Avanika Mahajan

Dihydroxy-cholecalciferol stimulates adipocytic differentiation of porcine mesenchymal stem cells.

Dihydroxy-cholecalciferol [1,25(OH)2D3] has been shown to have pleiotropic effects on the differentiation of mesenchymal stem cells (MSC) based on species and culture conditions. We have examined the effects of 1,25(OH)2D3 on the differentiation of porcine MSC under culture conditions designed to promote proliferation in order to attempt to mimic the conditions in young, rapidly growing animals. The MSC were isolated from bone marrow of a young pig and grown in basal media (BM) containing DMEM+10% fetal bovine serum and antibiotics. Cells received either BM, BM+10(-8) M 1,25(OH)2D3 or BM+10(-7) M 1,25(OH)2D3 with complete media changes every 3 days for a total of 12 days of culture. On days 3, 6, 9 and 12, viable cell numbers were determined, and samples were collected for gene expression analysis and cytochemical staining. There was a treatment-based reduction in cell numbers on 6, 9 and 12 days (P<.05). The concentrations of mRNAs encoding peroxisome proliferator-activated receptor gamma, lipoprotein lipase, and adipocyte-binding protein 2 were increased (P<.05) in a manner indicative of adipocytic differentiation by treatment with 1,25(OH)2D3 in a dose-dependent manner. However, the mRNA levels of osteocalcin, a late stage marker of osteoblastic differentiation, was also increased (P<.05) by treatment with 1,25(OH)2D3. An increased percentage of lipid filling, based on Oil Red O staining, and decreased alkaline phosphatase activity, was also seen with 1,25(OH)2D3 treatment. These data suggest that 1,25(OH)(2)D(3) stimulates the differentiation of porcine MSC towards an adipocytic phenotype.

Response to dietary phosphorus deficiency is affected by genetic background in growing pigs

Concern over the environmental effect of P excretion from pig production has led to reduced dietary P supplementation. To examine how genetics influence P utilization, 94 gilts sired by 2 genetic lines (PIC337 and PIC280) were housed individually and fed either a P-adequate diet (PA) or a 20% P-deficient diet (PD) for 14 wk. Initially and monthly, blood samples were collected and BW recorded after an overnight fast. Growth performance and plasma indicators of P status were determined monthly. At the end of the trial, carcass traits, meat quality, bone strength, and ash percentage were determined. Pigs fed the PD diet had decreased (P < 0.05) plasma P concentrations and poorer G:F (P < 0.05) over the length of the trial. After 4 wk on trial, pigs fed the PD diet had increased (P < 0.05) plasma 1,25(OH)(2)D(3) and decreased (P < 0.05) plasma parathyroid hormone compared with those fed the PA diet. At the end of the trial, pigs fed the PD diet had decreased (P < 0.05) BW, HCW, and percentage fat-free lean and tended to have decreased LM area (P = 0.06) and marbling (P = 0.09) and greater (P = 0.12) 10th-rib backfat than pigs fed the PA diet. Additionally, animals fed the PD diet had weaker bones and also decreased (P < 0.05) ash percentage and increased (P < 0.05) concentrations of 1alpha-hydroxylase and parathyroid hormone receptor mRNA in kidney tissue. Regardless of dietary treatment, PIC337-sired pigs consumed more feed and gained more BW than their PIC280-sired counterparts (P < 0.05) during the study. The PIC337-sired pigs also had greater (P < 0.05) HCW, larger (P < 0.01) LM area, and tended to have (P = 0.07) greater dressing percentage. Meat from the PIC337-sired pigs also tended to have greater (P = 0.12) concentrations of lactate but decreased (P = 0.07) concentrations of total glucose units 24 h postslaughter. Although plasma 1,25(OH)(2)D(3) concentrations were elevated (P < 0.05) in all the animals fed the PD diet, this elevation due to P deficiency tended (P = 0.09) to be greater in the PIC337-sired pigs after 12 wk on the treatment. The PIC337-sired pigs had stronger (P < 0.01) bones with greater ash percentage than the PIC280-sired pigs. The difference in the strength of the radii between the PIC337-sired pigs fed the PA and PD diets was greater than their PIC280-sired counterparts, which resulted in sire line x treatment interactions (P < 0.05). These data indicate differing mechanisms of P utilization between these genetic lines. Elucidating these mechanisms may lead to strategies to increase efficiency of growth in a more environmentally friendly manner.

Dietary Phosphate Restriction Decreases Stem Cell Proliferation and Subsequent Growth Potential in Neonatal Pigs

Although mesenchymal stem cells (MSC) and satellite cells are essential for postnatal muscle and bone development and phosphate (PO(4)) restriction reduces both muscle and skeletal tissue growth, no research to our knowledge has investigated the possible mechanism by which this mineral may affect early cell programming. Twenty piglets obtained at 1 d of age (1.8 +/- 0.3 kg) received either a PO(4)-adequate diet or a 25% less PO(4)-available diet over a 15-d trial. Feed intake and body weight were recorded daily and blood samples collected every 5 d. After 15 d, pigs were given an intraperitoneal injection of bromodeoxyuridine 4 h prior to tissue collection. As expected, PO(4) deficiency resulted in reduced growth (P < 0.05), feed conversion efficiency (P < 0.05), and bone mineral content (P < 0.05), as well as lower plasma concentrations of both PO(4) (P < 0.01) and parathyroid hormone (P < 0.05). In addition to these classical indicators of PO(4) deficiency, there was also reduced proliferation of both MSC (P < 0.01) and satellite cells (P < 0.05) in vivo. The expression of osteocalcin mRNA in bone marrow was also 2-fold greater (P < 0.01) within the PO(4)-adequate treatment group. These data indicate that in addition to reductions in muscle and bone growth, dietary PO(4) affects proliferation of tissue-specific stem cells in vivo. Nutritional programming of tissue-specific stem cells by dietary PO(4) may have profound implications for life-long growth potential.

A calcitonin receptor (CALCR) single nucleotide polymorphism is associated with growth performance and bone integrity in response to dietary phosphorus deficiency

Although concerns over the environmental impact of excess P in the excreta from pig production and governmental regulations have driven research toward reducing dietary supplementation of P to swine diets for over a decade, recent dramatic increases in feed costs have further motivated researchers to identify means to further reduce dietary P supplementation. We have demonstrated that genetic background impacts P utilization in young pigs and have identified genetic polymorphisms in several target genes related to mineral utilization. In this study, we examined the impact of a SNP in the calcitonin receptor gene (CALCR) on P utilization in growing pigs. In Exp. 1, 36 gilts representing the 3 genotypes identified by this CALCR SNP (11, 12, and 22) were fed a P-adequate (PA) or a marginally P-deficient (approximately 20% less available P; PD) diet for 14 wk. As expected, P deficiency reduced plasma P concentration, bone strength, and mineral content (P < 0.05). However, the dietary P deficiency was mild enough to not affect the growth performance of these pigs. A genotype x dietary P interaction (P < 0.05) was observed in measures of bone integrity and mineral content, with the greatest reduction in bone strength and mineral content due to dietary P deficiency being associated with the allele 1. In Exp. 2, 168 pigs from a control line and low residual feed intake (RFI) line were genotyped for the CALCR SNP and fed a PA diet. As expected, pigs from the low RFI line consumed less feed but also gained less BW when compared with the control line (P < 0.05). Although ADFI did not differ between genotypes, pigs having the 11 genotype gained less BW (P < 0.05) than pigs having the 12 or 22 genotypes. Pigs of the 11 and 12 genotypes had bones that tolerated greater load when compared with animals having the 22 genotype (P < 0.05). A similar trend was observed in bone modulus and ash % (P < 0.10). These data are supportive of the association of this CALCR SNP with bone integrity and its response to dietary P restriction. Although the allele 1 is associated with greater bone integrity and mineral content during adequate P nutrition, it is also associated with the greatest loss in bone integrity and mineral content in response to dietary P restriction. Understanding the underlying genetic mechanisms that regulate P utilization may lead to novel strategies to produce more environmentally friendly pigs.

Dietary Calcium Restriction Affects Mesenchymal Stem Cell Activity and Bone Development in Neonatal Pigs

The effects of dietary calcium (Ca) deficiency on skeletal integrity are well characterized in growing and mature mammals; however, less is known about Ca nutrition during the neonatal period. In this study, we examined the effects of neonatal Ca nutrition on bone integrity, endocrine hormones, and mesenchymal stem cell (MSC) activity. Neonatal pigs (24 ± 6 h of age) received either a Ca-adequate (1.2 g/100 g) or an ~40% Ca-deficient diet for 18 d. Ca deficiency reduced (P < 0.05) bone flexural strength and bone mineral density without major differences in plasma indicators of Ca status. There were no meaningful differences in plasma Ca, phosphate (PO(4)), parathyroid hormone, or 1,25-dihydroxycholecalciferol due to Ca nutrition throughout the study. Calcium deficiency also reduced (P < 0.05) the in vivo proliferation of MSC by ~50%. In vitro studies utilizing homologous sera demonstrated that MSC activity was affected (P < 0.05) by both the Ca status of the pig and the sera as well as by their interaction. The results indicate that neonatal Ca nutrition is crucial for bone integrity and suggest that early-life Ca restriction may have long-term effects on bone integrity via programming of MSC.