PRELIMINARY STUDY OF TIME-DEPENDENT INFLUENCE OF MATERNAL NUTRITION WITH ADDITION OF β-HYDROXY- β -METHYLBUTYRATE ON THE BODY WEIGHT AND SELECTED ORGANS WEIGHT IN THE NEW-BORN SPINY MICE (ACOMYS CAHIRINUS) OFFSPRING

Siemowit Muszyński¹, Agnieszka Tomczyk², Piotr Dobrowolski³, Ewa Tomaszewska², Monika Hułas-Stasiak^3
1 Department of Physics, Faculty of Production Engineering, University of Life Sciences in Lublin, Poland
² Department of Animal Physiology, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Lublin, Poland
³ Department of Comparative Anatomy and Anthropology, Maria Curie-Skłodowska University, Lublin, Poland

ABSTRACT

Interferences into maternal nutrient are used in studies related to prenatal programming – the permanent changes in homeostasis of regulatory physiological mechanisms. In present study, the effect of HMB (β-hydroxy-β-methylbutyrate) maternal administration on body composition in newborn offspring of spiny mouse (Acomys cahirinus) was studied. The spiny mice were randomized into control (fed with a standard diet) and two experimental sub-groups additionally supplemented with HMB – 0.2 mg/kg of body weight per day in accordance with the time of HMB administration during gestation: period I lasting from the 13^th to the 26^th day of the gestation and period II lasting from day 26 to day 39 of gestation (lasting generally 39–40 days). Newborn spiny mice from the control group, the HMB I and HMB II groups were weighted and euthanized, and their organ weights were recorded. Maternal HMB-treatment did not significantly influence the body weight of newborns, although the offspring delivered by dams administered with HMB during middle gestation were significantly smaller compared to newborns delivered by dams treated with HMB during the last period of gestation. Lung and spleen weights were increased in the offspring delivered by HMB-treated dams irrespective of the period of administration. The weights of liver, kidneys and heart in the offspring delivered by HMB-treated dams in middle gestation were increased compared to the control group.
    This study showed a time-dependent maternal influence of HMB supplementation on the development of kidneys and liver. The increase in the weight of basal vital organs of newborn offspring in spiny mouse was not proportional to their body size.

Key words: prenatal programing, β-hydroxy β-methylbutyrate, spiny mouse, offspring development.

INTRODUCTION

Changes in the pattern of prenatal development influence postnatal development depending on the phase of development, genetic sensitivity of the foetus, and the condition of the pregnant dam. Adaptive, functional, structural, and metabolic changes appearing during prenatal development as an effect of interaction between genes and environmental or nutritional factors play a dual function. First, they increase the chance of survival of the foetus in suboptimal conditions during the prenatal period, and then influence the postnatal development [5, 21]. In studies concerning prenatal programming – permanent changes in homeostasis of regulatory physiological mechanisms – interference into maternal diet is applied [16, 17, 22].

The positive impact of specific compounds in food is evident. Leucine is a branched-chain amino acid most responsible for protein synthesis and muscle growth. Its bioactive metabolite, HMB (β-Hydroxy-β-methylbutyrate), is found in foods of both plant and animal origin, and it is also normally produced in the body. Depending on the nutritional program, 0.3 grams to 1 gram of HMB is produced in the body per day [14, 15]. It has been proven that HMB given to pregnant dams can program the postnatal development of their offspring [20]. Moreover, maternal administration with HMB has positive long-term effects on postnatal development, including the skeletal system in offspring, improving bone mineral density, as well as the geometric and mechanical properties [20]. Maternal supplementation of HMB affects the physical properties of the enamel surface of spiny mice offspring in dose-dependent manner. Powdered HMB given at the daily dosage of 0.2 g/kg of body weight from day 26 of gestation until parturition causes more significant changes in the enamel surface texture and results in more reduced roughness parameters compared to the administration of 0.02 g/kg of body weight during the same period [18]. It has been documented that HMB modulates protein turnover [14,15], and also serves as a key carbon source for de novo cholesterol synthesis in tissues, which is necessary for the maintenance of maximal cell function [14, 15, 24]. Other studies in humans and rats have shown that the supplementation of diet with HMB results in increased wound collagen deposition, increasing wound repair processes [24]. A study on chickens fed in ovo with HMB proved its influence on intestinal development [19]. On the other hand, there is a recent study, which demonstrates that HMB administrated to mice at a late stage of pregnancy disrupts the control and synchronisation of neuronal excitability in their male offspring via the influence on neurons with the calretinin expression in neurons of the dorsal hippocampus [10]. HMB at 0.2 g/kg of body weight per day administrated to pregnant mice (days 26-39 of pregnancy) causes a decrease in the density of neurons and the intensity of calretinin expression in the hilus of the dentate gyrus in newborn and weaned males [10].

Moreover, the efficacy of HMB has also been demonstrated in pathological conditions, where it has been reported to reduce muscle wasting associated with muscular dystrophies, trauma and cancer cachexia. HMB has been shown to reduce muscle atrophy and increase muscle hypertrophy by inhibiting muscle degradation involving stimulation of muscle cell proliferation and differentiation [9].

Acomys cahirinus is an unusual animal model characterized by rapid development and substantial autonomy shortly after birth, which makes it an extremely valuable research object. Moreover, the offspring are furred, have open eyes, and sophisticated locomotor capabilities, and organogenesis is largely complete by the end of gestation. Therefore, in the present study, we have chosen the spiny mouse as the most suitable mouse species for maternal programming [7, 8].

The aim of this study was to investigate the postnatal consequences of time-dependent HMB maternal administration on the body composition of newborn offspring of spiny mice (Acomys cahirinus).

MATERIAL AND METHODS

Ethical approval
The experiments were performed in accordance with the Polish legal requirements under the licence of the Local Ethical Committee of Maria Curie-Skłodowska University in Lublin, Poland.

Pregnant dams
The spiny mice used in this study were obtained from our own laboratory colony. Precise mating time is not possible to be determined in this species due to the lack of visible vaginal plugs; therefore, postpartum oestrus exhibited by the spiny mice within 24 h of giving birth was used to determine gestational ages.

The dams were randomized into control (n=10) and experimental groups (n=20) on the basis of body weight (40–50 g). Dams were paired at a ratio of 1 male to 1 female and they were allowed to naturally conceive and deliver their first litter with no human intervention. The first 24-h period after the birth of the first litter is deemed to be the day of conception of the second litter and the next day as day 1 of gestation. Pregnant spiny mice were housed singly in separate cages under constant conditions with a 12 h light/dark cycle at 22°C and 55–60% humidity level.

To investigate the detrimental effects of maternal nutrition treatment on the development of newborn offspring, HMB administration was performed in experimental dams during two different periods, lasting from day 13 to day 26 (period I) and from day 26 to day 39 (period II) of gestation in dams (39–40 days). HMB-treated dams were assigned into two groups according to the time of HMB administration: period I (the HMB I group; n=10) and period II (the HMB II group; n=10) and were fed with HMB (Sigma-Aldrich) in the morning (0.2 g/kg of body weight per day).

The total amount of food consumed by the dams was estimated before the start of the experiment and was calculated at 15 g of feeding stuff per day (three pellets). In the morning, the experimental group received 1 pellet with HMB and the control dams received the feed alone. At 12:00, the remaining part of the feed (2 pellets) was given to the control and the HMB-treated dams. The gestation length (39–40 days) and the number of newborn pups (all offspring were live-born; 2 to 5) did not differ between the HMB and the control group.

Offspring
Newborn offspring were randomly chosen from each mother to avoid litter and dam variability. In general, one female and one male pup was taken from each dam. However, if a litter consisted of a single pup, the dam was excluded. If all the pups were of the same gender, only one pup was taken. Each group of offspring was formed in such a way that each of them contained 5 females and 5 males. Finally, the number of offspring euthanized by CO₂ inhalation was 10 in the control, 10 in the HMB I group and 10 in the HMB II group. Internal organs such as the liver, heart, lungs, spleen, stomach, kidneys and brain were isolated and weighted. Relative organ weight [%] was calculated as a ratio of organ weight and body weight.

Statistical analysis
All the results are expressed as means ± SD (standard deviation). Differences between the means were tested with the One Way ANOVA and post hoc Tukey’s test as the correction for multiple comparisons. Normal distribution of data was examined using the W Shapiro-Wilk test and equality of variance was tested with the Brown-Forsythe test. In the case of a lack of normal distribution and/or unequal variance of data, the Kruskal-Wallis ANOVA was used. A P-value of less than 0.05 was considered statistically significant. All statistical analyses were carried out with the use of STATISTICA 12.0 software (StatSoft, Inc., Tulsa, OK, USA).

RESULTS

The HMB administration did not influence the gestation length, nor the mean number of live-born mice in litters delivered by HMB-treated dams.

Body and organ weight
Maternal HMB-treatment did not significantly influence the total body weight of newborn offspring, although offspring delivered by dams administered with HMB during middle gestation (HMB I) were significantly smaller compared to newborns delivered by dams treated with HMB during the last stage of gestation (HMB II), as presented in Figure 1.

Fig. 1. The weight of the offspring. Bars represent the data for the offspring of dams fed with the standard feed (Control) and fed with the diet containing 0.2 mg/kg of body weight/every day of HMB in the middle (HMB I) and the last period of gestation (HMB II). Data are the average from 10 animals (5 male and 5 female) in each group.  Error bars ± SD (standard deviation). Treatments with different letters (a, b) are significantly different (P<0.05, one-way ANOVA followed by Tukey’s post hoc test)

The weights of liver, heart, kidneys and spleen increased in the offspring delivered by dams treated with HMB during the first period compared to the control group. At the same time, the weight of lungs decreased and the weight of brain was not altered compared to the control group (Fig. 2). The same effects were observed when the comparison of relative weight of organs was done (Fig. 2). An opposite effect was observed after HMB-treatment administered during the second period. The weights of liver, heart, kidneys and brain of offspring delivered by dams HMB-treated in the second period did not change compared to the control group. However, the weight of lungs decreased (Fig. 2). In this case, the same effects were also observed when the comparison of relative weight of organs was done (Fig. 2).

Fig. 2. Organ weights of offspring from each group: liver (A), spleen (B), heart (C), kidneys (D), lungs (E) and brain (F). Each panel on the ride side shows mean organ weight of 5 male and 5 female offspring from each group expressed in grams. On the right side of each panel the organ weight is expressed as relative organ weight (mg/1g body weight). Error bars ± SD (standard deviation), a, b – the values with different letters are significantly different (P<0.05, one-way ANOVA followed by Tukey’s post hoc test)

Moreover, the weights of liver and lungs of offspring delivered by dams treated with HMB during the second period decreased, while the weight of spleen increased compared to the values noted in the group of offspring delivered by dams treated with HMB during the first period (Fig. 1). The weights of heart, kidneys and brain did not differ between both groups (Fig. 2). The relative weights of liver, heart and kidneys were lower in the period II offspring compared to data noted in the group of period I offspring. However, the weights of lungs, spleen and brain did not differ between both investigated groups (Fig. 2).

DISCUSSION

In terms of safety, a number of animal species were fed large amounts of HMB for different periods of time. In all animal studies, there were no adverse effects, increased death rates, or pathological changes in organs or tissues [15]. However, it is very important to analyse the safety of various dosages of HMB. Studies conducted so far have found no potential adverse side effects of supplementation with HMB in both humans consuming 3–6 grams daily and animals consuming different dosages [4, 11, 12, 15]. In fact, no adverse effects have been seen in animals consuming enormous amounts of HMB within a range between 8 and 5000 mg/kg of body weight per day for a period of 1–16 weeks, but all these studies were performed in postnatal life [13, 15].

In our study, HMB was administered to pregnant dams in the amount of 0.2 g/kg of body weight. In general, there was no influence of HMB administration on body weight in the spiny mice when HMB was added to the diet in the middle gestation. This study once again proved that HMB administration during the last period of the gestation resulted in an increase of body weight in newborns. However, in an earlier study, where newborns were 23% heavier compared to the control group, HMB was given to pregnant sows in the amount of 0.05 g/kg of body weight per day [20]. Nevertheless, HMB administration was performed through a very short period of gestation (two weeks before delivery) and in a completely different mammalian species. However, in agreement with our data, there is one study performed on sows supplemented with HMB from day 35 of gestation to parturition, which demonstrates that the birth weights of piglets increased in response to maternal HMB supplementation [23]. Nevertheless, it seems that in general maternal HMB supplementation leads to increased body weight of newborn piglets. Additionally, it is important that this increase applies to the lean body mass [20]. Numerous studies have demonstrated the possible mechanisms of HMB action. It appears that the decrease in proteolysis and not the increase in protein synthesis underlies the basal mechanism by which HMB can increase the gain of lean body mass. Moreover, changes in protein synthesis appear in specific tissues. Significant decreases in protein synthesis are found in the heart, colon, kidney, and the spleen. The exception is the liver, in which an increase in protein synthesis is observed [6].

Studies conducted so far have shown that supplementation with HMB is safe even with a supply exceeding the most often recommended dose and does not cause side effects in either humans or animals [6]. Many studies performed during postnatal life show a significant effect of HMB on the weight and body composition due to the anti-catabolic effect, stimulation of synthesis, and inhibition of the degradation of muscle protein [17, 25]. Nevertheless, HMB's mode of action in general, and during prenatal time in particular, is still unknown.

In our study, the increase in the weight of organs in newborn offspring was not proportional to the body size. Supplementation with HMB significantly increased the weight of livers and kidneys, although there was no difference in the consumed amount of food and water between the groups of pregnant dams. However, liver and kidney weight increased when HMB was given to pregnant dams during the middle period of gestation. Although the spiny mouse is characterized by rapid prenatal development leading to substantial autonomy shortly after the birth, internal organs are more immature during the middle period, compared to the last days of gestation. Nutrients that reach the foetus from the mother's blood are processed in the liver. The liver of a foetus in the middle of gestation is functionally too immature and inefficient. Thus, the increase of liver weight was observed because it became swollen. Moreover, as the liver is involved in the production of blood elements in the foetus, its impaired function could influence haematology. Also, all functions such as detoxification, metabolism, filtering, or storage could be changed in the offspring during postnatal life and influence their general development. This effect could be related to tissue specific differences in leucine and HMB metabolism. Moreover, this event was not observed in the offspring of the dams administered with HMB during the last days of pregnancy. On the other hand, the kidneys of the newborn offspring were also enlarged. Urine produced by the foetus gradually becomes the source of amniotic fluid. In addition, kidneys influence lung development. It is not known how such enlarged kidneys fulfil their role and influence postnatal development, but the decrease in the weight of lungs was evident. Perhaps this decrease was linked with the underdevelopment of the lungs. Additional studies are needed to clarify the mechanisms of the maternal action of HMB on the development of offspring, especially since the spleen was also enlarged. Furthermore, several studies demonstrated that supplemental HMB is well tolerated and has no adverse effects. The dietary supplementation of HMB is widely used as an ergogenic aid to increase muscular strength and lean body mass gains. To our knowledge, there is one study where the increase of the weight of kidneys is noted in females fed with the addition of HMB at the level of 5% in the diet. Moreover, rats of both sexes fed with HMB at the levels of 0, 1, 2 or 5% in the diet for 91 days do not show HMB-related effects for body weight or body weight gain. Also, there are no other HMB-related effects on absolute organ weights or relative organ weights, nor HMB treatment-related adverse effects on haematology, organ pathology, or histology. This alteration of kidneys weight is also not considered biologically significant because there is no effect on absolute kidney weight or kidney to-brain ratio and there are no associated histopathology findings [3]. Other study with approximately 20-kg pigs fed 100g of HMB per day for 4 days also shows no haematology and organ pathology [15]. Also, the relative weights of internal organs of newborn piglets are not affected by the dietary HMB treatment from day 35 of gestation to parturition [23].

A comparison of our findings with those from other studies is somewhat difficult because studies of the effects of HMB supplementation at different time of pregnancy are limited. However, it should be mentioned, that commonly available food stuffs are generally poor in β-hydroxy-β-methylbutyrate. The highest amount of this substance was found in grapefruit, alfalfa, and catfish. Other food stuffs like corn contain trace amounts of HMB in the product [1–3].

CONCLUSION

This study showed a time-dependent maternal influence of HMB supplementation on kidneys and liver development. Maternal administration of β-hydroxy-β-methylbutyrate (HMB) during different periods of gestation has been shown to influence the development of internal organs, but the final effect of this action is still unknown. The influence of a diet rich in the metabolite of leucine needs further investigation.

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Accepted for print: 12.12.2016

The publication on 26.01.2017 changes were made in numbering the references at the request of the authors.

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Siemowit Muszyński
Department of Physics, Faculty of Production Engineering, University of Life Sciences in Lublin, Poland


Agnieszka Tomczyk
Department of Animal Physiology, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Lublin, Poland


Piotr Dobrowolski
Department of Comparative Anatomy and Anthropology, Maria Curie-Skłodowska University, Lublin, Poland


Ewa Tomaszewska
Department of Animal Physiology, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Lublin, Poland
E-mail: ewaRST@interia.pl
Phone +48 4456973
Fax +48 4456973
email: ewa.tomaszewska@up.lublin.pl

Monika Hułas-Stasiak
Department of Comparative Anatomy and Anthropology, Maria Curie-Skłodowska University, Lublin, Poland

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