Validation of dual-energy X-ray absorptiometry in live White Leghorns.

Dual energy x-ray absorptiometry (DEXA) was evaluated for use as a noninvasive tool to monitor skeletal integrity in live laying hens. The objectives of the current study were 1) to validate the use of DEXA in evaluating bone integrity in live birds as compared with excised bones under a normal nutritional regimen as well as in hens fed varying levels of dietary Ca and 2) to correlate densitometric scans with other bone strength criteria and egg traits. Densitometric scans were conducted on the tibia and humerus of live hens at 10-wk intervals from 17 to 67 wk of age. After each scan, bones were excised from euthanized hens to measure breaking strength characteristics and bone ash (experiment 1). Similar measurements were collected at 38, 48, and 58 wk of age from hens fed hypercalcemic (5.4%), control (3.6%), and hypocalcemic (1.8%) diets from 32 to 58 wk of age (experiment 2). The bone mineral density (BMD) and bone mineral content (BMC) between live and excised bone scans were highly correlated (r = 0.85 and 0.92, respectively, P < 0.0001, experiment 1). Densitometric scans of live birds were positively correlated with bone breaking force and bone ash (r = 0.68 and 0.73, respectively, P < 0.001) with little to no correlation with shell traits. In experiment 2, the excised tibial scan had lower BMD and BMC than the live bird (P < 0.01), whereas no difference was detected in densitometric scans of the humerus. The live and excised BMD and BMC of the tibia (r = 0.87 and 0.82, respectively, P < 0.001) and humerus (r = 0.94 and 0.93, respectively, P < 0.001) were highly correlated. Due to the high correlations between live and excised bone scans and the significant correlations of live scans to more traditional invasive bone measurement tests such as bone breaking force and bone ash, we concluded that DEXA is a useful noninvasive tool for evaluating skeletal integrity in live birds.

Effects of ovulatory and egg laying cycle on bone mineral density and content of live White Leghorns as assessed by dual-energy X-ray absorptiometry.

Dual-energy X-ray absorptiometry has been validated in our laboratory as a noninvasive tool to assess skeletal integrity in live birds. The first objective of the current study was to determine if there were detectable changes in bone mineral density (BMD) and bone mineral content (BMC) while an egg was being formed in the oviduct. Implications from this experiment would define the time of day scans should be conducted for future experiments. Densitometric scans were conducted on the tibia and humerus of live hens undergoing active egg formation when hens were 0, 5, 15, and 20 h postoviposition at 24, 30, and 40 wk of age. No detectable changes in either the BMD or BMC of the tibia and humerus were observed as the egg was being formed in the reproductive tract at any age measured. These results suggest that densitometric scans may be conducted on bones in live birds at any time during the day, irrespective of the stage of egg formation. The second objective was to monitor the change and degree of variation in skeletal integrity of live birds during the first cycle of egg laying. The humerus and tibia of White Leghorns were scanned repeatedly at 10-wk intervals from 15 to 65 wk of age. The BMD of the humerus increased from 15 to 65 wk of age, whereas the BMD and BMC of the tibia increased from 15 to 55 wk of age, resulting in a bone-by-age interaction (P < 0.001). The BMC of the humerus did not change from 15 to 55 wk of age but increased at 65 wk of age. Age-related increases in BMD and BMC may be due to the inability of dual energy X-ray absorptiometry to distinguish medullary from structural bone. The CV for BMD and BMC of egg-type chickens was greater than 10% after 25 wk of age, which suggested that bone densitometry could be used as an indicator tool in genetic selection with a potential for improving skeletal integrity of birds.

Assessing bone mineral density in vivo: dual energy X-ray absorptiometry.

Dual-energy X-ray absorptiometry can be used as a noninvasive tool to monitor the skeletal integrity of live birds. A pDexa X-ray bone densitometer was used to determine bone mineral densities (BMD) of the left tibia together with the fibula and the humerus of live, unanesthetized birds. Densitometry effectively detected changes in bone integrity of live birds fed varying levels of dietary calcium. Hens consuming 1.8, 3.6, or 5.4% dietary calcium had BMD of 0.147, 0.157, and 0.176 g/cm2 (SEM = 0.005), respectively (linear effect, P < 0.001). Likewise, bone ash weight, breaking force, stress, modulus of elasticity, and eggshell traits also increased linearly in response to increased calcium in the diet (P < 0.05). Densitometric live scans for BMD were positively correlated (P < 0.001) with bone breaking force (r = 0.65) and bone ash (r = 0.77). We also monitored BMD in live Leghorn and broiler females during their life cycle. The tibial BMD of White Leghorns and broilers increased from 15 to 65 wk of age with the BMD of the broiler tibia increasing at a greater rate than that of the Leghorn tibia (line x age interaction, P < 0.0001). A precipitous drop in BMD occurred during an induced molt of Leghorns subjected to 10 d of feed withdrawal. Our long-term goal is to improve skeletal integrity in egg-type chickens by genetic selection for improved BMD. By crossing a broiler with an egg-laying line, an F2 resource population of birds has been developed to identify quantitative trait loci influencing BMD in chickens.

The use of densitometry to detect differences in bone mineral density and content of live White Leghorns fed varying levels of dietary calcium.

Densitometry was investigated as a noninvasive tool to monitor skeletal integrity in live White Leghorns as an indicator for osteoporosis, a noninfectious disease resulting in mineral loss from the bone. The objectives of the experiment were 1) to assess the ability of densitometry to detect differences in bone integrity in live White Leghorns fed varying concentrations of dietary calcium and 2) to correlate densitometric scans with other bone test methods and production parameters that are sensitive to calcium concentrations in the diet. Hens were fed hypercalcemic (5.4%), control (3.6%), or hypocalcemic (1.8%) diets from 32 to 58 wk of age. A Norland densitometer was used to assess bone mineral density (BMD) and bone mineral content (BMC) of the left tibia and humerus in restrained, unanesthetized hens at 36, 46, and 56 wk of age (experiment 1) and at 38, 48, and 58 wk of age (experiment 2). Bones were excised from hens at 38, 48, and 58 wk of age for breaking strength measurements. Results from the densitometric scans showed that BMD and BMC of the humerus and tibia of live hens decreased linearly when hens consumed diets with decreasing concentrations of calcium (experiment 2). Similar trends in BMD and BMC were detected in experiment 1 at 36 wk of age using BW as a covariate. The results from the densitometric scans were comparable to those obtained from other bone tests commonly used. For example, bone breaking force, stress, and modulus of elasticity decreased linearly as hens consumed decreasing concentrations of calcium. Bone breaking force was correlated with BMD (r=0.65, P<0.001). We concluded that densitometry accurately measures differences in BMD and BMC in live birds fed varying concentrations of dietary calcium.

The effect of dietary ascorbic acid on semen traits and testis histology of male turkey breeders.

A 9-mo field trial was conducted to evaluate the effects of dietary L-ascorbic acid (AA) on semen traits of 144 male turkey breeders. Dietary AA treatments were initiated when birds were 30 wk of age. Semen and blood collection began at 32 wk of age. Three treatments with four pens per treatment and 12 birds per pen were fed 0, 75, and 150 mg/kg AA during the first 4 mo of their reproductive cycle. Levels of AA were doubled in the supplemented diets to 150 and 300 mg/kg during Months 5 to 9. Semen traits and blood AA were unaffected by dietary AA. When birds were 65 wk of age, testes were removed from 12 birds per treatment for histological analysis. Multinucleated giant cells (MCG), indicative of degeneration, were observed in the testes of 7 of the 12 control birds but were absent from AA-supplemented birds (P < 0.02). The antioxidant properties of AA may delay formation of these degenerative cells. In conclusion, dietary AA levels employed in the current study did not affect semen traits or testis weight but were associated with reduced formation of MGC in the testes of 65 wk-old breeder toms.

Modification of the phosphoketolase assay for rapid identification of bifidobacteria.

The phosphoketolase assay is commonly used as a definitive criterion for identification of bifidobacteria. A limitation of the assay is the time-consuming process of cell disruption, either by use of the French Pressure Cell or by sonication. We have replaced the time consuming cell disruption process with a more rapid cell membrane disruption process by pretreating cells with the detergent hexadecyltrimethylammonium bromide (cetrimonium bromide, CTAB). The effect of no pretreatment, sonication or the addition of CTAB (0.45 mg/ml) on color development in the phosphoketolase assay was tested using pure cultures of bifidobacteria and lactobacilli. No phosphoketolase activity was observed with bifidobacterial cultures without cell disruption or with lactobicilli that had undergone cell disruption. All bifidobacterial cultures gave a similar color formation whether sonication or CTAB addition was used to disrupt cells. Use of CTAB to disrupt cell membranes is an effective alternative to the time consuming traditional cell disruption procedures and increases the number of cultures that can be simultaneously assayed and presumptively identified using the phosphoketolase assay.

Microbial phytase in finisher diets of White Pekin ducks: effects on growth performance, plasma phosphorus concentration, and leg bone characteristics.

Two experiments (Exp.) were conducted to determine the growth response of White Pekin ducks to inclusion of microbial phytase in finisher diet. In Exp. 1, 1-d-old male ducks (240 total) were reared in litter-floor pens and fed regular starter diet until 3 wk of age. At 3 wk of age, ducks were randomly divided into six groups of 10 ducks each and each group was fed one of four diets. Three finisher diets containing 16% CP and 0.18% available phosphorus (AP) without supplemental P were formulated with microbial phytase (Natuphos) added at 0, 750, or 1,500 phytase units/kg of diet. The fourth diet was a control finisher diet that was supplemented with dicalcium phosphate (DCP) to supply dietary AP of 0.41%. Group BW and feed intake were measured weekly to assess growth response. At 6 wk of age, leg bones (tibia, femur, metatarsus) from five randomly selected ducks were removed and analyzed for bone characteristics. In Exp. 2, a total of 120 ducks reared as in Exp. 1 were randomly divided into six groups of five ducks each and fed one of four diets. A basal finisher diet was formulated to contain 16% CP and 0.18% AP. Monosodium phosphate was added to the basal diet to give dietary AP levels of 0.18, 0.27, and 0.36%. The fourth diet was the basal diet supplemented with microbial phytase (750 phytase units/kg of diet). Ducks were fed these diets from 3 to 6 wk of age. At the end of the study, ducks were bled by cardiac puncture and blood plasma was analyzed for P concentration. Leg bones from all ducks were removed and analyzed for bone characteristics as in Exp. 1. Feed intake increased linearly with increased level of dietary phytase, whereas the weight gain response was quadratic only during the last week of Exp. 1. In Exp. 2, there was a quadratic response for weight gain due to dietary AP. Weight gain due to phytase (750 units) was not different from ducks fed diets at 0 or 0.18% AP. Plasma P concentration increased linearly as dietary AP increased. Plasma P levels of ducks fed phytase were similar to those of ducks fed 0.18% AP but lower than in ducks fed 0.27% AP. Estimates of AP resulting from the addition of 750 units of phytase to basal diet were 0.05 and 0.07% based on plasma P concentration and weight gain, respectively. Using regression analysis, the AP due to phytase effect in the diet was estimated to range from 0.06 to 0.08%. Results suggest that phytase can be used in finisher diets similar to the one used in this study for ducks from 3 to 6 wk of age to improve growth performance and leg bone development similar to ducks fed diets supplemented with P from inorganic sources.

Calcium utilization by quail embryos during activities preceding space flight and during embryogenesis in microgravity aboard the orbital space station MIR.

A series of studies were conducted to determine the effect of activities preceding spaceflight and during space-flight on calcium utilization during quail embryonic development. In the pre-space trials, fertile quail eggs were subjected to pre-flight dynamics including forces of centrifugation, vibration, or a combination of vibration and centrifugation prior to incubation for 6 or 16 days. Quail eggs were also tested for survivability in a refrigerator stowage kit for eggs (RSKE) which was subsequently used to transport the eggs to space. Eggs in the RSKE were subjected to shuttle launch dynamics including G force and random vibration profiles. The space-flight trial involved 48 quail eggs launched on space shuttle Flight STS-76 which were subsequently incubated in a Slovakian incubator onboard space station, MIR. Two ground control trials, each with 48 eggs with and without exposure to shuttle launch dynamics were initiated 5 days post-launch. Eggshells from all study trials were retrieved and analyzed for calcium content. Results showed that neither pre-flight activities nor shuttle launch dynamics had an effect on calcium utilization by developing embryos. However, calcium utilization by developing embryos incubated in microgravity was impaired by 12.6% when compared to embryos incubated on earth under laboratory control environment. This impairment was believed to be due to unidentified factors of the microgravity environment.

Effect of sucrose thermal oligosaccharide caramel, dietary vitamin-mineral level, and brooding temperature on growth and intestinal bacterial populations of broiler chickens.

Two experiments were conducted to determine the effect of sucrose thermal oligosaccharide caramel (STOC) and dietary vitamin-mineral (V/M) level on growth performance and intestinal microflora of broiler chickens. In Experiment 1, Peterson x Arbor Acres male broilers (n = 384) were randomly allocated into four groups that were fed either the control diet or diets containing the antibiotic virginiamycin (11 mg/kg), 3.7% STOC or 7.5% STOC for 4 wk at brooding temperatures of 32 to 29.7 C. Weight gains for broilers in Experiment 1 were greater (P < 0.001) for birds fed STOC diets, with weight gains of 763, 822, 1,124, and 1,080 g for birds on the control, antibiotic, 3.7% STOC, and 7.5% STOC diets, respectively. Feed intake and feed conversion by birds fed STOC diets were also significantly improved. Cecal bifidobacterial numbers were increased (P < 0.03) over the control diet with numbers being 5.98, 6.99, 7.47, and 7.39 log10 cfu/g cecal DM, respectively. In Experiment 2, Peterson x Hubbard male broilers (n = 384) were used in a 2 x 2 x 2 factorial arrangement with two levels of V/M premix (0.5 or 1% of the diet), two levels of STOC (0 or 3.5% of the diet), and two brooding temperatures, normal (32 to 23.6 C) or high (32 to 29.7 C) for 4 wk. Feeding the STOC diet improved (P < 0.05) weight gain, feed intake, and feed conversion of broilers. The effect of STOC on animal performance was less evident when broilers were fed twice the NRC recommended levels of V/M. Feeding the STOC diets resulted in a significantly greater increase in weight gain at high brooding temperatures than at normal brooding temperatures. There was also a reduction (P < 0.05) in numbers of total aerobes and coliforms in the ceca of birds fed diets containing STOC. Feeding STOC has potential to improve growth performance of broiler chickens.

Selective enrichment of bifidobacteria in the intestinal tract of broilers by thermally produced kestoses and effect on broiler performance.

A series of kestose oligosaccharides have been produced from pyrolysis of sucrose and the effects of feeding these thermal kestoses on broiler performance and cecal microbial populations were evaluated. Eighty-four broiler chicks (day-old Hubbard x Hubbard) were fed either a nutritionally complete basal starter diet (control), the starter diet dressed with 8% of other sugars found in the thermal kestoses mixture (glucose, sucrose, and fructose), or the starter diet dressed with 10% crude thermal kestoses (2% kestoses, 8% other sugars) for a 4-wk period. Body weight and feed intake were measured weekly. After 4 wk the birds were killed, cecal contents were collected, and selected microbial populations were enumerated. Weight gains were 938, 968, and 989 g for control, other sugars, and thermal kestoses groups, respectively. There were no dietary effects on weight gain, feed conversion, or concentrations of total aerobic bacteria, coliforms, aerobically enumerated lactobacilli, total anaerobes, or clostridia. Cecal bifidobacterial concentrations were increased (P < 0.001) 24-fold in kestose-treated birds compared with controls, with bifidobacterial concentrations being 8.98, 9.09, and 10.36 log10 cfu/g cecal DM in birds fed the control, other sugars, and thermal kestoses diets, respectively. Anaerobically enumerated lactobacilli concentrations in kestose-treated birds were increased (P < 0.007) sevenfold compared with controls, with lactobacilli concentrations being 9.56, 9.53, and 10.36 log10 cfu/g cecal contents, respectively. Thermally produced kestoses altered intestinal bacterial populations in broilers and may have potential to enhance health and performance under the appropriate conditions.

Growth performance and intestinal microbial populations of growing pigs fed diets containing sucrose thermal oligosaccharide caramel.

Four experiments were conducted to determine growth performance and changes in intestinal microbial populations of growing pigs fed diets containing sucrose thermal oligosaccharide caramel (STOC). Ninety-six barrows and 96 gilts were group-fed experimental nursery diets for 32 d after weaning in both Exp. 1 and 2. For each experiment, pigs were divided into four groups of 48 pigs and were fed either control, antibiotic (Apramycin sulfate, 34 mg/kg), 1% STOC, or 2% STOC diets for 32 d after weaning. Each diet was replicated six times with eight pigs per replication. Pigs were either orally gavaged (Exp 1) with water of STOC (2 g per pig) or pigs were creep-fed (Exp 2) either a control diet or a 2% STOC diet for 5 d before weaning (33 d). At the end of Exp 1 and 2, cecal material was collected for enumeration of total aerobes, total anaerobes, coliforms, lactobacilli, and bifidobacteria. Gilts (96 per experiment) used in Exp. 3 and 4 were weaned at 26 d and fed experimental nursery diets for 32 d. They were fed either a control or 1% STOC diet and were otherwise treated as previously described. There were no significant effects of STOC or antibiotic on ADG, ADFI, feed efficiency, or cecal microbial populations in pigs in this study. Feeding diets containing either antibiotic of STOC did not improve animal performance or change intestinal bacterial populations in the present study.

Age at sexual maturity influences the response of single comb White Leghorn pullets to marginal and low levels of dietary phosphorus.

This study was designed to determine the differences in response of early-maturing (EM) and late-maturing (LM) Single Comb White Leghorn pullets within a flock to marginal or low dietary phosphorus. Various levels of dietary phosphorus were fed to EM and LM Leghorn pullets from 18 wk of age until the age of peak egg production (24 wk). The dietary phosphorus levels were 0.40, 0.45, 0.50, 0.55 and 0.70 g total phosphorus (tP)/100 g diet, corresponding to calculated available phosphorus values of 0.18, 0.23, 0.28, 0.33 and 0.48 g/100 g, respectively. At 0.70 and 0.55 g tP/100 g, the plasma inorganic phosphorus, Ca++ and urine calcium concentrations did not differ between EM and LM pullets, whereas LM pullets had a better bone status than EM pullets as reflected by bone mineral content, bone density and bone breaking strength. As dietary phosphorus was lowered from 0.55 to 0.4 g tP/100 g, the plasma concentration of inorganic phosphorus dropped and that of Ca++ increased at greater rates in LM pullets than in EM pullets. The magnitude of decline in bone status was also greater in LM than in EM pullets when dietary phosphorus was lowered from 0.55 to 0.40 g tP/100 g. The maximum incidences of osteoporosis and mortality were observed in LM pullets fed 0.40 g tP/100 g followed by LM pullets fed 0.45 g tP/100 g diet. We conclude that when early layer diets contain marginal or low levels of phosphorus, the severity of adverse effects are greater in LM pullets than in EM pullets.

Influence of large doses of ascorbic acid on performance, plasma calcium, bone characteristics, and eggshell quality in broilers and Leghorn hens.

Four experiments were conducted using broilers (Experiments 1 and 2) or White Leghorn hens (Experiments 3 and 4) to determine the effects of large doses of dietary ascorbic acid on performance, plasma concentration of total and ionic calcium, bone characteristics, and eggshell quality. A total of 564 male broilers were fed diets containing ascorbic acid (ranging from 0 to 3,000 ppm) from 3 to 7 wk of age. Weight gain and feed conversion were measured, and blood plasma was analyzed for total and ionic calcium. Leg bones (femur, tibia, and metatarsus) were analyzed for bone mineral content, density, and breaking strength. In Experiments 3 and 4, a total of 484 Leghorn hens were fed diets containing ascorbic acid (ranging from 0 to 3,000 ppm) for 4 wk. Egg weight and specific gravity were determined, and plasma and tibiae were analyzed as in Experiments 1 and 2. Results of the broiler experiment (Experiment 2) indicated that plasma ionic calcium was significantly increased (P < .05) in ascorbic-acid-treated birds. Among leg bones examined, femur strength was improved by 16% in birds fed 2,000 ppm of ascorbic acid (Experiment 2). Other bone characteristics were not affected. In the layer experiments (Experiments 3 and 4), egg weight increased up to 5% and egg specific gravity was improved in hens fed 2,000 or 3,000 ppm of ascorbic acid, which also had increased calcium in the blood. Results suggest that large doses of ascorbic acid in the diet influence calcium metabolism, affecting bone and eggshell mineralization in chickens.

Sample size required for various methods of assessing bone status in commercial leghorn hens.

A study was conducted to determine the appropriate sample size required for various methods used to assess tibial bone status in commercial Leghorn hens. The methods used were in vivo bone mineral content (BMC), in vivo bone density (BD), in vitro BMC, in vitro BD, tibia bone breaking strength (TBS), and percentage bone ash (BA). Dietary total P levels of .4, .45, .5, .55, and .7% were used as treatment source of variation. Twenty hens were sampled randomly to represent each dietary treatment. The CV for each bone status comparison method was estimated and was used in a procedure to estimate the sample size requirement for detecting a difference of delta between treatments. The sample size required to detect the difference between treatment means varied depending on 1) the method used to compare bone status 2) the difference between the treatment means to be detected as significant (delta); and 3) the level of significance (alpha) assumed. The sample size required for various methods are tabulated at .01, .05, and .1 level of significance and for 2.5, 5,7.5, 10, 15, and 20% delta. To detect an actual difference of 5% from the mean to be significant, at the .05 level of significance, a sample size of 44, 22, 31, 23, 47, and 85 hens per treatment would be necessary for in vivo BMC, in vivo BD, in vitro BMC, in vitro BD, TBS, and BA methods, respectively. The estimated sample size values would help researchers in designing experiments that involve bone status comparison of commercial Leghorn hens.

Influence of dietary cholecalciferol, calcium, and phosphorus on urinary calcium in commercial Leghorn hens.

An experiment was conducted to determine the influence of various dietary levels of cholecalciferol, Ca, and P on urinary Ca and pH, and on plasma concentrations of inorganic P (Pi) and total Ca (TCa) in commercial Leghorn hens. All hens were fed a layer diet containing 500 ICU of cholecalciferol/kg for 30 days and then allocated equally to treatment diets. Twelve treatment diets were in a 3 x 2 x 2 factorial arrangement that comprised three levels of cholecalciferol (0, 2,200, and 4,400 ICU/kg), two Ca levels (3.75 and 5.75%) and two P levels (.3% and .7%). After 9 days of feeding, blood (12 hens per time per treatment) and urine (6 hens per time per treatment) were sampled at 8 and 16 h after oviposition. Urinary Ca and plasma TCa concentrations increased (P less than .05) when dietary cholecalciferol level was increased from 0 to 2,200 ICU/kg, but did not change when cholecalciferol was increased from 2,200 to 4,400 ICU/kg. High levels of dietary Ca elevated (P less than or equal to .05) the urinary Ca concentration of hens fed the low-P treatments. Low levels of dietary P reduced plasma P and increased urinary Ca and pH at all levels of cholecalciferol and Ca; however, the magnitude of increases in urinary Ca were not uniform across all levels of cholecalciferol and Ca. Increases in urinary Ca resulting from low dietary P were relatively small when the diet lacked cholecalciferol, but was increased three- to fourfold when the diet contained adequate or excess levels of Ca and excess but not toxic levels of cholecalciferol.

Response of four broiler strains to dietary phosphorus above and below the requirement when brooded at two temperatures.

Four strains of broilers (1,200 birds) were fed diets containing .14, .51, and .88% available phosphorus. The birds were raised for 3 wk at two brooding temperatures, 29.4 C and 35 C, with each being reduced 2.8 degrees per week. Feed consumption, feed conversion, body weight, bone weight (tibia), strength and percentage ash, and mortality were measured in order to assess broiler response. An increase or improvement in each measurement was observed from feeding diets containing .51% rather than .14% available phosphorus, but no further increase or improvement was observed in measurements by feeding diets containing .88% rather than .51% available phosphorus. The values for feed consumption, weight gain, and bone weight were significantly lower at the higher temperatures than at the lower temperatures. There were interactions of broiler strain-by-phosphorus with feed consumption, BW gain, and bone weight and strength; also, of temperature-by-phosphorus with bone strength, bone ash, and mortality. These interactions indicate that the bird's response to phosphorus is influenced by strain and environmental temperature.