A metabolite of leucine (b-hydroxy-b-methylbutyrate) given to sows during pregnancy alters intestine development of their newborn offspring

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A metabolite of leucine (b-hydroxy-b-methylbutyrate) given to sows during pregnancy alters intestine development of their newborn offspring

Ewa Tomaszewska

The effects of dietary f-hydroxy-f-methylbutyrate (HMB) supplementation during gestation on intestinal tract development in newborns were determined. At 70th day of gestation, a total of 12 sows (Large White Polish breed) were randomly assigned to two groups, with each group receiving either a basal diet or the same diet supplemented with 0.2 g/day HMB until the 90th day. Maternal HMB supplementation resulted in the widest myenteron, submucosa and mucosa thickness compared with the control group. Further, it resulted in significantly longer and wider villi, and the deepest crypts. The increase of total crypt number was observed after HMB supplementation. The absorptive surface of the small intestine was higher in newborn offspring from the HMB group. In summary, maternal HMB supplementation in the mid-gestation period significantly influenced intestinal tract development in the offspring.

Keywords: LEUCINE, METABOLITE, SOWS, PREGNANCY, INTESTINE. methylbutyrate small intestine newborn

Визначено вплив дієтичної добавки Р-гідрокси-Р-метилбутирату (НМВ) під час вагітності на розвиток кишкового тракту у новонароджених. На 70-й день вагітності в загальній кількості 12 свиноматок (велика біла польська порода) були випадковим чином розподілені на дві групи, кожна група отримувала або основну дієту, або таку ж дієту з додаванням 0,2 г / добу НМВ до 90-го дня. Додавання НМВ матерям призвело до більшої товщини м'язової, підслизової і слизової оболонки в порівнянні з контрольною групою. Крім того, це призвело до значно довших і ширших ворсинок, а також глибших крипт. Після додавання НМВ спостерігалося збільшення загального числа крипт. Поглинаюча поверхня тонкого кишечника була вищою у новонароджених із групи НМВ. Таким чином, додавання НМВ свиноматкам в середній період вагітності значно вплинули на розвиток кишкового тракту у потомства.

Ключові слова:ЛЕЙЦИН, МЕТАБОЛІТ, СВИНОМАТКИ, ВАГІТНІСТЬ,

Определено влияние диетической добавки Р-гидрокси-Р-метилбутирата (НМВ) во время беременности на развитие кишечного тракта в новорожденных. На 70-й день беременности в общем количестве 12 свиноматок (большая белая польская порода) было случайным образом распределенные на две группы, каждая группа получала или основную диету, или такую же диету с добавлением 0,2 г / время НМВ до 90-го дня. Добавление НМВ матерям привело к большей толщине мышечной, подслизистой и слизистой оболочки по сравнению с контрольной группой. Кроме того, это привело к значительно более длинным и более широким ворсинкам, а также более глубоким криптам. После добавления НМВ наблюдалось увеличение общего числа крипт. Поглощающая поверхность тонкого кишечника была выше у новорожденных из группы НМВ. Таким образом, добавление НМВ свиноматкам в средний период беременности значительно повлияли на развитие кишечного тракта в потомства.

Ключевые слова: ЛЕЙЦИН, МЕТАБОЛИТ, СВИНОМАТКИ, БЕРЕМЕННОСТЬ, КИШЕЧНИК.

Leucine (LEU) is the essential branched-chain amino acid, a potent regulator of protein metabolism, which shows anti-catabolic action [1, 2]. As a natural biologically active compound, HMB is also present in many food products such as catfish, alfalfa, asparagus, avocado or grapefruit [2-4]. Oral administration of LEU or HMB increases protein synthesis not only in skeletal muscle but also in other tissues [1, 5]. Observations in humans and studies conducted on animals explain that some of HMB effects on tissue is trigger by the growth hormone (GH) and insulin-like growth factor 1 (IGF-1) axis, which affects myoblast proliferation, differentiation and survival [2]. Moreover, animal studies show that prenatal administration of HMB has a positive impact on postnatal growth and development [7, 8]. It has been also proved that maternal HMB supplementation affects the physical properties of the enamel surface [9]. Adaptive, functional, structural or metabolic changes occurring during prenatal development as an effect of the interaction between genes and different factors, generate permanent alterations in the homeostasis in foetus [10-15].

The present study was conducted to test the influence of maternal HMB treatment on the development of the gastrointestinal tract of newborn offspring.

Material 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 24 piglets born by 12 sows of Large White Polish breed. The sows were randomly assigned into two groups (6 sows in

each group) and fed with a standard diet (control) or diet supplemented with HMB (0.2 g/kg of body weight/every day) from the 70^th to 90^th day of gestation.

All piglets born by physiological partum had no congenital changes. Newborn piglets born by sows fed with the control diet belonged to the control group (n=6), and piglets born by HMB- treated sows belonged to the HMB group (n=6). After birth, piglets were euthanized by intravenous injections of lethal doses of pentobarbitalum natrium (Morbital, Biowet, Pulawy, Poland).

Tissue collection and histomorphometry analysis. One 10 mm long segment of the small intestine from the duodenum (5 mm distal to the pylorus) was taken from each animal. They were opened along the mesenteric border and pinned flat, without stretching, on pieces of cork in physiological saline. Tissues were fixed in 4% buffered formaldehyde (pH 7.0) for 24 h, dehydrated in graded series of ethanol and embedded in paraffin. The tissue samples were subjected to histology as described previously, except the sections were cut at 4 pm thick [16]. Masson's trichrome staining was used to differentiate the small intestine wall layers more efficiently [17]. Microscopic images were collected using a microscope (AXIOVERT 200M, Carl Zeiss, Jena, Germany). An AxioCam HRc camera (Carl Zeiss, Jena, Germany) was also used for image acquisition. Objective magnifications of 4x, 10x, 20x, and 40x were used to show the different intestinal structures and to collect images. The structure of the small intestine wall was examined under microscopic observation and with the use of graphic analysis software Olympus cellSens Version 1.5 (Olympus, Tokyo, Japan). The following morphometric variables in the intestine were analyzed: mucosa, submucosa, and myenteron (longitudinal and transversal 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 [18]. Only vertically oriented villi and crypts were analysed.

Statistical analysis. Data were analyzed using Statistica 12 software (StatSoft, Inc., Tulsa, OK, USA). Comparison between variables from the control and HMB groups was carried out using Student's t test. For all tests P<0.05 was considered statistically significant. Data are presented as mean ± standard deviation.

Results and discussion. Gastro-intestinal tract morphology

Maternal HMB supplementation resulted in the widest myenteron, submucosa and mucosa thickness compared with the control group (table). Although HMB given to sows during pregnancy did not influence total villi number, but resulted in significantly longer and wider villi in their newborns. Further, HMB maternal treatment resulted in the deepest crypts, while crypts width was not altered compared to the control group. However, the increase of total crypt number was observed after HMB supplementation. The absorptive surface of the small intestine was higher in newborn offspring from the HMB group (table).

Table