Food-restricted Mothers Research Articles

Liver Proteome Profile of Growth Restricted and Appropriately Grown Newborn Wistar Rats Associated With Maternal Undernutrition.

BackgroundFetal growth restriction (FGR) has been associated with adverse perinatal outcomes and epigenetic modifications that impact gene expression leading to permanent changes of fetal metabolic pathways and thereby influence development of disease in childhood and adult life. In this study, we investigated the result of maternal food restriction on liver protein expression in Wistar male newborn pups.Materials & MethodsTen (n = 10) timed pregnant Wistar rats on their 14th day of gestation were randomly assigned to either control (n = 4) or food restricted group (n = 6). The control group had ad libitum access to food. In the food restricted group, maternal diet was limited in a moderate fashion (50%) from day 15 of pregnancy until delivery. All rats delivered spontaneously on day 21 and newborn pups were immediately weighed. Pups born to normally nourished mothers were considered as controls, while pups born to food restricted mothers were subdivided into two groups, based on their birth weight: growth restricted (FGR) and appropriately grown (non-FGR). Rats were euthanized immediately after birth and liver tissues of 11 randomly selected male offspring (FGR n = 4, non-FGR n = 4, control n = 3) were collected and analyzed using quantitative proteomics.ResultsIn total 6,665 proteins were profiled. Of these, 451 and 751 were differentially expressed in FGR and non-FGR vs. control, respectively, whereas 229 proteins were commonly expressed. Bioinformatics analysis of the differentially expressed proteins (DEPs) in FGR vs. control revealed induction of the super-pathway of cholesterol biosynthesis and inhibition of thyroid hormone metabolism, fatty acid beta oxidation and apelin liver signaling pathway. Analysis of DEPs in non-FGR vs. control groups showed inhibition of thyroid hormone metabolism, fatty acid beta oxidation, and apelin liver signaling pathway.ConclusionThis study demonstrates the impact of prenatal food restriction on the proteomic liver profile of FGR and non-FGR offspring underlying the importance of both prenatal adversities and birth weight on liver-dependent postnatal disease.

Impact of Maternal Food Restriction on Heart Proteome in Appropriately Grown and Growth-Restricted Wistar-Rat Offspring.

Objective: Fetal growth restriction is associated with increased postnatal cardiovascular morbidity. The alterations in heart physiology and structure caused by in utero nutrient deprivation have not been extensively studied. We aim to investigate the impact of maternal food restriction on the cardiac proteome of newborn rats with normal (non-fetal growth-restricted (FGR)) and reduced (FGR) birth weight. Methods: On day 14 of gestation, 10 timed pregnant rats were randomized into two nutritional groups: (a) Standard laboratory diet and (b) 50% global food restriction. Pups born to food-restricted mothers were subdivided, based on birthweight, into fetal growth-restricted (FGR) and non-FGR, while pups born from normally nourished mothers were considered controls. Rat neonates were euthanized immediately after birth and the hearts of 11 randomly selected male offspring (n = 4 FGR, n = 4 non-FGR, n = 3 control group) were analyzed using quantitative proteomics. Results: In total, 7422 proteins were quantified (q < 0.05). Of these, 1175 were differentially expressed in FGR and 231 in non-FGR offspring vs. control with 151 common differentially expressed proteins (DEPs) between the two groups. Bioinformatics analysis of DEPs in FGR vs. control showed decreased integrin and apelin cardiac fibroblast signaling, decreased muscle contraction and glycolysis, and over-representation of a protein network related to embryonic development, and cell death and survival. Conclusion: Our study illustrates the distinct proteomic profile of FGR and non-FGR offspring of food-restricted dams underlying the importance of both prenatal adversities and birth weight in cardiac physiology and development.

Gestation Food Restriction and Refeeding Compensate Maternal Energy Status and Alleviate Metabolic Consequences in Juvenile Offspring in a Rabbit Model.

Nutritional status during gestation can influence mother and offspring metabolism. Undernutrition in pregnancy affects women in both western and developing countries, and it is associated with a high prevalence of chronic diseases in later life. The present work was conducted in the rabbit model, as a longitudinal study, to examine the effect of food restriction during early and mid-gestation, and re-feeding ad libitum until the end of pregnancy on metabolic status and body reserves of mother and, its association with development and metabolism of fetuses and female offspring to the juvenile stage. Little changes in live body weight (LBW), compensatory feed intake, similar body reserves, and metabolism were observed in dams. Placenta biometry and efficiency were slightly affected, but fetal BW and phenotype were not modified. However, hyperinsulinemia, insulin resistance, and hypertriglyceridemia were demonstrated in pre-term fetuses. In the juvenile period, these changes were not evidenced, and a similar pattern of growth and serum metabolic parameters in offspring of food-restricted mothers were found, except in serum aminotransferases levels, which increased. These were associated with higher liver fibrosis. Maternal food restriction in the early and mid-pregnancy followed by re-feeding in our rabbit model established a compensatory energy status in dams and alleviated potential long-term consequences in growth and metabolism in the offspring, even if fetal metabolism was altered.

Liraglutide Enhances the Activity of the ACE-2/Ang(1-7)/Mas Receptor Pathway in Lungs of Male Pups from Food-Restricted Mothers and Prevents the Reduction of SP-A.

In utero growth restriction and being born small for gestational age are risk factors for respiratory morbidity. IUGR (in utero growth retardation) is associated to overall reduction in lung weight, surfactant content and activity, impaired maturation of the alveolar type II cells, and decreased alveolar formation. The renin-angiotensin system (RAS) may be a key target underlying pathophysiological lung alterations. GLP-1 and agonists of its receptor modulate the expression levels of different components of RAS and also are very important for lung maturation and the production of surfactant proteins. The aim of this study was to elucidate the effects of IUGR induced by perinatal food restriction of the mother in the lung function of pups at early stages of life (PD21) and to determine if liraglutide had any effect during gestational period. Sprague-Dawley pregnant rats were randomly assigned to 50% food restriction (MPFR) or ad libitum control (CT) groups at day of pregnancy 12 (GD12). From GD14 to parturition, pregnant MPFR and CT rats were treated with liraglutide or vehicle. At postnatal day 21 and before weaning, 20 CT and 20 FR male pups were sacrificed and lungs were analyzed by RT-PCR. Liraglutide restored surfactant protein A (SP-A) mRNA expression in pup lungs from food-restricted mothers. Surfactant protein B (SP-B) mRNA expression is not affected by neither IUGR nor liraglutide treatment. Moreover, liraglutide modulated different elements of RAS, increasing angiotensin-converting enzyme 2 (ACE2) and MasR mRNA expression only in pups from food-restricted mothers (MPFR), despite food restriction had not any direct effect at this early stage. Liraglutide also increased endothelial nitric oxide synthase (eNOS) expression in MPFR lungs, reflecting the activation of MasR by angiotensin 1–7. In conclusion, liraglutide prevented the alteration in lung function induced by IUGR and promoted the positive effects of ACE2-Ang(1–7)-MasR in restoring lung function.

Placental changes caused by food restriction during early pregnancy in mice are reversible.

In a previous study, 50% calorie restriction in mice from d1.5 to 11.5 of pregnancy resulted in reduced placental weights and areas,relative sparing of labyrinth zone area compared to junctional zone area, and dramatic changes in global gene expression profiles.However, little lasting effect was seen on adult offspring of these pregnancies, with a slight reduction in adiposity in males and some changes in liver gene expression in both sexes. The goals of the present study were to determine whether the placental changes induced by caloric restriction in early pregnancy had permanent, irreversible effects on the placenta, and whether the changes in liver gene expression in adult offspring were present before birth. There were no differences in placental weights or areas, or the areas of individual placental zones near term in mice that had previously been food restricted. Global gene expression profiles at d18.5 were indistinguishable in placentas from control and previously food-restricted mothers. In fetuses from restricted dams at d18.5, liver expression of Gck, a key regulator of glycogen synthesis, was reduced, whereas its expression was increased in livers from adult offspring of restricted dams. Ppara expression was also reduced in fetal livers from restricted dams at d18.5, but not in adult offspring livers. We conclude that alterations in the placenta caused by nutrient restriction in early pregnancy are reversible, and that alterations in gene expression in livers of adult offspring are not a result of changes initiated during pregnancy and maintained through adulthood.

Offspring's hydromineral adaptive responses to maternal undernutrition during lactation.

Early development, throughout gestation and lactation, represents a period of extreme vulnerability during which susceptibility to later metabolic and cardiovascular injuries increases. Maternal diet is a major determinant of the foetal and newborn developmental environment; maternal undernutrition may result in adaptive responses leading to structural and molecular alterations in various organs and tissues, such as the brain and kidney. New nephron anlages appear in the renal cortex up to postnatal day 4 and the last anlages to be formed develop into functional nephrons by postnatal day 10 in rodents. We used a model of undernutrition in rat dams that were food-restricted during the first half of the lactation period in order to study the long-term effects of maternal diet on renal development, behaviour and neural hydromineral control mechanisms. The study showed that after 40% food restriction in maternal dietary intake, the dipsogenic responses for both water and salt intake were not altered; Fos expression in brain areas investigated involved in hydromineral homeostasis control was always higher in the offspring in response to isoproterenol. This was accompanied by normal plasma osmolality changes and typical renal histology. These results suggest that the mechanisms for the control of hydromineral balance were unaffected in the offspring of these 40% food-restricted mothers. Undernutrition of the pups may not be as drastic as suggested by dams' restriction.

Maternal calorie restriction modulates placental mitochondrial biogenesis and bioenergetic efficiency: putative involvement in fetoplacental growth defects in rats

Low birth weight is associated with an increased risk for developing type 2 diabetes and metabolic diseases. The placental capacity to supply nutrients and oxygen to the fetus represents the main determiner of fetal growth. However, few studies have investigated the effects of maternal diet on the placenta. We explored placental adaptive proteomic processes implicated in response to maternal undernutrition. Rat term placentas from 70% food-restricted (FR30) mothers were used for a proteomic screen. Placental mitochondrial functions were evaluated using molecular and functional approaches, and ATP production was measured. FR30 drastically reduced placental and fetal weights. FR30 placentas displayed 14 proteins that were differentially expressed, including several mitochondrial proteins. FR30 induced a marked increase in placental mtDNA content and changes in mitochondrial functions, including modulation of the expression of genes implicated in biogenesis and bioenergetic pathways. FR30 mitochondria showed higher oxygen consumption but failed to maintain their ATP production. Maternal undernutrition induces placental mitochondrial abnormalities. Although an increase in biogenesis and bioenergetic efficiency was noted, placental ATP level was reduced. Our data suggest that placental mitochondrial defects may be implicated in fetoplacental pathologies.

Reproductive resilience to food shortage in a small heterothermic primate.

The massive energetic costs entailed by reproduction in most mammalian females may increase the vulnerability of reproductive success to food shortage. Unexpected events of unfavorable climatic conditions are expected to rise in frequency and intensity as climate changes. The extent to which physiological flexibility allows organisms to maintain reproductive output constant despite energetic bottlenecks has been poorly investigated. In mammals, reproductive resilience is predicted to be maximal during early stages of reproduction, due to the moderate energetic costs of ovulation and gestation relative to lactation. We experimentally tested the consequences of chronic-moderate and short-acute food shortages on the reproductive output of a small seasonally breeding primate, the grey mouse lemur (Microcebus murinus) under thermo-neutral conditions. These two food treatments were respectively designed to simulate the energetic constraints imposed by a lean year (40% caloric restriction over eight months) or by a sudden, severe climatic event occurring shortly before reproduction (80% caloric restriction over a month). Grey mouse lemurs evolved under the harsh, unpredictable climate of the dry forest of Madagascar and should thus display great potential for physiological adjustments to energetic bottlenecks. We assessed the resilience of the early stages of reproduction (mating success, fertility, and gestation) to these contrasted food treatments, and on the later stages (lactation and offspring growth) in response to the chronic food shortage only. Food deprived mouse lemurs managed to maintain constant most reproductive parameters, including oestrus timing, estrogenization level at oestrus, mating success, litter size, and litter mass as well as their overall number of surviving offspring at weaning. However, offspring growth was delayed in food restricted mothers. These results suggest that heterothermic, fattening-prone mammals display important reproductive resilience to energetic bottlenecks. More generally, species living in variable and unpredictable habitats may have evolved a flexible reproductive physiology that helps buffer environmental fluctuations.

Mixed sex allocation strategies in a polytocous mammal, the house mouse (Mus musculus)

The issue of adaptive adjustment of offspring sex ratio (proportion of male births) in polytocous mammals, producing several offspring per litter, is controversial because females of these species can maximize their fitness mainly by adjusting offspring number. To address this issue, we examined the effect of maternal condition at mating, experimentally decreased by pre-mating food restriction, on the sex ratio variation in 137 female mice. We tested two basic sex allocation hypotheses plausible for polytocous mammals: (1) the Myers hypothesis, predicting that cheaper sex should be favored in poor environmental conditions to maximize offspring number; and (2) the Williams hypothesis, predicting maximum fitness returns by adjusting size- and sex-specific composition of the litter according to the maternal condition. The food-restricted mothers produced larger litters with a higher proportion of cheaper daughters than the control mothers. By contrast, the control mothers optimized size and sex composition of the litter according to their weight at mating. In addition, the offspring of the food-restricted mothers suffered less from pre-weaning mortality than those of the control mothers. Therefore, when comparing the groups, the Myers hypothesis had a general significance while the Williams hypothesis was plausible only for the control mothers. Furthermore, some of the food-restricted mothers partly coped with the pre-mating food restriction and increased the proportion of sons in the litter with the increasing maternal weight loss (during the period of food restriction). The sex ratio variation was thus a result of three sex allocation strategies depending on the maternal condition at mating.

Influence of prenatal undernutrition on the effects of clozapine and aripiprazole in the adult male rats: Relevance to a neurodevelopmental origin of schizophrenia?

Epidemiological and experimental data indicate that maternal undernutrition may sensitize the offspring to the apparition of chronic diseases such as metabolic syndrome and schizophrenia, suggesting that these pathologies may have a developmental origin. To test this hypothesis, we have compared the effects of a 4 weeks treatment of clozapine (30 mg/kg once daily, p.o.) or aripiprazole (10 mg/kg once daily, p.o.) on metabolic and hormonal parameters in 4-month-old male animals from control or 70% prenatally food-restricted mothers (FR30 model). Both neuroleptics did not markedly modify body weight gain and food intake in both controls and FR30 rats. Clozapine decreased insulin secretion in both groups but significantly diminished leptin, corticosterone and glucose plasma levels only in FR30 animals. Aripiprazole decreased corticosterone plasma levels only in FR30 animals. Using quantitative RT-PCR array containing 84 obesity-related genes, we identified several genes involved in energy metabolism regulation whose expression was modified by clozapine or aripiprazole in adult male rat hypothalami. In addition, we demonstrated that expression of some of these genes was differentially affected by each neuroleptic in the hypothalamus of both FR30 and control animals. Although no marked metabolic alterations were observed in both control and FR30 animals after clozapine or aripiprazole treatment, our data indicate that offspring from undernourished mothers exhibit a modified sensitivity to atypical neuroleptics. Our results do not rule out a putative developmental origin of schizophrenia and may help to understand the way by which atypical neuroleptics, such as clozapine, sensitize schizophrenic patients to the development of metabolic disorders.

Transgenerational programming of hypertension by maternal food restriction during pregnancy

As human food poverty often spans generations, the aim was to determine whether the hypertensive influence of maternal food restriction during pregnancy is amplified across generations. Control Wistar rat mothers were exposed to ad libitum food intake in utero and then had free access to food while pregnant (CM); food restricted mothers were exposed to food restriction (50% of the pregnant control rat ad libitum intake from day 1 of pregnancy until birth) in utero and then had free access to food while pregnant (FRM); double food restricted mothers were exposed to 50% food restriction in utero and then were fed 50% of ad libitum while pregnant (DFRM). Systolic blood pressures (BP) did not differ between FRM (120.4 ± 1.7, n = 8) and DFRM (118.9 ± 1.0, n = 6) but were significantly higher than CM prior to mating (113.5 ± 1.0 mmHg, n = 6, P < 0.05). At 9 weeks, the BP of male offspring from FRM (120.1 ± 1.9, n = 15) was significantly higher than that of CM (109.3 ± 1.7, n = 11, P < 0.001); the BP of DRFM offspring was higher still (135.7 ± 1.5 mmHg, n = 12, P < 0.01 vs FRM). There was no correlation between maternal and offspring BP (r = 0.26, P = 0.12), implying that the higher BP of the DFRM offspring was not due to higher maternal BP. These data show that the programming effects of maternal food restriction are amplified across generations. Funded by the Fetal Programming of Diseases Research Chair, King Saud University.

Maternal food restriction during gestation elevates insulin-like growth factor I and insulin-like growth factor binding protein 1 in adult male rat offspring

Low birth weight due to maternal malnutrition is associated with increased risk of developing diseases in adulthood, for example, cardiovascular disease. Postnatal oxytocin treatment has previously been shown to have positive effects on blood pressure and corticosterone levels in adult offspring from malnourished dams. The aims of this study were to investigate if maternal food restriction during gestation alters plasma levels of insulin-like growth factor I (IGF-I), IGF binding protein 1 (IGFBP-1), glucose, and insulin in adult male rat offspring, and if postnatal oxytocin treatment has any effect on those changes. Adult offspring from food-restricted mothers had increased levels of IGF-I and IGFBP-1 compared with ad libitum–fed offspring. Postnatal oxytocin treatment had no effect on the IGF system in adulthood. In conclusion, maternal malnutrition during gestation altered the IGF system in adult rat offspring.

Oxytocin Treatment during Early Life Influences Reproductive Performance in ad libitum Fed and Food-Restricted Female Rats

Oxytocin treatment may permanently alter endocrine axes resulting in anti-stress and anabolic effects. However, the nutritional status influences the effects of oxytocin. The specific aims of this study were to investigate the effects of postnatal oxytocin treatment on reproductive performance in adult life, by studying maternal weight gain, adiposity, plasma levels of IGF-I as well as fetal and placental weights in the following groups of animals: (1) Ad libitum fed dams coming from ad libitum fed mothers. (2) Ad libitum fed dams coming from food-restricted mothers. (3) Food-restricted dams coming from ad libitum fed mothers. (4) Food-restricted dams coming from food-restricted mothers. Oxytocin treatment postnatally had long-term effects and increased adiposity in pregnant dams and stimulated placental and fetal growth relative to saline-treated dams. However, if the dams themselves had been exposed to food restriction during fetal life, the effect of postnatal oxytocin treatment changed. The oxytocin-treated mothers were still fatter but had smaller fetuses. In conclusion, postnatal oxytocin treatment influences reproductive performance in later life but is dependent on the mother’s previous and current nutritional experience.

Beta-cell mass and proliferation following late fetal and early postnatal malnutrition in the rat.

We have recently shown that maternal food restriction during late pregnancy in rats decreased beta-cell mass in the offspring at birth, without altering beta-cell proliferation. The aim of the present work was to determine: 1) whether sustained maternal undernutrition until weaning (R group) more dramatically alters beta-cell mass in the offspring and if normal food supply from weaning until adulthood could reverse the deleterious effects and; 2) if altered beta-cell proliferation was responsible for the decreased beta-cell mass. Beta-cell fraction and proliferative capacity were determined during the suckling period and at adult age after ad libitum feeding from weaning in the R animals and in age-matched controls (C group). At day 21, the offspring born and nursed by food-restricted mothers (R animals) showed a 66% reduction in beta-cell mass and number, which did not increase from birth to weaning, although beta-cell proliferation remained normal. At 3 months of age, R animals had 35% decreased beta-cell fraction, with a 50% decrease in the head of the pancreas. In that area, beta-cell proliferation was similar to that of the controls. In the tail of the pancreas, beta-cell fraction was only slightly impaired but beta-cell proliferation was increased by 37%, as compared with the controls. This increase was associated with a shift in islet size distribution towards medium and large islets compared with the head of pancreas from these R animals. No regional variations of beta-cell fraction, proliferation or islet size distribution were observed in adult control animals. In conclusion, prolonged malnutrition until weaning impairs beta-cell development but not beta-cell proliferation. Subsequent re-nutrition is followed by increased beta-cell proliferation but this is insufficient to fully restore beta-cell mass.

Sex-biased mortality in woodrats occurs in the absence of parental intervention

Male-biased mortality in young animals is often viewed as adaptive discrimination against male offspring by parents unable to raise reproductively competitive sons. Unequivocal evidence of the presence or absence of parental discrimination against males is lacking, however, and the adaptive interpretation of male-biased mortality is confounded by an alternative explanation that it reflects differential energetic requirements between the sexes (due to sexual selection for large size in mature males) independent of parental manipulation. To determine whether maternal discrimination against offspring explains postnatal mortality in a sexually dimorphic rodent, we examined patterns of growth and mortality in offspring of food-restricted and food-enriched lactating bushy-tailed woodrats,Neotoma cinerea. We also monitored mothers and their litters daily throughout lactation for evidence of maternal discrimination against offspring. Offspring of food-restricted mothers showed depressed growth, and mortality of offspring born to both food-restricted and food-enriched mothers was male-biased, but in the absence of maternal discrimination. Offspring that died were no less likely to be attached to their mother's teats in the 10 days prior to death than were offspring that successfully weaned. Similarly, offspring of food-restricted mothers were attached as often as were offspring of food-enriched mothers. In a series of behavioural arena trials in the first 10 days after birth, restricted mothers were no less attentive toward their sons than they were to their daughters, nor did mothers treat their offspring that did not survive to weaning differently from those that survived. Our findings provide empirical evidence that postnatal, sex-biased mortality in offspring is not necessarily due to parental intervention, and they call into question the adaptive interpretations of previous examples of sex-biased offspring mortality.

Distribution of energy between food-restricted dams and offspring.

Pregnant and nonpregnant rats were subjected to one of four food intake schedules: ad libitum, 20, 40 and 60% of food restriction, during 20 days, with the objective of studying the distribution of energy between food-restricted mothers and offspring. The animals were anesthetized and the conceptuses were removed and separated into male, female, and placental tissues. The rats were then killed and all carcasses were prepared for energy balance determination. A baseline group of weight-matched rats were killed in the first day of the experiment to evaluate the initial body energy. The results obtained showed that the energy balance of pregnant rats was impaired as a consequence of the food restriction imposed. The offspring from restricted mothers had lower body weight and energy content and were less in number than the ones from the control dams. It is also suggested that food restriction causes the mother to transfer similar percent energy to the offspring as in control animals which will deplete the mother energy stores more than in ad libitum fed rats.

Underfeeding of rat mothers during the first two trimesters of gestation does not alter insulin action and insulin secretion in the progeny

It has been suggested that impaired insulin action and/or insulin secretion in adult mammals could be a consequence of severe food restriction during fetal life. We have determined to what extent glucose homeostasis and insulin action are modified in male offspring of rats undernourished only during the first two trimesters of pregnancy. Pregnant females then were assigned to one of the following three experimental conditions. Rats in the first group had their food restricted to 50% of their pregnancy intake during the first 2 weeks of pregnancy, after which they were allowed to eat ad libitum. Rats in the second group were similarly restricted during the first 2 weeks, but beginning on day 14 of gestation were pair-fed to control rats until weaning on day 21 after birth: Such an experimental group was introduced because we observed that food-restricted mothers increased their food intake significantly above control levels in the last week of gestation and maintained this increase into the first postnatal week, when they were returned to ad libitum feeding on day 14 of gestation. Control rats (third group) were given access to food ad libitum throughout pregnancy and lactation. Offspring of mothers in the three groups are referred to as food-restricted/ad libitum refed (RA), food-restricted/pair-refed (RP) and control (C) groups, respectively. From 6 weeks of age, RA males ate significantly more food and gained significantly more weight on a standard laboratory diet than control offspring.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal state affects intestinal changes of rat pups at weaning

Two treatments of hooded rat (Rattus norvegicus) mothers after the birth of their pups affected the stage of development at which their pups started to take solid food and showed an increase in the expression of sucrase, an enzyme needed for digesting solid food. The pups of mothers that became pregnant in a post-partum oestrus showed the changes significantly earlier than those in the control group (Fig. 1A, B). The pups of mothers that had been mildly food-restricted after birth showed the changes significantly later than those in the control group. In brief, the pups of the pregnant mothers weaned first, the control pups next and those of food-restricted mothers weaned last. Differences in the timing of weaning between the three groups occurred in the absence of differences in pup body weight at the time of weaning. Within each experimental group, on the day in which sucrase expression showed the first detectable increase, sucrase activity was strongly predicted by the weight of the pups shortly after birth (Fig. 3). Pregnant mothers put on weight rapidly before implantation and their pups prepared for early weaning even though they did not differ in body weight from control pups (Fig. 4A, B). Food-restricted mothers were significantly lighter than control mothers and, with lighter pups before weaning started, settled for a longer period of suckling.