This study has shown that prenatal administration with AKG and HMB, individual or in combination, increases body weight of newborn piglets. The most notable increases of body weight at birth were seen in piglets born by sows with combined treatment of AKG and HMB than in HMB only group and reached 27 % and 23 %, respectively, while this parameter was improved by only 16 % in the AKG group. Similar effects were obtained for fattening time and daily body weight gain. The most advantageous effects were stated in the AKG and AH groups where fattening time was reduced by 25 and 27 days, and the final body weight in these groups was increased by 4.3 % and 5.8 %, respectively. Even though final body weight in pigs was not influenced by prenatal administration with HMB, beneficial effects in this group was expressed as accelerated postnatal growth rate and improved the daily body weight gain. These results are in accordance with previous investigations on pig model where administration of AKG during last 24 days of pregnancy increased body weight of newborns by 29 % . The 13 % difference between the scales of improved body weight of newborns in both these investigations may results form 10 day longer administration of AKG in previous studies, since the same daily dosage and form of AKG was used. In the other report on prenatal influence of AKG during the last 24 days of pregnancy, body weight in newborn and 14 day old piglets was improved by 29 % and 6 %, respectively. Unfortunately in that study, the offspring from sows receiving AKG were sacrificed at 2 weeks of life and constituted a short-term effect study, and so it was not possible to compare the results achieved there to our long-term effect results . Twenty-one and 24-day long neonatal treatment of piglets with AKG at the daily dosages of 0.1 and 0.3 g/kg of body weight increased growth rate in 10- and 21-day old females, whilst in males opposite effects were obtained and lighter body weights were recorded at these developmental stages. However, administration with AKG has not induced long-term effects on final body weights of the animals slaughtered at the age of 169 days of life, indicating positive effects lasting only as long as it is being administered . Investigations on HMB administration to sows during the last 2 weeks of pregnancy have showed birth body weight of male and female piglets increased by 21 % and 26 % when compared to the controls. Furthermore, as the consequence of prenatal influences of HMB, the fattening time to final body weight of the pigs was reduced by 11 days, and the body weights at slaughter in these pigs were not different from controls in both males and females . In other studies on chicken embryos model, in ovo administration of HMB increased body weight of hatched and 10-day chicks by 3.3 % and 4.7 %, respectively . Experiments performed on turkey embryos have also shown positive effects of in ovo feeding with HMB on body weight from hatch to 14 days of age when the study was finished . Similarly to the outcome in our study, the only one available report on combined treatment with AKG and HMB in growing fundectomized pigs has not revealed additive effects of these metabolites on final body weight that reached comparable values in 8-month old animals receiving AKG or HMB only. However, the final body weights of pigs growing over 6 months without the fundic part of the stomach and fed AKG, HMB or AKG and HMB per os were increased double when compared to the fundectomized group receiving placebo .
Effects of prenatal administration with AKG and HMB on systemic growth and development of piglets in postnatal life observed in our study were associated with increased plasma concentration of free amino acids. The most prominent effects of prenatal dietary manipulations on amino acid metabolism were observed in the group being under combined influence of AKG and HMB. In this group, 18 amino acids such as aspartate, threonine, serine, glutamine, proline, glycine, citrulline, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ornithine, lysine, histidine and arginine were found to be increased in plasma of newborns in comparison to the control group. In case of threonine, serine, glutamine, proline, citrulline, valine, methionine, isoleucine, leucine, phenylalanine, ornithine, lysine, histidine and arginine, the obtained results indicate an additive anabolic effects of AKG and HMB on amino acid metabolism, since the amino acid plasma concentrations were significantly higher than in the groups receiving solely AKG or HMB. In the AKG group, concentrations of 10 amino acids such as aspartate, serine, glutamine, proline, glycine, alanine, valine, ornithine, lysine and arginine were elevated above the control values. Noteworthy is the fact that in the AH and AKG groups, the final body weights as well as daily body weight gain and fattening time reached the most desirable characteristics. In the HMB group, relatively moderate effects of the dietary manipulation on plasma amino acid concentration were observed and the level of glutamine, glycine, valine and tyrosine was increased in comparison to the control group. Results obtained in our study are in accordance with the previous investigations on weaned pigs where an enteral 6-hour continuous infusion of 930 μmol/kg/h of AKG improved net portal absorption rate of proline, leucine and lysine by 64 %, 49 % and 36 %, respectively. The concentrations of all these amino acids were increased in our study in newborn piglets being under individual or combined prenatal influence of AKG . Moreover, in 17-week old turkeys, plasma concentrations of proline and leucine were elevated by 53 % and 25 % after 14-week long oral administration with AKG at the same dosage as in the current study . Available data from studies on growing turkeys have shown that 15-week administration with HMB increased concentration of cysteic acid, glutamine, valine, aspartate, glutamate, proline, alanine, isoleucine, leucine and phenylalanine, whilst body weights were not different in comparison to control birds . In studies on fundectomized pigs, HMB improved plasma concentration of methioine, threonine, valine, leucine, tyrosine, tryptophan and arginine, depending on blood sampling time since last oral dosage of HMB . However, simultaneous administration with AKG and HMB improved concentration of histidine, methionine and threonine when evaluated in plasma of fundectomized 8-month old pigs subjected to 24-hour fasting .
The current study also indicates that positive effects of prenatal administration with AKG and HMB were associated with improved somatotrophic axis function due to increased serum levels of GH and IGF-1. In the newborns, GH level was increased in animals being under influence of HMB only or HMB introduced simultaneously with AKG, while IGF-1 reached higher values in all the investigated groups. In 90-day old piglets, GH was found to be increased only in the group born by the sows receiving combined treatment of AKG and HMB but IGF-1 concentration was found to be significantly elevated in all the experimental groups. These data suggest that higher serum concentration of IGF-1 in all the groups receiving experimental treatments may results from enhanced somatotrophic axis function and liver production of IGF-1 as well as locally produced IGF-1 and its autocrine or paracrine actions . Recent experiments on rats performed by Gerlinger-Romero and colleagues (2011) have shown that chronic supplementation of HMB increases the activity of the GH/IGF-1 axis and induces increased content of IGF-1 mRNA in the liver . It was shown in previous studies on humans that AKG administered as ornithine salt (OKG) in multiple trauma adult patients, who were highly catabolic and hypermetabolic, increases plasma levels of IGF-1 and GH by 41 % and 82 %. Similarly to the results in this study, OKG administration in those patients enhanced glutamine, proline and ornithine levels by 25 %, 39 % and 234 %, respectively . In children suffering from growth retardation, 5-month long OKG administration (15 g/day) induced growth acceleration in association with increased plasma IGF-1 level. Moreover, these effects were related to significantly higher plasma glutamine concentration in children receiving AKG . Similarly to studies on humans, elevated levels of GH and IGF-1 in newborn piglets from AKG-treated sows during the last 24 days of pregnancy were stated . As opposite to studies on pigs, 14-day neonatal treatment with AKG in lambs has not induced effects on circulating IGF-1 level. However, 4 times lower dosage of AKG was administered to sheep than in the current study . Investigations on animals with maternal and neonatal administration of HMB have also shown improved somatotrophic axis function. HMB treatment in pregnant sows for 2 weeks before the partum increased serum GH and IGF-1 levels by 38 % and 20 % in the offspring . In lambs, oral administration with HMB during the first 21 days of life also increased GH and IGF-1 concentration by 70 % as determined at 3 weeks of life, while at the age of 130 days of life both these hormones reached similar values to those in controls . Thus, it may be postulated that prenatal treatment with HMB may induce long-term effects on secretive function of the somatotrophic axis, while neonatal or postnatal administration improves temporary its function and brings short-term effects.
Next to the observed beneficial effects of prenatal influences of AKG and HMB on increased growth rate of pigs and hormonal and amino acid status, this study demonstrated improved morphometric, densitometric and mechanical properties of bones after the experimental treatments. Both these metabolites induced individually or in combination long-term anabolic effects on bone tissue metabolism which were expressed as increased values of bone formation marker – BAP in newborn and 90-day old piglets. Increased serum concentration of OC in 90-day old pigs in the AH group also confirmed continuous acceleration of bone formation processes resulting from the performed dietary manipulation in pregnant sows. However, the observed in this study difference of serum OC concentration between AKG and AH groups of newborns need further investigation to be explained. Among morphological traits, weight of femur was improved in all the experimental groups and the most readable effects were induced after exclusive or combined administration with HMB, while bone length was improved by nearly 2 % in case of the combined treatment. Improved weight of femur may results from both the observed increased vBMD and bone geometry. It is noteworthy that cross-sectional area was enhanced in all the experimental groups, while second moment of inertia reached higher values in the HMB and AH groups. As the result of improved periosteal and endoosteal bone deposition, MRWT and CI of femur were higher after exclusive treatment with AKG or HMB. Prenatal treatment with AKG and HMB increased also vBMD within both cancellous and compact compartments in all the experimental groups. It is surprising that the highest values of Td and Cd were stated in animals receiving HMB only, and the additive effect of AKG and HMB on bone mineralization was not induced. Evaluation of mechanical endurance of femur has not revealed additive effects of AKG and HMB; however, improved maximum elastic strength and ultimate strength were observed in all the experimental groups. The improved mechanical characteristic of femur observed in all the experimental groups seems to be result of improved bone geometry and vBMD since both these factors determine bone rigidity and mechanical endurance to acting forces [46–48]. The observed anabolic effects of AKG and HMB on skeleton in the current study are in accordance to the results obtained on the fundectomized pig model, where severe osteopenia development of femur and tibia was inhibited by a 6-month administration with these metabolites. In AKG-treated animals, significant increase of weight, length, bone mineral density (BMD), bone mineral content (BMC), cross-sectional area, second moment of inertia, mean relative wall thickness, cortical index, maximum elastic strength and ultimate strength of the bones was associated with improved serum concentration of IGF-1 and serum BAP activity when compared to the fundectomized group . Results of long bone analysis in slaughter pigs treated during 21 and 24 days of neonatal life with AKG revealed its positive effects on length, Cd, Wy, Wf and Young’s modulus that was connected with elevated plasma estrogens level . In studies on growing turkeys, 14-week long administration with AKG eliminated neurectomy-induced osteopenia of radius increasing its weight, vBMD, A, Ix, MRWT, Wy and Wf. These advantageous effects were combined with higher serum concentration of proline and leucine in comparison to placebo-treated birds . In other studies on sheep, two week long neonatal treatment with AKG improved Td, Cd and Wf of femur as well as increasing weight, length, Cd, Wy and the moments of maximum elastic strength and ultimate strength [12, 15]. Furthermore, it was shown that 7-week administration with OKG in growing female turkeys induces higher Td, Cd, Wy and Wf of tibia without any changes of bone geometry. However, increased synthesis of aspartate, proline, alanine, valine, leucine, isoleucine and ornithine was postulated to be responsible for enhanced skeletal properties . Using fendectomized pig model, it was revealed that HMB inhibits fundectomy-induced osteopenia in axial skeleton (lumbar vertebrae) improving weight, total volume, mean vBMD, Td, calcium hydroxyapatite density of trabecular and cortical bone, BMD, BMC, ultimate force, ultimate stress, Young’s modulus, stiffness and work to the ultimate force point. Together with the beneficial influence of HMB on bone tissue and skeletal characteristic, the improved synthesis of threonine, glycine, alanine, valine, methionine, leucine, tyrosine, tryptophan and arginine was observed, confirming importance of amino acid metabolism for bone formation and skeletal homeostasis maintenance . In studies on growing meat-type male turkeys, HMB administration from 6 to 20 week of life positively influenced geometry, vBMD and mechanical endurance of tibia. It was shown that improved Td, Cd, A, Ix, Wy and Wf of tibia was related to higher plasma concentration of cysteic acid, aspartate, glutamate, alanine, valine, leucine, isoleucine and phenylalanine . Unfortunately, the comparison of the bone properties determined in this study on pigs from the AH group is not possible with other experimental data, since effects of combined treatment with AKG and HMB on bone tissue metabolism and skeletal system were not previously studied, either in animals or in humans.
Considering the results obtained in the current study, it should be emphasized that prenatal dietary manipulation with AKG and HMB influenced systemic development of pigs stimulating different physiological mechanism. It may be postulated that increased birth and final body weight, as well as accelerated systemic growth and development of pigs were induced via stimulated secretory function of the somatotrophic axis by AKG and HMB. This hypothesis is supported by the data showing that endocrine gland function can be programmed in utero at critical periods of prenatal development with metabolic pre-partum and post-partum consequences on the cellular, tissue and system levels . It may be proposed that the improved skeletal properties in AKG and/or HMB treated animals result from positive prenatal programming of the somatotrophic axis, especially when one considers decisive role of GH and IGF-1 for skeletal growth, osteoblasts function, bone mineralization and calcium-phosphate homeostasis maintenance . The other mechanism positively influencing prenatal development of piglets, their birth and final body weight, as well as daily body weight gain and bone tissue properties may result from improved amino acid metabolism. It must be underlined that plasma glutamine concentration was increased in newborns from all the experimental groups. Besides its building function in protein and polypeptides structures, glutamine - being a functional amino acid - determines key metabolic pathways necessary to provide optimal growth rate and immunity. It maximizes efficiency of feed utilization, enhances protein accretion and improves health status influencing effectives in animal production. Furthermore, glutamine, as the most abundant alpha-amino acid in skeletal muscles, regulates nutrient metabolism, gene expression and protein synthesis – the main factors influencing muscle mass and body weight . Improved metabolism of glutamine would also provide benefit for skeletal formation, especially when one considers its role as the main substrate for synthesis of proline – the amino acid undergoing hydroxylation process to form hydroxyproline. Both proline and hydroxyproline are considered as integral constituents of collagen helix and its protectors against proteases. While proline and hydroxyproline contribute to two-thirds of the collagen structure, one-third is ascribed to glycine . Except for proline concentration in the HMB group of newborns, both proline and glycine were increased in all the experimental groups. Thus, AKG- and HMB induced increase of the amount of collagen in bones of the newborns from experimental groups would provide steady advantage in bone mass of the skeleton maintained or increased during postnatal growth and development in relation to control animals. It can not be excluded that higher muscle mass since birth of piglets has also induced positive effects on skeleton through higher life-long tension and biomechanical stimulation of bones and their positive response in terms of enhanced mineralization, morphological traits and mechanical endurance. Better muscle function resulting from direct and indirect influences of the other amino acids (mostly leucine, isoleucine, valine, arginine and methionine) increased in AKG- and HMB-treated piglets would also be effective in relation to improved muscle-bone interactions . Due to observed higher body weights since birth in all the experimental groups of pigs than in the control animals, positive effects in bones may results partially from heavier skeletal loading, since adaptive response of bone tissue to loading increases bone mass, size and mineral density [55, 56].
In response to the question whether additive effects of maternal treatment with AKG and HMB on prenatal programming of growth, development and bone tissue metabolism exist, which arises from the current study, the complete explanation of this issue seems to be difficult. It must be underlined that in the AH group, the highest plasma concentration of 16 amino acids such as threonine, serine, glutamine, proline, citruline, valine, cysteine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ornithine, lysine, histidine and arginine in comparison to all other groups indicates on additive effects of these metabolites in relation to prenatal programming of growth, development and bone tissue metabolism. The observed secretive response of the somatotrophic axis to combined maternal treatment with AKG and HMB and the highest concentration of GH and IGF-1 in the AH group confirm additive effects of these metabolites in relation to systemic growth and development. However, except for bone length, the evaluation of systemic growth parameters and bone properties in pigs at slaughter age has not shown additive effects of prenatal AKG and HMB application.