Artificially Reared Pups Research Articles

The Effect of Artificial Rearing on Gut Microbiota in a Mouse Pup-in-a-Cup Model

In this paper, the mouse pup-in-a-cup model was improved for younger mouse pups, and the effect of artificial rearing on gut microbiota development was evaluated. Intragastric cannulas were placed through the esophagus into 3-day-old C57BL/6J mice (n=48), and the mice were artificially reared (AR) with mouse milk substitute (MMS). Littermate pups (n=20) were maternally reared (MR) as controls. The feces of 3-day-old pups were analyzed by combining the PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting technique and sequencing of 16s rRNA gene fragments. After 11 days of artificial rearing, 37 of 48 pups were still alive. There were no significant changes in the number of DGGE bands or the Shannon index between the two groups. However, several bands in the AR group were obviously different from those in the MR group in the DGGE profile. These results demonstrate that it is possible to implant intragastric cannulas into 3-day-old C57BL/6J mice pups. However, the variation in the gut microbiota composition is non-negligible, even though the AR pups grow well.

Artificial Rearing of Rat Pups Reveals the Beneficial Effects of Mother Care on Neonatal Inflammation and Adult Sensitivity to Pain

Repeated pain during brain development can have long-term consequences in both humans and animals. We previously showed that maternal care provided to pups experiencing pain reduced adult pain sensitivity. This study tested whether sensory stimulation was responsible for this effect. Rat pups were either mother-reared controls (MR-CON) or artificially reared (AR) with minimal (AR-MIN) or maximal (AR-MAX) stimulation provided daily. In each rearing condition, pups were either uninjected or injected from postnatal day (PND) 4 to 14 with saline (0.9%) or formalin (0.2-0.4%). Pain behavior and paw inflammation were scored. Thermal sensitivity and responses to formalin were tested in adulthood (PND 70). AR neonates, irrespective of sensory stimulation received, exhibited a pain response (p < 0.001), even with a mild formalin dose. Maternal rearing reduced inflammation during the second week of life compared with AR pups (p < 0.05). Early pain exposure did not modify adult pain sensitivity. However, rearing altered adult pain sensitivity such that uninjected MR-CON rats had lower pain sensitivities than uninjected AR rats (p < 0.05). This suggests that the beneficial effects of maternal rearing can be obliterated if additional stimulation/stress occurs during the early neonatal period. In addition, this suggests that optimal level of maternal stimulation exists that determines adult pain sensitivity.

Adrenocortical response to stress in fasted and unfasted artificially reared 12‐day‐old rat pups

Recent studies have compared artificially reared (AR) rats with dam-reared rats on behavioral outcomes but, despite the fact that they are deprived of their mothers during the stress hyporesponsive period (SHRP), little is known about the effects of AR on the development of the stress response. In this study, the corticosterone (CORT) response to a stressor (saline injection ip) on postnatal Day 12 was assessed in rat pups that had been either dam-reared (DR) or artificially reared since Day 5. In the preceding 24 hr, half the pups in the DR group were maternally deprived (DEP). To control for the food deprivation consequent to maternal deprivation in these groups, half the pups in the AR groups also underwent 24-hr food deprivation (DEP). In the nondeprived condition AR pups did not differ from DR pups on untreated CORT levels or on levels at 1-hr poststress (i.e., all rats demonstrated low levels of CORT characteristic of the SHRP). In contrast, both maternally deprived DR pups and food-deprived AR pups exhibited increased untreated CORT levels as well as a significant increase at 30-min poststress, but CORT elevations were lower in the AR groups than in the DR groups. Thus, long-term maternal deprivation through artificial rearing in rats does not affect the reduced CORT levels and reduced CORT responsiveness associated with the SHRP; however, if animals are food deprived, then all show increased basal CORT levels and a greater CORT response to stress, although this response is lower in AR groups than in DR groups. These results suggest that rat pups artificially reared with adequate nutrition will still exhibit the SHRP.

Thymic stress in artificially reared and maternally reared rat pups

It is well documented that the hypothalamic-pituitary-adrenal (HPA) axis influences immunological responses to stress. Maternal factors have been shown to be necessary for appropriate modulation of the HPA axis in the developing rat. The purpose of this study was to determine whether artificially reared (AR) infant rat pups (a procedure whereby infant rats are gastrostomized and reared independently of maternal factors) have an altered function of the HPA axis in terms of thymocyte apoptosis (programmed cell death) and other indices of thymic stress. AR and maternally reared (MR) Long–Evans rat pups were randomized to control, fasted, stressed, and fasted+stressed treatment groups, as well as an unhandled, MR naive group that served as a baseline control. AR rat pups were significantly heavier than MR ( p<0.001). AR rat pups had significantly lighter thymuses than did the MR pups ( p<0.001) and fasted pups had significantly lighter thymuses than unfasted pups, regardless of whether they were in the AR or MR condition ( p<0.005). AR pups had significantly lower thymic cell numbers and a greater percent of necrotic cells than did MR pups. There were no significant effects of rearing condition on the percent of apoptotic thymocytes. The thymocyte alterations observed in this study between the two rearing conditions suggest that AR reduces thymic weight and cell numbers, which may have consequences for the development of adult cellular immunity.

Effect of intermittent feeding on the development of disaccharidase activities in artificially reared rat pups

We investigated the effect of an intermittent feeding schedule on the development of disaccharidase activities in the small intestine of artificially reared (AR) rat pups. Rat pups were fitted with an intragastric cannula at 5 days of age. A milk formula similar to the composition of rat milk was supplied by intermittent gastric infusion over the following 15–19 days. The body weight gain and plasma corticosterone levels of the AR pups matched those of pups reared naturally by dams (MR pups). At 10, 15 and 19 days of age, the small intestine from the ligament of Treitz to the ileocecal junction was divided into three segments of equal length and enzyme activities were measured in each. At the age of 10 and 15 days, sucrase and isomaltase activities were undetectable in AR pups fed according to a controlled schedule from the early postnatal period. These activities were first detected in the middle segment of the small intestine at 19 days of age in both AR and MR pups. Sucrase and isomaltase activities at the age of 19 days were diurnal in AR pups, but arrhythmic in MR pups. We conclude that artificial rearing via the intermittent gastric infusion of a milk formula containing only lactose as the carbohydrate source did not prematurely increase intestinal sucrase and isomaltase activities. Diurnal changes started from the beginning of development of these enzyme activities in AR pups.

Simplified preparation of a refined milk formula comparable to rat's milk: influence of the formula on development of the gut and brain in artificially reared rat pups.

Milk formulas for artificially reared (AR) rat pups are mostly based on complex cow's milk products, prepared by laborious and time-consuming processes. The aim of this study was to develop a simplified procedure for preparing a refined formula and to examine its influences on gut and brain development. The formula comprised a combination of purified cow's casein and whey proteins, five kinds of edible oil, minerals, and vitamins. Detailed analyses showed that the composition of macro- and micro-nutrients, osmolarity, and pH of the new formula closely resembled those of rat's milk. Rat pups, each with an intragastric cannula implanted at age 5 days, were artificially reared for the following 10-15 days. The body weight gain of AR pups matched that of mother-reared (MR) pups. Histoplanimetrical analyses showed that the small intestine in AR pups was more developed in relation to area of a transverse section, number and length of villi, and thickness of tunica muscularis than that of MR pups. Fat components in the formula influenced the fatty acid composition and the cholesterol-to-phospholipid ratio in the small intestinal microvillus membrane (MVM) of AR pups, but not the MVM fluidity. Brain weight was not significantly different between the two groups at age 15-20 days. This formula is useful for artificial rearing of rats and for identifying dietary components contributing to metabolic adaptation during the suckling period.

Is docosahexaenoic acid necessary in infant formula? Evaluation of high linolenate diets in the neonatal rat.

Neural accretion of docosahexaenoic acid (DHA) is thought to play an important role in the neural development of human infants. The lack of DHA in infant formulas contributes to the lowered neural accretion of DHA observed in formula-fed infants relative to those breast-fed. We hypothesized that lowering the dietary linoleic acid (LA) to alpha-linolenic acid (LNA) ratio may lead to increases in the level of DHA in the developing brain and retina. Lowering the LA to LNA ratio from 10:1 to 1:1 and to 1:12 in the artificially reared (AR) neonatal rat pup resulted in a significant increase in the percentage of brain DHA between AR dietary groups. The brain level of DHA in the AR group fed a 1:12 ratio was similar to that of a dam-reared reference group. However, levels of DHA in the retina of all AR groups were significantly lower than that of the (chow fed) dam-reared group. It appears that LNA may serve as an adequate substrate for the accretion of DHA in the brain, but not the retina of the developing rat. In both the brain and the retina, levels of arachidonic acid in the AR pups fed the 1:1 ratio were similar to that of the dam-reared group. However, levels in the 1:12 group were significantly reduced. The addition of long chain n-3 polyunsaturates such as DHA to infant formula may therefore be necessary for adequate neural DHA accretion and optimal neural development.

Artificial rearing of rat pups using rat milk

The contribution of diet and surgery to the brain weight deficits observed in artificially reared rats was investigated. Four day old Long Evans rat pups were assigned to an artificially reared (AR) or mother reared (MR) group. AR pups were encannulated and fed either rat milk (AR-MOM) or replacement formula (AR-MES). MR pups received a sham encannulation (MR-SHAM) or no surgery (MR-CONT) before being returned to their dam for rearing. On day 7 all the animals were killed. Brain weights and visceral organ weights were obtained. There was no significant difference between the MR groups on any measure except stomach weights. AR-MOM pups had larger visceral organ weights than pups in the other groups. AR-MOM and AR-MES pups had similar whole brain weights, smaller than those of the MR pups. However, the cerebellar weights, and to a lesser extent, brainstem weights, showed improvements in the AR-MOM group, over the AR-MES group. Neither the effect of surgery nor of diet alone can account for the organ weight differences that have been described in AR rats. The possibility that normal growth may be primarily dependent on diet at one stage of development, with other factors gaining importance at later stages is discussed.

Hormonal regulation of adaptive intestinal growth in artificially reared rat pups.

Adaptive growth and precocious expression of sucrase activity occur in the small intestine of artificially reared (AR) rat pups fed a hormone-free diet. The physiological mechanisms underlying adaptive intestinal growth were studied. Day 12 rat pups that received jejunal isografts subcutaneously on day 0 were subjected to artificial feeding and were killed on day 16. Crypt cellularity and DNA labeling index in isografts from AR, but not from mother-fed, rats increased significantly to levels found in in situ host jejunum of AR rats, indicating that humoral regulatory mechanisms are responsible for intestinal cell proliferation in AR pups. Radioimmunoassays of serum corticosterone, thyroxine, insulin, and gastrin and of gastric gastrin contents revealed that only serum corticosterone concentrations were significantly elevated, suggesting that corticosterone plays a critical role for intestinal growth. To examine this possibility directly, day 12 rats were adrenalectomized (ADX) and AR by continuous infusion of diets containing 0, 10, or 50 micrograms/ml corticosterone. Serum corticosterone concentrations paralleled the infused doses of corticosterone. Jejunal DNA labeling index increased in all ADX AR rats at day 13 in a dose-dependent manner. Increased jejunal DNA labeling index was maintained on day 14 in intact AR rats and ADX AR rats fed 10 micrograms/ml corticosterone but not in ADX AR rats receiving 0 or 50 micrograms/ml. We conclude that endogenous corticosterone is one of the systemic factors responsible for the adaptive increase in intestinal growth of AR rats.

Effects of early-life undernutrition in artificially reared rats: subsequent body and organ growth.

1. Four groups of rat pups were reared: mother-reared (MR) control (well-fed) and undernourished (MRC and MRU respectively) and artificially reared (AR) control and undernourished (ARC and ARU respectively). Pups for artificial rearing were fitted with a gastric cannula on postnatal day 5 and were fed, by intermittent gastric infusion, expressed rats' milk (days 5-7), mixtures of rats' milk and milk-substitute (days 8-16), and milk-substitute only (days 17-20). Solid food was available to MR pups throughout and to AR pups from day 14. Undernutrition, imposed from postnatal days 5 to 25, was effected initially by underfeeding the mother (MRU) or by infusing restricted quantities of milk (ARU). Weaning was at 21 d and undernutrition from day 21 to day 25 was by restricting the supply of solid food. All rats were fed ad lib. from 25 d. 2. The developmental milestone, eye-opening, was delayed by undernutrition but unaffected by artificial rearing. 3. Growth curves in body-weight during the refeeding phase were influenced most by previous undernutrition and to a lesser extent (also negatively) by artificial rearing. 4. Fourteen measures of body and organ growth were taken at autopsy at 39 weeks. Twelve measures were affected by nutrition and only four by rearing (weight of whole body, epididymal fat pads, renal fat pads and adrenals). 5. AR rats had lighter epididymal and renal fat pads than MR rats perhaps due to the low fat content of the expressed milk they received early in artificial rearing.

A phenomenon: left-biassed asymmetrical eye-opening in artificially reared rat pups

From 4 or 5 postnatal days, rat pups were either reared by their mothers (MR) or artificially reared (AR) without their mothers by infusion of ‘milk’ through a gastric cannula. In AR, but not MR, pups the left eye was more likely to open first than the right eye ( P < 0.001). The left-biassed eye-opening of AR pups may be a manifestation of stress-induced lateralisation of their brains.

Type of Milk Substitute Influences Growth of the Gastrointestinal Tract in Artificially Reared Rat Pups

Summary:Growth of the gastrointestinal (GI) tract was studied in rat pups that were reared normally (motherreared, MR) or were artificially reared (AR) by intragastric infusion of milk substitutes from postnatal day 5. Two mitk substitutes were used: one high in carbohydrate and low in protein compared with rat's milk (Messer) and the other close in composition to rat's milk (Auestad). Pups reared on these formulae are termed ARM and ARA, respectively. Pups were killed at 7, 12, and 20 days for the determination of the wet and dry weights of stomach and caecum and the lengths of the small and large intestines (SI and LI). At 7 days AR pups showed deficits in several GI measures compared with MR rats. However, by 20 days they showed enhanced growth of various parts of the GI tract. Stomach weight was greatest in ARA rats; caecum weight was greater in AR than in MR rats; SI was longer in AR than MR rats, with the effect more pronounced in ARM rats. These effects were already evident, or becoming so, by 12 days. In an additional experiment, rats were AR on rat's milk from 5 to 12 days. Their GI tracts did not differ from MR, suggesting that the AR procedure per se was not responsible for the above GI growth effects.

Somatic and central nervous system growth in artificially reared rat pups

Rearing preweanling rat pups away from their mothers by feeding through chronic intragastric cannulas has been shown to result in alterations in the growth of specific organs. In the present study, artificially reared (AR) rats and their normally reared (NR) siblings were sacrificed at various ages during this procedure to determine the time course of these alterations. Brain growth deficits were detected within 24 hours, peaked after 8 days of artificial rearing and showed some recovery by the end of the study. By day 18, the livers, kidneys and spleens of the AR pups were significantly larger than those of their NR siblings. The spleens showed an initial decrease in weight compared to the spleens of the NR pups. However, by day 18, the spleens of the AR group were significantly larger than those of the NR group.