Impact of the arginine silicate inositol complex on bone metabolism in broiler chickens with tibial dyschondroplasia caused by manganese deficiency.

1. Tibial dyschondroplasia (TD) is a skeletal disorder in broilers that has financial implications, necessitating dietary modifications to reduce the prevalence of this disease. This study explored how arginine silicate inositol complex (ASI) supplementation affected tibial growth plate (TGP) and overall bone health in broilers with manganese (Mn) deficiency-induced TD.2. A total of 240 broiler chicks were divided into four groups, each consisting of 60 birds (15 replicates of four broilers each) as follows: i) Control, with 60 mg Mn per kg of diet; ii) ASI, with 60 mg Mn and 1 g ASI per kg of diet; iii) TD, with 22 mg Mn per kg of diet, and iv) TD+ASI, with 22 mg Mn and 1 g ASI per kg of diet.3. It was found that ASI supplementation increased tibial bone length in Mn-deficient TD broilers (p = 0.007). There was no Mn x ASI interaction for other bone morphometry variables (p > 0.05). However, both tibial bone mineral content and density were affected by Mn and ASI (p < 0.05). With ASI supplementation, serum bone-specific alkaline phosphatase and osteocalcin levels were elevated in the TD+ASI group compared to the TD group (p < 0.001). In the TD group, osteoprotegerin (OPG) levels in the TGP decreased compared to the control groups (p < 0.001).4. In contrast, ASI supplementation in the TD broilers counteracted the decrease in OPG compared to TD broilers without ASI supplementation (p < 0.001). The Mn level and ASI supplementation significantly influenced the OPG/receptor activator of the nuclear factor-κB ligand ratio (p < 0.001).5. In conclusion, the results demonstrated that inclusion of ASI in broiler diets could enhance bone formation variables by controlling OPG levels in the TGP, potentially serving as an effective method to decrease the occurrence of TD.

Lactobacillus rhamnosus JYLR-005 Prevents Thiram-Induced Tibial Dyschondroplasia by Enhancing Bone-Related Growth Performance in Chickens.

Tibial dyschondroplasia (TD) is a leg disorder caused by the abnormal development of the tibia in fast-growing poultry. Lactobacillus rhamnosus (L. rhamnosus) strains have been reported to have effects on increasing bone growth and improving osteoporosis in animals. However, whether L. rhamnosus JYLR-005 can improve bone growth in TD chickens remains unclear. In this study, we noted that L. rhamnosus JYLR-005 could not reduce the suppression of the production performance of TD broilers (p > 0.05) but had a slight protective effect on the broiler survival rate (χ2 = 5.571, p = 0.062). However, for thiram-induced TD broiler chickens, L. rhamnosus JYLR-005 could promote tibia growth by increasing tibia-related parameters, including the tibia weight (day 11, p = 0.040), tibia length (day 15, p = 0.013), and tibia mean diameter (day 15, p = 0.035). Moreover, L. rhamnosus JYLR-005 supplementation improved the normal growth and development of the tibial growth plate by maintaining the morphological structure of the chondrocytes and restored the balance of calcium and phosphorus. Taken together, these findings provide a proof of principle that L. rhamnosus JYLR-005 may represent a therapeutic strategy to treat leg disease in chickens.

Morinda officinalis polysaccharides improve meat quality by reducing oxidative damage in chickens suffering from tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is the common leg disease in commercial broilers. However, the effects of TD on meat quality and the protective of Morinda officinalis polysaccharide (MOP) are largely unknown. Three hundred broiler chicks (one-day-old) were equally allocated into control (CON), TD and MOP-treated groups for 15 days. The results indicated that TD influenced morphology and meat quality-related parameters of the breast muscle, and changed the activity and mRNA expression of antioxidant enzymes in plasma and breast muscles. Moreover, metabolomics profiling of breast muscle revealed that the main altered metabolites 4-guanidinobutyric acid and chenodeoxycholic acid, which are related to meat quality and oxidative stress. Additionally, 500 mg/L MOP effectively restored the content of meat metabolites and oxidative damage. These findings suggest that oxidative damage caused by TD may affect meat quality in broilers by changing the content of breast muscle metabolites and that MOP supplementation has a restorative effect.

In Vitro Effect of Apigenin and Danshen in Tibial Dyschondroplasia Through Inhibition of Heat-Shock Protein 90 and Vascular Endothelial Growth Factor Expressions in Avian Growth Plate Cells.

Tibial dyschondroplasia (TD) is one of the common skeletal abnormalities in fast-growing birds, and it is characterized by nonvascularized, unmineralized, and nonviable cartilage in the tibial growth plate that fails to form bone. The aim of this study was to check the in vitro effect of apigenin and danshen on heat-shock protein 90 (Hsp90) and vascular endothelial growth factor (VEGF) expressions in avian growth plate cells treated with sublethal concentration of thiram. Initially, chondrocytes from chicken growth plates were isolated on culturx ed medium with and without various concentration of thiram to determine the sublethal dose. Then, to check the effect of apigenin and danshen, the chondrocytes were treated first with a sublethal (2.5 µM) concentration of thiram and then with different doses (10, 20, 40, and 80 µM) of apigenin and danshen. The mRNA expression levels of Hsp90 and VEGF genes were evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results showed that the expression levels of Hsp90 and VEGF mRNA transcripts were increased significantly (P < 0.05) in thiram-treated chondrocytes culture medium up to 1.5-fold, whereas apigenin and danshen therapy to chondrocytes in culture medium significantly (P < 0.05) reduced the Hsp90 and VEGF expression levels. In conclusion, up-regulation of both (Hsp90 and VEGF) genes and damage to chondrocytes in culture medium caused by thiram can be restored by using apigenin and danshen. Therefore, apigenin and danshen therapies are suggested and encouraged as a promising approach to control TD in broiler chickens.

A recessive skeletal dysplasia, SEMD aggrecan type, results from a missense mutation affecting the C-type lectin domain of aggrecan.

Analysis of a nuclear family with three affected offspring identified an autosomal-recessive form of spondyloepimetaphyseal dysplasia characterized by severe short stature and a unique constellation of radiographic findings. Homozygosity for a haplotype that was identical by descent between two of the affected individuals identified a locus for the disease gene within a 17.4 Mb interval on chromosome 15, a region containing 296 genes. These genes were assessed and ranked by cartilage selectivity with whole-genome microarray data, revealing only two genes, encoding aggrecan and chondroitin sulfate proteoglycan 4, that were selectively expressed in cartilage. Sequence analysis of aggrecan complementary DNA from an affected individual revealed homozygosity for a missense mutation (c.6799G --> A) that predicts a p.D2267N amino acid substitution in the C-type lectin domain within the G3 domain of aggrecan. The D2267 residue is predicted to coordinate binding of a calcium ion, which influences the conformational binding loops of the C-type lectin domain that mediate interactions with tenascins and other extracellular-matrix proteins. Expression of the normal and mutant G3 domains in mammalian cells showed that the mutation created a functional N-glycosylation site but did not adversely affect protein trafficking and secretion. Surface-plasmon-resonance studies showed that the mutation influenced the binding and kinetics of the interactions between the aggrecan G3 domain and tenascin-C. These findings identify an autosomal-recessive skeletal dysplasia and a significant role for the aggrecan C-type lectin domain in regulating endochondral ossification and, thereby, height.

Cartilage dysplasia and tissue mineralization in the rat following administration of a FGF receptor tyrosine kinase inhibitor.

PD176067 is a reversible and selective inhibitor of fibroblast growth factor receptor tyrosine kinase, and was in preclinical development as an angiogenesis inhibitor for the treatment of solid tumors. A 14-day oral toxicity study of PD176067 in young female rats (7 weeks old) was conducted at doses of 2.5, 5, and 10 mg/kg/day (15, 30, and 60 mg/m(2), respectively). Skeletal changes, and vascular and soft tissue mineralization were observed as primary drug-related toxicities. To determine if these changes are specific to young, rapidly growing animals with increased vascular and osseous development, PD176067 was administered to mature (11 months old) rats. Female rats received PD176067 by gavage for 14 days at doses of 2.5, 5, and 10 mg/kg/day and necropsied on day 15. Clinical signs of toxicity were seen at > or =5 mg/kg and one death occurred at 10 mg/kg. Physeal dysplasia (distal femur, proximal tibia, sternum) occurred in all drug-treated animals and was characterized by dose-related increased thickness of the zones of chondrocyte proliferation and hypertrophy, and marked thickening of the zone of ossification. Cartilage hyperplasia was characterized by proliferation of chondrocytes along margins of the synchondrosis and subperiosteum of sternebrae. Serum phosphorus levels increased 47% and 166% at 5 and 10 mg/kg, respectively. Mineralization of cardiac myocytes, aorta, various arteries, renal tubules, and gastric mucosa and muscularis was seen at 10 mg/kg, and consistent with the presence of calcium-phosphorus deposition. Physeal changes occurred at similar plasma PD176067 exposures in young and mature rats (AUC > or = 4.83 microg.hr/mL). PD176067 produced morphologically similar lesions in young and adult rats.

Disease-causing mutations in cartilage oligomeric matrix protein cause an unstructured Ca2+ binding domain.

Chondrocytes from pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) patients display an enlarged rough endoplasmic reticulum that accumulates extracellular matrix proteins, including cartilage oligomeric matrix protein (COMP). Mutations that cause PSACH and EDM1 are restricted to a 27-kDa Ca(2+) binding domain (type 3 repeat). This domain has 13 Ca(2+)-binding loops with a consensus sequence that conforms to Ca(2+)-binding loops found in EF hands. Most disease-causing mutations are found in the 11-kDa C-terminal region of this domain. We expressed recombinant native and mutant forms of the type 3 repeat domain (T3) and its 11-kDa C-terminal region (T3-Cterm). T3 and T3-Cterm bind approximately 13 and 8 mol of Ca(2+)/mol of protein, respectively. CD, one-dimensional proton, and two-dimensional (1)H-(15)N HSQC spectra of Ca(2+)-bound T3-Cterm indicate a distinct conformation that has little helical secondary structure, despite the presence of 13 EF hand Ca(2+)-binding loops. This conformation is also formed within the context of the intact T3. 19 cross-peaks found between 9.0 and 11.4 ppm are consistent with the presence of strong hydrogen bonding patterns, such as those in beta-sheets. Removal of Ca(2+) leads to an apparent loss of structure as evidenced by decreased dispersion and loss of all down field resonances. Deletion of Asp-470 (a mutation found in 22% of all PSACH and EDM1 patients) decreased the Ca(2+)-binding capacity of both T3 and T3-Cterm by about 3 mol of Ca(2+)/mol of protein. Two-dimensional (1)H-(15)N HSQC spectra of mutated T3-Cterm showed little evidence of defined structure in the presence or absence of Ca(2+). The data demonstrate that Ca(2+) is required to nucleate folding and to maintain defined structure. Mutation results in a partial loss of Ca(2+)-binding capacity and prevents Ca(2+)-dependent folding. Persistence of an unstructured state of the mutated Ca(2+) binding domain in COMP is the structural basis for retention of COMP in the rough endoplasmic reticulum of differentiated PSACH and EDM1 chondrocytes.

Decreasing ascorbate intake does not affect the levels of glutathione, tocopherol or retinol in the ascorbate-requiring osteogenic disorder shionogi rats.

Levels of glutathione in liver and kidney, and other nutrients in plasma were evaluated in male and female ascorbate-requiring osteogenic disorder Shionogi (ODS) rats fed semipurified diets in which the concentrations of ascorbate were gradually decreased from 1965 to 180 mg/kg. Plasma ascorbate levels in ODS rats were unaffected when ascorbate levels in the diet were decreased from 1965 to 768 mg/kg. However, plasma ascorbate levels decreased progressively when levels of ascorbate in the diet were decreased from 527 to 180 mg/kg. Plasma ascorbate levels decreased up to 77% when the dietary ascorbate concentration decreased from 1965 to 180 mg/kg. Ascorbate levels in liver and kidney fell as much as 60-70% when the dietary ascorbate levels were reduced from 1965 to 180 mg/kg. However, the glutathione levels in these tissues were not affected. Plasma retinol and vitamin E levels were not affected by decreasing dietary ascorbate intake. Total cholesterol levels increased significantly in female rats as dietary ascorbate intake declined. Levels of glycated hemoglobin decreased significantly when dietary ascorbate levels decreased from 1965 to 527 mg/kg. This study suggests that levels of vitamin E, retinol and glutathione are not affected by decreased dietary intake of ascorbate under nonscorbutic conditions, whereas elevated ascorbate intake is associated with a decrease in levels of plasma cholesterol in female ODS rats. However, excessive intake of ascorbate may be associated with elevated glycation of hemoglobin. To achieve the maximal health benefit of ascorbate supplementation, further studies are necessary to define optimal ascorbate intakes.

Evidence of increased cholecalciferol requirement in chicks with tibial dyschondroplasia.

A series of experiments was conducted to test the hypothesis that vitamin D utilization may not be as efficient in chicks with tibial dyschondroplasia (TD). The basal diet contained 1.0% Ca and 0.45% available P with no supplemental cholecalciferol (D3). Chicks from low TD (LTD) and high TD (HTD) selected lines were fed diets supplemented with various levels of vitamin D compounds and examined for rickets and TD. When chicks were fed a D3-deficient diet containing only 1.25 micrograms/kg added D3, HTD chicks had a greater incidence of severe rickets than LTD chicks (P < 0.05). The LTD chicks did not exhibit TD when fed a diet containing adequate (20 micrograms/kg) D3. The LTD chicks fed a diet supplemented with 5 micrograms/kg D3, however, had 22% incidence of TD. When HTD chicks were fed diets supplemented with 5 micrograms/kg D3 [control diet that meets NRC (1994) requirement for D3], 20 micrograms/kg D3, 5 micrograms/kg 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] or the combination of both D3 (20 micrograms/kg) and 1,25-(OH)2D3 (5 micrograms/ kg), TD incidence was highest in HTD chicks fed the control diet. When HTD chicks were fed diets with an increased dietary level of 1,25-(OH)2D3 (10 micrograms/kg) further reduction of TD incidence (P < 0.05) occurred. A potentially toxic level (Soares et al., 1983) of 1,25-(OH)2D3 (15 micrograms/kg) fed to HTD chicks resulted in still greater suppression of incidence of TD even though growth and feed intake in HTD chicks was greater than those of LTD chicks. It is concluded that the development of TD in HTD chicks is associated with subnormal ability to metabolize vitamin D.

Non-reducible collagen cross-linking in cartilage from broiler chickens with tibial dyschondroplasia.

Recent work has shown that hydroxylysylpyridinoline (HP), a non-reducible cross-link that stabilizes the collagen fibril network, is significantly greater (over 10-fold) in dyschondroplastic cartilage than in normal growth-plate cartilage in the tibiotarsi of chickens with homocysteine-induced tibial dyschondroplasia (TD). In the present study, broiler chicks with a genetic disposition to TD, as well as normal broiler chicks on a copper-deficient diet alone or supplemented with copper and thiram, were raised for 3 to 4 weeks (Expts. 1 and 2). Their dyschondroplastic cartilage from the proximal tibiotarsus was collected and analyzed for HP as well as lysylpyridinoline (LP) cross-links. Normal growth plate cartilage was obtained from chicks on the copper-deficient diet supplemented with copper. In a third experiment, another set of broiler chicks was raised on a corn/soybean meal-based diet with or without homocysteine, and their articular and sternal cartilage were isolated for cross-link analysis. In the first two experiments, dyschondroplastic cartilage from all birds with induced TD had higher HP and LP concentrations than growth-plate cartilage from birds without TD, although the ratios of HP to LP varied. In the third experiment, the sternal and articular cartilage from birds with homocysteine-induced TD appeared normal, having similar HP concentrations in the same types of cartilage in birds without TD.

Schwartz-Jampel syndrome: II. Na+ channel defect causes myotonia.

Skeletal muscle fibers from a patient with Schwartz-Jampel syndrome were studied in vitro. The fibers had normal resting membrane potentials, but their resting [Ca2+]i was elevated. The resting potentials were unstable and spontaneous depolarizations caused twitching in all fibers. Stimulated contractions were characterized by markedly slowed relaxation which was due to electrical after-activity. Neither curare (0.7 microM), tocainide (50 microM), nor phenytoin (80 microM) had an effect on the myotonic activity. In contrast, procainamide (200 microM) suppressed the hyperexcitability without affecting the twitch amplitude. The steady-state current-voltage relation was normal in 5 fibers, but altered in 3 others. These latter fibers had an increased specific membrane resistance owing to a decreased Cl- conductance. The Na+ channels were investigated in the cell-attached patch clamp mode. In all patches on either type of fiber, depolarizing pulses elicited delayed, synchronized openings of Na+ channels. These abnormal openings occurred even after the surface membrane repolarized. We hypothesize that these altered membrane conductances are responsible for the hyperexcitability and the associated slowed relaxation.

Effect of dietary calcium, phosphorus, chloride, and zeolite on the development of tibial dyschondroplasia.

The effect of synthetic zeolite was investigated on the development of tibial dyschondroplasia in young broilers fed diets in which the dietary levels of calcium, phosphorus, and chloride ranged from adequate to deficient. In the first two experiments the calcium level was maintained at .65% and four combinations were fed of .30% and .15% chloride and .75% and .50% phosphorus with and without 1% zeolite. Feeding the high phosphorus diet caused a high incidence of tibial dyschondroplasia that was lowered by feeding 1% zeolite. In both experiments the feeding of zeolite at a low level of dietary phosphorus caused a significant lowering of 16-day weight and bone ash. In the third experiment, when diets containing all combinations of .65% and .80% calcium and .50% and .60% phosphorus were fed, the addition of 1% zeolite caused a significant lowering of the 16-day weight, bone ash, and incidence, score, and percentage severe tibial dyschondroplasia. Once again as in the previous two experiments, there was a significant interaction between dietary phosphorus level and zeolite and 16-day weight and bone ash. Feeding zeolite significantly decreased phytate phosphorus retention.

Effect of dietary selenium on the development of Fusarium-induced tibial dyschondroplasia in broiler chickens.

A trial was conducted to determine the effects of dietary level of selenium on the pathogenesis of Fusarium-induced tibial dyschondroplasia (FITD) in broiler chicks, and to assess the applicability of FITD as an animal model of Kashin-Beck disease of humans. Day-old female broilers were fed diets that were deficient in selenium (0.02 ppm Se), adequate in selenium (0.15 ppm Se), or generous in selenium (0.50 ppm Se). TDP-1, the toxic component of the fungus, was administered to 15 of 26 chicks in each dietary group starting at 1 week of age and continuing until the chicks were killed at 24-30 days of age. Plasma selenium levels and hepatic glutathione peroxidase activity were significantly lower in the selenium-deficient group than in other dietary groups; these parameters were not affected by treatment with TDP-1. The mortality rate of the TDP-1-treated selenium-generous group was significantly less than that in the other TDP-1-treated groups, but there were no differences in the incidence, severity, or character of the FITD lesions among the groups. Thus, the interaction of selenium and TDP-1 did not include an effect on FITD.