Effects of dietary calcium (Ca) deficiency on skeletal integrity and endocrine parameters are well characterized in growing and mature mammals; however, little work has examined Ca nutrition during the neonatal period. In this study, we examined the effects of neonatal Ca nutrition on bone integrity, endocrine parameters, and in vivo mesenchymal stem cell (MSC) activity. Neonatal pigs (24 ± 6h post‐partum) were pair‐fed either a Ca adequate or a 30% Ca deficient liquid formula diet for 18 days. There were no differences in growth rate or feed conversion efficiency based on dietary Ca level and all pigs grew at a rate similar to sow‐reared pigs. As anticipated, dietary Ca deficiency reduced (P < 0.05) both BMD and bone flexural strength. The anticipated increase (P < 0.05) in plasma PTH levels in pigs fed the Ca deficeint diet was not evident until the end of the study. Surprisingly, dietary Ca level did not affect plasma Ca or 1,25(OH) 2 vitamin D concentrations throughout the study. Calcium deficiency reduced (P < 0.05) the in vivo proliferation of MSC isolated from bone marrow by approximately 50%. These 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 its effects on MSC activity.
ABSTRACT In an effort to develop a sustainable platform for manufacturing protein-based vaccine candidates, we expressed a triple mutant of staphylococcal enterotoxin B carrying the L45R, Y89A, and Y94A modifications in transgenic soybean seeds (soy-mSEB). Soy-mSEB possessed no detectable superantigen activity in vitro . We found that this soybean-derived, nontoxic mutant of SEB could be stably expressed, stored in seeds for extended periods at room temperature without degradation, and easily purified from contaminating soy proteins. Vaccination of pigs with purified soy-mSEB, or the identical triple mutant expressed in Escherichia coli ( E. coli -mSEB), resulted in high antibody titers against the native toxin in immunized animals. In fact, titers were indistinguishable regardless of the immunogen used, demonstrating the equivalence of soy-mSEB and E. coli -mSEB vaccinations. Antisera from either immunized group were able to block native SEB superantigen activity in an in vitro neutralization assay. Similar results were obtained when immunized animals were challenged with a sublethal dose of native toxin. Significant reductions in toxin-induced serum cytokine levels were observed in soy-mSEB- and E. coli -mSEB-immunized pigs compared to control animals. The reductions in SEB-induced cytokine responses were similar regardless of the immunogen used for vaccination. Surprisingly, however, some clinical symptoms, such as prostration, lethargy, emesis, and/or diarrhea, were still observed in all immunized animals. These studies demonstrate the potential for soybean-derived proteins as a platform technology for sustainable vaccine manufacturing and the usefulness of a sublethal challenge model in pigs for evaluating the efficacy of potential SEB vaccine candidates.
Satellite cell activity is necessary for postnatal skeletal muscle growth. Severe phosphate (PO4) deficiency can alter satellite cell activity, however the role of neonatal PO4 nutrition on satellite cell biology remains obscure. Twenty-one piglets (1 day of age, 1.8 ± 0.2 kg BW) were pair-fed liquid diets that were either PO4 adequate (0.9% total P), supra-adequate (1.2% total P) in PO4 requirement or deficient (0.7% total P) in PO4 content for 12 days. Body weight was recorded daily and blood samples collected every 6 days. At day 12, pigs were orally dosed with BrdU and 12 h later, satellite cells were isolated. Satellite cells were also cultured in vitro for 7 days to determine if PO4 nutrition alters their ability to proceed through their myogenic lineage. Dietary PO4 deficiency resulted in reduced (P < 0.05) sera PO4 and parathyroid hormone (PTH) concentrations, while supra-adequate dietary PO4 improved (P < 0.05) feed conversion efficiency as compared to the PO4 adequate group. In vivo satellite cell proliferation was reduced (P < 0.05) among the PO4 deficient pigs, and these cells had altered in vitro expression of markers of myogenic progression. Further work to better understand early nutritional programming of satellite cells and the potential benefits of emphasizing early PO4 nutrition for future lean growth potential is warranted.
Rapid growth during infancy has been associated with increased risk of childhood obesity. We examined the effects of increased growth rate caused by overfeeding on growth, bone integrity, and activity of MSC in neonatal piglets. Limit fed (LF) animals received a standard piglet milk replacer at a rate to achieve normal growth, while the overfed (OF) group voluntarily consumed nearly 60% more of the same diet for 21 days. Overfed piglets had higher growth rate (P < 0.001) and feed intake (P < 0.05), and lower feed efficiency (P < 0.001) compared to LF piglets. The OF animals had heavier, larger bones with greater bone mineral content than the LF group (P < 0.05). Surprisingly, MSC isolated from OF animals adopted an adipocytic lineage less readily than those isolated from LF animals after induction with adipocytic media. This was indicated by lower gene expression of PPARG, LPL and AP2 (P < 0.05) as well as by decreased lipid accumulation determined by Oil Red O staining. Increased (P < 0.2) gene expression of Runx2 following osteogenic induction was also seen in MSC from OF pigs. Accelerated growth during infancy led to a decreased response of MSC to an adipocytic environment, as well as an increased response to osteogenic induction. These results indicate the potential for neonatal dietary interventions to influence growth and body composition later in life via programming of MSC. Grant Funding Source : NC Agricultural Research Service
Ossabaw swine are an exciting model to study the effects of diet induced obesity. We examined the affects of an obesigenic diet on bone integrity and on mesenchymal stem cells (MSC) activity, which underpins bone integrity. Pigs fed an obesigenic diet had greater (P < 0.05) body fat % and insulin resistance. Obese pigs had larger and heavier bones (P < 0.05), however the percentage of mineral in the bone did not differ between obese and lean pigs. In order to evaluate alterations in MSC activity, MSC from obese and lean pigs were cultured under adipocytic and osteogenic conditions. Under osteogenic conditions, the expression of genes indicative of osteoblastic differentiation (RunX2 and osteocalcin) were greater (P<0.1) in MSC isolated from obese pigs. Under adipogenic conditions, increased gene expression of markers of adipocytic differentiation (PPARG, AP2, and LPL), as well as greater lipid accumulation (Oil Red O staining), were seen in MSC isolated from the lean pigs. While the increased osteogenic activity of MSC from obese pigs supports the bone data, the reduced adipogenic activity of these cells seems counterintuitive. However, the reduced in vitro adipocytic differentiation of MSC from obese pigs may be a result of the insulin resistance seen in these pigs. These results suggest that obesity and insulin resistance may have long‐term effects on bone development and MSC activity.
Neonatal Ca nutrition affects bone development and mesenchymal stem cell (MSC) activity; however, its effect on MSC differentiation potential has not been fully characterized. MSC were isolated from neonatal pigs fed either a Ca deficient, adequate, or excessive diet. After in vitro adipogenic induction, PPARG and AP2 expression was up‐regulated in MSC from Ca deficient (P<0.1) and Ca excess (P<0.05) groups. Under osteogenic induction, osteocalcin (OC) gene expression was depressed in MSC from Ca deficient pigs. To further evaluate the impact of Ca status on MSC activity, 20 individual colonies were obtained from each pig. From these colonies, we selected the most and least osteogenic (O+/O−) and the most and least adipogenic (A+/A−) clones based on functional staining. During osteogenesis, increased (P < 0.1) gene expression of Runx2 and OC was seen among O+ colonies. Increased expression of OC was seen among Ca adequate (P<0.01) and excess (P=0.07) colonies compared with the Ca deficient colonies. During adipogenesis, A+ colonies had greater expression (P < 0.1) of AP2 and LPL. No interaction between osteogenic or adipogenic capability and Ca status was seen. This suggests that the differences in adipocytic and osteogenic differentiation of MSC based on the Ca status of the pig is due to alterations in the number of highly osteogenic and adipogenic MSC in the heterogeneous population. Grant Funding Source : NC Agricultural Research Service
We evaluated the preferences of nursery pigs for diets containing increasing distillers dried grains with solubles (DDGS), varying in color, or high-protein distillers dried grains (HP-DDG) and the effects of flavor supplementation on pig preference and growth performance. In Exp. 1 through 5, diet preference was determined in weanling pigs adjusted to a commercial diet for at least 10 d, and then housed individually for a 2-d double-choice preference test. In Exp. 1, a total of 60 pigs (11.6 ± 0.3 kg of BW) were given a choice between a reference diet (0% DDGS) and test diets containing 0, 10, 20, or 30% DDGS. In Exp. 2, a total of 80 pigs (10.8 ± 0.1 kg of BW) were given a choice between a reference diet (0% HP-DDG) and diets containing 0, 10, 20, or 30% HP-DDG. In Exp. 3, a total of 80 pigs (10.3 ± 0.2 kg of BW) were given a choice between a reference diet (0% DDGS) and a diet containing 0%, 30% light, or 30% dark DDGS. In Exp. 4, a total of 80 pigs (11.2 ± 0.2 kg of BW) were given a choice between a reference diet without DDGS and a diet containing either 0% DDGS, 10 or 20% light DDGS, or 10 or 20% dark DDGS. In Exp. 5, a total of 108 pigs (9.0 ± 0.2 kg of BW) were given a choice between a reference diet (0% DDGS and no flavor) and a diet without or with flavor and containing 0, 10, or 20% DDGS. In Exp. 1 and 2, DDGS and HP-DDG, respectively, linearly decreased (P < 0.01) pig preference. In Exp. 3, dark DDGS were preferred (P < 0.05) compared with light DDGS. In Exp. 4, preferences were linearly reduced (P < 0.01) with DDGS inclusion, and dark DDGS tended (P = 0.06) to be preferred compared with light DDGS. In Exp. 5, DDGS reduced preference (P < 0.01) and flavor reduced preference (P < 0.01) regardless of DDGS level. In Exp. 6, a total of 192 pigs (6.7 ± 0.1 kg of BW) were fed starter 1 diets without or with flavor for 1 wk. Subsequently, pigs were fed starter 2 and 3 diets (2 wk each) containing 0, 10, or 20% DDGS while continuing to receive their respective flavor treatment. Flavor addition during the starter 1 phase increased ADFI (P = 0.02), and DDGS inclusion tended to decrease ADG (P = 0.06) and decreased ADFI (P = 0.03) during the starter 2 phase. Volatile components in DDGS and HP-DDG varied greatly depending on the source. Nursery pigs preferred a diet without DDGS or HP-DDG, and this appeared to be unrelated to color differences between sources. Knowledge of volatile compounds that enhance or suppress the palatability of feed may lead to further development of feed additives for masking relatively unpalatable, albeit cost-effective, ingredients.
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