The effect of prenatal treatment with B-hydroxy-B-methylbutyrate on the small intestine morphology in a mink model

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The effect of prenatal treatment with B-hydroxy-B-methylbutyrate on the small intestine morphology in a mink model

The effect of prenatal dietary supplementation with f-hydroxy-f-methylbutyrate (HMB) on the intestinal morphology of American mink offspring was determined. A total of 14 primiparous minks were randomly assigned to two groups receiving either a basal diet or the same diet supplemented with 0.02 g/kg b.w./day HMB during gestation period. Maternal HMB supplementation resulted in significant increase of absorptive surface of duodenum in females. Male offspring had thinner mucosa, submucosa and myenteron, shorter and wider villi as well as narrower and dipper crypts, although not significantly. Almost opposite situation was noticed in female offspring, where villi were longer and thinner, and crypts were dipper, as well as moderate effect on myenteron. In summary, maternal HMB supplementation during gestation period affected intestinal structure in the offspring and the observed effect was gender dependent.

The development of gastrointestinal tract as well as nutrition decides on a health status and the growth of an organism. Since the fetal live is one of the most critical stage of development and can be subjected to regulation by hormones and nutritional status of mother it may be possible to improve the development of digestive system by prenatal programming [1-3]. Breeding animals like minks are subjected to intensive production regimes achieved through proper nutrition which involves properly arranged feed rations. However, diseases in the perinatal period and metabolic diseases associated with the rearing period are the consequences of improper animal feeding and/or nutrients absorption related with the structure of small intestine mucosa. Therefore there is a search for methods or substances which would improve the health status of the digestive system [3, 4]. One of such substances may be P-hydroxy-P-methylbutyrate (HMB) which plays a key role in animal metabolism as a metabolite of leucine. Leucine, in turn, an essential branched-chain amino acid acts as a substrate for protein and cholesterol biosynthesis and as a signaling molecule. HMB ameliorates loss in body weight, lean mass and reduction in the muscle fiber cross-sectional area [4-6]. Dietary supplementation with HMB improved bone and dental development, immune function and health status as well as increased the fat content of milk in lactating animals [6-9]. Recent studies also showed a positive impact of prenatal administration of HMB on postnatal growth and development [10]. Thus the aim of this study was to elucidate the effect of maternal HMB treatment on the development of selected morphological parameters of small intestine of mink offspring.

Materials and methods. The experiment was approved by The Local Ethics Committee on Animal Experimentation of University of Life Sciences in Lublin, Poland.

Animals, breeding and experimental design. The study was performed on 40, clinically healthy, 8 months old offspring of 14 primiparous, 10 months old, American minks (Neovison vison) of the standard dark brown type. Animals were housed singly in separate cages under standard breeding/farming conditions and under natural daylight conditions with free access to fresh water. The animals were fed with a wet standard ranch feed mixture based on fish, fish and animal by-products, heat-treated barley, animal fat and vegetable oil (rapeseed oil). To investigate the effects of the maternal nutritional treatment on offspring intestinal morphology, HMB administration was performed in female minks since day 1 after the mating until the end of the gestation (ca. 46 days). The minks were randomly assigned into 2 groups: a control group not supplemented (n=7; Control) and group supplemented with a daily dose of 0.02 g/kg of body weight of powdered HMB (Lonza, Basel, Switzerland) (n=7; HMB). On the day of parturition, the supplementation was finished. Mink kits were kept as family groups with their dams until approximately 2 months of age, and then were placed into the separate cages. Kits delivered by the control dams belonged to the control group (n=10 of each sex), kits delivered by the dams supplemented with HMB belonged to the HMB group (n=10 of each sex). In accordance with the farm procedures and national Polish legislation, all the offspring (at the age of 8 months) were euthanized by carbon dioxide inhalation, and after harvesting pelt, skinned carcasses were delivered to the laboratory. Tissue collection andhistomorphometry analysis. A 20 mm long segment of the small intestine from the duodenum (15 mm distal to the pylorus) was taken from each animal. The fragments were cut open along the mesentery and placed flat in a histopathological cassettes, without stretching. The tissues were fixed in 4% buffered formaldehyde (pH 7.0) for 24 h, dehydrated in graded ethanol solutions, and embedded in paraffin. The tissue samples were subjected to histology as described previously, except the sections were cut at 4 ^m thick [11]. Goldner's trichrome staining was used to differentiate the small intestine wall layers more efficiently [12]. Microscopic images were collected using a confocal microscope AXIOVERT 200M (Carl Zeiss, Jena, Germany) and AxioCam HRc camera (Carl Zeiss, Jena, Germany). Objective magnifications of 10x, 20x, and 63x were used to show the different intestinal structures. The structure of the small intestinal wall was examined under microscopic observation and with the use of graphic analysis software ImageJ 1.51n (National Institutes of Health, Bethesda, MD, USA; available at: http://rsb.info.nih.gov/ij/index.html). The following morphometric variables in the intestine were analyzed: mucosa, submucosa, and myenteron (longitudinal and circular lamina) thickness, crypt depth (defined as the depth of the invagination between adjacent villi from the bottom of the crypt to the base of villi); crypt width (measured in the middle of the crypt depth); the number of crypts; villar length (from the tip of the villi to the villous- crypt junction); villar thickness (measured in the middle of villar height); the number of villi; small intestine absorptive surface [11,13]. Only vertically oriented villi and crypts were analyzed.

Statistical analysis. All results are expressed as mean ± standard deviation. Differences between means were tested with the use of two-way analysis of variance (ANOVA) and a post hoc Tukey's honest significant difference test as a correction for multiple comparisons. Normal distribution of data was examined using the W. Shapiro-Wilk test and equality of variance was tested by the Brown-Forsythe test. When there was a lack of a normal distribution and/or an unequal variance of data, the Kruskal-Wallis test was used to analyze the differences between means and post hoc tests for mean range for every pair of groups. The two-sided significance level (P value) of less than 0.05 was considered statistically significant. All statistical analyses were carried out by means of STATISTICA (data analysis software system), version 12 (StatSoft, Inc. (2014). STATISTICA).

Results and discussion. All results of measurements are presented in table. Although prenatal treatment with HMB did not exert strong effect on examined parameters of the histological structure of duodenum, significant increase of absorptive surface was noticed in females. On the other hand male offspring of mothers treated with studied substance had thinner mucosa, submucosa and myenteron, shorter and wider villi as well as narrower and dipper crypts, although not significantly.

Almost opposite situation was noticed in female offspring, where villi were longer and thinner, and crypts were dipper, as well as moderate effect on myenteron.

Anti-catabolic effect of HMB was confirmed in studies on animals and humans [4, 14, 15]. However, there is still little research on the prenatal effect of HMB on postnatal life. Result obtained in the present study broaden the knowledge on the effect of dietary supplementation on postnatal development and especially in the case of digestive system. HMB given to pregnant minks affected structure of small intestine of their offspring. Which is of importance, the effects of prenatal treatment were observed after relatively long period of growth. Besides, to some extent the effect of HMB was gender dependent since greater changes were observed in female offspring. Additionally the most important aspect was the strong increase of absorption surface od duodenum observed in females. This increase was evident although there was no chance in number of villi and crypts between the HMB and the control group. Thus one may speculate that anabolic effect of administered substance was evident. HMB administered in the diet of pregnant minks accelerated later development of duodenal mucosa of their offspring. Although there was no observed symptoms of diarrhea, dehydration or intestinal infections it can be speculated that observed changes in the structure of duodenal wall could lower the risk of such diseases. Obtained results are promising in the view of the use of HMB as a feed additive in intensive breeding, where it is of importance for the welfare of animals and their productivity. More detailed studies are necessary to elucidate further the effects of HMB in other organs and systems especially prenatal effects of this substance.

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prenatal morphology mink

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