Expression of constitutively activated Akt in the mammary gland leads to excess lipid synthesis during pregnancy and lactation

Expression of constitutively activated Akt in the mammary glands of transgenic mice results in a delay in post-lactational involution. We now report precocious lipid accumulation in the alveolar epithelium of mouse mammary tumor virus-myr-Akt transgenic mice accompanied by a lactation defect that results in a 50% decrease in litter weight over the first 9 days of lactation. Although ductal structures and alveolar units develop normally during pregnancy, cytoplasmic lipid droplets appeared precociously in mammary epithelial cells in early pregnancy and were accompanied by increased expression of adipophilin, which is associated with lipid droplets. By late pregnancy the lipid droplets had become significantly larger than in nontransgenic mice, and they persisted into lactation. The fat content of milk from lactating myr-Akt transgenic mice was 65-70% by volume compared to 25-30% in wild-type mice. The diminished growth of pups nursed by transgenic mothers could result from the high viscosity of the milk and the inability of the pups to remove sufficient quantities of milk by suckling. Transduction of the CIT3 mammary epithelial cell line with a recombinant human adenovirus encoding myr-Akt resulted in an increase in glucose transport and lipid biosynthesis, suggesting that Akt plays an important role in regulation of lipid metabolism.     

Effects of uteroplacental insufficiency and reducing litter size on maternal mammary function and postnatal offspring growth

Human intrauterine growth restriction is often associated with uteroplacental insufficiency and a decline in nutrient and oxygen supply to the fetus. This study investigated the effects of uteroplacental insufficiency and intrauterine growth restriction (Restricted) or reducing litter size for normally grown pups (Reduced Litter) on maternal mammary development and function, milk composition, offspring milk intake, and their resultant effects on postnatal growth. Uteroplacental insufficiency was surgically induced by bilateral uterine vessel ligation on day 18 of gestation in the Wistar Kyoto rat. At birth, a group of sham control rats had their litter size reduced to five (Reduced Litter) to match that of the Restricted group. Cohorts of rats were terminally anesthetized on day 20 of gestation or day 6 of lactation, and a third group was studied throughout lactation. Restricted pups had a lower birth weight (by 16%) and litter size (by 36%) compared with controls, as well as reduced mammary parathyroid hormone-related protein content and milk ionic calcium concentrations associated with reduced total pup calcium. Restricted dams with lower circulating progesterone experienced premature lactogenesis, producing less milk per pup with altered composition compared with controls, further slowing growth during lactation. Reducing litter size of pups born of normal birth weight (Reduced Litter) was associated with decreased pup growth, highlighting the importance of appropriate controls. The present study demonstrates that uteroplacental insufficiency impairs mammary function, compromises milk quality and quantity, and reduces calcium transport into milk, further restraining postnatal growth.     

Ambient temperature affects postnatal litter size reduction in golden hamsters

Background

To better understand how different ambient temperatures during lactation affect survival of young, we studied patterns of losses of pups in golden hamsters (Mesocricetus auratus) at different ambient temperatures in the laboratory, mimicking temperature conditions in natural habitats. Golden hamsters produce large litters of more than 10 young but are also known to wean fewer pups at the end of lactation than they give birth to. We wanted to know whether temperature affects litter size reductions and whether the underlying causes of pup loss were related to maternal food (gross energy) intake and reproductive performance, such as litter growth. For that, we exposed lactating females to three different ambient temperatures and investigated associations with losses of offspring between birth and weaning.

Results

Overall, around one third of pups per litter disappeared, obviously consumed by the mother. Such litter size reductions were greatest at 30 °C, in particular during the intermediate postnatal period around peak lactation. Furthermore, litter size reductions were generally higher in larger litters. Maternal gross energy intake was highest at 5 °C suggesting that mothers were not limited by milk production and might have been able to raise a higher number of pups until weaning. This was further supported by the fact that the daily increases in litter mass as well as in the individual pup body masses, a proxy of mother’s lactational performance, were lower at higher ambient temperatures.

Conclusions

We suggest that ambient temperatures around the thermoneutral zone and beyond are preventing golden hamster females from producing milk at sufficient rates. Around two thirds of the pups per litter disappeared at high temperature conditions, and their early growth rates were significantly lower than at lower ambient temperatures. It is possible that these losses are due to an intrinsic physiological limitation (imposed by heat dissipation) compromising maternal energy intake and milk production.

     

The response of plasma prolactin to suckling during normal and prolonged lactation in the rat

In dams which had been kept isolated from pups for 8-10 h, the magnitude of the suckling-induced prolactin rise in the plasma was studied in relation to intensity of suckling stimulus and lactational age of the mother. At midlactation the response of prolactin evoked by suckling was enhanced as litter size increased. Suckling of 2 pups induced a greater prolactin rise in dams adjusted to 2 pups than in dams adjusted to 8 pups. Suckling of 8 pups caused a greater prolactin rise in dams which had been adjusted to an 8-pup litter, than in rats with a 2-pup litter. At late and prolonged lactation the rise of prolactin in the plasma induced by the suckling stimulus of 8 pups was significantly lower than at midlactation. Injection of perphenazine after a period of suckling induced a moderate increase of plasma prolactin in dams at midlactation, and a similar increase in dams at late lactation and at day 42 of lactation. It is concluded that in the first half of lactation the number of pups, i.e. the intensity of the suckling stimulus, is an important factor in determining the magnitude of the prolactin response to suckling. The lower response of plasma prolactin to suckling in late lactation is neither caused by a decrease in suckling stimulus from the pups nor by an increase in prolactin clearance; it is probably due to a gradual reduction in prolactin synthesizing and releasing capacity of the pituitary, brought on by a desensitization of the neural or neuroendocrine system to suckling stimuli as lactation proceeds.     

Lactational performance, consummatory behavior, and suppression of estrous cyclicity in rats suckling underfed pups

The role of pups' appetite in the regulation of maternal consummatory behavior (food intake of nursing mothers), lactational performance and postpartum diestrus was studied over a period of 45 days postpartum in rats chronically exposed to either underfed or normally fed pups. Experimental rats (n = 10) daily received 5 pups, 4-10-days-old, that had been deprived of food for the preceding 24 h while under the care of nonlactating foster mothers. Control rats (n = 10) received normally fed pups obtained daily from lactating foster mothers. Throughout the experimental period, the daily milk yield (estimated by litter weight gain), the intake of food and water by the mother, as well as the ratio of litter weight gain to mother's intake of food and water were all markedly higher in rats nursing underfed pups than in rats nursing normally fed pups. After a peak in lactation around Day 15 postpartum, experimental rats produced the same amount of milk during extended lactation as they did in the beginning of lactation, while control rats produced only half the amount of milk during extended lactation as they did in early lactation. Regardless of the nutritional state of the suckling pups, maternal body weight increased progressively over the first four weeks of lactation and remained unchanged during the time of extended lactation. The postpartum diestrus and the subsequent diestrous phase in the time of extended lactation were considerably longer in duration in rats that nursed underfed pups. On Day 45 of lactation, prolactin levels were higher and the adrenal glands were larger in experimental rats than in controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Weaning and Parent-Offspring Conflict in the Domestic Dog

Weaning and signs of a parent-offspring conflict were studied in four females of the Swedish Dachsbracken breed of domestic dog and their pups. The animals were observed from the second to the seventh week of age of the pups. In addition to regular weighing, measurements of milk and solid food intake per pup and meal were also made, and samples of milk from the mothers were collected and analysed. The most important mechanism for weaning seemed to be on the behavioural level. The time that the mother spent with her pups decreased continuously from week 2 to week 7, as did the number of sucklings per hour. Furthermore, both duration of suckling and the number of sucklings initiated by the mother decreased during the period, while the proportion of sucklings where the mother was standing increased steadily. The weights of mothers stayed rather constant during the period and there was no difference in the amount of milk given per suckling or in the composition of milk between the early and late weeks of lactation. Consequently, costs for the mother, in terms of loss of weight, were negligible as she was able to compensate for the increased energy demand of lactation with an increased food intake. There was a tendency for care-giving behaviour to decrease and aggression from the mother to increase at the same time as there was a tendency for care-seeking and contact-seeking behaviour from the pups to increase. These changes, together with the less frequent initiation of suckling by the mother, could perhaps be seen as signs of conflict. Conflict was defined according to TRIVERS' theory (1974) and referred to the disagreement between the female and her pups about the amount of care given. However, although the animals were kept in a way that allowed them to perform as much as possible of their natural behaviour, the good nutritional conditions, one of the characteristics of captive life, may have reduced overt parent-offspring conflict.     

Milking strategy in subantarctic fur seals Arctocephalus tropicalis breeding on Amsterdam Island: evidence from changes in milk composition.

Milk composition was investigated throughout the 10-mo pup-rearing period in subantarctic fur seals (Arctocephalus tropicalis) breeding on Amsterdam Island. The mean milk composition was 42.8% +/- 5.7% lipid, 12.1% +/- 1.5% protein, and 42.6% +/- 7.3% water. Subantarctic fur seals breeding on Amsterdam Island produced one of the richest milks ever reported in otariids (20.4 +/- 2.9 kJ/g), with lipid content contributing 85% of total gross energy. The high lipid levels measured in the milk of subantarctic fur seals breeding on Amsterdam Island is consistent (i) with the relatively long time lactating females spend at sea, due to the relatively poor local trophic conditions near the colony that necessitate that they travel long distances to reach the foraging grounds, and (ii) with the consequently short time mothers spend with their pups ashore. Milk composition changed according to the time mothers were fasting ashore: milk produced during the first 2 d spent ashore, when more than 80% of milk transfer occurred, had higher levels of lipids, proteins, and gross energy than milk produced later during the visit ashore, suggesting that the pups were fed with two types of milk during a suckling period. Throughout the year, mothers in good condition produced milk of higher lipid content than others, suggesting that individual foraging skills contribute to enhance milk quality. Milk lipid and gross energy content varied with pup age, according to quadratic relationships, increasing during the earlier stages of lactation before reaching asymptotic values when pups were 180 d old. The stage of lactation appears to be a better predictor of milk lipid content than the duration of the preceding foraging trip, suggesting that either changes in the nutritional requirements of the pup and/or seasonal changes in trophic conditions act on milk composition. These changes in milk quality may also be related to changes in maternal care; lactating subantarctic fur seals apparently reallocate their body reserves toward gestation rather than lactation at the end of the pup-rearing period.     

Bioavailability of lead from various milk diets studied in a suckling rat model

The bioavailability of lead from various milk diets was studied in 14 day old suckling rats. Human milk, infant formula, cow's milk, rat milk and deionized water labeled with ²⁰³Pb were given to rat pups by gastric intubation. Animals were killed after 2 or 6 h and the radioactivity in the tissues was measured. At 2 h after administration the lead bioavailability, defined as lead uptake in the body, excluding the gastrointestinal tract, was 47% from water, 42% from human milk, 40% from infant formula, 31% from cow's milk and 11% from rat milk. After 6 h the bioavailability of lead was about 50% from water and human milk, 45% from infant formula and cow's milk, and 36% from rat milk. The blood lead levels in the pups reflected the total body uptake and were also correlated to the brain lead levels. Thus, rat pups given lead in human milk had approximately twice as high lead levels in blood and brain than pups given lead in rat milk. The intestinal absorption of lead was dependent on the milk diet given to the sucklings. In duodenum, the highest uptake of lead was found in rats given water or human milk, whereas in rats given rat or cow's milk the highest uptake of lead was found in ileum. The distribution of lead in cream, whey and casein fractions of the milk diets after in vitro labeling with ²⁰³Pb was also studied. The casein fraction in cow's and rat milk contained 90–96% of the total amount of lead in the diet. In infant formula and human milk, 77 and 56% lead was found in the casein fraction, respectively. The higher lead bioavailability observed in the suckling rat fed human milk than in those fed rat and cow's milk may partly be explained by a lower proportion of lead bound to casein in human milk.     

Initiation of fatty acid synthesis in rat mammary glands.

The rate of fatty acid synthesis from [6-14C]glucose in mammary tissue remained low until parturition at 22 days of gestation and increased 10-fold at 1 day post partum. Administration of progesterone on days 20 and 21 or removal of pups at parturition abolished this increase. In the latter case, administration of prolactin, corticosterone or oxytocin had no stimulatory effect; tissue from suckled glands in which the ducts had been ligated at parturition also showed no increased rate in 24 h. Foetoplacentectomy on day 18 did not stimulate fatty acid synthesis but subsequent suckling by foster pups did. Whereas lactose synthesis is initiated by withdrawal of progesterone from the circulation, a further stimulus related to removal of milk by suckling is required to initiate fatty acid synthesis.     

Tenacious nipple attachment in rodents: the sibling competition hypothesis

Tenacious attachment to the mother's nipples by suckling pups has been observed in many rodent species, but apeears to be absent in others. The natural history of this behaviour is reviewed, along with the possible adaptive value. Four hypotheses are extracted from the literature: the antipredator, anti-falling, anti-drowning, and infant-nipple contact hypotheses. These hypotheses are found to be ad hoc and supported by only anecdotal evidence. An alternative hypothesis is presented, that tenacious nipple attachment is an adaptation to sibling competition for maternal resources (nipples and milk). Morphological specialization, developmental time course, and patterns of pup mortality support the new model. The behaviour has been noted only in the Muridae. Quantitative interspecific comparisons within the Muridae demonstrate that tenacious nipple attachment is associated with reproductive tactics that promote sibling competition: breeding by means of litter overlap, low mammary number, and more offspring per mammary.     

Effect of short-term fasting/refeeding on epidermal growth factor content in the gastrointestinal tract of suckling rats.

Epidermal growth factor (EGF) is trophic for varying regions of the developing gastrointestinal tract (GIT) of suckling rats. The presence of large amounts of EGF in milk from various species, combined with low production of EGF by suckling animals, led to speculation that milk is a major source of EGF for suckling rats. We report that short-term fasting (8 hr) of 12-day-old suckling rats resulted in a significant decrease in the levels of immunoreactive EGF (irEGF) in the GIT. Pups refed by lactating mothers for 1 to 4 hr exhibited an increase in irEGF to original levels, whereas pups fed a rat milk substitute by gastric gavage did not have an increase in irEGF content. The irEGF levels in the GIT of pups that were manually fed normal rat milk, or rat milk substitute supplemented with EGF, returned to the prefasted levels. Fasted suckling rats refed 2 ml of rat milk in 2 h exhibited significantly higher level of irEGF in the GIT than did those refed with 0.5 ml in 45 min. Since rat milk irEGF exists in three distinct forms (A, B, and C; C is equal to authentic submandibular gland EGF, the irEGF forms in the GIT were characterized by native polyacrylamide gel electrophoresis. In the stomach luminal contents of the fed suckling rats, only the larger form, Peak B, was observed. Both the luminal content and the mucosa scrapings of all other segments of all groups contained only Form D (comigrating with desarginyl EGF), a metabolic derivative of EGF. All forms were immunoreactive, exhibited receptor binding, and stimulated DNA synthesis in growth-arrested fibroblasts. The rapid changes in EGF within the GIT of suckling rats suggest the EGF can acutely modify some GIT functions of suckling rats.     

The effect of environmental temperature on the production of ultrasounds by isolated non-handled albino mouse pups

Albino mouse pups develop homoiothermy during the first 19 days of their life and show three distinct stages during this period. From day 1 to day 6 they are almost completely poikilothermic at low ambient temperatures; from day 7 to day 14 or 15 is a transitional period during which they develop homoiothermy at medium temperatures (e.g. 22°C); from day 15 or 16 to day 19 or 20 is the final stage when homoiothermy at very low ambient temperatures is developed. These changes are very closely correlated with changes in the ultrasounds produced by isolated pups within the 19 day period. Thus their acoustic response to cold is weak from day 1 to day 5 or 6 and very strong from day 6 or 7 to day 12 or 13, on exposure even to medium temperatures. From day 14 onwards the pups progressively stop producing ultrasounds depending on the ambient temperatures, and there is complete cessation of calling after day 19 or 20 even on exposure to temperatures as low as 2°C. In general, decreasing the ambient temperatures increases the rate and intensity of ultrasounds produced but very low ambient temperatures have an inhibiting effect and at 2–3°C breathing and calling cease completely after a time which varies with age of the pups.     

Leptin levels in rat offspring are modified by the ratio of linoleic to alpha-linolenic acid in the maternal diet

The supply of polyunsaturated fatty acids (PUFA) is important for optimal fetal and postnatal development. We have previously shown that leptin levels in suckling rats are reduced by maternal PUFA deficiency. In the present study, we evaluated the effect of maternal dietary intake of (n-3) and (n-6) PUFA on the leptin content in rat milk and serum leptin levels in suckling pups. For the last 10 days of gestation and throughout lactation, the rats were fed an isocaloric diet containing 7% linseed oil (n-3 diet), sunflower oil (n-6 diet), or soybean oil (n-6/n-3 diet). Body weight, body length, inguinal fat pad weight, and adipocyte size of the pups receiving the n-3 diet were significantly lower during the whole suckling period compared with n-6/n-3 fed pups. Body and fat pad weights of the n-6 fed pups were in between the other two groups at week one, but not different from the n-6/n-3 group at week 3. Feeding dams the n-3 diet resulted in decreased serum leptin levels in the suckling pups compared with pups in the n-6/n-3 group. The mean serum leptin levels of the n-6 pups were between the other two groups but not different from either group. There were no differences in the milk leptin content between the groups. These results show that the balance between the n-6 and n-3 PUFA in the maternal diet rather than amount of n-6 or n-3 PUFA per se could be important for adipose tissue growth and for maintaining adequate serum leptin levels in the offspring.     

Lifespan ontogeny of breeding and reproductive success in Mongolian gerbils

An analysis was made of demographic data collected from a colony of Mongolian gerbils (Meriones unguiculatus). The data on births of over 3800 gerbils provide evidence on several aspects of development and fecundity. Approximately 4% more females than males survived to weaning. Mean number of pups per dam per year was estimated to be 48.3, greater reproductive success than reported previously. The shortest period of gestation resulting in viable pups was 25 days. Implantation was delayed by the presence of suckling pups. When gestation was prolonged, there was a direct relationship between the number of pups being suckled concurrently and the length of the delay. Length of gestation did not affect the size of the subsequent litter. Female gerbils gave birth as early as 72 days of age. Gerbils continued to reproduce until after 2 years of age in some cases. There were fewer pups per litter born to older females, fewer pups per litter with increasing parity, and longer intervals between litters with increasing age.     

Fasting and lactation effect fat-soluble vitamin A and E levels in blood and their distribution in tissue of grey seals (Halichoerus grypus)

Grey seals among other phacoids represent a good model to study the mobilisation, transfer and deposition of fat-soluble components such as vitamins in lactating females and suckling pups because during the lactation period mothers may fast completely while secreting large quantities of high fat milks, and pups deposit large amounts of fat as blubber. The level of vitamins A and E in different tissues (liver, adipose tissue, kidney, heart, skeletal muscle, testis) and blood plasma of adult grey seal females and males changed as a result of fasting and lactation; changes were also observed in pups. The most obvious effects were a significant increase of retinol and a decrease of vitamin E levels in plasma of females with the onset of lactation as well as a substantial decrease in liver vitamin E. In suckling pups both retinol and vitamin E levels in plasma increased with the onset of suckling; after weaning no changes in retinol but a significant decrease in plasma vitamin E was observed. While liver vitamin A levels tended to be unaffected by suckling or post-weaning fast, liver vitamin E levels increased with the uptake of milk substantially (P<0.01) and returned at weaning to low levels similar to that in fetuses. Adipose tissue levels of vitamin A and E in both females and pups were only marginally affected by lactation, suckling or post-weaning fast. Results indicate that both plasma and liver levels of vitamin A and E are affected by the mobilisation, absorption and deposition of these components during lactation in seals to a much greater extent than adipose tissue, from which fat-soluble vitamins are mobilized at rates similar to that of lipids.     

Somatosensory Contributions to c-fosActivation within the Caudal Periaqueductal Gray of Lactating Rats: Effects of Perioral, Rooting, and Suckling Stimuli from Pups

In lactating rats, the immediate-early gene c-foswas previously shown to be highly activated in several brain sites by physical interaction with either suckling or nonsuckling pups but not by distal stimuli from pups, a nonpup stimulus, or no stimulation. Further, even greater levels of Fos-immunoreactivity (ir) occurred following suckling versus nonsuckling contact with pups in only 1 of over 25 sites—the caudal periaqueductal gray (cPAG) at an intercollicular level, lesions of which severely reduced the typical suckling-induced kyphotic nursing posture. Herein we further evaluated the effects and site-specificity of various somatosensory cues received from pups during 60 min on Fos-ir in the PAG of day 7 postpartum rats after a 48-h dam–litter separation. Dams interacting with suckling versus nonsuckling pups showed relatively high numbers of Fos-ir cells in the intercollicular cPAG site identified earlier, but not in three other rostrocaudal planes of the PAG. Elimination of rooting on the dam's ventrum by use of fully anesthetized pups did not further diminish Fos-ir in maternally behaving, nonsuckled dams. Perioral anesthesia of dams prior to reunion with the litter prevented retrieval and licking of pups but not pup-initiated nursing behavior, the duration of which was positively correlated with Fos-ir levels within the intercollicular cPAG. Thus, various somatosensory stimuli from pups activate c-fosin a discrete region of the cPAG but only interactions that include suckling and its behavioral consequences elicit maximal expression, consistent with a role for this midbrain site in the sensorimotor control of kyphotic nursing in rats.     

Changes in sialic acid and fucose contents of enterocytes across the crypt-villus axis in developing rat intestine

Alterations in sialic acid and fucose contents of different populations of epithelial cells have been studied in suckling and adult rat intestine. The progression of cells from crypt base to villus tip is associated with an increase in sialic acid and a decrease in fucose levels of the cells in adult rats. In suckling pups, sialic acid is uniformly distributed along the length of villi, and fucose is richly (P less than 0.01) present in cryptic cells compared to that at the villus tip. Adult-type changes in sialylation and fucosylation of enterocytes across the crypt-villus axis were precociously produced by cortisone administration to suckling pups. Thyroxine treatment was less effective in influencing the glycosylation process in rat intestine.     

Involvement of neuromedin S in the oxytocin release response to suckling stimulus

We recently identified neuromedin S (NMS) from the rat hypothalamus as an endogenous ligand for the FM-4/TGR-1 receptor distinct from neuromedin U. In the present study, we examined the role of NMS in the oxytocin release response to suckling stimulation by rat pups. Intracerebroventricular (icv) injection of NMS induced cFos expression in the paraventricular nucleus and supraoptic nucleus. Double immunohistochemical analysis revealed induction of cFos expression in a proportion of oxytocinergic neurons in both nuclei. In addition, icv injection of NMS stimulated oxytocin release dose-dependently in intact rats, and increased milk secretion in lactating rats. On the other hand, icv injection of anti-NMS antiserum into lactating rats significantly suppressed suckling-induced milk ejection. These results suggest that, in the rat, endogenous NMS plays an important role in the oxytocin release response to the suckling stimulus.     

Supplementation of suckling rats with cow's milk induces hyperphagia and higher visceral adiposity in females at adulthood,but not in males

In humans, complementary feeding should be started after 6 months-old; the introduction of any food or water before this time is considered early weaning, which is associated with health problems in adulthood. Cow's milk is a common food introduced to children less than 6 months that has inadequate nutritional composition mainly due to a worse casein: whey protein ratio compared to human milk. We hypothesized that suckling rats fed with cow's milk, rich in bioactive peptides, develop further metabolic dysfunctions. From postnatal day (PN) 14 to 20, Wistar rat pups were divided into 3 groups: rat milk (RM) – pups received rat milk orally in a syringe; cow's milk (CM), pups received cow's milk; CM with high protein (CM-H), CM with twice protein amount of rat milk. Pups were killed on PN21 and PN180. At PN21, CM males had lower visceral fat mass compared with other groups. Serum corticosterone was higher in CM-H males, despite no change in glucocorticoid metabolism in liver and visceral fat. At PN180, CM and CM-H females had greater fat depots and hyperphagia, although no alteration in leptinemia and leptin signaling in hypothalamus. CM-H females had a trend of hypoinsulinemia and significant decrease in HOMA-β, suggesting lower insulin secretion. Males from CM-H group had only lower total body protein mass. CM males had hypercorticosteronemia associated with lower expression of 11βHDS1 in visceral fat. In conclusion, early introduction of cow's milk in neonate rats leads to gender-dependent differences in metabolic and endocrine parameters in the short- and long-term.     

High fat feeding of lactating mice causing a drastic reduction in fat and energy content in milk without affecting the apparent growth of their pups and the production of major milk fat globule membrane components MFG-E8 and butyrophilin

Lactating mice were fed either a low fat or a high fat diet. Milk samples were collected and the composition was examined. Triglyceride and free fatty acid contents were greatly reduced in the milks of high fat diet group, while protein and lactose contents were almost the same between both diet groups. Although the energy content of each component was also lower in milk of high fat diet group, there was apparently no significant difference in the growth of the pups raised by either diet group. This discrepancy might be in part explained by a hypothesis that the pups might monitor calorie content in milk and keep suckling until the energy intake reaches their satisfaction. Moreover, nearly the same amounts of major milk fat globule membrane proteins MFG-E8 and butyrophilin were shown to be present in the milks from both diet groups and gene expression of both proteins in the mammary glands were also indistinguishable, suggesting that production of major MFGM components is not simply related to fat production and secretion.