Nutritional influences on sexual maturation in the rat

The effect of altered nutrition on sexual maturation may depend in part on the nature and timing of the dietary change. The data are conflicting as to whether rats undernourished before weaning but normally fed after weaning have delayed puberty, but such undernourished rats clearly weigh less at vaginal opening than do normally fed animals. Altered nutrition after weaning can change the timing of puberty, and in such cases the body weight at puberty of the animals given the modified diet is frequently abnormal. The factors regulating the age and weight at puberty of rats fed altered diets seem to include the degree of underfeeding, as reflected in the growth rate, and the composition of the diet. Undernourished immature male rats have low serum testosterone secondary to gonadotropin deficiency. Basal luteinizing hormone (LH) in these animals is either low or "inappropriately normal" relative to their hypoandrogenic state (low serum testosterone and sexual accessory gland weights), and serum LH increases after luteinizing hormone-releasing hormone (LHRH) or castration are normal or minimally reduced. Serum follicle-stimulating hormone (FSH) in undernourished rats is subnormal basally and after administration of LHRH, but not after castration, which suggests that the low basal serum FSH is due to inhibition of FSH output by a testicular factor. Spermatogenesis may be unaltered by dietary changes severe enough to cause hypoandrogenism, although very severe under-nutrition will impair sperm production.     

Purine metabolizing enzyme activities in radiosensitive tissues of mice after sublethal whole-body irradiation

The activities of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were determined between days 1-14 in the spleen, thymus and femoral bone marrow of mice subjected to whole-body gama irradiation with a dose of 5.5 Gy. In control animals, the highest activity of ADA (as related to 10(6) cells) was recorded in the thymus (58.9 pmol.s-1), the lowest one in the femur (34.8 pmol.s-1), the PNP activity was the lowest in the thymus (14.5 pmol.s-1) and the highest in the femur (96.0 pmol.s-1). In the spleen, an elevation of ADA activity (up to 379%) was observed during the first postirradiation days; PNP activity was reduced (to 58%) on postirradiation day 3, followed by the return and even elevation on day 14 (265%). In the thymus, a parallel reduction of the activities of both enzymes appeared during the first postirradiation days, with a subsequent increase during the regeneration phase. In the femoral bone marrow, ADA and PNP activities were increased on postirradiation day 1 (275% and 201%, respectively). Reference is made to the possible relationship between the observed characteristic changes in activities and the degree of damage and/or renewal of cell population in the hemopoietic tissues after irradiation.     

The effect of cyclophosphamide and gamma irradiation on adenosine deaminase and purine nucleoside phosphorylase in mice

Changes in ADA and PNP activities in the spleens and thymuses of mice were studied after a single administration of cyclophosphamide (CY, 200 mg/kg) and after whole-body gamma irradiation (5.5 Gy), applied alone or three days after CY application. In the first days after the treatment the enzyme activities were significantly depressed (p less than 0.01) with the exception of ADA in the spleen, where a high elevation (220-380%) in relation to controls was observed. During the regeneration period a pronounced rise of PNP activity in the spleen occurred mainly after a combined application of CY and irradiation (270%). In the thymus the regeneration was manifested by a mild increase of both ADA and PNP activities towards control values. The findings suggest that the expressive changes of ADA and PNP activities, participating in the purine salvage pathway, may, after a cytotoxic treatment, influence the nucleotide pool and DNA synthesis in lymphoid organs.     

Early weaning programs rats to have a dietary preference for fat and palatable foods in adulthood

The objective of this work was to study the effect of early weaning on alimentary preference for the macronutrients protein, carbohydrate and fat in adult rats. Male Wistar rat pups were weaned by separation from the mother at 15 (D15) or 30 (D30) days old. Body weight and food intake were measured every 30 days until pups were 150 days old. At 110 days of age, the alimentary preference was evaluated for 1 h on 3 consecutive days. At 120 days of age, the palatable diet test was conducted during 3 consecutive 24-h periods. Body weight and food intake were not altered, but early weaning in rats induced an alimentary preference to fat and hyperphagia of a palatable diet. In conclusion, early weaning, although did not modify body weight or basal food intake, promoted an increased preference for palatable and fatty foods. This demonstrates that early weaning is not capable of promoting perceptible alterations of alimentary behavior under normal laboratory conditions. However, in the presence of a stimulating factor such as a choice of nutrients or a palatable diet, a possible latent effect on dietary preferences may become apparent. Over the long term, this preference for foods with high caloric density can lead to obesity and metabolic perturbations.     

Insulin secretion and GLUT-2 expression in undernourished neonate rats

In previous studies, we verified increased insulin sensitivity in adult male offspring of lactating rats readjusting to lack of insulin secretion reduction brought about by protein restriction during lactation. The present study aims to evaluate the effects of maternal protein undernutrition during lactation on glucose-induced insulin secretion and GLUT-2 expression in beta-cells of neonate male and female rats. Lactating Wistar rats were given a protein-free diet during the first 10 days and a normal diet (22% of protein) until weaning. The neonates were separated at birth by sex and diet and studied at 4, 8 and 21 days of lactation. Glucose-induced insulin secretion by pancreatic islets was analyzed by radioimmunoassay and GLUT-2 expression in beta-cells by Western blot. Glucose-induced insulin secretion of the undernourished groups was higher than in the control groups except among females. When comparing the male and female groups and the control and undernourished groups, female neonates showed significantly greater insulin secretion than the male group. Also it was noted that undernutrition induced greater GLUT-2 expression. For instance, comparing the undernourished male and female neonates there was an increase in female GLUT-2 expression on day 4. On the other hand, in undernourished male neonates a GLUT-2 expression increased later in lactation. In conclusion, during a short term, maternal undernutrition induces an increase of the glucose-induced insulin secretion only in male neonates and is associated with an increase in GLUT-2 expression in the beta-cell.     

Effects of undernutrition during suckling and of training on the hypothalamic beta-endorphin of young and adult rats

The involvement of the hypothalamic beta-endorphinergic system in behavioral processes has previously been studied in adult rats. In the present report, we studied the effects of undernutrition and of inhibitory avoidance training on the hypothalamic beta-endorphin-like immunoreactivity of 21-day-old and adult rats. Rats were undernourished by feeding their dams an 8% protein diet from the day of delivery until weaning (21 days of age). The beta-endorphin-like immunoreactivity was measured by radioimmunoassay. In adult rats, undernutrition decreased the basal level hypothalamic beta-endorphin. Avoidance training decreased the content of beta-endorphin in the hypothalamus of well-nourished adults, but had no effect on the levels of previously undernourished rats. In 21-day-old rats, neither undernutrition nor avoidance training altered the levels of beta-endorphin. These results suggest that the hypothalamic beta-endorphinergic system of weaning rats is not yet functional in relation to the parameters analyzed. Probably, other developmental factors are necessary for the emergence of the effects of undernutrition found in adult rats and for the emergence of the response of this system to training (novelty).     

Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

Effects of pre- and postnatal protein deprivation and postnatal refeeding on myenteric neurons of the rat large intestine: a quantitative morphological study

We investigated the effect of protein deprivation and refeeding on weight gain, the size of the colon, and the numbers and sizes of enteric neurons. Neurons were located by reduced nicotinamide adenine dinucleotide (NADH) diaphorase staining. Protein deprivation of the mother throughout pregnancy, and the mother and unweaned rat pups in the first 21 postnatal days, reduced the weights of pups to about 50% of control. The size of the colon was also reduced, by about 40%. Despite this, total numbers of neurons in the colon were not reduced. However, there was a small, but significant, 15% reduction in the areas of neuron profiles. After 21 days the remaining pups were removed from the mothers, and either maintained on the control diet, maintained on the protein-deprived diet, or changed from the protein-deprived diet to a normal diet (refed group). These rats were examined after a further 21 days. Refeeding restored body weight to 20% below control, restored colon size, and restored nerve cell size. After a total of 42 days of protein deprivation, nerve cell numbers were not significantly different from control. In undernourished rats at 21 and 42 days, neurons were less well stained than control for NADH diaphorase. Refeeding between 21 and 42 days restored the normal appearance of the neurons. It is concluded that enteric neurons are protected from loss even when there is a substantial reduction in body weight and organ size caused by protein deprivation. The neurons become smaller, but recover size after refeeding.     

Suckling Behavior of the Collared Peccary (Tayassu tajacu)

Based on Trivers' (1974) theory of parent/offspring conflict, we predicted that weaning would be accelerated under poor dietary conditions in an aseasonally breeding, monomorphic ungulate, the collared peccary (Tayassu tajacu). Suckling behavior was observed in 18 captive litters under a dietary treatment of high or low protein formulated to mimic forage under ecological conditions of normal rainfall vs. drought. Litters on the low protein diet weaned later (mean of 27 weeks) than litters on the high protein diet (mean of 21 weeks). Other indices of cumulative suckling effort (weaning weight, cumulative minutes suckling, cumulative bouts) did not differ between dietary treatments. No differences in suckling effort were found relative to offspring sex or litter size. Milk did not completely compensate for the low protein diet affecting growth of young in the second and third phases of lactation. Some nursers did not conserve bodily resources as would be expected if the benefits of short interbirth intervals outweighed the costs of lactation during unpredictable drought conditions.     

Low Tryptophan and Protein in the Diet During Development Increase the Susceptibility to Convulsions in Adult Rats

Tryptophan (TRY) is the precursor for serotonin (5-HT) synthesis. Common maize has low protein content with low concentration of TRY and lysine. A diet based on two strains of corn differing in their TRY content were given to adult female rats, prior mating, during pregnancy and lactation. Same diets were offered to their male offspring after weaning until reaching 60-days old. The pattern and severity of the convulsive phenomenon induced by monosodium glutamate (MSG) in a well established model of Status epilepticus were evaluated in comparison with data from animals of two control groups: (a) rats fed a hypoproteic (8% protein) diet, and (b) rats fed a normal Purina chow diet (23% protein). Significant increased susceptibility to convulsions was observed in both groups of rats fed the corn-based diets. However, the animals fed the common corn-based diet (8–9% protein; 0.058% TRY) showed a higher susceptibility to convulsions than what was registered in animals fed a Quality Protein Maize (QPM)-based diet (8–9% protein; 0.1% TRY). It is concluded that low TRY concentration in the diet during development, produces lower rate of brain 5-HT synthesis, affecting development and maturation of GABAergic inhibitory cortical interneurons, with alteration of cortical excitability, contributing in part, to the increased susceptibility to convulsions, as shown in the experiments here reported. Special issue article in honor of Dr. Ricardo Tapia.     

Adaptive changes in translation initiation activities for rat pancreatic protein synthesis with feeding of a high-protein diet

We have previously demonstrated that dietary protein induced pancreatic hypergrowth in pancreaticobiliary diverted (PBD) rats. Dietary protein and dietary amino acids stimulate protein synthesis by regulating translation initiation in the rat skeletal muscle and liver. The aim of the present study was to determine whether feeding a high-protein diet induces activation of translation initiation for protein synthesis in the rat pancreas. In PBD rats in which the bile-pancreatic juice was surgically diverted to the upper ileum for 11-13 days, pancreatic dry weight and protein content were doubled compared with those in sham rats and further increased with feeding of a high-protein diet (60% casein diet) for 2 days. These pancreatic growth parameters were maintained at high levels for the next 5 days and were much higher than those of sham rats fed a high-protein diet. In both sham and PBD rats, feeding of a high-protein diet for 2 days induced phosphorylation of eukaryotic initiation factor 4E-binding protein 1 and 70-kDa ribosomal protein S6 kinase, indicating the activation of the initiation phase of translation for pancreatic protein synthesis. However, this increased phosphorylation returned to normal levels on Day 7 in PBD but not in sham rats. We concluded that feeding a high-protein diet induced pancreatic growth with increases in the translation initiation activities for pancreatic protein synthesis within 2 days and that prolonged feeding of a high-protein diet changed the initiation activities differently in sham and PBD rats.     

Birth weight and postnatal dietary protein level affect performance, muscle metabolism and meat quality in pigs

Intrauterine growth restriction (IUGR), resulting in low birth body weight (LBW) occurs naturally in pigs. However, IUGR may also cause persistent changes in physiology and metabolism resulting in poorer performance, organogenesis and meat quality. As IUGR pigs have a lower daily gain from birth to slaughter they may differ in utilization of nutrients and requirements for dietary protein compared with their larger littermates. Thus, the objective in this study was to examine the interaction between birth body weight (BW) and the postnatal dietary protein level, in relation to postnatal performance, organogenesis, muscle metabolism and meat quality. The experiment was carried out with offspring from 16 purebred Danish Landrace gilts mated to Danish Landrace boars. The female and entire male pigs with LBW that survived at weaning were compared with the female and male pigs with the highest/high birth body weight (HBW) within each litter. The offspring were reared individually from weaning and were fed ad libitum a diet containing either a normal level of protein (NP) for optimal growth or an isocaloric diet containing a 30% lower protein content (LP) from 3 weeks to 150 days of age. At slaughter, we found no interactions between birth weight group and dietary protein level for any of the measured traits. The relative crown-rump length (cm/kg) at birth indicates that LBW pigs were thinner than HBW pigs. Daily gain and feed intake were reduced by 14% and 10%, respectively, while the kg feed/kg gain was slightly increased by 3% in LBW pigs compared with HBW pigs. The LP diet reduced daily gain by 27% due to reduced feed intake and increased kg feed/kg gain by 12% and 21%, respectively compared with the NP diet. LBW male pigs produced meat with a higher shear force than male HBW pigs and also LP pigs produced meat with higher shear force than NP pigs. The activity of lactate dehydrogenase in the Longissimus dorsi muscle (LD) was reduced in pigs fed the LP diet. Calpastatin was increased in LD of LBW pigs and decreased in pigs fed the NP diet. In conclusion, these results suggest a rejection of our hypothesis that low birth weight littermates have a lower requirement for dietary protein compared with heavy weight littermates. Furthermore, LBW male pigs and LP fed pigs of both genders produced less tender meat than HBW pigs or NP fed pigs, respectively.     

Dietary regulation of adenosine deaminase activity in stomach, small intestine and spleen of mice

Activity of adenosine deaminase (ADA) and its regulation by dietary restriction were studied in the stomach, small intestine and spleen of mice. ADA activity (U/mg protein) was highest in the stomach, followed by small intestine and spleen of mice on normal diet. The activity decreased significantly in the stomach (41%) and small intestine (45%) of 24 hr fasted mice, when compared to mice fed ad-libitum. However, ADA activity in spleen did not show any change by dietary intervention. Refeeding of fasted mice for 24 hr restored the activity of ADA in tissues. In addition, dietary restriction (alternate days of feeding for three months) had a cumulative effect, whereby ADA activity decreased significantly in the stomach (53% on the day of feeding and 60% on the day of fasting) and small intestine (50% and 54% on the day of feeding and fasting, respectively) without any change in activity in spleen. These findings indicate that dietary restriction reduces ADA activity in a tissue-specific manner. Long-term dietary restriction leads to a cumulative adaptation in lowering the ADA activity of GIT, but not in spleen.     

Use of soybean flour (dehulled, solvent-extracted soybean) as a fish meal substitute in practical diets for African catfish, Clarias gariepinus (Burchell 1822): growth, feed utilization and digestibility

Growth and digestibility trials were conducted using African catfish, Clarias gariepinus (Burchell 1822): (1) to obtain apparent digestibility coefficient (ADC) values for capelin fish meal, soybean flour and corn meal; (2) to formulate diets based on ADC values of the protein feedstuffs; and (3) to evaluate the effects of replacing 25%, 50% and 75% of fish meal in control diets with soybean flour on growth, feed utilization efficiency and carcass composition. Supplemental methionine was added to the diet formulation in which soybean flour replaced 75% of the diet. Diets were formulated (400 g digestible protein kg⁻¹ and 15 kJ digestible energy g⁻¹ dry diet) and fed to catfish fingerlings (13.1 ± 0.5 g) to apparent satiation twice daily for 70 days. The protein and energy digestibilities of fish meal and soybean flour were high (>90% and >80%, respectively; P < 0.05). At 75% fish meal replacement with soybean flour (without methionine supplementation), growth and feed utilization efficiency indicators were depressed compared with other diet treatments which had a similar (P > 0.05) growth and feed utilization efficiency to those fed the control diet. The carcass compositions of catfish in all diets were similar (P > 0.05) and the liver histology of catfish fed any of the diets showed no alterations. The results obtained indicate that 50% of fish meal protein in practical catfish diets can be replaced with soybean flour and that catfish can effectively utilize supplemental methionine, thereby allowing up to 75% of the dietary fish meal protein to be replaced by soybean flour.     

Early exposure to a high-fat diet has more drastic consequences on metabolism compared with exposure during adulthood in rats

The aim of this study was determine whether the introduction of a high-fat diet during the peripubertal phase induces significant changes in body weight control, glucose homeostasis and the parasympathetic tonus compared with the administration of this diet to adult rats. High-fat diet was offered to male Wistar rats at weaning or during adulthood. A group of rats received high-fat diet for 60 days, from weaning to 81-day-old (HF81) or from 60 to 120-day-old (HF120), whereas 2 other groups received a normal-fat diet (i. e., NF81 and NF120). We analyzed adiposity, glucose homeostasis, insulin sensitivity, and vagal nerve activity. High-fat diet increased the accumulation of adipose tissue in all of the rats, but the difference was greater in the rats that were fed the high-fat diet since weaning (p<0.001). The HF rats showed glucose intolerance with high levels of insulin secretion during the glucose tolerance test (p<0.01). Rats that were fed the high-fat diet presented severe insulin resistance, indicated by a low K itt (p<0.01). Interestingly, the HF81 rats exhibited greater insulin resistance compared with the HF120 rats (p<0.05). The recordings of vagus nerve activity showed that the HF rats had higher parasympathetic activity than the NF rats irrespective of age (p<0.01). Our results show that a high-fat diet offered to rats just after weaning or in adulthood both cause impairment of glycemic homeostasis and imbalance in parasympathetic activity. Importantly, the consumption of high-fat diet immediately after weaning has more drastic consequences compared with the consumption of the same diet during adulthood.     

Effects of dietary protein supply, weaning age and experimental enterotoxigenic Escherichia coli infection on newly weaned pigs: health

Weaning is often associated with post-weaning colibacillosis (PWC), caused by enterotoxigenic Escherichia coli (ETEC). The objective was to investigate the effects of manipulating dietary protein supply and increasing weaning age on enteric health and ETEC shedding of newly weaned pigs exposed to an experimental ETEC challenge. The experiment consisted of a complete 2 × 2 × 2 factorial combination of weaning age (4 v. 6 weeks), dietary protein content (H, 230 g crude protein (CP)/kg v. L, 130 g CP/kg) and experimental ETEC challenge (+ v. -); all foods were free from in-feed antimicrobial growth promoters (AGP). An additional four treatments were added to allow the effect of protein source (DSMP, dried skimmed milk powder v. SOYA, soybean meal) and AGP inclusion (yes v. no) to be investigated in challenged pigs of both weaning ages. On day 3 post-weaning challenged pigs were administered per os with 109 cfu ETEC O149. A subset of pigs was euthanased on days 0 and 6 post weaning to assess enteric health and small intestine morphology. Both weaning age and dietary protein content affected the consequences of ETEC challenge. ETEC excretion persisted longer in the 4-week-weaned pigs than those weaned at 6 weeks. Although not significant, the numbers of ETEC shed in the faeces post infection (days 4 to 14) were higher on the H than L diet, especially in the 4-week-weaned pigs (P = 0.093). Lowering CP level led to significantly firmer faeces post challenge (days 3 to 6) and decreased colonic digesta pH. Protein level had no effect on small intestine villous heights or crypt depths. There was no significant effect of protein source on ETEC excretion or enteric health. Results suggest that increasing weaning age and decreasing the level of dietary protein, especially in earlier weaned pigs, may help to maintain enteric health and minimise the effects of PWC.     

Behavioural responses of rats to early housing conditions and to protein-energy malnutrition

Lactating rats were fed either the commercial diet (CO) or the low protein diet which induced symptoms of protein - energy deprivation (PD). They were housed in cages either individually (IH) together with eight offspring or in a large community cage (COM) shared by six dams and 48 youngs. After weaning all rats lived in cages by 4-5 animals. The PD terminated at the age of 49 days. Behaviour was tested on the 10th, 42nd and 150 th days of life. Body weight was recorded during nursing period both in dams and pups, after weaning in male rats. The low protein diet affected both body weight and maternal behaviour of the dams. In PD + IH pups growth as well as behavioural development was retarded. Behavioural alterations persisted even after the PD had been treated. The COM housing improved the body weight in the PD + COM dams, behaviour of both the PD + COM dams ond the pups was less affected than of the PD + IH group. The effect of the early housing was long lasting, manifested itself in elevated exploratory activity and in the decreased emotional responses in both the PD + COM and the CO + COM groups at the age of 42 and 150 days.     

Inverse relationship between adenosine deaminase and purine nucleoside phosphorylase in rat lymphocyte populations

The levels of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were measured in rat lymphoid cell populations. The specific activities of the two enzymes in thymus, lymph node, spleen, and bone marrow were found in inverse proportion to each other. Thy mocytes had the highest ADA activity and the lowest PNP activity, whereas spleen and bone marrow lymphocytes had the lowest ADA activity (six- to sevenfold lower than thymocytes) and the highest PNP activity (fourfold higher than thymocytes). This reciprocal relationship was also found in cells of the T lymphocyte lineage at various stages of differentiation. These results suggest that specific stages of T-cell development may be characterized by the levels of the two enzymes, and that deficiencies of each of these enzymes might affect T cells at separate stages of differentiation.     

Importance of Dietary Cholesterol for the Maturation of Mouse Brain Myelin

The effect of nil (control), 1% (CH-l) and 5% (CH-5) dietary cholesterol on the myelination of mouse brain, and its deposition in the heart and liver were investigated during infancy. Swiss Webstar female mice were given formulated diets from early gestation, and their pups were weaned on the same diet as that of the individual mothers up to 60 days after birth. The test diets increased the liver weight and cholesterol content compared to the control even in suckling pups (20 days), but did not significantly influence the heart weight until 60 days. The cholesterol content of the heart was not increased by the CH-l diet throughout the feeding period, but it did increase the mole ratio of major myelin lipids and hastened its maturation. Myelin cholesterol was 10% higher in 20-day-old suckling pups in the CH-5 group compared to the control. Data indicate that dietary cholesterol altered the brain myelination rate of weaning mice, and that the mother’s dietary cholesterol influenced myelination of the suckling pups.     

Importance of dietary cholesterol for the maturation of mouse brain myelin.

The effect of nil (control), 1% (CH-1) and 5% (CH-5) dietary cholesterol on the myelination of mouse brain, and its deposition in the heart and liver were investigated during infancy. Swiss Webstar female mice were given formulated diets from early gestation, and their pups were weaned on the same diet as that of the individual mothers up to 60 days after birth. The test diets increased the liver weight and cholesterol content compared to the control even in suckling pups (20 days), but did not significantly influence the heart weight until 60 days. The cholesterol content of the heart was not increased by the CH-1 diet throughout the feeding period, but it did increase the mole ratio of major myelin lipids and hastened its maturation. Myelin cholesterol was 10% higher in 20-day-old suckling pups in the CH-5 group compared to the control. Data indicate that dietary cholesterol altered the brain myelination rate of weaning mice, and that the mother's dietary cholesterol influenced myelination of the suckling pups.