minerals, protein, carbohydrate and fat. J. Nufr. 111, 226-236. Keen C. L., Lonnerdal B., Sloan M. V. and Hurley L. S. (1980) Effects of milking procedure on rat milk procedure. Physiology and Behaviour 24, 6 13-6 15.
Kulski J. K. and Hartmann P. E. (1981) Changes in human milk composition during the initiation of lactation. AKU. J. Exp. Sci. Med. Sci. 59, 101-114. Leurquin J. and Delville J. P. (1950) Modification de I’appareil a aeration de Van Slyke-Cullen et son adaption au microdosage de I’azote proteique apres Kjeldahlisation et entrainement a de ammoniaque. Experenria 6.274-275. Linzell J. L. and Peaker M. (197la) Mechanism of milk secretion. Physiol. Rev. 51, 564-597. Linzell J. L. and Peaker M. I I97 I b) The effects of oxvtocin and milk removal on milk secretion in the goat. J. Piysiol. 216, 717-734. Linzell J. L. and Peaker M. (1974) Changes in colostrum composition and in the permeability of the mammary epithelium at about the time of parturition in the goat. J. Physiol. 243, 129-151.
Linzell J. L.. Peaker M. and Taylor J. C. (1975) The effects of prolactin and oxytwin on milk secretion and the permeability of the mammary epithelium in the rabbit. J. Physiol. 253, 547-563.
Lowry 0. H., Rosebrough N. F., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Luckey T. D., Mende T. J. and Pleasants T. J. (1954) The physical and chemical characterization of rat’s milk. J. Nutr. 54, 345-359. Mancini G., Carbonara A. 0. and Heremans J. F. (1965) Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2, 235-254. Nicholas K. R. and Hartmann P. E. (1981) Progesterone control of the initiation of lactose synthesis in the rat. AIM. J. Biol. Sci. 34, 435443.
Nicholas K. R., Hartmann P. E. and McDonald B. L. (1981) a-Lactalbumin and lactose concentrations in rat milk during lactation. Biochem. J. 194, 149-154. Peaker M. (1975) Recent advances in the study of monovalent ion movements across the mammary epithelium: relation to onset of lactation. J. Dairy Sci. !t8, 1042-1062. Peaker M. (1976) Lactation: some cardiovascular and metabolic consequences, and the mechanisms of lactose and ion secretion into milk. In Breast Feeding and the Mother [Ciba Foundation Symposium 451, (Lloyd J. K., Chairman), pp. 87-101. Elsevier, Excerpta Mediea, North Holland.
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Peaker M. (1983) Secretion of ions and water. In Eiochemistry of Lactation (Edited by Mepham T. B.), pp. 285-305. &&cr, Amsterdam. Feaker M., Jones C. D. R., Goode J. A. and Linzell J. L. (1975) Changes in milk composition during lactation in the guinea-pig and the effect of prolactin. J. Endow. 67, 307-308. Peaker M. and Tavlor J. C. (1975) Milk secretion in the rabbit: changes during lactaiion and the mechanism of ion transport. J. Physiol. W, 521-545. Prosser C. G., Saint L. and Hartmann P. E. (1984) Mammary gland function during gradual weaning and early gestation in women. Aust. J. Exp. Sci. Med. Sci. 62, 215-228. Rook J. A. F. and Wheelock J. V. (1967) The secretion of water and water-soluble constituents in milk. J. Dairy Sci. 34, 273-287.
Sampson D. A. and Jansen G. R. (1984) Protein and energy nutrition during lactation. Ann. Rev. Mutr. 4,441. Schanbacher F. L. and Smith K. L. (1975) Formation and role of unusual whey proteins and enzymes; relation to mammary function. J. &airy Sci. St& 1048-1062. Stern I. and Shapiro B. (1953) A rapid and simple method for the determin&ion of e&&d-fatty acids-and for total fattv acids in blood. J. C/in. Puthol. 6. IS&-160. Wheeiock J. V., Smith A., Dodd F. H. ahd Lystet P. L. J. (1967) Changes in the quantity and composition of mammary gland secretion in the dry period between lactations-I. The beginning of the dry period. J. Dairy Research 34, l-12.
Zilversmith D. B. and Davis A. K. (1950) Microdetermination of plasma phospholipids by trichloroacetic acid precipitation. J. Lab. Clin. Med. 35, 155-160.
Physiol.
Vol.98A,
No. 314, pp. 535-542,
0300-9629/91
1991
$3.00 + 0.00
0 1991Pergamon Press plc
Printed in Great Britain
MILK SECRETION IN THE RAT: PROGRESSIVE CHANGES IN MILK COMPOSITION DURING LACTATION AND WEANING AND THE EFFECT OF DIET KEVINR. NICHoLAs*t and PETERE. HARTMANN Department of Biochemistry, University of Western Australia, Nedlands, W.A. 6009, Australia; and *Division of Wildlife and Ecology, CSIRO, PO Box 84, Lyneham, ACT. 2602, Australia, Telephone: (06) 242-1600, Fax; (06) 241-3343 (Received 26 June 1990) Abstract-l. Progressive changes in the composition of milk from rats has been studied from day 0 to 20 of lactation and for 3 days following separation of the dams and pups at day 20 post partum. 2. The changes in concentration of Na, K and lactose suggested that secretion both prepartum and following weaning occurred by a paracellular mechanism whereas a transcellular pathway existed during established lactation. 3. The concentration of total protein and casein increased gradually throughout lactation. In contrast, the concentration of serum albumin increased and transferrin decreased markedly during early lactation. The fat content of milk declined 3-fold within 5 days of birth but the concentration of Ca, Mg and inorganic P increased. The concentration of each of these milk constituents remained constant during established lactation. 4. Following weaning the pronounced decline in lactose, K and inorganic P was negatively correlated with an increase in all other milk constituents except fat. 5. Rats fed a low energy diet produced milk with a lower fat content but with an unaltered concentration of protein and carbohydrate. The growth rate of these litters was similar for the first 5 days of lactation when compared to litters from dams fed a high energy diet. The growth rate of litters thereafter and following weaning was greater for rats fed a high energy diet.
Hartmann et al., 1985). In view of the intensive use of the rat as a model for the study of lactation it is surprising that there is a lack of comprehensive analysis of the relative changes in the concentration of milk constituents throughout the lactation cycle (Luckey et ul., 1954; Chalk and Bailey, 1979; Keen et al., 1981; Godbole et al., 1981). This may reflect the difficulty in obtaining samples since. the volume of milk required for comprehensive analysis precludes the possibility of intensive sequential sampling and often results in analysis of milk at only a few time points. Indeed, Keen et al. (1980) have provided evidence to show that sequential sampling leads to erroneous values for the concentration of Zn in milk. This paper describes the progressive changes in the major milk constituents in rats during the first 20 days of lactation and for 3 days following the cessation of suckling at 20 days post partum. These changes are discussed in terms of what is known about the mechanisms of milk secretion in other species. In addition, the effects of energy content of the diet on milk composition and growth rate of the young before and after weaning is assessed.
INTRODUCTION The study of progressive changes in milk composition during initiation, maintenance and cessation of lactation can provide useful information on mammary gland metabolism and the process of secretion. For example, the differences in the relationships between the concentration of Na, K and lactose in milk during the initiation and cessation of lactation compared to established lactation suggests that different mechanisms of secretion operate during these stages of the lactation cycle (see Linzell and Peaker, 197 1a; Peaker, 1975, 1983). Additional factors also can contribute to changes in milk composition. For example, it is now clear that variation in the amount of food and quality of the diet can have profound influences on the composition of the major milk constituents and the rate at which milk is produced (Grigor and Warren, 1980; Grigor and Gain, 1983; Sampson and Jansen, 1984; Hartmann et al., 1985; Grigor et al., 1987; Grigor and Thompson, 1987; Geursen et al., 1987). The majority of studies on changes in milk composition have been limited to large domestic and laboratory animals such as the cow, goat, rabbit and guinea pig, and to the woman (Cowie, 1969; Peaker and Taylor, 1975; Peaker et al., 1975; Kulski and Hartmann, 1981; Peaker, 1983; Prosser et al., 1984;
MATERIALS
tAddress all corresnondence . Nicholas. _.._..-.-_, ___... R. ______r--------- to: Dr Kevin - ------- -_. Division of Wildlife and Ecology, CSIRO, PO Box 84, Lyneham ACT 2602, Australia.
AND METHODS
Materials Lactase (grade I 1, from Sacchuromyces fiugilis), peroxidase (type lj from_ __-.___-_____,, horseradish). ______ nlucose oxidase (tvne 11. \-,r- --, from Aspergillis niger) and tributyrin (glyceryl tributyrate, grade 1, approx. 99%) were obtained from the Sigma
535
KEVINR. NICHOLASand
536
Table 1. Comoosition of animal diets Fat Carbohydrate Protein Fibre Ca P Ash Moisture Metabolizable energy (kJ/a)
Diet A
Diet B
4.0 43.0 ii.0 5.0 1.3 0.9 5.0 9.0
1.5 56.3 i9.0 2.0 0.6 0.6 6.25 9.0
11.93
15.02
Analysis of the diets was provided by the companies from which the food was purchased. Metabolizable energy was calculated (Davidson er al., 1975)from the fat, carbohydrate and protein content of each diet.
Chemical Co. (St. Louis, MO). Oxytocin (Pitocin) was purchased from Park-Davis and Co. (Sydney, Australia). Animals
Rats were from the Wistar albino strain of Rarru.r norwegicus. Milk samples were expressed at O-5, 8, 10, 15, and 20 days of lactation and at days 1, 2 and 3 after separation of the pups and dam at day 20. During lactation the dam was separated from the litter for 0.5-l .Ohr, administered 0.1 iu of oxytocin (SC) and a milk sample collected within 5min under light ether anaesthesia. Milk was collected at day 0 within 2 hr of parturition. Dams which had been weaned also received 0.1 iu of oxytocin before milking. No rat was milked on more than one occasion. Whenever possible each milk sample was analysed for all parameters studied. The influence of diet on milk composition was studied in rats fed the 2 diets shown in Table 1. Diet A (Westfeeds Ptv Ltd, Perth, Western Australia) was formulated for rats. Diet B (W. H. Milne and Co Pty Ltd, Perth, Western Australia) was formulated as a creep ration for piglets. This latter diet was chosen as it represented the only other suitable commercial diet available which appeared to meet the nutritional requirements of the rat. Rats were maintained on each diet during mating, pregnancy and for the first 20 days of lactation. Litters weaned at day 20 continued with the same diet as their respective dams. The number and weight of pups in each litter was recorded. In addition, the food consumption (g food consumed/kg body wt/24 hr) of adult male rats fed each diet was assessed during a 7 day trial.
PETER
E. HARTMANN
water, dissolved by adjusting the pH to 8.0 and the phosphorus content determined as above. Protein content of the pellet was estimated by the method of Lowry et al. (1951) using crystalline lyophilized bovine serum albumin as standard, The concentration of casein in milk was calculated from the determination of protein phosphorus and assuming the phosphorus content of casein was 1.61 + 0.15% (mean f SD, n = 7). Inorganic phosphorus was measured in the protein free supematant and the method was similar to that for protein phosphorus except that the digestion procedure was omitted and all tubes (including standards) were extracted with one volume of ether before the absorbance was measured. (5) Serrun albumin and transferrin. Milk samples were diluted in 0.9% (w/v) NaCl and the concentration of serum albumin and transferrin determined by single radial immunodiffusion (Mancini er al., 1965). The serum albumin, transferrin and the corresponding antisera were kindly provided by Professor E. H. Morgan (Department of Physiology, University of Western Australia). (6) Zen confenr. Sodium and potassium were determined by flame photometry and calcium and magnesium by atomic absorption spectrophotometry. (7) Milk energy content. The metabolizable energy was calculated (Davidson et al., 1975) from the fat, carbohydrate and protein content of the milk. RESULTS (1) Changes in milk composition during the first 20 days of lactation Lactose, potassium and sodium. The concentration
of lactose remained relatively constant at approximately 73 mM during the first 5 days of lactation and then increased to a maximum value of 126 mM at 15 days (Fig. 1). There was a significant negative correlation (r = -0.42, P < 0.02, n = 30) between the increase in potassium and decline in sodium concentration during the first 5 days of lactation.
Milk analyses
(1) Lactose. Milk samples were diluted in water and assayed enzymatically as described previously (Nicholas et al., 1981). (2) Fa;. Milk fat was estimated as total esterified fatty acid by the method of Stem and Shapiro (1953). A standard curve of tributyrin was included with each set of detenninations. Milk fat (triacylglycerols) was calculated by assuming the average molecular weight of fatty acids in rat milk to be 245 (Glass et al., 1967). (3) Protein. Total protein nitrogen was determined by a micro-Kjeldahl method using the distillation apparatus designed by Leurquin and Delville (1950). Milk protein was calculated as N x 6.38. No correction was applied for non-protein nitrogen. (4) Casein and inorganic phosphorus. Casein was estimated as protein phosphorus by the method of Zilversmit and Davis (1950). The phosphorus content of casein was estimated in a total of seven milk samples collected at 5, 10, 15 and 20 days of lactation. Milk was centrifuged at 3000g for 10 min to remove fat and cell debris. The supematant was centrifuged at 45,000g for 2 hr at 4°C and the casein pellet retained and washed in water. The pellet was suspended in
o0
5
10
15
2c
tdays) Fig. 1. Changes in the concentration of lactose, sodium and potassium in milk during the first 20 days of lactation. The concentration of Na and K (flame photometry) and lactose was determined in each milk sample. Each value represents the mean + SEM for four to seven observations. TIME AFTER PARTURITION
537
Milk secretion in the rat
10 -
I
L 0
0
5
10
2( 1
15
TIME AFTER PARTURITION (days1 3 rhcanaee in the mnrentrst;nn V. nf tntal .Fio .e. 1. _.L..‘.b” ..1 .I._ ~“I.-_...L...L”.. .“I... nrntpin p”.-*.., carpin ‘Y”_.l.,
serum albumin and transferrin in milk during the first 20 days of lactation. The concentration of total protein was measured by the Kjeldahl method. Casein was measured as protein phosphorus by the method of Zilversmit and Davis (1950) and by assuming the phosphorus content of casein was 1.61%.Transferrin and serum albumin were estimated by single radial immunodiffusion as described by Mancini er ol. (1965).
or 5
10
TIME AFTER PARTURITION
15
20
(days)
Fig. 3. Changes in the concentration of fat in milk during the first 20 days of lactation. Fat was estimated as total esteritied fatty acid using the method of Stem and Shapiro (1953). Milk fat (triglyceride) was calculated by assuming the average molecular weight of fatty acids in rat milk to be 245 (Glass er al., 1967). Each point represents the mean f SEM for four to eight observations.
5 10 15 TIME AFTER PARTURITION(days1
2(
Fig. 4. Changes in the concentration of calcium, magnesium and inorganic phosphorus in milk during the first 20 days of lactation. The concentration of Ca and Mg was determined by atomic absorption spectrophotometry in each milk sample. The concentration of inorganic phosphorus was measured by the method of Zilversmit and Davis (1950). Each point represents the mean + SEM for four to
Thereafter the concentration of both sodium and potassium decreased gradually until day 20 of lactation. During this time there was a significant negative correlation (P < 0.01) between the concentration of lactose and potassium (r = -0.54, n = 22) and lactose and sodium (r = -0.57, n = 22). Total protein, casein, serum albumin and transferrin. A slight decrease (approx. 2 g/100 ml) in total protein from 8 to 6 g/ 100 ml during the first 2 days after birth was followed by a gradual increase to constant levels of 7-9g/lOOml for the remainder of lactation (Fig. 2). Casein concentration increased gradually from 2.5 g/100 ml at birth to a maximum of 4 g/100 ml at day 20 of lactation. In contrast, the changes in the concentration of transferrin and serum albumin were more pronounced. Serum albumin increased 2-fold in the first 2 days after birth but remained constant for the remaining 18 days. On the other hand, the concentration of transferrin decreased l-fold during the first day of lactation, remained constant until day IO and then increased to reach a maximum concentration on day 20. Fat. The concentration of fat (triacylglycerols) in milk declined 3-fold during the first 5 days post pat-turn and then remained constant for the remaining 15 days (Fig. 3). Calcium, magnesium and inorganic phosphorus. The level of Ca increased abruptly from 25 mM to 50 mM in the 24 hr after birth but thereafter more gradually to a maximum (70 mM) at mid-lactation (Fig. 4). The pattern of changes in concentration of inorganic P was similar to that for Ca. An initial increase from about 20 to 100 mM within the first 2 days of lactation was followed by relatively constant levels for the remainder of lactation. The concentration of Mg increased from approximately 17 to 25 mM
KEVIN
538
R.
NICHOLAS
and
PETER
E. HARTMANN
80 60 40 20
i
125 1
y/h4 Inorganic
P
P4
:
P- --__
--__
Ca
4
Fat
-
I
15
I
I
201
1
c
(
2 3
1
15
LACTATlONMlEANlNG
.
201
1
1
I
2 3
(days)
Fig. 5. Progressive changes in milk composition for 3 days after the cessation of suckling at day 20 of lactation. The dam and litter were separated at day 20 of lactation and milk samples collected on the subsequent 3 days. Each value represents the mean + SEM for four or five observations. during the first 5 days of lactation but thereafter did not change significantly. (2) Changes in milk composition after the cessation of suckling at day 20 of lactation The change in concentration of milk constituents during the cessation of lactation is shown in Fig. 5. Lactose, potassium and sodium. The concentration of lactose declined abruptly from 117 mM at day 20 of lactation to 15 mM 3 days after separation of the dam and the litter. In the corresponding time interval the concentration of K decreased 4-fold whereas the concentration of Na increased 2-fold. There were significant (P < 0.001) negative correlations between the concentration of lactose and Na (r = -0.87, n = 17) and K and Na (r = -0.82, n = 17) and a significant (P < 0.001) positive correlation between the concentration of lactose and K (r = 0.8 1, n = 17). Total protein, casein, transferrin and serum albumin. The concentration of total protein declined from 7.8 to 6.7 g/lOOml after the pups and dam had been separated for 1 day but thereafter the concentration increased markedly to 13 g/lOOml at day 3. The casein content of milk did not change significantly in the first 24 hr but doubled from 4.5 to 9.0 g/100 ml during the following two days. In contrast, the concentration of transferrin and serum albumin in-
creased 43% and 20% respectively in the first 24 hr after weaning and the concentration of both proteins increased further on each of the next 2 days. There were significant (r < -0.72, P < 0.001, n = 17) negative correlations between the concentration of lactose and the concentration of total protein, casein, serum albumin and transferrin. Fat. The concentration of fat in milk collected after the cessation of suckling did not change significantly from the concentration (11 g/l00 ml) at day 20 of lactation. There was no significant correlation between the concentration of fat and lactose. Calcium, magnesium and inorganic phosphorus. The concentration of Ca in milk increased abruptly from 60 mM to 90 mM during the first 2 days after the removal of the pups but remained constant for the next 24 hr. On the other hand, the concentration of inorganic P declined from 120 mM to 75 mM after 24 hr and remained relatively constant for the next 2 days. Magnesium concentration increased only slightly from 9.5 mM at day 20 of lactation to 11.2 mM after separation of the dam and litter for 2 days. (3) Assessment of diet Milk composition. The effects of diets A and B on the concentration of the major milk constituents during lactation is shown in Table 2. Whereas there
Milk secretion in the rat
539
Table 2. Effect of diet on milk comwsition
Fat
LXt0Se
Diet
A
B
Protein
A
B
A
B
Lactation (Day) 0 5 IO I5 20
2.56 k 0.1 I (3) 2.47 + 0.1 I (5) 3.21kO.29 (5) 4.31kO.21 (4) 4.04+ 0.48(4)
2.22 f 0.09 (5) 23.19 + 3.2 (5) 2.79 f 0.15 (5) l7.65 + 2.1(5) 3.51kO.16 (5) l8.% + 1.8(5) 4.30kO.18 (4) *IO.93f 1.7(8) 4.41f 0.27(5) 11.06a2.9 (4)
28.9 f 0.61 (5) 14.59f 0.48(5) 16.69k 1.46(5) 18.56kO.38(4) 13.10~0.71(5)
7.75 f 0.82 (5) 6.95f 0.42(5) 8.21kO.40 (5) 9.19kO.85 (4) 8.2820.28 (4)
8.18 20.35 (5) 7.002 0.18(5) 8.18kO.17 (5) 9.30f 0.41(4) 9.57f 0.32(5)
Rats were maintained on each diet during mating, pregnancy and lactation. Each vale nzpresents mean + SEM (g/100 ml) with the number of milk samples analysed shown in parentheses. The valuea obtained were analysed by a one-way analysis of variance. l, indicates values were significantly dilTercnt at the 5% level.
was no prolonged effect of diet on either protein or carbohydrate, milk from rats fed diet B had an elevated level of fat at days 5, IO and I5 of lactation. Body weight of pups during lactation and after weaning. The change in the mean body weight of pups
which suckled from dams fed each of the diets A and B is shown for the first 20 days of lactation in Fig. 6a. The mean body weight increased from approximately 5.5 g at birth to 9.0 g at day 5 of lactation in both experimental groups. However, from day 5 to day 20 of lactation the rate of gain in body weight was greater in the pups from dams fed diet B. The increase in body weight after the pups were removed from the dam at day 20 of lactation is seen in Fig. 6b. The weaned pups were maintained on the same diet as their respective dams. The body weight of rats fed diet B increased 3.5-fold after 23 days whereas the body weight of animals fed diet A increased 3.1 fold. Energy content. The energy content of milk expressed from rats fed diet A and B was 606 and 718 mJ/lOO ml, respectively, at day 10 and 523 and 810 mJ/lOO ml, respectively, at day 20 of lactation. Food consumption.The main food consumption (g food consumed/kg body weight/24 hr) in two groups of 3 adult rats, one group fed diet A and the other group fed diet B for a 7 day trial was 91.5 + 5.4 and 55.3 + 3. I (mean + SEM) respectively. This food consumption represents 1091.6 kJ and 830.6 kJ of metabolizable energy for rats consuming diet A and diet B respectively.
The transition from colostrum to milk is characterized by a number of compositional changes in the mammary secretion. The increase in the concentration of K and lactose and decline in the concentration of Na in the mammary secretion from 12 hr before (Nicholas and Hartmann, 1981) to 48 hr after parturition in the rat is consistent with the change from colostrum to milk over this period observed in other species (Peaker, 1975). The 2-fold increase in concentration of serum albumin in milk within 2 days of parturition in the rat is similar to that reported by Geursen and Grigor (1987) and is in contrast to the decline observed during the transition from colostrum to milk in the
(a)
30 -
20-
DlSCUS!SlON
Recent studies have stressed that the dose of oxytocin administered and the time interval between removal of the young and collection of the milk sample must be carefully controlled in order to obtain a physiologically represenative sample. Several studies in the past have reported separating the dam and pups for 4-12 hr and the administration of 0.3-2.5 units of oxytocin prior to collection of milk samples. Oxytocin administration to goats (Linzell and Peaker, 1971b) and rabbits (Linzell et al., 1975) has been shown to evoke a rapid change (less than 60 min) in milk composition and the response was accentuated following administration of increased levels of the hormone. It should be emphasized that in the present study rats were separated from the pups for OS-l.0 hr and were administered only 0.1 iu of oxytocin 5 min before a milk sample was collected, ensuring a sample accurately reflecting the physiological status of the mammary gland.
I1 2o 0
I
t
I
I
20 TIME AFTER PARTURITtONIWEANINQ(days) 5
10
15
Fig. 6. The effect of diet on the rate of gain in body weight of pups during the first 20 days of lactation and after weaning. (a) Lactation. Each value represents the mean f SEM for 3-7 litters. The litter size was 10.5 f 0.3 (50) and 9.8 f 0.4 (47) (mean f SEM) from rats fed diet A (0-e) and diet B (A---A) respectively. (b) Weating. Litters were separated from the dams at day 20 of lactation and the pups divided into groups of 10 animals. Each group continued to feed from the diet previously fed to their respective dams. The values from rats fed diet A (@-a) and diet B (A---A) represent the mean f SEM for three groups of rats and the individual values from two groups of rats respectively.
540
KEVINR. NICHOLAS and
cow (Schanbacher and Smith, 1975) and woman (Kulski and Hartmann, 1981). In these species the post partum decline in serum albumin concentration in milk is negatively correlated to the change in the concentration of lactose and probably results from the dilution by newly synthesized milk. The mRNA for serum albumin has not been detected in the rat mammary gland (Geursen and Grigor, 1987) and consequently the concentration of serum albumin in milk can be used as an index of diffusion from the blood, reflecting the integrity of the permeability barrier between the extracellular fluid and the milk. Therefore, the increase in both the synthesis of lactose and the accumulation of serum albumin in the mammary secretion in early lactation suggests that a specific mechanism for the transport of serum albumin from the extracellular fluid to the milk may become operational in the rat after parturition. In contrast to serum albumin the concentration of transferrin in milk declined after parturition and remained low until day 10, after which it increased to day 20. These changes are consistent with those reported by Grigor et al. (1988) and Jorden and Morgan (1967). Studies in vitro have shown that rat mammary tissue will incorporate 14C-leucine into a protein which will precipitate with an antiserum specific for serum transferrin (Grigor et al., 1988; Jorden and Morgan, 1969). The respective contributions of transferrin to the milk from the mammary gland and the serum throughout lactation remains to be determined. However, Grigor et al. (1988) have observed low levels of accumulation of transferrin mRNA in the mammary gland early in lactation with a significant increase late in lactation. During established lactation in the rat (day 5 to 20) the increase in lactose concentration is negatively correlated with the concentration of both Na and K. A similar result has been supported for the goat (Linzell and Peaker, 197lb), cow (Rook and Wheelock, 1967) and woman (Hartmann and Prosser, 1982) and is consistent with the transfer of lactose and ions between the extracellular fluid and the milk by a transcellular pathway (Linzell and Peaker, 197la; Peaker, 1975, 1983). The concentration of casein in milk increased gradually from approximately 30% of the total protein at term to approximately 50% at day 20 of lactation. Whereas the concentration of casein (3-4g/lOOml) was lower than reported by Cox and Meuller (1937) Luckey et al. (1954) and Dymsza et al. (1964), the concentration of total protein (7-9 g/l00 ml) was similar to the range reported by Luckey et al. (1954), Dymsza et al. (1964), Chalk and Bailey (1979) Godbole et al. (1981) and Keen et al. (1981). The decline in the concentration of fat in early lactation in the rat is in agreement with the report of Luckey et al. (1954) and Godbole et al. (1981) and similar to that observed during early lactation in other Rodentia (Cowie, 1969; Peaker et al., 1975). In contrast, the studies of Chalk and Bailey (1979) showed an increase in the concentration of milk fat during the first 10 days of lactation. However, they also reported that if rats progressed through two additional, successive lactations, the concentration of milk triglyceride did not change. Keen et al. (1981)
PETERE. HARTMANN reported that the concentration of fat in milk did not change significantly throughout lactation. The ratio of the concentration of Ca, Mg and inorganic phosphorus and that for lactose in the milk from rats is in agreement with the reciprocal relationship which exists between lactose and the concentration of salts in other species. For example, species such as women and cows with a high concentration of lactose in the milk have less salt than milks lower in lactose such as in the guinea pig and the rabbit (Peaker et al., 1975; Davies et al., 1983). Thus, a general reciprocal relationship between lactose and salt concentration helps maintain the total osmotic power of milk close to that of blood. The cessation of suckling on day 20 of lactation in the rat preceded an abrupt decline in the concentration of lactose and K together with an increase in concentration of Na in the milk. Similarly, milk expressed from rats at term had an elevated concentration of Na and a decreased concentration of K suggesting that secretion at both these times occurred by the paracellular pathway. This relationship is also observed during involution in the cow (Wheelock et al., 1967) and woman (Hartmann and Kulski, 1978) and is believed to result from changes in the zonulae occludentes which connect neighbouring secretory cells. Consequently, lactose and K pass out of, and Na and Cl into the extracellular fluid through leaky ‘tight’ junctions according to their concentration gradients (see Peaker, 1975, 1976, 1983). Further examples of this paracellular secretion have been reported in late pregnancy in the goat (Linzell and Peaker, 1974) during lactation in the rabbit (Peaker and Taylor, 1975) and the latter stages of lactation in the guinea pig (Peaker et al., 1975). The rapid decline in the concentration of lactose in the milk during the first 3 days after the cessation of suckling was accompanied by an increase in concentration of casein and the whey proteins. This is consistent with the removal of lactose, the major osmole in milk, to the extracellular fluid by the paracellular pathway. The inadequacies by the low energy diet A is reflected in the elevated food consumption of male rats during a 7-day trial and the diminished weight gain of pups after weaning. On the other hand the protein and carbohydrate content of milk from lactating rats fed each of the diets remained essentially unchanged. This is consistent with studies in other species (see Davies et al., 1983) showing that changes in diet, including the energy content, does not have significant effects on protein and carbohydrate content of milk. The major difference between milks in the present study was the concentration of fat which was largely responsible for the variation in energy content. Whereas the fat content of the diet may influence the fatty acid composition of the milk, the effect of energy content of the diet on milk composition is less clear (Davies et al., 1983). Milk yield, which can be estimated indirectly in the rat by measuring the rate of gain in the body weight of the pups (see Cowie and Tindal, 1971), did not differ significantly for the first 5 days of lactation in rats fed each of the diets. However the body weight of pups from dams fed diet B was higher than pups from dams fed diet A at day 20 of lactation. In the
Milk secretion in the rat
latter stages of lactation the young begin to sup plement the mother’s milk with solid food which most likely would contribute to the differences observed in pup weight. These results emphasize the importance of choosing the correct diet with a suitable quality control for animal experimentation. An earlier report has further emphasized this fact showing that rats fed diet A demonstrated an increasing concentration of glucose in the mammary gland just prior to lactogenesis (Nicholas and Hartmann, 1981). However, the concentration of mammary glucose in rats fed diet B remained low throughout the peripartum period. Furthermore, the choice of animal diet is important from an economic point of view since rats consumed twice as much of diet A (a low energy diet) when compared to the other diet. Acknowledgement-This
project was supported by a grant from the National Health and Medical Research Council.
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