Gene expression of matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 13 (MMP13), vascular endothelial growth factor (VEGF) and fibroblast growth factor 23 (FGF23) in femur and vertebra tissues of the hypovitaminosis D kyphotic pig model.

The hypovitaminosis D kyphotic pig provides a reliable model to study the initiation of bone lesions caused by maternal vitamin D (D) deficiencies. Matrix metalloproteinases (MMP; specifically, MMP9 and MMP13) and vascular endothelial growth factor (VEGF) are important in endochondral ossification and are potentially regulated by D. Fibroblast growth factor 23 (FGF23) is interrelated with D homoeostasis and bone mineralisation. Relative mRNA expression of MMP9, MMP13, VEGF and FGF23 was measured in pig femur and vertebra. Sows (n 37) were fed diets with 0 (-D), 8·125 (+D) or 43·750 (++D) µg D3/kg throughout gestation and lactation. At weaning (3 weeks), pigs were fed diets with 0 (-D) or 7·0 (+D) µg D3/kg, each with 75 and 95 % (LCaP) or 150 and 120 % (HCaP) of the Ca and P requirements. Pigs at birth (n 27), 3 weeks (n 27) and after the nursery period (7 weeks; n 72) were euthanised for analysis. At 3 weeks, femur MMP9 expression of pigs produced by +D or ++D sows was reduced (P<0·05) to 0·5-fold and VEGF expression to 0·4-fold compared with pigs from -D sows. At 7 weeks, MMP9 expression was reduced (P<0·05) to 0·45-fold in femur and 0·58-fold in vertebra from pigs produced by +D or ++D sows compared with pigs from -D sows. Pig femur VEGF expression was reduced to 0·75-fold in pigs produced by ++D sows. MMP9 and VEGF mRNA expression offer potential markers for the initiation of bone lesions in the hypovitaminosis D kyphotic pig model.

Serum and tissue 25-OH vitamin D3 concentrations do not predict bone abnormalities and molecular markers of vitamin D metabolism in the hypovitaminosis D kyphotic pig model.

The hypovitaminosis D kyphotic pig provides a model to study maternal vitamin D (D) carryover on gross and molecular characteristics of bone abnormalities in offspring. Excess maternal D is proposed to protect offspring under nutritional challenges from developing bone abnormalities. Relationships between D sufficiency parameters and bone abnormalities were characterised. Sows (n 37) were fed diets with 0 (-D), 8·125 (+D) or 43·750 (++D) µg D3/kg throughout gestation and lactation. At weaning (3 weeks) pigs were fed diets with 0 (-D) or 7·0 (+D) µg D3/kg, each with 75 and 95 % (LCaP) or 150 and 120 % (HCaP) of the Ca and P requirements. Pigs were euthanised before colostrum consumption at birth (n 27), 3 weeks (n 27) or after the nursery period (7 weeks, n 71) for tissue analysis. At 7 weeks, differences due to maternal D were detected (P≤0·05) in pig growth, serum parameters and mRNA expression regardless of nursery diet. Prevalence of kyphosis in pigs at 13 weeks was affected by maternal D, but not prevented by only HCaP or +D nursery diets. Increased (P≤0·05) serum 25-OH-D3 concentrations in sows fed +D or ++D diets were not reflected by similar magnitudes of 25-OH-D3 in colostrum, 18-d milk, or serum and tissue concentrations in pigs. The mode of action by which maternal dietary D influences development of skeletal abnormalities warrants further investigation.

Maternal dietary vitamin D carry-over alters offspring growth, skeletal mineralisation and tissue mRNA expressions of genes related to vitamin D, calcium and phosphorus homoeostasis in swine.

Maternal dietary vitamin D carry-over effects were assessed in young pigs to characterise skeletal abnormalities in a diet-induced model of kyphosis. Bone abnormalities were previously induced and bone mineral density (BMD) reduced in offspring from sows fed diets with inadequate vitamin D3. In a nested design, pigs from sows (n 23) fed diets with 0 (-D), 8·125 (+D) or 43·750 (++D) µg D3/kg from breeding through lactation were weaned and, within litter, fed nursery diets arranged as a 2×2 factorial design with 0 (-D) or 7·0 (+D) µg D3/kg, each with 95 % (95P) or 120 % (120P) of P requirements. Selected pigs were euthanised before colostrum consumption at birth (0 weeks, n 23), weaning (3 weeks, n 22) and after a growth period (8 weeks, n 185) for BMD, bone mechanical tests and tissue mRNA analysis. Pigs produced by +D or ++D sows had increased gain at 3 weeks (P<0·05), and at 8 weeks had increased BMD and improved femur mechanical properties. However, responses to nursery diets depended on maternal diets (P<0·05). Relative mRNA expressions of genes revealed a maternal dietary influence at birth in bone osteocalcin and at weaning in kidney 24-hydroxylase (P<0·05). Nursery treatments affected mRNA expressions at 8 weeks. Detection of a maternal and nursery diet interaction (P<0·05) provided insights into the long-term effects of maternal nutritional inputs. Characterising early stages of bone abnormalities provided inferences for humans and animals about maternal dietary influence on offspring skeletal health.

Copper metabolism and its implications for canine nutrition.

Canine copper nutrition has received increased attention due to recent reports of apparent copper-associated hepatitis in the USA and European Union. In order to properly address the need to modify the U.S. National Research Council and Association of American Feed Control Officials canine copper recommendations that will have implications for all dogs, it is important to understand the complexities of copper metabolism, confounding variables affecting copper status, and the available research on this topic in dogs. Recent trends in consumer preference for dog diets, supplements, and functional treats introduce another layer of complexity, as most ingredients used in these formulations provide vastly different proportions of essential nutrients, thus resulting in great variation in nutrient profiles available to the animal. Although controlled research addressing copper metabolism in dogs is limited, there are lessons to be learned from other monogastric species as well as canine case studies that can provide a base for increasing knowledge to address this issue. Copper metabolism and status in animals is affected by a multitude of factors including absorption, storage, excretion, and nutrient interactions. Given its vital role in many physiological processes, it is important that both nutritional deficiencies and toxicities be avoided. Additionally, another challenge for proper copper nutrition in dogs is the known genetic predispositions of some breeds for copper storage and excretion abnormalities. Therefore, it is imperative that veterinarians, nutritionists, and pet food manufacturers collaborate with the shared goal of providing dog food options that supply the essential nutrients at adequate concentrations to support an active and healthy life. Many questions remain regarding copper metabolism and proper diet formulation for dogs. Future research efforts should focus on discovering reliable, non-invasive methods for evaluating canine copper status, a deeper understanding of genetic predispositions of certain breeds, increased knowledge of copper contributions from various ingredients, and the role of unpredictable physiological stressors on copper metabolism.

Effect of mineral diets on the development of cartilage material properties.

Vitamin D and minerals, including zinc (Zn) and manganese (Mn), are vital in the development of bones, but their roles in the development of articular cartilage material behavior are not well understood. In this study, articular cartilage material properties from a hypovitaminosis D porcine model were evaluated. Pigs were produced by sows fed vitamin D deficient diets during gestation and lactation, and the offspring were subsequently fed vitamin D deficient diets for 3 weeks during the nursery period. Pigs were then assigned to dietary treatment groups with inorganic minerals only or inorganic plus organic (chelated) minerals. Humeral heads were harvested from pigs at 24 weeks of age. Linear elastic modulus and dissipated energy were measured under compression to 15% engineering strain at 1 Hz. Anatomical location within the humeral head affected elastic modulus. Diet significantly affected linear modulus and dissipated energy. The largest modulus and highest energy dissipation was in the inorganic zinc and manganese group; the lowest modulus and the least energy dissipation was in the organic (chelated) zinc and manganese group. Pairwise results between the control group and all vitamin D deficient groups were not statistically significant. Overall, these results suggest that mineral availability during rapid growth subsequent to a vitamin-D deficiency during gestation and lactation had minimal effects on articular cartilage material properties in young growing pigs. Though not statistically significant, some of the numerical differences between mineral sources suggest the potential importance of mineral availability during cartilage formation and warrant further study.

Maturity-dependent cartilage cell plasticity and sensitivity to external perturbation.

OBJECTIVE: Articular cartilage undergoes biological and morphological changes throughout maturation. The prevalence of osteoarthritis in the aged population suggests that maturation predisposes cartilage to degradation and/or impaired regeneration, but this process is not fully understood. Therefore, the objective of this study was to characterize the cellular and genetic profile of cartilage, as well as biological plasticity in response to mechanical and culture time stimuli, as a function of animal maturity. METHODS/DESIGN: Porcine articular cartilage explants were harvested from stifle joints of immature (2-4 weeks), adolescent (5-6 months), and mature (1-5 years) animals. Half of all samples were subjected to a single compressive mechanical load. Loaded samples were paired with unloaded controls for downstream analyses. Expression of cartilage progenitor cell markers CD105, CD44, and CD29 were determined via flow cytometry. Expression of matrix synthesis genes Col1, Col2, Col10, ACAN, and SOX9 were determined via qPCR. Tissue morphology and matrix content were examined histologically. Post-loading assays were performed immediately and following 7 days in culture. RESULTS: CD105 and CD29 expression decreased with maturity, while CD44 expression was upregulated in cartilage from mature animals. Expression of matrix synthesis genes were generally upregulated in cartilage from mature animals, and adolescent animals showed the lowest expression of several matrix synthesizing genes. Culture time and mechanical loading analyses revealed greater plasticity to mechanical loading and culture time in cartilage from younger animals. Histology confirmed distinct structural and biochemical profiles across maturity. CONCLUSION: This study demonstrates differential, nonlinear expression of chondroprogenitor markers and matrix synthesis genes as a function of cartilage maturity, as well as loss of biological plasticity in aged tissue. These findings have likely implications for age-related loss of regeneration and osteoarthritis progression.

Maternal Diets Deficient in Vitamin D Increase the Risk of Kyphosis in Offspring: A Novel Kyphotic Porcine Model.

BACKGROUND: The purpose of this study was to explore the role of perinatal vitamin-D intake on the development and characterization of hyperkyphosis in a porcine model. METHODS: The spines of 16 pigs were assessed at 9, 13, and 17 weeks of age with radiography and at 17 weeks with computed tomography (CT), magnetic resonance imaging (MRI), histology, and bone-density testing. An additional 169 pigs exposed to 1 of 3 maternal dietary vitamin-D levels from conception through the entire lactation period were fed 1 of 4 nursery diets supplying different levels of vitamin D, calcium, and phosphorus. When the animals were 13 weeks of age, upright lateral spinal radiography was performed with use of a custom porcine lift and sagittal Cobb angles were measured in triplicate to determine the degree of kyphosis in each pig. RESULTS: The experimental animals had significantly greater kyphotic sagittal Cobb angles at all time points when compared with the control animals. These hyperkyphotic deformities demonstrated no significant differences in Hounsfield units, contained a slightly lower ash content (46.7% ± 1.1% compared with 50.9% ± 1.6%; p < 0.001), and demonstrated more physeal irregularities. Linear mixed model analysis of the measured kyphosis demonstrated that maternal diet had a greater effect on sagittal Cobb angle than did nursery diet and that postnatal supplementation did not completely eliminate the risk of hyperkyphosis. CONCLUSIONS: Maternal diets deficient in vitamin D increased the development of hyperkyphosis in offspring in this model. CLINICAL RELEVANCE: This study demonstrates that decreased maternal dietary vitamin-D intake during pregnancy increases the risk of spinal deformity in offspring. In addition, these data show the feasibility of generating a large-animal spinal-deformity model through dietary manipulation alone.

Effect of induced hypocalcemia in nonlactating, nonpregnant Holstein cows fed negative DCAD with low, medium, or high concentrations of calcium.

The main objective of this study was to determine how feeding different dietary calcium (Ca) concentrations in combination with a negative dietary cation-anion difference (DCAD) would affect the cow's response to induced hypocalcemia. We conducted an experiment with multiparous, nonlactating, nonpregnant Holstein cows fed a negative DCAD (average -18.2 across all diets) for 21 d with low (LC; 0.45% Ca; n = 5), medium (MC; 1.13% Ca; n = 6), or high (HC; 2.02% Ca; n = 6) concentrations of dietary Ca. Urine and blood samples were collected and urine pH measured daily during the 21-d feeding period prior to hypocalcemia challenge. Cows were then subjected to a controlled induction of hypocalcemia to determine how dietary Ca intake affected the response to a hypocalcemia challenge. On days 22, 23, and 24, hypocalcemia was induced with an intravenous infusion of 5% EGTA in 2 different cows from each treatment daily. During infusion, blood samples were collected every 15 min until 60% of prechallenge ionized calcium (iCa) concentrations were achieved. Samples were collected postinfusion at 0, 2.5, 5, 10, 15, 30, and every 30 min thereafter until 90% of prechallenge iCa was reached. Blood pH, hematocrit, and serum total Ca (tCa), sodium (Na), potassium (K), phosphorous (P), magnesium (Mg), and serotonin did not differ (P > 0.05) among treatments during the feeding period. Blood iCa (P = 0.04) and glucose (P = 0.03) were significantly elevated in HC compared with LC and MC cows during the feeding period. Urine pH was less than 6.0 in all cows, but was lowest in LC (P = 0.02) compared with MC and HC cows during the feeding period. Urine Ca, P, Mg, and deoxypyridinoline did not differ among treatments (P > 0.05). Cows fed HC maintained higher concentrations of iCa (P = 0.03) during the challenge period than MC (P = 0.04), and LC (P = 0.004), and required a longer time to reach 60% of whole blood iCa, and required more EGTA to reach 60% iCa than MC or LC cows (P = 0.01). Serum tCa decreased in all cows during infusion (P < 0.0001) but did not differ among treatments. Serotonin concentrations were elevated in MC cows compared with HC and LC cows during EGTA infusion (P = 0.05), suggesting an interdependent relationship between iCa and serotonin. Cows fed HC had a slower rate of decrease in iCa, but not tCa, when induced with hypocalcemia, indicating potential metabolic benefits of feeding higher dietary Ca in combination with a negative DCAD.