Ashing of Bone: Errors Due to Loss of CO2 and Its Correction

Background: Ashing is widely used to determine weight fraction of water-free bone that is mineral, but no standard procedure exists and the range of techniques used spans a range of temperatures and times over which the amount of weight loss is variable. We show that variability is largely due to progressive loss of CO2 from CO3 2- ions in the apatite crystal lattice, beginning at 600⁰C, typically used for ashing. We test the effect of varying temperature, time and weight of sample and develop a reliable method, using small samples.Methods: Replicate samples of bovine cortical bone were tested at 500⁰, 600⁰ and 700⁰C for times ranging up to 24h. We also tested samples of multiple humans at what we concluded to be the optimal conditionsResults: Varying conditions of ashing resulted in variations in apparent ash weight % by up to 7%. Samples between 5 and 20 mg heated to 600⁰C for 1 h gave results agreeing with generally accepted values, but with much smaller variability. Ash wt% values for multiple human bone samples differed by up to 4.8% but replicate data for individuals agree to ± 1 wt%. Conclusions: A satisfactory method is given for ash weight determination using small samples, and yielding highly reproducible data. If accepted widely, ash weight values between laboratories could be used to study variations due to diet, age, drug treatment and disease.

Nutritional, Textural, and Sensory Quality of Aloe Vera Leaf Gel Powder Fortified Plain Cake

Aloe Vera leaves have a great potential as an economic supplement with an adequate nutritional profile. In this study, Aloe Vera leaf gel (AVG) powder was used to fortify plain cakes. Freeze drying of AVG was performed for the production of Aloe Vera powder (ALP) and four plain cakes were prepared with different proportions of ALP for further investigation. Analysis suggested that ALP contained significantly (p<0.05) higher amount of protein (22.23 vs 12.24), ash (19.83 vs 0.64) and iron (175 vs 3.05) content than refined wheat flour (RWF). ALP also contained significant amount of total polyphenols and antioxidant. Moisture, protein, ash, weight, and minerals (Fe, Ca) content were higher (p<0.05) in ALP-cakes; whereas fat, volume, specific volume, height, baking loss, and total carbohydrate content were higher (p<0.05) in RWF-cakes. Incorporation of 6 and 8% ALP in the formulation increased the total polyphenols and anti-oxidant activity in plain cakes. Texture analysis revealed that hardness and chewiness increased in ALP-cakes but decreased in RWF-cakes, however, cohesiveness, springiness, and chewiness decreased in ALP-cakes. Sensory attributes suggested that 4% ALP incorporated cake was attributed as the best formulation. In conclusion, ALP can be supplemented in cakes up to 8% to improve the nutrient value.

Determining the phosphorus release of GraINzyme phytase in diets for nursery pigs

The objective of this study was to determine the available P (aP) release curve for a new phytase source, GraINzyme Phytase (Agrivida Inc., Woburn, MA), which is expressed in corn containing an engineered Escherichia coli phytase called Phy02. Plant-expressed phytases are created by inserting phytase-encoding genes into plants resulting in their ability to produce seeds with increased concentrations of phytase. A total of 360 pigs (Line 200 × 400, DNA, Columbus, NE, initially 9.9 ± 0.19 kg) were used in a 21-d growth study. Pigs were weaned at approximately 21-d of age, randomly allotted to pens based on initial body weight (BW) and fed common starter diets. From d 18 to 21 post-weaning, all pigs were fed a diet containing 0.11% aP. On d 21 post-weaning, considered d 0 of the study, pens were blocked by BW and randomly allotted to 1 of 8 dietary treatments with 5 pigs per pen and 9 pens per treatment. Dietary treatments were formulated to include increasing aP derived from either an inorganic P source (0.11, 0.19, or 0.27% from monocalcium P) or increasing phytase (150, 250, 500, 1,000, or 1,500 FTU/kg). Diets were corn-soybean meal-based and contained 1.24% standardized ileal digestible (SID) Lys. On d 21 of the trial, 1 pig per pen (weighing closest to the mean pen BW) was euthanized and the right fibula was collected to determine bone ash using the non-defatted processing method. Overall (d 0 to 21), pigs fed increasing aP from inorganic P or phytase had increased (linear, P < 0.002) ADG, ADFI, and G:F (quadratic, P < 0.05). Bone ash weight (g) and percentage bone ash increased (linear, P < 0.001) with increasing inorganic P or added phytase. Based on the composition of the diets used in this study, the release equations developed for GraINzyme for ADG, G:F, bone ash weight, and percentage bone ash are: aP = (0.255 × FTU) ÷ (1299.969 + FTU), aP = (0.233 × FTU) ÷ (1236.428 + FTU), aP = (45999.949 × FTU) ÷ (462529200 + FTU), and aP = (0.272 × FTU) ÷ (2576.581 + FTU), respectively.

Irisin Has a Protective Role against Osteoporosis in Ovariectomized Rats

The reduction in estrogen levels results in a decrease in bone density at menopause. Irisin is a myokine that modulates the benefits of exercise, which may include bone health. This study was planned to examine irisin’s impact in preventing osteoporosis after ovariectomy. 4 groups of female albino rats (10 rats/group): control, sham-operated, ovariectomized (OVX-control), and OVX-irisin-treated. Serum levels of bone markers [osteocalcin (OC), bone alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP), calcium (Ca++), phosphorus (P)], glucose, and insulin were being measured. Body mass index, Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), dry and ash femur weight, and bone contents of Ca++ and P were investigated. The femur was examined histopathologically. The OVX-control group showed an increase in serum levels of OC, BALP, TRAP, calcium, phosphorus, BMI, glucose, insulin, and HOMA-IR ( P < 0.05 ) and a reduction in dry and ash weight of the femur, the concentration of calcium and phosphorus content in bone ash ( P < 0.05 ). The OVX-irisin-treated group exhibited a decrease in serum levels of OC, BALP and TRAP, calcium, phosphorus, BMI, glucose, insulin, HOMA-IR ( P < 0.05 ), and a rise in dry and ash weight of the femur, the concentration of calcium and phosphorus in bone ash ( P < 0.05 ). Histological examination of the distal femur diaphysis of the OVX-irisin-treated group exhibited proper bone architecture and density compared with that of the OVX-control group. It is concluded that irisin treatment in the OVX rats safeguarded the regular bone architecture and normal levels of serum bone biomarkers. Irisin may be a possible novel target in the prohibition of postmenopausal osteoporosis.

Relationship between Bone Quality, Egg Production and Eggshell Quality in Laying Hens at the End of an Extended Production Cycle (105 Weeks)

(1) Background: Nowadays the industry aims to improve lay persistency for extended cycles (100 weeks or longer) to make egg production more sustainable. However, intensive egg production challenges hen health, inducing severe osteoporosis and the incidence of bone fractures. In this study, the relationship between bone quality and egg production, and/or eggshell quality, was evaluated at the end of an extended laying cycle of 100 weeks, comparing groups of hens with different production and eggshell quality parameters; (2) Methods: Quality parameters of egg (as weight, egg white height), eggshell (as thickness, weight, breaking strength, elasticity and microstructure) and tibiae bone (weight, diameter, cortical thickness, ash weight, breaking strength, medullary bone) were determined; (3) Results: Hens from groups with a high egg production and good eggshell quality have poorer bone quality (lower ash weight and lesser amount of medullary bone). However, Pearson’s correlation analysis shows no clear relationship between bone and egg/eggshell parameters. (4) Conclusions: Bone and egg production/eggshell quality are independent and can be improved separately. Medullary bone has an important contribution to bone mechanical properties, being important to accumulate enough bone medullary bone early in life to maintain skeletal integrity and eggshell quality in old hens.

Assessing calcium availability from limestone sources through bone and blood status of chickens

Calcium (Cu) availability from limestone (procured from different cement factories) was assessed through bone and blood status of chickens using the completely randomized design. Five hundred and twenty five (525) straight run broiler chicks that were 28 days Of age (trial l), four hundred and twenty (420) broiler finishers that were 56 days of age (trial 2), and 210 layers that had been laying for six months (trial 3) were used in the study. Six limestone dietary treatments and a control accounted for the seven diets that were assessed Twelve birds per treatment and 4 per replicate that had their weights close to the mean of the pen were selected in the broiler trials (trials I and 2) and three birds per pen and nine per diet were selected in the layer trial (trial 3) for bone and blood samples analysis. Bone samples were analyzed for bone weight, bone length, ash weight, percentage ash, Ca and phosphorus contents of ash and blood samples were analyzed for plasma Ca and alkaline phosphates activity. Results showed similar influence of sources on bone weight, bone length, ash weight and percentage ash. Ca and P contents of ash were significantly (P<0.05) affected by limestone sources but generally. Limestone sources produced mean values that were equal to or than the control diet in the starter phase (trail 1). In the finisher phase (trial 2), only Ca content of ashy varied significantly where Jakura source produced the least mean value. The layer trial (trial 3) also produced mean values that were significant (P <0.05) for Ca content of ash with the Sokoto source producing the least mean value. Plasma Ca and alkaline phosphates enzyme activity attained published values for chickens. It was concluded based on the information from this study that the (h from tested limestone sources was generally available for chicken production.

Murine Fetal Serum Phosphorus is Set Independent of FGF23 and PTH, Except in the Presence of Maternal Phosphate Loading

Fibroblast growth factor 23 (FGF23) appears to play no role until after birth, given unaltered phosphate and bone metabolism in Fgf23- and Klotho-null fetuses. However, in those studies maternal serum phosphorus was normal. We studied whether maternal phosphate loading alters fetal serum phosphorus and invokes a fetal FGF23 or parathyroid hormone (PTH) response. C57BL/6 wild-type (WT) female mice received low (0.3%), normal (0.7%), or high (1.65%) phosphate diets beginning 1 week prior to mating to WT males. Fgf23+/- female mice received the normal or high-phosphate diets 1 week before mating to Fgf23+/- males. One day before expected birth, we harvested maternal and fetal blood, intact fetuses, placentas, and fetal kidneys. Increasing phosphate intake in WT resulted in progressively higher maternal serum phosphorus and FGF23 during pregnancy, while PTH remained undetectable. Fetal serum phosphorus was independent of the maternal phosphorus and PTH remained low, but FGF23 showed a small nonsignificant increase with high maternal serum phosphorus. There were no differences in fetal ash weight and mineral content, or placental gene expression. High phosphate intake in Fgf23+/- mice also increased maternal serum phosphorus and FGF23, but there was no change in PTH. WT fetuses remained unaffected by maternal high-phosphate intake, while Fgf23-null fetuses became hyperphosphatemic but had no change in PTH, skeletal ash weight or mineral content. In conclusion, fetal phosphate metabolism is generally regulated independently of maternal serum phosphorus and fetal FGF23 or PTH. However, maternal phosphate loading reveals that fetal FGF23 can defend against the development of fetal hyperphosphatemia.

Technical Note: Assessment of two methods for estimating bone ash in pigs

Data from three experiments conducted to evaluate the effects of increasing available P in swine diets were used to compare two different bone processing methods. Our objective was to determine if the procedures influenced treatment differences and the ability to detect changes in the percentage bone ash. In each experiment, pigs (nursery pigs in experiments 1 and 2, and finishing pigs in experiment 3) were fed a wide range of available phosphorus levels provided from either increasing monocalcium P or added phytase. At the completion of each experiment, a subset of pigs was euthanized, and either fibulas (experiments 1 and 2) or metacarpals (experiment 3) were collected to determine the percentage bone ash. Bones were processed by cleaning away all soft tissues followed by ether extraction for 7 d (defatted), or no lipid extraction (non-defatted), and then ashed. In nursery and finishing pigs, defatted bones had increased (P &lt; 0.001) percentage bone ash compared with non-defatted bones. No evidence of a method × treatment interaction or linear and quadratic interactions were observed in bone ash weight and percentage bone ash (P &gt; 0.10) for nursery pigs; however, a linear interaction was detected (P &lt; 0.05) in percentage bone ash for grow-finish pigs. This response was minimal and likely due to increased variation observed in grow-finish pigs when bones were not defatted. The processing method did not affect the ability to detect differences among treatments as a result of changing dietary P concentrations in the nursery or grow-finish pigs. In summary, either non-defatted or defatted bone processing methods can be used to determine bone ash weight and percentage bone ash as a way to assess bone mineralization and dietary treatment differences in nursery pigs; however, the increased variation observed in mature pigs suggests that defatted bone processing is the preferred method for grow-finish pigs.

Determining the phosphorus release of Smizyme TS G5 2,500 phytase in diets for nursery pigs

Two experiments were conducted to determine the available P (aP) release of Smizyme TS G5 2,500 (Origination, LLC., Maplewood, MN) phytase. Pigs were weaned at approximately 21-d of age, randomly allotted to pens based on initial body weight (BW) and fed a common diet. On d 21 post-weaning, pens were blocked by BW and randomly allotted to 1 of 8 (experiment 1) or 7 (experiment 2) dietary treatments with five pigs per pen and eight pens per treatment. Treatments were formulated to include increasing aP from either inorganic P (0.12%, 0.18%, or 0.24% in experiment 1 and 0.11%, 0.19%, or 0.27% in experiment 2 from monocalcium P) or increasing phytase (150, 250, 500, 750, or 1,000 FTU/kg in experiment 1 and 250, 500, 1,000, or 1,500 FTU/kg in experiment 2). Prior to beginning the 21-d studies, all pigs were fed the lowest inorganic P diet for a 3-d period. At the conclusion of each experiment, the pig closest to the pen mean BW was euthanized and fibulas were collected to determine bone ash weight and percentage bone ash. Fibulas were processed using defatted bone mineral procedures. In both experiments, pigs fed increasing aP from inorganic P had increased (linear, P &lt; 0.01) average daily gain (ADG), G:F, and final BW. Additionally, pigs fed diets with increasing phytase had increased (experiment 1 linear, P &lt; 0.01; experiment 2 linear and quadratic, P &lt; 0.05) performance across all growth response criteria. For bone composition, pigs fed increasing aP from inorganic P had increased bone ash weights (linear, P &lt; 0.01) and percentage bone ash (experiment 1 quadratic, P &lt; 0.01; experiment 2 linear, P &lt; 0.01). Similarly, pigs fed increasing phytase had increased bone ash weights (linear, P &lt; 0.01) and percentage bone ash (experiment 1 linear, P &lt; 0.01; experiment 2 linear and quadratic, P &lt; 0.05). The percentage aP released from Smizyme TS G5 2,500 for both experiments varied depending on the response criteria used. As the amount of phytase in the diet increased, the calculated aP release increased when ADG (experiment 1 linear, P &lt; 0.01; experiment 2 linear and quadratic, P &lt; 0.01), G:F (linear, P &lt; 0.01), or percentage bone ash (experiment 1 linear and quadratic, P &lt; 0.05; experiment 2 linear, P &lt; 0.01) were used the predictor variable. When combining the data from experiment 1 and 2, the aP release prediction equations for Smizyme TS G5 2,500 are aP = (0.197 × FTU)/(584.956 + FTU), aP = (0.175 × FTU)/(248.348 + FTU), and aP = (0.165 × FTU)/(178.146 + FTU) when using ADG, G:F, and percentage bone ash, respectively as the predictor variable.

Chitin-chitosan complex from Rhizopus oryzae obtained on a pea culture medium, and some of its physicochemical properties

The article provides information about the way of obtaining chitin-chitosan biosorbents from the fungus Rhizopus oryzae, using mild deacetylation and demineralization conditions. Such physicochemical properties as deacetylation degree, total ash, weight loss on drying etc. were determined. Method of obtaining the chitin-chitosan complex, shown in the current study, allows to obtain a pure biosorbent with a high degree of deacetylation possible to be further used as entero-and hemosorbent.