Pup Milk Research Articles

Effects of maternal protein restriction during pregnancy and lactation on milk composition and offspring development.

Before weaning, breast milk is the physiological form of neonatal nutrition, providing pups with all nutrient requirements. Maternal low-protein diet (LPD) during pregnancy and lactation induces adverse changes in key maternal organs, which have negative effects on pup development. We studied the effects of maternal LPD on liver weight, mammary gland (MG) cell differentiation, milk composition and production and pup development throughout lactation. We fed rats with control (C) or LPD (R) during pregnancy and lactation. At 7 d early, 14 d mid and 21 d late lactation stages, maternal biochemical parameters, body, liver and MG weights were analysed. MG cell differentiation was analysed by haematoxylin and eosin staining; milk nutrient composition and production were studied; pup body, liver and brain weights, hippocampal arachidonic acid (AA) and DHA were quantified. Results showed lower body and liver weights, minor MG cell differentiation and lower serum insulin and TAG in R compared with C. R milk contained less protein and higher AA at early and mid stages compared with C. R pup milk and fat intake were lower at all stages. R protein intake at early and mid stages and DHA intake at mid and late stages were lower compared with C. In R pups, lower body, liver and brain weights were associated with decreased hippocampal AA and DHA. We conclude that maternal LPD impairs liver and MG function and induces significant changes in maternal milk composition, pup milk intake and organ development.

Attractive and appetitive odor factors in murine milk: Their fade-out time and differential cryo-preservation

Murine milk conveys an odor factor that is both attractive and appetitive to conspecific newborns. Up to now, little is known about the temporal dynamic of this odor factor and about the stability of its behavioral activity after milk ejection. We aim to characterize the conditions in which the attractive and appetitive potency of milk to newborns is best conserved and, as a logical outcome, at standardizing conditions in which milk varies in reactogenic potency for newborns. Milk was collected and conserved in two conditions of cold (4 °C, −80 °C) for several durations (3 and 24 h, and 1, 2 and 8 months). The reactogenic potency of milk was assayed in 2 day-old mouse pups. We found that milk remains olfactorily attractive and appetitive to newborns after 3 h of storage at 4 °C, but it completely loses reactogenic potency on newborn pups after 24 h of storage at 4 °C. Storage at −80 °C preserves the behavioral activity of milk up to 1 month, but milk stored for 2 months at this temperature remains appetitive but not attractive to pups. Finally, the reactogenic potency of murine milk in pups is abolished after 8 months of storage at −80 °C. This study highlights that attractive and appetitive factors of milk appear dissociable and, in any case, highly labile. It provides, for two different storage temperatures, a temporal window in which milk remains behaviorally active on pups. These results will allow designing a contrastive chemical approach to identify the reactogenic compounds of milk.

PC.129 Use of breast milk fortifier in a preterm baby post discharge to avoid use of formula

A baby girl was born at 34 weeks gestation weighing 1400 g. Mother had not planned to breast feed but managed to express milk for all her baby’s needs. The...

Maternal Protein Deficiency during a Gastrointestinal Nematode Infection Alters Developmental Profile of Lymphocyte Populations and Selected Cytokines in Neonatal Mice

Neonatal immune development begins in pregnancy and continues into lactation and may be affected by maternal diet. We investigated the possibility that maternal protein deficiency (PD) during a chronic gastrointestinal (GI) nematode infection could impair neonatal immune development. Beginning on d 14 of pregnancy, mice were fed protein-sufficient (PS; 24%) or protein-deficient (PD; 6%) isoenergetic diets and were infected weekly with either 0 (sham) or 100 Heligmosomoides bakeri larvae. Pups were killed on d 2, 7, 14, and d 21 and dams on d 20 of lactation. Lymphoid organs were weighed. Cytokine concentration in maternal and pup serum and in milk from pup stomachs and lymphoid cell populations in pup spleen and thymus were determined using luminex and flow cytometry, respectively. GI nematode infection increased Th2 cytokines (IL-4, IL-5, IL-13), IL-2, IL-10, and eotaxin in serum of dams whereas PD reduced IL-4 and IL-13. The lower IL-13 in PD dams was associated with increased fecal egg output and worm burdens. Maternal PD increased vascular endothelial growth factor in pup milk and eotaxin in pup serum. Maternal infection increased eotaxin in pup serum. Evidence of impaired neonatal immune development included reduced lymphoid organ mass in pups associated with both maternal infection and PD and increased percentage of T cells and T:B cell ratio in the spleen associated with maternal PD. Findings suggest that increases in specific proinflammatory cytokines as a result of the combination of infection and dietary PD in dams can impair splenic immune development in offspring.

Maternal Progesterone Treatment Rescues the Mammary Impairment Following Uteroplacental Insufficiency and Improves Postnatal Pup Growth in the Rat

Uteroplacental insufficiency in the rat reduces maternal progesterone and impairs mammary function and pup milk intake, compromising postnatal growth. We determined whether progesterone administration to rat dams progesterone-deficient following uteroplacental insufficiency improves lactation and pup growth. Uteroplacental insufficiency (Restriction) or sham surgery (Control) was performed on day 18 of pregnancy in WKY rats. Pregnant dams were injected with progesterone or vehicle, and Control mothers with vehicle for three days and killed on day 20 of pregnancy or day 6 of lactation. Progesterone treatment in the Restricted group restored maternal progesterone with no effect on mammary Pgr mRNA expression. Uteroplacental insufficiency triggered early lactogenesis, with increased mammary Csn2, Lalba and Wap mRNA. Progesterone treatment following uteroplacental insufficiency increased mammary alveolar number and area. Pups from progesterone treated mothers had increased body weight when compared to Controls. Overall, maternal progesterone treatment following uteroplacental insufficiency improved postnatal growth by rescuing the mammary impairment.

Reproductive safety studies with genistein in rats

Genistein is a phytoestrogen that occurs naturally in the diet and is found in a wide variety of plant-derived foods especially in soybeans and soy-based foods. There is wide spread interest in genistein and related phytoestrogens as chemopreventive agents for a variety of human diseases and cancers based on epidemiologic evidence of reduced cancer rates in populations with a high intake of soy. Soy, and hence its constituents, such as genistein, have been consumed at high levels in several Asian populations for many centuries without any apparent adverse effects and to the contrary, many health benefits have been associated with the ingestion of soy based foods. Concern has been raised, however, of potential adverse effects due to the estrogenic and other activities of the isoflavones and thus a comprehensive series of safety studies was performed with genistein. To assess the teratogenic and fetal toxic potential of genistein, several studies were conducted. Genistein was tested in an in vitro rat whole embryo culture assay (WEC), which is a preliminary screen, for fetotoxic and teratogenic potential, over a concentration range of from 1 to 100 μg/mL. Treatment related anomalies were observed at concentrations of ⩾10 μg and at 100 μg/mL, all embryos were malformed. Two in vivo embryo fetal developmental safety studies were conducted with genistein by oral administration (gavage and dietary admix) in which there was no evidence for a teratogenic effect. In an oral (gavage) embryonic and fetal development pilot study, genistein was administered to rats at dose levels of 0, 20, 150 and 1000 mg/kg/day from days 6–20 of gestation to females that were allowed to litter and rear their offspring up to day 7 of lactation. A slight maternal toxicity at 1000 mg/kg/day was observed as indicated by decreased body weight and food consumption and at this dose, adverse effects in the pups were observed including increased pup mortality, poor general condition, reduced pup body weights, and reduced pup milk uptake. At the high dose of 1000 mg/kg, no external malformations were noted, however some minor visceral and skeletal variations were observed. At the low dose of 20 mg/kg/day, an increased mortality, reduced milk uptake, a decreased % male sex ratio, and decreased body weights during lactation were observed. Due to lack of effects at the mid dose and the small number of animals, a relationship to treatment was considered unlikely. In an oral (dietary admix) Prenatal developmental safety study, genistein was administered to rats at dose levels of 0, 5, 50, 100 and 500 mg/kg/day from day 5–21 of gestation. At 500 mg/kg, maternal body weight and food consumption were markedly reduced. The incidence of resorptions was markedly increased with a corresponding decrease in the number of live fetuses per dam. Fetal body weights were also reduced. No treatment-related teratogenic effects were noted during external, visceral and skeletal examination of fetuses or in body weight normalized anogenital distance. On the basis of these studies, it is concluded that genistein has no teratogenic potential in vivo at very high doses of up to 1000 mg/kg/day by oral gavage in the embryo-fetal toxicity pilot study or up to 500 mg/kg/day by dietary admix in the Prenatal developmental study even though these doses were maternally toxic and fetal-toxic. In vitro, genistein had teratogenic potential at high concentrations in the WEC screening assay, however this was not predictive of the in vivo findings. On the basis of the definitive Prenatal development study, the NOAEL for maternal toxicity and adverse effects on embryonic development was considered to be 100 mg/kg/day when administered orally by dietary admix.

Dietary Bisphenol A Suppresses the Growth of Newborn Pups by Insufficient Supply of Maternal Milk in Mice

Bisphenol A (BPA), a monomer used for the production of polycarbonate, is known to have estrogen activity. In this study, pregnant mice were fed chow diet containing 1% BPA, and we examined the influence of dietary BPA on delivery and growth of newborn pups in mice. When pregnant mice were fed BPA diet, the mice normally delivered pups on day 21 of gestation. The number of offspring pups in maternal mice fed BPA diet was same to those fed normal diet. Therefore, the growth of fetuses and the process of delivery were not influenced by dietary BPA. However, the growth of newborn pups was markedly suppressed when maternal mice were fed BPA diet. The growth of neonatal mice depends on breast milk, and stomach was filled with milk in pups. In newborn pups from maternal mice supplemented with BPA diet, the weight of stomach was lower than that from maternal mice with normal diet. Since the serum level of prolactin was limited in maternal mice supplemented with BPA diet, the suppressed growth of newborn pups by dietary BPA may be due to the insufficient supply of breast milk. These results suggest that the influence of BPA on the growth of newborn pups is related to hormonal condition in maternal mice.

Transfer of polychlorinated biphenyls and chlorinated pesticides from mother to pup in relation to cytochrome P450 enzyme activities in harp seals (Phoca groenlandica) from the Gulf of St. Lawrence, Canada

Congener-specific transfer of polychlorinated biphenyls (PCBs) and chlorinated pesticides from female to pup was studied in harp seals from eastern Canada. Possible effects on hepatic cytochrome P450 enzymes (CYP450) due to contaminant mobilization from blubber lipids in females and ingestion of contaminated milk in pups were studied. Contaminant transfer from blubber to milk in females favored the more polar compounds (lower chlorinated PCBs, toxaphenes, hexachlorocyclohexanes, and hexachlorobenzene) relative to more lipophilic compounds (higher chlorinated PCBs, dichlorodiphenyltrichloroethane [DDT], chlordane). In spite of substantial contaminant mobilization from blubber in females and ingestion of contaminated milk by pups, CYP450 activities were low in all animals. Possibly, increased plasma estradiol concentrations, involved in breeding after lactation, suppressed CYP450 directly. Although the pups were exposed to contaminants in milk, CYP450 activities were low, resulting in low contaminant metabolism. This was confirmed by similar contaminant patterns in milk and pups. A strong positive relation between CYP1A-like activities and body weight in the pups suggested not yet fully developed CYP1A enzymes. A negative association between CYP3A and pesticides in females and pups was hypothesized to be a result of metabolic inactivation of CYP450. The CYP450 enzyme activities were considered unsuitable indicators for contaminant mobilization and transfer in harp seals.

Maternal influences on milk intake in SHR and WKY pups

Mother to pup milk transfer was examined in the spontaneously hypertensive rat (SHR) and its normotensive progenitor, the Wistar-Kyoto (WKY). Litters of SHR and WKY pups were either reared by their natural mothers or cross-fostered to mothers of the opposite strain shortly after birth. At postpartum days 7 and 14, pup body weights were monitored throughout a 2-h maternal separation period followed by a 3-h refeeding period during which pups remained with their mothers. Weight increases of pups over the refeeding period served as an indirect measure of milk ingestion. At postpartum day 7, both SHR and WKY pups nursed by SHR mothers received lower quantities of milk compared to their counterparts nursed by WKY mothers. Maternal strain differences in milk transfer were largely attenuated at postpartum day 14. The different nutritional environments provided by SHR and WKY mothers may mediate the alterations in offspring cardiovascular physiology and physical development that occur with reciprocal cross-fostering.

Maternal Energy Investment in Elephant Seal Pups: Evidence for Sexual Equality?

Labeled water methodology was used to quantify maternal energy investment in northern elephant seal (Mirounga angustirostris) pups. Pup milk intake, field metabolic rate, mass change, and body composition were monitored during the nursing period and throughout the postweaning fast. Maternal investment did not vary significantly as a function of pup sex. There was no evidence for sex differences in growth rate or milk intake rate, and mean metabolic rate of suckling pups did not differ significantly between the sexes. Males and females appeared to be in similar condition at the end of the postweaning fast, just prior to their first trip to sea. Some male pups may attain greater size relative to females without additional cost to their mothers. These data do not provide evidence to reject R. A. Fisher's theory of equal investment in the sexes.

Measurement of milk yield in the lactating rat from pup weight and weight gain.

A new method was developed that enables milk yield to be estimated from pup weight and pup weight gain in well-nourished rats. Daily milk yield, body weight, and daily weight gain were measured over 4-5-day periods in 38 suckling pups aged 3-13 days postpartum. Each 24-h period was divided into consecutive 6-h cycles, during which dams and pups were separated for 4 h and then reunited for 2-h suckling intervals. Daily milk yield per pup was calculated from pup weight gain during the four suckling intervals for each 24-h period, and corrected for insensible weight loss of the pups. The following equation, relating pup milk yield to pup weight and weight gain, was obtained by stepwise forward multiple regression analysis: yield = 0.0322 + 0.0667 (weight) + 0.877 (gain); where yield is daily yield per pup (g/pup/day), weight is pup weight (g), and gain is pup weight gain per day (g/day) (n = 174; F = 552.3; p less than 0.001; r2 = 0.866). Per litter milk yield can be obtained by multiplying per pup yield by litter size. Using this equation, dams fed a stock diet and nursing eight pups were estimated to have produced 29.5 ml of milk on day 10 of lactation. This value is similar to yields measured by more laborious methods. SEs of yield estimates calculated with this method represent 1-3% of predicted yield values. The new method gives useful estimates of rat milk yield during the first 2 weeks of lactation from measurements that are more easily obtained than is the case with other methods.

Measurement of Milk Yield in the Lactating Rat from Pup Weight and Weight Gain

A new method was developed that enables milk yield to be estimated from pup weight and pup weight gain in well‐nourished rats. Daily milk yield, body weight, and daily weight gain were measured over 4–5‐day periods in 38 suckling pups aged 3–13 days post‐partum. Each 24‐h period was divided into consecutive 6‐h cycles, during which dams and pups were separated for 4 h and then reunited for 2‐h suckling intervals. Daily milk yield per pup was calculated from pup weight gain during the four suckling intervals for each 24‐h period, and corrected for insensible weight loss of the pups. The following equation, relating pup milk yield to pup weight and weight gain, was obtained by stepwise forward multiple regression analysis: yield = 0.0322 + 0.0667 (weight) + 0.877 (gain); where yield is daily yield per pup (g/pup/day), weight is pup weight (g), and gain is pup weight gain per day (g/day) (n = 174; F = 552.3; p < 0.001; r2 = 0.866). Per litter milk yield can be obtained by multiplying per pup yield by litter size. Using this equation, dams fed a stock diet and nursing eight pups were estimated to have produced 29.5 ml of milk on day 10 of lactation. This value is similar to yields measured by more laborious methods. SEs of yield estimates calculated with this method represent 1–3% of predicted yield values. The new method gives useful estimates of rat milk yield during the first 2 weeks of lactation from measurements that are more easily obtained than is the case with other methods.