Calcium particle size effects on plasma, excreta, and urinary Ca and P changes in broiler breeder hens

Calcium particle size effects on plasma, excreta, and urinary Ca and P changes in broiler breeder... Abstract An experiment was conducted using non-colostomized and colostomized broiler breeder hens to determine the effects of feeding limestone of 2 different mean particle sizes (185 microns and 3490 microns) on P excretion, total P and Ca retention, and urinary P and Ca excretion during a 6-week feeding study. Additionally, changes in plasma inorganic P (iP) and ionic Ca (Ca++) and urinary excretion of P and Ca were determined in one egg laying cycle of 24 hours. One-hundred-fifty non-colostomized and 6 colostomized broiler breeder hens, 30 wk of age, were divided into 2 groups and fed broiler breeder diets supplemented with either small particle or large particle limestone. Two % acid insoluble ash (Celite) was added to the feed as a marker. Diets, excreta, and urine samples were analyzed for total P and Ca by ionic coupling plasma (ICP) analysis. The non-colostomized breeders fed large particle limestone compared to small limestone particles produced a significant increase in percent tibia ash (P < 0.0001) and egg specific gravity (P = 0.0382), but P excretion approached a tendency of being reduced (P = 0.1585). The urinary total P and Ca (∼18 and 9%, respectively) of total P and Ca excretion for breeders fed both sizes of limestone was not significantly different in the colostomized breeders. In plasma, both iP and Ca++ reached a peak during 18 to 20 h and 20 to 24 h post oviposition for smaller and larger particle sized limestone fed groups, respectively. The maximal excretion of urinary P was found during 11 to 20 h post oviposition, whereas urinary Ca peaked during 0 to 11 h post oviposition for both smaller and larger particle sized limestone supplemented groups. In summary, the findings indicate that the particle size (smaller and larger) of calcium source did not significantly influence the quantitative total urinary excretion of Ca and P but did influence the timing of Ca and P excretion. INTRODUCTION An equivalent of about 10% of the total Ca present in the skeleton of a hen is in the eggshell. Each eggshell contains approximately 2.3 g of Ca, which comes from the diet through intestinal absorption and bone mobilization (Bar, 2009). The skeletal Ca utilization is directly related to the time and level of Ca intake. Dependency on skeletal Ca for eggshell formation has been shown to decrease the quantity of Ca deposited in the egg shell (Farmer et al., 1986). In order to increase the availability of Ca for shell calcification from the diet rather than from bone mobilization, hens should have a constant source of dietary Ca. This can be achieved either by feeding during the time of eggshell calcification or by retaining the Ca source for a longer period. Feeding larger particle sized limestone has been previously shown to increase the retention of limestone particles for a longer time in the gizzard and also increase the in vivo solubility of the limestone (Zhang and Coon, 1997). The Ca and P content of blood in layers may vary depending on the timing of active shell formation. The literature indicate a marked rise in blood P levels of laying hens during the period of rapid shell deposition (11.5 to 13 h post oviposition), while the Ca in blood remains steady during egg formation with a tendency to fall slowly towards the end of shell calcification (Feinberg et al., 1937; Peterson and Parrish, 1939; Paul and Snetsinger, 1969; Miller et al., 1977; Lambert et al., 2014). The cyclic changes in serum P over time have been mainly attributed to changes in shell calcification, bone resorption, remineralization, and renal clearance (De Vries et al., 2010). Wideman (1987) reported that dietary Ca as high as 3.25% with 0.6% P inclusion or low dietary P as low as 0.4% with Ca inclusion (1%) can suppress the urinary excretion of P in domestic fowls. Rao and Roland (1990) indicate that low dietary P has a greater influence on elevating urinary Ca in laying hens than excess dietary Ca based on urine samples. There is a lack of information on the relationship between dietary Ca and its particle size, and P on urinary Ca and P excretion in broiler breeder hens. Because of the limitation and difficulty in separating urine from the feces, colostomized broiler breeder hens were used to determine total urinary excretion of Ca and P for a 24-hour period or one egg laying cycle. The objectives of broiler breeder studies were to determine the effects of 2 different Ca particle sizes on: 1) Ca and P retention in normal broiler breeder hens, 2) urinary total Ca (tCa) and total P (tP) excretion in colostomized broiler breeder hens, 3) changes in plasma tCa, ionic Ca (Ca++), tP, and inorganic P (iP) in relation to oviposition in one egg laying cycle, 4) eggshell quality, and 5) percent tibia ash. MATERIALS AND METHODS Experimental Birds, Test Diets, and Husbandry Three hundred broiler breeder pullets (Cobb 700), 23 wk of age, were offered a breeder diet with standard nutrient specifications (NRC, 1994) in individual cages until 30 wk of age. Six broiler breeder hens (25 wk of age) of uniform body weights were used for colostomy, utilizing the technique of Manangi et al. (2007). During 31st wk, 150 hens were divided into 2 groups of 75 each, and offered the test diets (Table 1) until 36 wk of age (6-week experimental period). The colostomized birds also were divided into 2 groups (3 birds per treatment) and offered the same test diets as normal birds. The Ca added to the basal diets consisted of 2 different particle sizes of limestone. The smaller particle sized limestone (Unical S, ILC Resources, Des Moines, IA) had an average particle size of 185 microns (solubility of 58.8%), whereas the larger particle sized limestone (Shell and Bone Builder, ILC Resources, Des Moines, IA) had an average particle size of 3,489 microns (solubility of 38.5%). Solubility was determined using the method of Zhang and Coon (1997). Table 1. Composition of breeder diet. Ingredients % Corn, 9.2% CP 72.21 Soybean meal, 49.5% CP 17.20 Fat-poultry 0.73 Dical phosphate 1.59 Limestone1 7.12 Methionine, 99% 0.16 Lysine 0.07 Chol chloride-60 0.20 Se premix, 0.06% 0.02 Copper sulfate 0.05 Ethoxyquin, 66% 0.02 Mold curb, 50% propionic acid 0.05 Salt 0.38 Threonine, 98% 0.04 Trace minerals2 0.06 Breeder premix3 0.10 Calculated nutrients (air dried basis) Protein 16.00 MEn, kcal/kg 2915.00 TSAA 0.75 Lysine 0.84 Total P 0.60 Ca 3.10 Analyzed nutrients (air dried basis) Total P 0.58 Phytate P 0.19 NPP 0.39 Ca 3.11 Protein 15.20 Dry matter 89.50 Ingredients % Corn, 9.2% CP 72.21 Soybean meal, 49.5% CP 17.20 Fat-poultry 0.73 Dical phosphate 1.59 Limestone1 7.12 Methionine, 99% 0.16 Lysine 0.07 Chol chloride-60 0.20 Se premix, 0.06% 0.02 Copper sulfate 0.05 Ethoxyquin, 66% 0.02 Mold curb, 50% propionic acid 0.05 Salt 0.38 Threonine, 98% 0.04 Trace minerals2 0.06 Breeder premix3 0.10 Calculated nutrients (air dried basis) Protein 16.00 MEn, kcal/kg 2915.00 TSAA 0.75 Lysine 0.84 Total P 0.60 Ca 3.10 Analyzed nutrients (air dried basis) Total P 0.58 Phytate P 0.19 NPP 0.39 Ca 3.11 Protein 15.20 Dry matter 89.50 1Limestone was replaced with 2 different particle sizes for 2 experimental diets. 2Provided per kilogram of diet: Manganese, 180 mg; zinc, 150.6 mg; iron, 20.16 mg; copper, 2.04 mg; iodine, 1.26 mg; Se, 0.3 mg. 3Provided per kilogram of diet: Vitamin A, 13200IU; vitamin E, 66IU; vitamin D3, 4950ICU; niacin, 74.25 mg; D-pantothenic acid, 33 mg; riboflavin, 19.8 mg; pyridoxine, 5000 mg; thiamine, 3.3 mg; menadione, 3.3 mg; folic acid, 3.3 mg; biotin, 0.33 mg; vitamin B12, 0.0297 mg. View Large Table 1. Composition of breeder diet. Ingredients % Corn, 9.2% CP 72.21 Soybean meal, 49.5% CP 17.20 Fat-poultry 0.73 Dical phosphate 1.59 Limestone1 7.12 Methionine, 99% 0.16 Lysine 0.07 Chol chloride-60 0.20 Se premix, 0.06% 0.02 Copper sulfate 0.05 Ethoxyquin, 66% 0.02 Mold curb, 50% propionic acid 0.05 Salt 0.38 Threonine, 98% 0.04 Trace minerals2 0.06 Breeder premix3 0.10 Calculated nutrients (air dried basis) Protein 16.00 MEn, kcal/kg 2915.00 TSAA 0.75 Lysine 0.84 Total P 0.60 Ca 3.10 Analyzed nutrients (air dried basis) Total P 0.58 Phytate P 0.19 NPP 0.39 Ca 3.11 Protein 15.20 Dry matter 89.50 Ingredients % Corn, 9.2% CP 72.21 Soybean meal, 49.5% CP 17.20 Fat-poultry 0.73 Dical phosphate 1.59 Limestone1 7.12 Methionine, 99% 0.16 Lysine 0.07 Chol chloride-60 0.20 Se premix, 0.06% 0.02 Copper sulfate 0.05 Ethoxyquin, 66% 0.02 Mold curb, 50% propionic acid 0.05 Salt 0.38 Threonine, 98% 0.04 Trace minerals2 0.06 Breeder premix3 0.10 Calculated nutrients (air dried basis) Protein 16.00 MEn, kcal/kg 2915.00 TSAA 0.75 Lysine 0.84 Total P 0.60 Ca 3.10 Analyzed nutrients (air dried basis) Total P 0.58 Phytate P 0.19 NPP 0.39 Ca 3.11 Protein 15.20 Dry matter 89.50 1Limestone was replaced with 2 different particle sizes for 2 experimental diets. 2Provided per kilogram of diet: Manganese, 180 mg; zinc, 150.6 mg; iron, 20.16 mg; copper, 2.04 mg; iodine, 1.26 mg; Se, 0.3 mg. 3Provided per kilogram of diet: Vitamin A, 13200IU; vitamin E, 66IU; vitamin D3, 4950ICU; niacin, 74.25 mg; D-pantothenic acid, 33 mg; riboflavin, 19.8 mg; pyridoxine, 5000 mg; thiamine, 3.3 mg; menadione, 3.3 mg; folic acid, 3.3 mg; biotin, 0.33 mg; vitamin B12, 0.0297 mg. View Large During the 6-week experimental period, the birds were housed in individual cages of 47 cm height x 30.5 cm width x 47 cm length, and the environmental temperature was maintained between 22.2 and 24.4°C. The breeder hens were provided a 16 h light: 8 h dark program during the experimental period. Nutrient Retention Study Ten birds from non-colostomized and 6 birds from the colostomized breeder hens were fed the experimental diets with added acid insoluble ash (AIA) product (CeliteTM5). Two-percent of the AIA product was added to the feed and used as a marker. The AIA mixed feed was fed to the hens that were acclimated to the diets for 4 d prior to a one-day excreta collection period. The excreta from individual birds were collected on trays on d 15 and d 42 and then frozen (–20 °C) and freeze dried for further analysis. Blood, Urine, Bone, and Egg Sample Collection Blood, urine, bone, and egg samples were collected. On d 14 and d 42 of the experiment, blood (plasma) and urine samples were collected. Venous blood (wing vein) was collected from 6 birds using heparinized syringes at 5 different time intervals with respect to oviposition (30 birds total). The 5 different time intervals were 0 to 6, 6 to 11, 11 to 18, 18 to 20, and 20 to 24 h after oviposition. Urine was collected over a 24-hour period from colostomized birds during the same time intervals as blood sample collection. Eggs were collected for 3 d before d 42. Egg weights were recorded after each collection and were kept at room temperature for 3 to 4 h before determination of specific gravity. Five birds on d 14 and 25 birds on d 42 from each treatment group were euthanized by CO2 inhalation for tibiae collection. Tibiae (right and left) were taken from each bird and cleaned of all exterior tissue and frozen until analysis. Samples Analyses Diets, excreta, and plasma samples were analyzed for tP and tCa by inductively coupled plasma (ICP) emission spectroscopic method as mentioned by Leske and Coon (2002). AIA was determined in experimental diets and excreta samples, using dry ash and hydrochloric acid digestion technique of Scott and Balnave (1991). Nitrogen and moisture in both the feed and excreta were determined by standard AOAC procedures (AOAC, 1990). Diet and excreta phytate P (PP) were measured as inositol hexa-phosphate by using ion-exchange chromatography as described by Bos et al. (1991). The gross energy of test feed and gross energy of excreta were measured with a Parr Bomb Calorimeter. PP disappearance and retention values of tP, non-phytate P (NPP), K, Ca, N, and energy were determined using the method of Scott and Balnave (1991). Plasma Ca++ concentrations were determined by using an electrolyte analyzer (Nova-8, NOVA Biomedical, Waltham, MA), and plasma iP was measured using iP reagent kit (Bayer Healthcare LLC, Tarrytown, NY). For tibia ash, tibiae were cut lengthwise and defatted in refluxing petroleum ether in Soxhlet apparatus for 48 hours. The defatted tibiae were oven dried and ashed in ceramic crucibles for 16 h 600°C. Ash content was expressed as percent tibia ash on defatted dried basis. The specific gravity of each egg was determined by floatation method, using gradational salt solutions with specific gravity ranging from 1.06 to 1.10 at increments of 0.05 (Moreng and Avens, 1985). Shell surface area was calculated by using the formula developed by Carter (1975) using fresh egg weights. Shell weight per unit surface area (SWUSA) was determined using shell surface area and dried shell weights (Hamilton, 1978). The collected urine samples were measured for pH and then centrifuged at 2,500 rpm for 15 minutes. The precipitate was freeze-dried after taking out the supernatant from each sample. Both the supernatant and the precipitate were analyzed for Ca and P quantification using ICP. Statistical Analysis Data from this experiment were collected for smaller particle sized limestone and larger particle sized limestone as 2 independent groups for different parameters measured in normal (non-colostomized) and colostomized birds, and mean values were obtained for both groups. Mean values from each of the groups for normal and colostomized birds were separated using “2 group t test” to determine the statistical significance or a tendency at P < 0.05 and P < 0.09, respectively (SAS Institute, 2012). RESULTS Excreta P, PP, NPP, and tP Retention, PP Disappearance, K Retention, GE Retention, and N Retention The mean comparisons of the effect of Ca particle size on excreta P, PP, NPP, and tP retention, PP disappearance, K retention, GE retention, N retention, and Ca retention in broiler breeder hens (non-colostomized) indicate no significant differences (P > 0.05) in feeding smaller or larger particle sized limestone for both 2- and 6-week periods (Tables 2 and 3). Similar results also were obtained from feeding colostomized birds except for excreta P (P < 0.0055). Only a numerical improvement of 2.09% P retention and 3.69% Ca retention was obtained for breeders fed larger sized limestone particles compared to breeders fed smaller particle sized limestone (Table 3). There was a significant (P < 0.0001) improvement of 3.22% in tibia ash content in the group fed larger particle sized limestone compared to the group of breeders fed smaller particle sized limestone for the 6-week collection period (Table 3). Table 2. Ca effects on broiler breeder hens fed small or large limestone particles after 2-week feeding period. Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 15.53 ± 1.29 18.53 ± 1.29 0.1249 15.92 ± 0.54 18.87 ± 0.64 0.0055 Excreta PP, mg/g DM 1.24 ± 0.82 2.96 ± 0.92 0.2047 1.25 ± 0.64 2.0 ± 0.91 0.5201 Excreta NPP, mg/g DM 14.17 ± 1.43 15.75 ± 1.60 0.4855 14.50 ± 0.91 15.52 ± 1.29 0.5418 Total P retention, % 36.59 ± 3.08 36.58 ± 3.08 0.9991 55.54 ± 1.91 51.55 ± 2.26 0.2076 PP disappearance, % 79.03 ± 10.45 67.48 ± 11.68 0.4853 88.78 ± 5.06 84.50 ± 7.15 0.64 Potassium retention, % 26.90 ± 5.77 34.28 ± 4.71 0.3504 62.37 ± 2.71 62.59 ± 3.21 0.9631 GE retention, % 81.00 ± 1.49 82.06 ± 1.38 0.6153 86.87 ± 0.42 88.16 ± 0.50 0.0752 N retention, % 44.10 ± 6.22 45.07 ± 5.76 0.9108 87.64 ± 0.72 87.02 ± 0.85 0.5923 Ca retention, % 40.55 ± 10.21 62.32 ± 8.34 0.1372 54.07 ± 4.69 63.22 ± 5.55 0.2365 Bone (tibiae) ash, % 52.52 ± 1.85 53.74 ± 1.85 0.6535 Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 15.53 ± 1.29 18.53 ± 1.29 0.1249 15.92 ± 0.54 18.87 ± 0.64 0.0055 Excreta PP, mg/g DM 1.24 ± 0.82 2.96 ± 0.92 0.2047 1.25 ± 0.64 2.0 ± 0.91 0.5201 Excreta NPP, mg/g DM 14.17 ± 1.43 15.75 ± 1.60 0.4855 14.50 ± 0.91 15.52 ± 1.29 0.5418 Total P retention, % 36.59 ± 3.08 36.58 ± 3.08 0.9991 55.54 ± 1.91 51.55 ± 2.26 0.2076 PP disappearance, % 79.03 ± 10.45 67.48 ± 11.68 0.4853 88.78 ± 5.06 84.50 ± 7.15 0.64 Potassium retention, % 26.90 ± 5.77 34.28 ± 4.71 0.3504 62.37 ± 2.71 62.59 ± 3.21 0.9631 GE retention, % 81.00 ± 1.49 82.06 ± 1.38 0.6153 86.87 ± 0.42 88.16 ± 0.50 0.0752 N retention, % 44.10 ± 6.22 45.07 ± 5.76 0.9108 87.64 ± 0.72 87.02 ± 0.85 0.5923 Ca retention, % 40.55 ± 10.21 62.32 ± 8.34 0.1372 54.07 ± 4.69 63.22 ± 5.55 0.2365 Bone (tibiae) ash, % 52.52 ± 1.85 53.74 ± 1.85 0.6535 S-smaller particle size calcium source; SSB-larger particle size calcium source; phytate P (PP). View Large Table 2. Ca effects on broiler breeder hens fed small or large limestone particles after 2-week feeding period. Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 15.53 ± 1.29 18.53 ± 1.29 0.1249 15.92 ± 0.54 18.87 ± 0.64 0.0055 Excreta PP, mg/g DM 1.24 ± 0.82 2.96 ± 0.92 0.2047 1.25 ± 0.64 2.0 ± 0.91 0.5201 Excreta NPP, mg/g DM 14.17 ± 1.43 15.75 ± 1.60 0.4855 14.50 ± 0.91 15.52 ± 1.29 0.5418 Total P retention, % 36.59 ± 3.08 36.58 ± 3.08 0.9991 55.54 ± 1.91 51.55 ± 2.26 0.2076 PP disappearance, % 79.03 ± 10.45 67.48 ± 11.68 0.4853 88.78 ± 5.06 84.50 ± 7.15 0.64 Potassium retention, % 26.90 ± 5.77 34.28 ± 4.71 0.3504 62.37 ± 2.71 62.59 ± 3.21 0.9631 GE retention, % 81.00 ± 1.49 82.06 ± 1.38 0.6153 86.87 ± 0.42 88.16 ± 0.50 0.0752 N retention, % 44.10 ± 6.22 45.07 ± 5.76 0.9108 87.64 ± 0.72 87.02 ± 0.85 0.5923 Ca retention, % 40.55 ± 10.21 62.32 ± 8.34 0.1372 54.07 ± 4.69 63.22 ± 5.55 0.2365 Bone (tibiae) ash, % 52.52 ± 1.85 53.74 ± 1.85 0.6535 Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 15.53 ± 1.29 18.53 ± 1.29 0.1249 15.92 ± 0.54 18.87 ± 0.64 0.0055 Excreta PP, mg/g DM 1.24 ± 0.82 2.96 ± 0.92 0.2047 1.25 ± 0.64 2.0 ± 0.91 0.5201 Excreta NPP, mg/g DM 14.17 ± 1.43 15.75 ± 1.60 0.4855 14.50 ± 0.91 15.52 ± 1.29 0.5418 Total P retention, % 36.59 ± 3.08 36.58 ± 3.08 0.9991 55.54 ± 1.91 51.55 ± 2.26 0.2076 PP disappearance, % 79.03 ± 10.45 67.48 ± 11.68 0.4853 88.78 ± 5.06 84.50 ± 7.15 0.64 Potassium retention, % 26.90 ± 5.77 34.28 ± 4.71 0.3504 62.37 ± 2.71 62.59 ± 3.21 0.9631 GE retention, % 81.00 ± 1.49 82.06 ± 1.38 0.6153 86.87 ± 0.42 88.16 ± 0.50 0.0752 N retention, % 44.10 ± 6.22 45.07 ± 5.76 0.9108 87.64 ± 0.72 87.02 ± 0.85 0.5923 Ca retention, % 40.55 ± 10.21 62.32 ± 8.34 0.1372 54.07 ± 4.69 63.22 ± 5.55 0.2365 Bone (tibiae) ash, % 52.52 ± 1.85 53.74 ± 1.85 0.6535 S-smaller particle size calcium source; SSB-larger particle size calcium source; phytate P (PP). View Large Table 3. Ca effects on broiler breeder hens fed small or large limestone particles after 6-week feeding period. Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 18.55 ± 0.87 16.72 ± 0.93 0.1585 12.25 ± 0.46 13.58 ± 0.54 0.0770 Excreta PP, mg/g DM 3.23 ± 0.35 3.04 ± 0.47 0.7533 3.91 ± 0.47 4.13 ± 0.54 0.7612 Excreta NPP, mg/g DM 15.32 ± 0.90 14.69 ± 1.20 0.6766 8.33 ± 0.61 9.45 ± 0.71 0.2503 Total P retention, % 31.00 ± 3.30 33.09 ± 3.72 0.6777 59.09 ± 2.09 54.84 ± 2.56 0.2208 PP disappearance, % 60.78 ± 4.30 61.32 ± 5.95 0.9422 55.99 ± 4.31 50.73 ± 5.28 0.4537 Potassium retention, % 18.90 ± 2.27 18.67 ± 2.72 0.9483 52.01 ± 2.42 50.61 ± 2.96 0.7190 GE retention, % 80.80 ± 0.73 80.45 ± 0.83 0.752 83.74 ± 0.58 82.58 ± 0.71 0.2261 N retention, % 42.85 ± 1.88 42.10 ± 2.28 0.7996 83.33 ± 0.86 84.27 ± 1.05 0.5030 Ca retention, % 49.86 ± 3.26 53.55 ± 4.15 0.4883 55.66 ± 3.57 61.98 ± 4.37 0.2825 Bone (tibiae) ash, % 54.75 ± 0.50 57.97 ± 0.57 <0.0001 Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 18.55 ± 0.87 16.72 ± 0.93 0.1585 12.25 ± 0.46 13.58 ± 0.54 0.0770 Excreta PP, mg/g DM 3.23 ± 0.35 3.04 ± 0.47 0.7533 3.91 ± 0.47 4.13 ± 0.54 0.7612 Excreta NPP, mg/g DM 15.32 ± 0.90 14.69 ± 1.20 0.6766 8.33 ± 0.61 9.45 ± 0.71 0.2503 Total P retention, % 31.00 ± 3.30 33.09 ± 3.72 0.6777 59.09 ± 2.09 54.84 ± 2.56 0.2208 PP disappearance, % 60.78 ± 4.30 61.32 ± 5.95 0.9422 55.99 ± 4.31 50.73 ± 5.28 0.4537 Potassium retention, % 18.90 ± 2.27 18.67 ± 2.72 0.9483 52.01 ± 2.42 50.61 ± 2.96 0.7190 GE retention, % 80.80 ± 0.73 80.45 ± 0.83 0.752 83.74 ± 0.58 82.58 ± 0.71 0.2261 N retention, % 42.85 ± 1.88 42.10 ± 2.28 0.7996 83.33 ± 0.86 84.27 ± 1.05 0.5030 Ca retention, % 49.86 ± 3.26 53.55 ± 4.15 0.4883 55.66 ± 3.57 61.98 ± 4.37 0.2825 Bone (tibiae) ash, % 54.75 ± 0.50 57.97 ± 0.57 <0.0001 S-smaller particle size calcium source; SSB-larger particle size calcium source; phytate P (PP). View Large Table 3. Ca effects on broiler breeder hens fed small or large limestone particles after 6-week feeding period. Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 18.55 ± 0.87 16.72 ± 0.93 0.1585 12.25 ± 0.46 13.58 ± 0.54 0.0770 Excreta PP, mg/g DM 3.23 ± 0.35 3.04 ± 0.47 0.7533 3.91 ± 0.47 4.13 ± 0.54 0.7612 Excreta NPP, mg/g DM 15.32 ± 0.90 14.69 ± 1.20 0.6766 8.33 ± 0.61 9.45 ± 0.71 0.2503 Total P retention, % 31.00 ± 3.30 33.09 ± 3.72 0.6777 59.09 ± 2.09 54.84 ± 2.56 0.2208 PP disappearance, % 60.78 ± 4.30 61.32 ± 5.95 0.9422 55.99 ± 4.31 50.73 ± 5.28 0.4537 Potassium retention, % 18.90 ± 2.27 18.67 ± 2.72 0.9483 52.01 ± 2.42 50.61 ± 2.96 0.7190 GE retention, % 80.80 ± 0.73 80.45 ± 0.83 0.752 83.74 ± 0.58 82.58 ± 0.71 0.2261 N retention, % 42.85 ± 1.88 42.10 ± 2.28 0.7996 83.33 ± 0.86 84.27 ± 1.05 0.5030 Ca retention, % 49.86 ± 3.26 53.55 ± 4.15 0.4883 55.66 ± 3.57 61.98 ± 4.37 0.2825 Bone (tibiae) ash, % 54.75 ± 0.50 57.97 ± 0.57 <0.0001 Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 18.55 ± 0.87 16.72 ± 0.93 0.1585 12.25 ± 0.46 13.58 ± 0.54 0.0770 Excreta PP, mg/g DM 3.23 ± 0.35 3.04 ± 0.47 0.7533 3.91 ± 0.47 4.13 ± 0.54 0.7612 Excreta NPP, mg/g DM 15.32 ± 0.90 14.69 ± 1.20 0.6766 8.33 ± 0.61 9.45 ± 0.71 0.2503 Total P retention, % 31.00 ± 3.30 33.09 ± 3.72 0.6777 59.09 ± 2.09 54.84 ± 2.56 0.2208 PP disappearance, % 60.78 ± 4.30 61.32 ± 5.95 0.9422 55.99 ± 4.31 50.73 ± 5.28 0.4537 Potassium retention, % 18.90 ± 2.27 18.67 ± 2.72 0.9483 52.01 ± 2.42 50.61 ± 2.96 0.7190 GE retention, % 80.80 ± 0.73 80.45 ± 0.83 0.752 83.74 ± 0.58 82.58 ± 0.71 0.2261 N retention, % 42.85 ± 1.88 42.10 ± 2.28 0.7996 83.33 ± 0.86 84.27 ± 1.05 0.5030 Ca retention, % 49.86 ± 3.26 53.55 ± 4.15 0.4883 55.66 ± 3.57 61.98 ± 4.37 0.2825 Bone (tibiae) ash, % 54.75 ± 0.50 57.97 ± 0.57 <0.0001 S-smaller particle size calcium source; SSB-larger particle size calcium source; phytate P (PP). View Large Urinary P, Ca, and K Feeding 2 different particle sizes of limestone did not have significant influence (P > 0.05) on total quantity of urine excreted or urinary tP, tCa, or K after the 2 and 6 wk of feeding study (Table 4). Table 4. Ca effects on urine and urinary mineral excretion in colostomized broiler breeder hens* fed small or large particle limestone. After 2 wk After 6 wk S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Total quantity of urine, mL 394.0 ± 57.4 399.0 ± 49.7 0.9508 136.0 ± 35.6 171.0 ± 29.8 0.4719 Total P excreted, mg 73.6 ± 20.4 101.0 ± 17.6 0.3551 80.4 ± 17.5 90.2 ± 14.7 0.6744 Total Ca excreted, mg 245.0 ± 20.1 203.3 ± 17.4 0.1778 186.0 ± 30.9 167.7 ± 25.8 0.6557 Total K excreted, mg 223.5 ± 38.2 205.6 ± 33.1 0.7372 246.7 ± 31.7 235.8 ± 26.5 0.7965 After 2 wk After 6 wk S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Total quantity of urine, mL 394.0 ± 57.4 399.0 ± 49.7 0.9508 136.0 ± 35.6 171.0 ± 29.8 0.4719 Total P excreted, mg 73.6 ± 20.4 101.0 ± 17.6 0.3551 80.4 ± 17.5 90.2 ± 14.7 0.6744 Total Ca excreted, mg 245.0 ± 20.1 203.3 ± 17.4 0.1778 186.0 ± 30.9 167.7 ± 25.8 0.6557 Total K excreted, mg 223.5 ± 38.2 205.6 ± 33.1 0.7372 246.7 ± 31.7 235.8 ± 26.5 0.7965 *All the values in Table 4 are average values for ∼24-hour period or one egg laying cycle. S-smaller particle size calcium source; SSB-larger particle size calcium source. View Large Table 4. Ca effects on urine and urinary mineral excretion in colostomized broiler breeder hens* fed small or large particle limestone. After 2 wk After 6 wk S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Total quantity of urine, mL 394.0 ± 57.4 399.0 ± 49.7 0.9508 136.0 ± 35.6 171.0 ± 29.8 0.4719 Total P excreted, mg 73.6 ± 20.4 101.0 ± 17.6 0.3551 80.4 ± 17.5 90.2 ± 14.7 0.6744 Total Ca excreted, mg 245.0 ± 20.1 203.3 ± 17.4 0.1778 186.0 ± 30.9 167.7 ± 25.8 0.6557 Total K excreted, mg 223.5 ± 38.2 205.6 ± 33.1 0.7372 246.7 ± 31.7 235.8 ± 26.5 0.7965 After 2 wk After 6 wk S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Total quantity of urine, mL 394.0 ± 57.4 399.0 ± 49.7 0.9508 136.0 ± 35.6 171.0 ± 29.8 0.4719 Total P excreted, mg 73.6 ± 20.4 101.0 ± 17.6 0.3551 80.4 ± 17.5 90.2 ± 14.7 0.6744 Total Ca excreted, mg 245.0 ± 20.1 203.3 ± 17.4 0.1778 186.0 ± 30.9 167.7 ± 25.8 0.6557 Total K excreted, mg 223.5 ± 38.2 205.6 ± 33.1 0.7372 246.7 ± 31.7 235.8 ± 26.5 0.7965 *All the values in Table 4 are average values for ∼24-hour period or one egg laying cycle. S-smaller particle size calcium source; SSB-larger particle size calcium source. View Large Eggshell Traits Average hen day egg production for a period of 6 wk showed no difference (P > 0.05) between the 2 groups of broiler breeder hens fed 2 different particle sizes of limestone (Table 5). There were about 8 to 9 shell-less and double yolk eggs recorded during the 6-week experimental period in both groups. Three-day average egg weight, percent shell weight, and SWUSA did not change (P > 0.05) between the 2 groups. Feeding a larger particle size of limestone significantly (P < 0.05) increased the specific gravity of eggs as compared to feeding breeders smaller particle sized limestone (1.087 vs. 1.085). There was no change (P > 0.05) in the egg weight, percent shell weight, SWUSA, or specific gravity of eggs from the 2 groups of colostomized breeder hens fed larger and smaller particle sized limestone (Table 5). Table 5. Ca effects on egg production and shell quality traits for broiler breeder hens* fed small or large particle limestone. Non-colostomized birds Colostomized birds S SSB P > | t | S SSB P > | t | Average hen day egg number 36.5 ± 0.80 36.9 ± 0.80 0.7454 – – Egg weight, g 60.6 ± 0.40 59.5 ± 0.40 0.0612 60.8 ± 0.6 58.8 ± 0.6 0.1486 % Dry shell weight 9.10 ± 0.08 9.22 ± 0.08 0.3010 9.6 ± 0.2 9.8 ± 0.2 0.4226 SWUSA, mg/cm2 76.72 ± 0.72 77.31 ± 0.75 0.5652 80.1 ± 5.42 81.4 ± 5.42 0.8808 Specific Gravity 1.085 ± 0.0006 1.087 ± 0.0006 0.0382 1.086 ± 0.0032 1.087 ± 0.0029 0.8801 Non-colostomized birds Colostomized birds S SSB P > | t | S SSB P > | t | Average hen day egg number 36.5 ± 0.80 36.9 ± 0.80 0.7454 – – Egg weight, g 60.6 ± 0.40 59.5 ± 0.40 0.0612 60.8 ± 0.6 58.8 ± 0.6 0.1486 % Dry shell weight 9.10 ± 0.08 9.22 ± 0.08 0.3010 9.6 ± 0.2 9.8 ± 0.2 0.4226 SWUSA, mg/cm2 76.72 ± 0.72 77.31 ± 0.75 0.5652 80.1 ± 5.42 81.4 ± 5.42 0.8808 Specific Gravity 1.085 ± 0.0006 1.087 ± 0.0006 0.0382 1.086 ± 0.0032 1.087 ± 0.0029 0.8801 *n = 135 per treatment for non-colostomized birds; n = 6 per treatment for colostomized birds. S-smaller particle size calcium source; SSB-larger particle size calcium source. SWUSA-Shell weight per unit surface area. View Large Table 5. Ca effects on egg production and shell quality traits for broiler breeder hens* fed small or large particle limestone. Non-colostomized birds Colostomized birds S SSB P > | t | S SSB P > | t | Average hen day egg number 36.5 ± 0.80 36.9 ± 0.80 0.7454 – – Egg weight, g 60.6 ± 0.40 59.5 ± 0.40 0.0612 60.8 ± 0.6 58.8 ± 0.6 0.1486 % Dry shell weight 9.10 ± 0.08 9.22 ± 0.08 0.3010 9.6 ± 0.2 9.8 ± 0.2 0.4226 SWUSA, mg/cm2 76.72 ± 0.72 77.31 ± 0.75 0.5652 80.1 ± 5.42 81.4 ± 5.42 0.8808 Specific Gravity 1.085 ± 0.0006 1.087 ± 0.0006 0.0382 1.086 ± 0.0032 1.087 ± 0.0029 0.8801 Non-colostomized birds Colostomized birds S SSB P > | t | S SSB P > | t | Average hen day egg number 36.5 ± 0.80 36.9 ± 0.80 0.7454 – – Egg weight, g 60.6 ± 0.40 59.5 ± 0.40 0.0612 60.8 ± 0.6 58.8 ± 0.6 0.1486 % Dry shell weight 9.10 ± 0.08 9.22 ± 0.08 0.3010 9.6 ± 0.2 9.8 ± 0.2 0.4226 SWUSA, mg/cm2 76.72 ± 0.72 77.31 ± 0.75 0.5652 80.1 ± 5.42 81.4 ± 5.42 0.8808 Specific Gravity 1.085 ± 0.0006 1.087 ± 0.0006 0.0382 1.086 ± 0.0032 1.087 ± 0.0029 0.8801 *n = 135 per treatment for non-colostomized birds; n = 6 per treatment for colostomized birds. S-smaller particle size calcium source; SSB-larger particle size calcium source. SWUSA-Shell weight per unit surface area. View Large Plasma iP, tP, Ca++, and tCa Blood plasma at both 2 and 6 wk, at 5 different periods related to oviposition, revealed no significant (P > 0.05) change in the mean iP, tP, or tCa (Figures 1 and 2) when each period was compared between groups of breeders fed smaller and larger particle sized limestone. At 2 wk (Figure 1a), the 24 h plasma iP ranged from ∼2 to 6 mg/dL for both smaller and larger particle sized limestone fed groups, with a tendency of higher values before (at 20 to 24 h) and after (at 0 to 6 h) oviposition. At 6 wk (Figure 1b), the 24 h plasma iP range was ∼5 to 7 mg/dL with the peak value for breeders fed smaller particle sized limestone observed during 20 to 24 h post oviposition and larger particle sized limestone fed group during 18 to 20 h post oviposition. At 2 wk (Figure 2a), plasma Ca++ was significantly higher (P < 0.05) by 0.5 mg/dL for the larger particle sized limestone fed group compared to the smaller particle size limestone fed group during 20 to 24 h post oviposition (4 vs. 3. 5 mg/dL). At 6 wk (Figure 2b), plasma Ca++ was significantly higher (P < 0.05) by 1 mg/dL for the larger particle sized limestone fed group compared to the smaller particle sized limestone fed group during 18 to 20 h post oviposition. Figure 1. View largeDownload slide Plasma inorganic P (iP) and total P (tP) profile (with error bars for SEM) in broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; no significant differences were observed in mean iP or tP at 5 different periods when compared at each period between S and SSB fed groups. Figure 1. View largeDownload slide Plasma inorganic P (iP) and total P (tP) profile (with error bars for SEM) in broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; no significant differences were observed in mean iP or tP at 5 different periods when compared at each period between S and SSB fed groups. Figure 2. View largeDownload slide Plasma ionic Ca (Ca++) and total Ca (tCa) profile (with error bars for SEM) in broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; * = significant differences; differences were observed in mean plasma ionic Ca++ at 20 to 24 h (2-week feeding) and 18 to 20 h (6-week feeding) periods between S and SSB fed groups; no differences were observed in mean tCa at 5 different periods when compared at each period between smaller and larger particle size limestone fed groups. Figure 2. View largeDownload slide Plasma ionic Ca (Ca++) and total Ca (tCa) profile (with error bars for SEM) in broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; * = significant differences; differences were observed in mean plasma ionic Ca++ at 20 to 24 h (2-week feeding) and 18 to 20 h (6-week feeding) periods between S and SSB fed groups; no differences were observed in mean tCa at 5 different periods when compared at each period between smaller and larger particle size limestone fed groups. Urinary pH The total collection of urine, from the colostomized birds during all the 5 periods, at 2 and 6 wk, indicated higher urine pH of ∼7.5 to 8 and 6.5 to 7.5, respectively, during 0 to 11 h post oviposition for both the smaller and larger particle sized limestone fed groups (Figure 3a and 3b.). The urine pH levels showed a declining trend to a low of 5.5 during 11 to 20 h post oviposition followed by a shift towards increasing pH values during 20 to 24 h post oviposition, for both smaller and larger particle sized limestone fed groups. None of the pH values was significantly different (P > 0.05) at each period between the 2 groups fed different particle sizes of limestone, except during 6 to 11 h post oviposition where the larger particle sized limestone fed group showed an increase (by 0.99) in urine pH. Figure 3. View largeDownload slide Urine pH profile (with error bars for SEM) of broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; * = significant difference; no differences were observed in mean urine pH at 5 different periods when compared at each period between S and SSB fed groups except at 6 to 11 h at 6 wk feeding. Figure 3. View largeDownload slide Urine pH profile (with error bars for SEM) of broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; * = significant difference; no differences were observed in mean urine pH at 5 different periods when compared at each period between S and SSB fed groups except at 6 to 11 h at 6 wk feeding. Excretion Pattern of Urinary P and Ca The diurnal patterns of urinary tCa and tP excretion by broiler breeder hens are shown in Figure 4a and 4b. The tP excretion at both 2 and 6 wk collection remained low during 0 to 11 h (<3 mg/dL for both particle sizes). The P excretion was at maximum during 11 to 20 h post oviposition (43.90 mg/dL for breeders fed smaller sized particles and 57.4 mg/dL for breeders fed larger sized particles). This rise in P excretion was later followed by decreased excretion at 20 to 24 h (∼25 mg/dL for breeders fed large particles of limestone vs. ∼5 mg/dL or less for breeders fed smaller particle sizes of limestone) post oviposition. The tCa excretion was at maximum during 0 to 6 h post oviposition at 2 wk (125. 2 mg/dL for breeders fed smaller sized limestone particles and 76.4 mg/dL for breeders fed larger sized limestone particles) and during 0 to 11 h at 6 wk (average of ∼50 mg/dL for breeders fed both particle sizes of limestone). The excretion continued to decline during shell formation post 11 h sampling time for breeders fed both particle sizes of limestone. Figure 4. View largeDownload slide Diurnal pattern of urinary total P (tP) and total Ca (tCa) excretion (with error bars for SEM) for broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; * = significant difference; similar trend for tP and tCa excretion pattern was observed for both 2-week feeding and 6-week feedings at 5 different periods of urine sampled with no difference determined when compared within each period (except tCa at 2-week feeding) for S and SSB. Mean values for tP and tCa excretion between the periods were different after 2-week and 6-week feedings. Figure 4. View largeDownload slide Diurnal pattern of urinary total P (tP) and total Ca (tCa) excretion (with error bars for SEM) for broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; * = significant difference; similar trend for tP and tCa excretion pattern was observed for both 2-week feeding and 6-week feedings at 5 different periods of urine sampled with no difference determined when compared within each period (except tCa at 2-week feeding) for S and SSB. Mean values for tP and tCa excretion between the periods were different after 2-week and 6-week feedings. DISCUSSION Urinary and Excreta P, Ca, and K, and Shell Quality Though the quantity of urine excreted at 2 wk collection was about 2.5 times more compared to the quantity excreted at 6 wk (Table 4), the magnitude of change in Ca, P, and K excretion at 2 and 6 wk was not proportional to the quantity of urine excreted. The decreased urine water concentration at 6 wk (Table 4) indicates the birds’ ability to adapt in conserving water during the post-colostomy period. Previous reports (Hester et al., 1940; Colvin et al., 1966; Paulson, 1969) indicated polyurea or diuresis to occur for 3 d after operation. The surgical exteriorization of the colon (colostomy), however, did not affect feed consumption, egg size, or egg laying compared to normal birds. The trend in tP, tCa, and K excretion at both 2 and 6 wk (Table 4) suggests that the larger particle sized limestone results in less excretion of Ca, indicating more retention. The observation of increased Ca retention for breeders fed larger limestone particle sizes in the present study was similar to findings in a previous study by Ekmay and Coon (2010a) conducted with broiler breeder pure lines fed 2 different limestone particle sizes. The researchers observed 26.72% Ca retention for breeders fed a larger limestone particle size (3489.7 microns, 38.5% solubility) compared to 19.50% Ca retention for breeders fed a smaller limestone particle size (185.5 microns, 58.8% solubility). Further, they also mentioned that the retention percentages varied between the pure lines. Larger particle sized limestone has been shown to have higher in vivo solubility (Zhang and Coon, 1997), which would potentially lead to increased Ca retention. It could be difficult to detect an effect of limestone particle size on P retention in breeders, as breeder hens do not lay eggs as often (when compared to commercial laying hens) and would exert less pressure on dietary Ca for shell deposition. This would decrease the amount of bone P that would be mobilized with resultant loss of P in urine. Considering the egg laying biology of breeder hens, urine P may need to be evaluated over a 2- to 3-day period to see the dynamics of Ca and P losses in breeder hens as compared to the 24-hour period conducted in this study. Another factor that may have impacted the P retention in the present study was NPP levels in the diet. Ekmay and Coon (2011) conducted a balance study in 31 wk broiler breeders and found that there was not an increment in P retention above 30%, as the NPP levels were increased above 0.25% (to dietary NPP levels of 0.30, 0.35, or 0.40%). Inclusion of NPP levels above 0.25% resulted in a significant increase in P excretion. In the present study, NPP levels in the diets were analyzed at 0.39%, which would decrease the opportunity to see less P loss caused by breeders consuming large particle limestone. At 6 wk (Table 3), the tP retention increased by 2% and Ca retention by 3.69%, supporting the significant increase in tibia ash % and specific gravity of eggs for breeders fed larger particle sized limestone compared to breeder hens fed smaller particle sized limestone. The improved bone ash and egg specific gravity also was noticed for broiler breeders fed larger particle sized limestone in a previous breeder study by Ekmay and Coon (2010a). The P retention in colostomized birds did not show a trend similar to normal birds because the average values may have been impacted by variation differences from the small number of colostomized hens (10 normal birds vs. 3 colostomized birds) in the present study. Considering the recovery period of colostomized breeders, it is difficult to have a large number of viable colostomized hens. Comparing the % PP disappearance from the present study (Table 3) to % PP disappearance from the laying hens at peak egg production (Leske and Coon, 1999), about 35% more PP disappearance was obtained in the present study. This increase in PP disappearance in breeder hens could be due to relatively higher bone mobilization during egg production. This would lead to improved efficiency in hydrolyzing dietary PP to replenish depleted bone reserves (Ekmay and Coon, 2010b). When the excretion patterns of tCa and tP are analyzed (Figure 4a and 4b), it is very evident that the Ca excretion was at its peak during the first 11 h post oviposition, and at its lowest during 11 to 20 h post oviposition, in both larger and smaller particle sized limestone fed colostomized hens. During 20 to 24 h of post oviposition (only h before the next egg is laid), tCa excretion continued to decrease in hens fed the smaller particle sized limestone, whereas in hens fed the larger particle size, the Ca excretion started increasing at both 2 and 6 wk collection. The P excretion followed the trend that is expected in relation to Ca excretion. The P excretion was at its basal level during first 11 h post oviposition and suddenly increased to its maximum during 11 to 20 h post oviposition. During 20 to 24 h post oviposition, P excretion again declined; however, there was a tendency of higher urinary P loss during this period with the breeder hens fed small particle sized limestone at both 2 wk and 6 wk sampling periods. Breeders fed larger particle sized limestone, as discussed earlier, show increased dietary Ca retention, leading to reduced dependency on bone Ca for shell deposition. This consequently decreases quantity of P loss that is potentially generated from bone demineralization. Feeding 31 wk breeder hens with larger particle limestone (3489.7 microns, 38.5% solubility) decreased the daily P excretion by ∼50 mg compared to breeders fed with smaller particle sized limestone (185.5 microns, 58.8% solubility) (Ekmay and Coon, 2011). The overall higher urinary P excretion from 2.4 mg P/g DM to 4.1 mg P/g DM was reported in a 16-week feeding study with laying hens fed with small particle Ca size (0.5 to 0.8 mm) vs. larger Ca particle sized limestone (3.3 to 4.7 mm) at 0.128% P and 3.5% Ca basal dietary inclusion with NPP levels added from 0.128 to 0.33% (Coon et al., 2011). Wideman (1987) reported that Ca excretion increases while iP excretion decreases when the dietary Ca intake exceeds the rate of Ca utilization by the shell glands. The report was based on a study of Fussell (1960) that quantified total excretion of urinary Ca using colostomized laying hens fed diets containing 0.67% total P and 2% Ca for d 1 to 8, 0.75% Ca from d 9 to 17, and 5% Ca on d 18 to 27. In the present study, the larger particle size might have continued to provide the Ca for absorption from the gut during calcification, but the amount of Ca that was required for shell calcification could have been greater than the amount that could be absorbed during the rapid period of calcium deposition in eggshell. This rapid requirement could possibly have triggered the bone mobilization to release more Ca coupled with its counter ion P (Kerschnitzki et al., 2014; Taylor and Belanger, 1969). The increased Ca retention coupled with decreased average Ca excretion could be the reason for significant increase in bone ash concentration and specific gravity of eggs, and a tendency to increase percent shell weight and SWUSA for breeder hens fed larger particle sized limestone compared to that of smaller particle sized limestone fed birds. During shell calcification, decreased blood Ca++ results in increased parathyroid hormone (PTH) secretion from the parathyroid gland. PTH increases bone mineral mobilization, resulting in increased Ca and P concentration in the blood. The increased iP accumulation in the blood is prevented by stimulation of urinary iP excretion by PTH stimulation, and simultaneous decrease of Ca excretion due to reduced Ca filtration rates (Wideman, 1987). Plasma iP, tP, Ca++, and tCa The plasma iP, tP, and Ca++ for the smaller particle sized limestone fed group showed peak values during 20 to 24 h post oviposition (Figure 1a and 1b). Plasma tCa for the smaller particle sized limestone fed group showed peak values at 0 to 6 h post oviposition (Figure 2a), whereas the plasma iP, tP, Ca++, and tCa for the larger particle sized limestone fed group showed peak values during 18 to 20 h post oviposition (Figure 1a and 1b). The Miller et al. (1977) study conducted to measure changes in the serum P level of laying hens over a 24-hour period found similar results as in this study in which a predicted peak was at 3 to 4 h prior to oviposition in 63-week-old laying hens fed 3.25% Ca and 0.75% total P. The present study contrasts with Fienberg et al. (1937) and Peterson and Parrish (1939), who reported serum P levels peaked during 11.5 to 13 h of egg formation, and Ca was relatively constant during the various stages of egg formation in a single egg laying cycle. Fienberg et al. (1937) and Peterson and Parrish (1939) utilized different bleeding times that may have contributed to the differences. The early researchers reported bleeding times that were 1 to 2 h (time immediately following oviposition), 11.5 to 13 h, and 26 h (about time for the next oviposition) after oviposition. Paul and Snetsinger (1969) observed the highest plasma Ca++ 1 h post oviposition, falling slowly towards the later part of shell calcification, whereas the plasma P levels reached peak around 16 h post oviposition, in 60-week-old hens fed 2.5% Ca. Paul and Snetsinger (1969) reported bleeding times selected at 1, 9, 16, and 23 h post oviposition. Rao and Roland (1990) reported plasma peak iP at 14 h post oviposition, and plasma Ca++ peaks at 0 and 21 h post oviposition, in 30-week-old laying hens fed diets with 4% Ca and 0.6% total P for 3 d, with selected bleeding time periods of 0, 7, 14, and 21 h post oviposition. The Miller et al. (1977) report was based on 10 different time periods conducted in 3 trials during a 24-hour egg laying cycle, whereas Feinberg et al. (1937), Peterson and Parrish (1939), Paul and Snetsinger (1969), and Rao and Roland (1990) reports were based on 3, 3, 4, and 4 different time periods, respectively. The discrepancy in blood iP or Ca values during the egg formation cycle could be attributed to a seasonal factor (Peterson and Parrish, 1939), amount of dietary P and Ca, limestone solubility due to particle size, hen's Ca status (Rao and Roland, 1990), age factor, breed factor, and time of estimating blood P or Ca levels during egg laying cycle. Urine pH The pH of urine at both 2 and 6 wk (Figure 3a and 3b) was at its peak during 0 to 11 h post oviposition. The pH was lowest at 11 to 20 h post oviposition, and rose in 20 to 24 h collection. Rao and Roland (1990) reported pH peaked at 0 h (pH ∼ 7.7) and 7 h (pH ∼ 8.2) post oviposition. The decreased pH (lowest) was then noticed at 14 h (pH ∼ 4.5) post oviposition and again increased pH at 21 h (pH ∼ 6.4) post oviposition in 30-week-old white leghorn laying hens fed a diet containing 0.6% total P and 4% Ca for 3 days. The proportionate decrease in pH at 6 wk compared to 2 wk collection in the present study could be attributed to the reduced excretion of Ca, which is in support of the observation by Wideman et al. (1985) who reported increased urine pH with increased urinary Ca concentration. The pH during the first 11 h in both groups was higher and in proportion to increased urinary Ca concentration (Figure 3a and 3b). Later, during shell formation period, the concentration of urine started declining, and pH also started declining. The decline in urine pH also could be attributed to urinary acid buffering by phosphaturic response to PTH (Wolbach, 1955; Prashad and Edwards, 1973). During shell calcification, both hydrogen ions and carbonate ions are co-generated, creating metabolic acidosis. This metabolic acidosis is compensated by renal clearance of acid leading to decrease in urinary pH. In summary, feeding larger particle sized limestone compared to smaller particle sized limestone to 30-week-old normal broiler breeder hens for 6 wk resulted in a significant increase in tibia ash and specific gravity, whereas tP retention and tCa retention increased numerically, and excreta P decreased. Of the tP excreted from feces and urine for one egg laying cycle of 24 h, the urinary tP contribution for smaller and larger particle sized limestone fed colostomized broiler breeder hens was about 18%. Of the tCa excreted from both feces and urine for one egg laying cycle of 24 h, the urinary Ca contributions for smaller and larger particle sized limestone fed colostomized broiler breeder hens were similar at ∼9%. 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Google Scholar CrossRef Search ADS PubMed Wideman R. F. Jr. 1987 . Renal regulation of avian calcium and phosphorus metabolism . J. Nutr. 117 : 808 – 815 . Google Scholar CrossRef Search ADS PubMed Zhang B. , Coon C. N. . 1997 . The relationship of calcium intake, source, size, solubility in vitro and in vivo and gizzard limestone retention in laying hens . Poult. Sci. 76 : 1702 – 1706 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Calcium particle size effects on plasma, excreta, and urinary Ca and P changes in broiler breeder hens

Poultry Science , Volume Advance Article (8) – Jul 11, 2018

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Abstract

Abstract An experiment was conducted using non-colostomized and colostomized broiler breeder hens to determine the effects of feeding limestone of 2 different mean particle sizes (185 microns and 3490 microns) on P excretion, total P and Ca retention, and urinary P and Ca excretion during a 6-week feeding study. Additionally, changes in plasma inorganic P (iP) and ionic Ca (Ca++) and urinary excretion of P and Ca were determined in one egg laying cycle of 24 hours. One-hundred-fifty non-colostomized and 6 colostomized broiler breeder hens, 30 wk of age, were divided into 2 groups and fed broiler breeder diets supplemented with either small particle or large particle limestone. Two % acid insoluble ash (Celite) was added to the feed as a marker. Diets, excreta, and urine samples were analyzed for total P and Ca by ionic coupling plasma (ICP) analysis. The non-colostomized breeders fed large particle limestone compared to small limestone particles produced a significant increase in percent tibia ash (P < 0.0001) and egg specific gravity (P = 0.0382), but P excretion approached a tendency of being reduced (P = 0.1585). The urinary total P and Ca (∼18 and 9%, respectively) of total P and Ca excretion for breeders fed both sizes of limestone was not significantly different in the colostomized breeders. In plasma, both iP and Ca++ reached a peak during 18 to 20 h and 20 to 24 h post oviposition for smaller and larger particle sized limestone fed groups, respectively. The maximal excretion of urinary P was found during 11 to 20 h post oviposition, whereas urinary Ca peaked during 0 to 11 h post oviposition for both smaller and larger particle sized limestone supplemented groups. In summary, the findings indicate that the particle size (smaller and larger) of calcium source did not significantly influence the quantitative total urinary excretion of Ca and P but did influence the timing of Ca and P excretion. INTRODUCTION An equivalent of about 10% of the total Ca present in the skeleton of a hen is in the eggshell. Each eggshell contains approximately 2.3 g of Ca, which comes from the diet through intestinal absorption and bone mobilization (Bar, 2009). The skeletal Ca utilization is directly related to the time and level of Ca intake. Dependency on skeletal Ca for eggshell formation has been shown to decrease the quantity of Ca deposited in the egg shell (Farmer et al., 1986). In order to increase the availability of Ca for shell calcification from the diet rather than from bone mobilization, hens should have a constant source of dietary Ca. This can be achieved either by feeding during the time of eggshell calcification or by retaining the Ca source for a longer period. Feeding larger particle sized limestone has been previously shown to increase the retention of limestone particles for a longer time in the gizzard and also increase the in vivo solubility of the limestone (Zhang and Coon, 1997). The Ca and P content of blood in layers may vary depending on the timing of active shell formation. The literature indicate a marked rise in blood P levels of laying hens during the period of rapid shell deposition (11.5 to 13 h post oviposition), while the Ca in blood remains steady during egg formation with a tendency to fall slowly towards the end of shell calcification (Feinberg et al., 1937; Peterson and Parrish, 1939; Paul and Snetsinger, 1969; Miller et al., 1977; Lambert et al., 2014). The cyclic changes in serum P over time have been mainly attributed to changes in shell calcification, bone resorption, remineralization, and renal clearance (De Vries et al., 2010). Wideman (1987) reported that dietary Ca as high as 3.25% with 0.6% P inclusion or low dietary P as low as 0.4% with Ca inclusion (1%) can suppress the urinary excretion of P in domestic fowls. Rao and Roland (1990) indicate that low dietary P has a greater influence on elevating urinary Ca in laying hens than excess dietary Ca based on urine samples. There is a lack of information on the relationship between dietary Ca and its particle size, and P on urinary Ca and P excretion in broiler breeder hens. Because of the limitation and difficulty in separating urine from the feces, colostomized broiler breeder hens were used to determine total urinary excretion of Ca and P for a 24-hour period or one egg laying cycle. The objectives of broiler breeder studies were to determine the effects of 2 different Ca particle sizes on: 1) Ca and P retention in normal broiler breeder hens, 2) urinary total Ca (tCa) and total P (tP) excretion in colostomized broiler breeder hens, 3) changes in plasma tCa, ionic Ca (Ca++), tP, and inorganic P (iP) in relation to oviposition in one egg laying cycle, 4) eggshell quality, and 5) percent tibia ash. MATERIALS AND METHODS Experimental Birds, Test Diets, and Husbandry Three hundred broiler breeder pullets (Cobb 700), 23 wk of age, were offered a breeder diet with standard nutrient specifications (NRC, 1994) in individual cages until 30 wk of age. Six broiler breeder hens (25 wk of age) of uniform body weights were used for colostomy, utilizing the technique of Manangi et al. (2007). During 31st wk, 150 hens were divided into 2 groups of 75 each, and offered the test diets (Table 1) until 36 wk of age (6-week experimental period). The colostomized birds also were divided into 2 groups (3 birds per treatment) and offered the same test diets as normal birds. The Ca added to the basal diets consisted of 2 different particle sizes of limestone. The smaller particle sized limestone (Unical S, ILC Resources, Des Moines, IA) had an average particle size of 185 microns (solubility of 58.8%), whereas the larger particle sized limestone (Shell and Bone Builder, ILC Resources, Des Moines, IA) had an average particle size of 3,489 microns (solubility of 38.5%). Solubility was determined using the method of Zhang and Coon (1997). Table 1. Composition of breeder diet. Ingredients % Corn, 9.2% CP 72.21 Soybean meal, 49.5% CP 17.20 Fat-poultry 0.73 Dical phosphate 1.59 Limestone1 7.12 Methionine, 99% 0.16 Lysine 0.07 Chol chloride-60 0.20 Se premix, 0.06% 0.02 Copper sulfate 0.05 Ethoxyquin, 66% 0.02 Mold curb, 50% propionic acid 0.05 Salt 0.38 Threonine, 98% 0.04 Trace minerals2 0.06 Breeder premix3 0.10 Calculated nutrients (air dried basis) Protein 16.00 MEn, kcal/kg 2915.00 TSAA 0.75 Lysine 0.84 Total P 0.60 Ca 3.10 Analyzed nutrients (air dried basis) Total P 0.58 Phytate P 0.19 NPP 0.39 Ca 3.11 Protein 15.20 Dry matter 89.50 Ingredients % Corn, 9.2% CP 72.21 Soybean meal, 49.5% CP 17.20 Fat-poultry 0.73 Dical phosphate 1.59 Limestone1 7.12 Methionine, 99% 0.16 Lysine 0.07 Chol chloride-60 0.20 Se premix, 0.06% 0.02 Copper sulfate 0.05 Ethoxyquin, 66% 0.02 Mold curb, 50% propionic acid 0.05 Salt 0.38 Threonine, 98% 0.04 Trace minerals2 0.06 Breeder premix3 0.10 Calculated nutrients (air dried basis) Protein 16.00 MEn, kcal/kg 2915.00 TSAA 0.75 Lysine 0.84 Total P 0.60 Ca 3.10 Analyzed nutrients (air dried basis) Total P 0.58 Phytate P 0.19 NPP 0.39 Ca 3.11 Protein 15.20 Dry matter 89.50 1Limestone was replaced with 2 different particle sizes for 2 experimental diets. 2Provided per kilogram of diet: Manganese, 180 mg; zinc, 150.6 mg; iron, 20.16 mg; copper, 2.04 mg; iodine, 1.26 mg; Se, 0.3 mg. 3Provided per kilogram of diet: Vitamin A, 13200IU; vitamin E, 66IU; vitamin D3, 4950ICU; niacin, 74.25 mg; D-pantothenic acid, 33 mg; riboflavin, 19.8 mg; pyridoxine, 5000 mg; thiamine, 3.3 mg; menadione, 3.3 mg; folic acid, 3.3 mg; biotin, 0.33 mg; vitamin B12, 0.0297 mg. View Large Table 1. Composition of breeder diet. Ingredients % Corn, 9.2% CP 72.21 Soybean meal, 49.5% CP 17.20 Fat-poultry 0.73 Dical phosphate 1.59 Limestone1 7.12 Methionine, 99% 0.16 Lysine 0.07 Chol chloride-60 0.20 Se premix, 0.06% 0.02 Copper sulfate 0.05 Ethoxyquin, 66% 0.02 Mold curb, 50% propionic acid 0.05 Salt 0.38 Threonine, 98% 0.04 Trace minerals2 0.06 Breeder premix3 0.10 Calculated nutrients (air dried basis) Protein 16.00 MEn, kcal/kg 2915.00 TSAA 0.75 Lysine 0.84 Total P 0.60 Ca 3.10 Analyzed nutrients (air dried basis) Total P 0.58 Phytate P 0.19 NPP 0.39 Ca 3.11 Protein 15.20 Dry matter 89.50 Ingredients % Corn, 9.2% CP 72.21 Soybean meal, 49.5% CP 17.20 Fat-poultry 0.73 Dical phosphate 1.59 Limestone1 7.12 Methionine, 99% 0.16 Lysine 0.07 Chol chloride-60 0.20 Se premix, 0.06% 0.02 Copper sulfate 0.05 Ethoxyquin, 66% 0.02 Mold curb, 50% propionic acid 0.05 Salt 0.38 Threonine, 98% 0.04 Trace minerals2 0.06 Breeder premix3 0.10 Calculated nutrients (air dried basis) Protein 16.00 MEn, kcal/kg 2915.00 TSAA 0.75 Lysine 0.84 Total P 0.60 Ca 3.10 Analyzed nutrients (air dried basis) Total P 0.58 Phytate P 0.19 NPP 0.39 Ca 3.11 Protein 15.20 Dry matter 89.50 1Limestone was replaced with 2 different particle sizes for 2 experimental diets. 2Provided per kilogram of diet: Manganese, 180 mg; zinc, 150.6 mg; iron, 20.16 mg; copper, 2.04 mg; iodine, 1.26 mg; Se, 0.3 mg. 3Provided per kilogram of diet: Vitamin A, 13200IU; vitamin E, 66IU; vitamin D3, 4950ICU; niacin, 74.25 mg; D-pantothenic acid, 33 mg; riboflavin, 19.8 mg; pyridoxine, 5000 mg; thiamine, 3.3 mg; menadione, 3.3 mg; folic acid, 3.3 mg; biotin, 0.33 mg; vitamin B12, 0.0297 mg. View Large During the 6-week experimental period, the birds were housed in individual cages of 47 cm height x 30.5 cm width x 47 cm length, and the environmental temperature was maintained between 22.2 and 24.4°C. The breeder hens were provided a 16 h light: 8 h dark program during the experimental period. Nutrient Retention Study Ten birds from non-colostomized and 6 birds from the colostomized breeder hens were fed the experimental diets with added acid insoluble ash (AIA) product (CeliteTM5). Two-percent of the AIA product was added to the feed and used as a marker. The AIA mixed feed was fed to the hens that were acclimated to the diets for 4 d prior to a one-day excreta collection period. The excreta from individual birds were collected on trays on d 15 and d 42 and then frozen (–20 °C) and freeze dried for further analysis. Blood, Urine, Bone, and Egg Sample Collection Blood, urine, bone, and egg samples were collected. On d 14 and d 42 of the experiment, blood (plasma) and urine samples were collected. Venous blood (wing vein) was collected from 6 birds using heparinized syringes at 5 different time intervals with respect to oviposition (30 birds total). The 5 different time intervals were 0 to 6, 6 to 11, 11 to 18, 18 to 20, and 20 to 24 h after oviposition. Urine was collected over a 24-hour period from colostomized birds during the same time intervals as blood sample collection. Eggs were collected for 3 d before d 42. Egg weights were recorded after each collection and were kept at room temperature for 3 to 4 h before determination of specific gravity. Five birds on d 14 and 25 birds on d 42 from each treatment group were euthanized by CO2 inhalation for tibiae collection. Tibiae (right and left) were taken from each bird and cleaned of all exterior tissue and frozen until analysis. Samples Analyses Diets, excreta, and plasma samples were analyzed for tP and tCa by inductively coupled plasma (ICP) emission spectroscopic method as mentioned by Leske and Coon (2002). AIA was determined in experimental diets and excreta samples, using dry ash and hydrochloric acid digestion technique of Scott and Balnave (1991). Nitrogen and moisture in both the feed and excreta were determined by standard AOAC procedures (AOAC, 1990). Diet and excreta phytate P (PP) were measured as inositol hexa-phosphate by using ion-exchange chromatography as described by Bos et al. (1991). The gross energy of test feed and gross energy of excreta were measured with a Parr Bomb Calorimeter. PP disappearance and retention values of tP, non-phytate P (NPP), K, Ca, N, and energy were determined using the method of Scott and Balnave (1991). Plasma Ca++ concentrations were determined by using an electrolyte analyzer (Nova-8, NOVA Biomedical, Waltham, MA), and plasma iP was measured using iP reagent kit (Bayer Healthcare LLC, Tarrytown, NY). For tibia ash, tibiae were cut lengthwise and defatted in refluxing petroleum ether in Soxhlet apparatus for 48 hours. The defatted tibiae were oven dried and ashed in ceramic crucibles for 16 h 600°C. Ash content was expressed as percent tibia ash on defatted dried basis. The specific gravity of each egg was determined by floatation method, using gradational salt solutions with specific gravity ranging from 1.06 to 1.10 at increments of 0.05 (Moreng and Avens, 1985). Shell surface area was calculated by using the formula developed by Carter (1975) using fresh egg weights. Shell weight per unit surface area (SWUSA) was determined using shell surface area and dried shell weights (Hamilton, 1978). The collected urine samples were measured for pH and then centrifuged at 2,500 rpm for 15 minutes. The precipitate was freeze-dried after taking out the supernatant from each sample. Both the supernatant and the precipitate were analyzed for Ca and P quantification using ICP. Statistical Analysis Data from this experiment were collected for smaller particle sized limestone and larger particle sized limestone as 2 independent groups for different parameters measured in normal (non-colostomized) and colostomized birds, and mean values were obtained for both groups. Mean values from each of the groups for normal and colostomized birds were separated using “2 group t test” to determine the statistical significance or a tendency at P < 0.05 and P < 0.09, respectively (SAS Institute, 2012). RESULTS Excreta P, PP, NPP, and tP Retention, PP Disappearance, K Retention, GE Retention, and N Retention The mean comparisons of the effect of Ca particle size on excreta P, PP, NPP, and tP retention, PP disappearance, K retention, GE retention, N retention, and Ca retention in broiler breeder hens (non-colostomized) indicate no significant differences (P > 0.05) in feeding smaller or larger particle sized limestone for both 2- and 6-week periods (Tables 2 and 3). Similar results also were obtained from feeding colostomized birds except for excreta P (P < 0.0055). Only a numerical improvement of 2.09% P retention and 3.69% Ca retention was obtained for breeders fed larger sized limestone particles compared to breeders fed smaller particle sized limestone (Table 3). There was a significant (P < 0.0001) improvement of 3.22% in tibia ash content in the group fed larger particle sized limestone compared to the group of breeders fed smaller particle sized limestone for the 6-week collection period (Table 3). Table 2. Ca effects on broiler breeder hens fed small or large limestone particles after 2-week feeding period. Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 15.53 ± 1.29 18.53 ± 1.29 0.1249 15.92 ± 0.54 18.87 ± 0.64 0.0055 Excreta PP, mg/g DM 1.24 ± 0.82 2.96 ± 0.92 0.2047 1.25 ± 0.64 2.0 ± 0.91 0.5201 Excreta NPP, mg/g DM 14.17 ± 1.43 15.75 ± 1.60 0.4855 14.50 ± 0.91 15.52 ± 1.29 0.5418 Total P retention, % 36.59 ± 3.08 36.58 ± 3.08 0.9991 55.54 ± 1.91 51.55 ± 2.26 0.2076 PP disappearance, % 79.03 ± 10.45 67.48 ± 11.68 0.4853 88.78 ± 5.06 84.50 ± 7.15 0.64 Potassium retention, % 26.90 ± 5.77 34.28 ± 4.71 0.3504 62.37 ± 2.71 62.59 ± 3.21 0.9631 GE retention, % 81.00 ± 1.49 82.06 ± 1.38 0.6153 86.87 ± 0.42 88.16 ± 0.50 0.0752 N retention, % 44.10 ± 6.22 45.07 ± 5.76 0.9108 87.64 ± 0.72 87.02 ± 0.85 0.5923 Ca retention, % 40.55 ± 10.21 62.32 ± 8.34 0.1372 54.07 ± 4.69 63.22 ± 5.55 0.2365 Bone (tibiae) ash, % 52.52 ± 1.85 53.74 ± 1.85 0.6535 Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 15.53 ± 1.29 18.53 ± 1.29 0.1249 15.92 ± 0.54 18.87 ± 0.64 0.0055 Excreta PP, mg/g DM 1.24 ± 0.82 2.96 ± 0.92 0.2047 1.25 ± 0.64 2.0 ± 0.91 0.5201 Excreta NPP, mg/g DM 14.17 ± 1.43 15.75 ± 1.60 0.4855 14.50 ± 0.91 15.52 ± 1.29 0.5418 Total P retention, % 36.59 ± 3.08 36.58 ± 3.08 0.9991 55.54 ± 1.91 51.55 ± 2.26 0.2076 PP disappearance, % 79.03 ± 10.45 67.48 ± 11.68 0.4853 88.78 ± 5.06 84.50 ± 7.15 0.64 Potassium retention, % 26.90 ± 5.77 34.28 ± 4.71 0.3504 62.37 ± 2.71 62.59 ± 3.21 0.9631 GE retention, % 81.00 ± 1.49 82.06 ± 1.38 0.6153 86.87 ± 0.42 88.16 ± 0.50 0.0752 N retention, % 44.10 ± 6.22 45.07 ± 5.76 0.9108 87.64 ± 0.72 87.02 ± 0.85 0.5923 Ca retention, % 40.55 ± 10.21 62.32 ± 8.34 0.1372 54.07 ± 4.69 63.22 ± 5.55 0.2365 Bone (tibiae) ash, % 52.52 ± 1.85 53.74 ± 1.85 0.6535 S-smaller particle size calcium source; SSB-larger particle size calcium source; phytate P (PP). View Large Table 2. Ca effects on broiler breeder hens fed small or large limestone particles after 2-week feeding period. Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 15.53 ± 1.29 18.53 ± 1.29 0.1249 15.92 ± 0.54 18.87 ± 0.64 0.0055 Excreta PP, mg/g DM 1.24 ± 0.82 2.96 ± 0.92 0.2047 1.25 ± 0.64 2.0 ± 0.91 0.5201 Excreta NPP, mg/g DM 14.17 ± 1.43 15.75 ± 1.60 0.4855 14.50 ± 0.91 15.52 ± 1.29 0.5418 Total P retention, % 36.59 ± 3.08 36.58 ± 3.08 0.9991 55.54 ± 1.91 51.55 ± 2.26 0.2076 PP disappearance, % 79.03 ± 10.45 67.48 ± 11.68 0.4853 88.78 ± 5.06 84.50 ± 7.15 0.64 Potassium retention, % 26.90 ± 5.77 34.28 ± 4.71 0.3504 62.37 ± 2.71 62.59 ± 3.21 0.9631 GE retention, % 81.00 ± 1.49 82.06 ± 1.38 0.6153 86.87 ± 0.42 88.16 ± 0.50 0.0752 N retention, % 44.10 ± 6.22 45.07 ± 5.76 0.9108 87.64 ± 0.72 87.02 ± 0.85 0.5923 Ca retention, % 40.55 ± 10.21 62.32 ± 8.34 0.1372 54.07 ± 4.69 63.22 ± 5.55 0.2365 Bone (tibiae) ash, % 52.52 ± 1.85 53.74 ± 1.85 0.6535 Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 15.53 ± 1.29 18.53 ± 1.29 0.1249 15.92 ± 0.54 18.87 ± 0.64 0.0055 Excreta PP, mg/g DM 1.24 ± 0.82 2.96 ± 0.92 0.2047 1.25 ± 0.64 2.0 ± 0.91 0.5201 Excreta NPP, mg/g DM 14.17 ± 1.43 15.75 ± 1.60 0.4855 14.50 ± 0.91 15.52 ± 1.29 0.5418 Total P retention, % 36.59 ± 3.08 36.58 ± 3.08 0.9991 55.54 ± 1.91 51.55 ± 2.26 0.2076 PP disappearance, % 79.03 ± 10.45 67.48 ± 11.68 0.4853 88.78 ± 5.06 84.50 ± 7.15 0.64 Potassium retention, % 26.90 ± 5.77 34.28 ± 4.71 0.3504 62.37 ± 2.71 62.59 ± 3.21 0.9631 GE retention, % 81.00 ± 1.49 82.06 ± 1.38 0.6153 86.87 ± 0.42 88.16 ± 0.50 0.0752 N retention, % 44.10 ± 6.22 45.07 ± 5.76 0.9108 87.64 ± 0.72 87.02 ± 0.85 0.5923 Ca retention, % 40.55 ± 10.21 62.32 ± 8.34 0.1372 54.07 ± 4.69 63.22 ± 5.55 0.2365 Bone (tibiae) ash, % 52.52 ± 1.85 53.74 ± 1.85 0.6535 S-smaller particle size calcium source; SSB-larger particle size calcium source; phytate P (PP). View Large Table 3. Ca effects on broiler breeder hens fed small or large limestone particles after 6-week feeding period. Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 18.55 ± 0.87 16.72 ± 0.93 0.1585 12.25 ± 0.46 13.58 ± 0.54 0.0770 Excreta PP, mg/g DM 3.23 ± 0.35 3.04 ± 0.47 0.7533 3.91 ± 0.47 4.13 ± 0.54 0.7612 Excreta NPP, mg/g DM 15.32 ± 0.90 14.69 ± 1.20 0.6766 8.33 ± 0.61 9.45 ± 0.71 0.2503 Total P retention, % 31.00 ± 3.30 33.09 ± 3.72 0.6777 59.09 ± 2.09 54.84 ± 2.56 0.2208 PP disappearance, % 60.78 ± 4.30 61.32 ± 5.95 0.9422 55.99 ± 4.31 50.73 ± 5.28 0.4537 Potassium retention, % 18.90 ± 2.27 18.67 ± 2.72 0.9483 52.01 ± 2.42 50.61 ± 2.96 0.7190 GE retention, % 80.80 ± 0.73 80.45 ± 0.83 0.752 83.74 ± 0.58 82.58 ± 0.71 0.2261 N retention, % 42.85 ± 1.88 42.10 ± 2.28 0.7996 83.33 ± 0.86 84.27 ± 1.05 0.5030 Ca retention, % 49.86 ± 3.26 53.55 ± 4.15 0.4883 55.66 ± 3.57 61.98 ± 4.37 0.2825 Bone (tibiae) ash, % 54.75 ± 0.50 57.97 ± 0.57 <0.0001 Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 18.55 ± 0.87 16.72 ± 0.93 0.1585 12.25 ± 0.46 13.58 ± 0.54 0.0770 Excreta PP, mg/g DM 3.23 ± 0.35 3.04 ± 0.47 0.7533 3.91 ± 0.47 4.13 ± 0.54 0.7612 Excreta NPP, mg/g DM 15.32 ± 0.90 14.69 ± 1.20 0.6766 8.33 ± 0.61 9.45 ± 0.71 0.2503 Total P retention, % 31.00 ± 3.30 33.09 ± 3.72 0.6777 59.09 ± 2.09 54.84 ± 2.56 0.2208 PP disappearance, % 60.78 ± 4.30 61.32 ± 5.95 0.9422 55.99 ± 4.31 50.73 ± 5.28 0.4537 Potassium retention, % 18.90 ± 2.27 18.67 ± 2.72 0.9483 52.01 ± 2.42 50.61 ± 2.96 0.7190 GE retention, % 80.80 ± 0.73 80.45 ± 0.83 0.752 83.74 ± 0.58 82.58 ± 0.71 0.2261 N retention, % 42.85 ± 1.88 42.10 ± 2.28 0.7996 83.33 ± 0.86 84.27 ± 1.05 0.5030 Ca retention, % 49.86 ± 3.26 53.55 ± 4.15 0.4883 55.66 ± 3.57 61.98 ± 4.37 0.2825 Bone (tibiae) ash, % 54.75 ± 0.50 57.97 ± 0.57 <0.0001 S-smaller particle size calcium source; SSB-larger particle size calcium source; phytate P (PP). View Large Table 3. Ca effects on broiler breeder hens fed small or large limestone particles after 6-week feeding period. Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 18.55 ± 0.87 16.72 ± 0.93 0.1585 12.25 ± 0.46 13.58 ± 0.54 0.0770 Excreta PP, mg/g DM 3.23 ± 0.35 3.04 ± 0.47 0.7533 3.91 ± 0.47 4.13 ± 0.54 0.7612 Excreta NPP, mg/g DM 15.32 ± 0.90 14.69 ± 1.20 0.6766 8.33 ± 0.61 9.45 ± 0.71 0.2503 Total P retention, % 31.00 ± 3.30 33.09 ± 3.72 0.6777 59.09 ± 2.09 54.84 ± 2.56 0.2208 PP disappearance, % 60.78 ± 4.30 61.32 ± 5.95 0.9422 55.99 ± 4.31 50.73 ± 5.28 0.4537 Potassium retention, % 18.90 ± 2.27 18.67 ± 2.72 0.9483 52.01 ± 2.42 50.61 ± 2.96 0.7190 GE retention, % 80.80 ± 0.73 80.45 ± 0.83 0.752 83.74 ± 0.58 82.58 ± 0.71 0.2261 N retention, % 42.85 ± 1.88 42.10 ± 2.28 0.7996 83.33 ± 0.86 84.27 ± 1.05 0.5030 Ca retention, % 49.86 ± 3.26 53.55 ± 4.15 0.4883 55.66 ± 3.57 61.98 ± 4.37 0.2825 Bone (tibiae) ash, % 54.75 ± 0.50 57.97 ± 0.57 <0.0001 Non-colostomized birds Colostomized birds S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Excreta P, mg/g DM 18.55 ± 0.87 16.72 ± 0.93 0.1585 12.25 ± 0.46 13.58 ± 0.54 0.0770 Excreta PP, mg/g DM 3.23 ± 0.35 3.04 ± 0.47 0.7533 3.91 ± 0.47 4.13 ± 0.54 0.7612 Excreta NPP, mg/g DM 15.32 ± 0.90 14.69 ± 1.20 0.6766 8.33 ± 0.61 9.45 ± 0.71 0.2503 Total P retention, % 31.00 ± 3.30 33.09 ± 3.72 0.6777 59.09 ± 2.09 54.84 ± 2.56 0.2208 PP disappearance, % 60.78 ± 4.30 61.32 ± 5.95 0.9422 55.99 ± 4.31 50.73 ± 5.28 0.4537 Potassium retention, % 18.90 ± 2.27 18.67 ± 2.72 0.9483 52.01 ± 2.42 50.61 ± 2.96 0.7190 GE retention, % 80.80 ± 0.73 80.45 ± 0.83 0.752 83.74 ± 0.58 82.58 ± 0.71 0.2261 N retention, % 42.85 ± 1.88 42.10 ± 2.28 0.7996 83.33 ± 0.86 84.27 ± 1.05 0.5030 Ca retention, % 49.86 ± 3.26 53.55 ± 4.15 0.4883 55.66 ± 3.57 61.98 ± 4.37 0.2825 Bone (tibiae) ash, % 54.75 ± 0.50 57.97 ± 0.57 <0.0001 S-smaller particle size calcium source; SSB-larger particle size calcium source; phytate P (PP). View Large Urinary P, Ca, and K Feeding 2 different particle sizes of limestone did not have significant influence (P > 0.05) on total quantity of urine excreted or urinary tP, tCa, or K after the 2 and 6 wk of feeding study (Table 4). Table 4. Ca effects on urine and urinary mineral excretion in colostomized broiler breeder hens* fed small or large particle limestone. After 2 wk After 6 wk S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Total quantity of urine, mL 394.0 ± 57.4 399.0 ± 49.7 0.9508 136.0 ± 35.6 171.0 ± 29.8 0.4719 Total P excreted, mg 73.6 ± 20.4 101.0 ± 17.6 0.3551 80.4 ± 17.5 90.2 ± 14.7 0.6744 Total Ca excreted, mg 245.0 ± 20.1 203.3 ± 17.4 0.1778 186.0 ± 30.9 167.7 ± 25.8 0.6557 Total K excreted, mg 223.5 ± 38.2 205.6 ± 33.1 0.7372 246.7 ± 31.7 235.8 ± 26.5 0.7965 After 2 wk After 6 wk S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Total quantity of urine, mL 394.0 ± 57.4 399.0 ± 49.7 0.9508 136.0 ± 35.6 171.0 ± 29.8 0.4719 Total P excreted, mg 73.6 ± 20.4 101.0 ± 17.6 0.3551 80.4 ± 17.5 90.2 ± 14.7 0.6744 Total Ca excreted, mg 245.0 ± 20.1 203.3 ± 17.4 0.1778 186.0 ± 30.9 167.7 ± 25.8 0.6557 Total K excreted, mg 223.5 ± 38.2 205.6 ± 33.1 0.7372 246.7 ± 31.7 235.8 ± 26.5 0.7965 *All the values in Table 4 are average values for ∼24-hour period or one egg laying cycle. S-smaller particle size calcium source; SSB-larger particle size calcium source. View Large Table 4. Ca effects on urine and urinary mineral excretion in colostomized broiler breeder hens* fed small or large particle limestone. After 2 wk After 6 wk S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Total quantity of urine, mL 394.0 ± 57.4 399.0 ± 49.7 0.9508 136.0 ± 35.6 171.0 ± 29.8 0.4719 Total P excreted, mg 73.6 ± 20.4 101.0 ± 17.6 0.3551 80.4 ± 17.5 90.2 ± 14.7 0.6744 Total Ca excreted, mg 245.0 ± 20.1 203.3 ± 17.4 0.1778 186.0 ± 30.9 167.7 ± 25.8 0.6557 Total K excreted, mg 223.5 ± 38.2 205.6 ± 33.1 0.7372 246.7 ± 31.7 235.8 ± 26.5 0.7965 After 2 wk After 6 wk S SSB S SSB Mean ± SE Mean ± SE P > | t | Mean ± SE Mean ± SE P > | t | Total quantity of urine, mL 394.0 ± 57.4 399.0 ± 49.7 0.9508 136.0 ± 35.6 171.0 ± 29.8 0.4719 Total P excreted, mg 73.6 ± 20.4 101.0 ± 17.6 0.3551 80.4 ± 17.5 90.2 ± 14.7 0.6744 Total Ca excreted, mg 245.0 ± 20.1 203.3 ± 17.4 0.1778 186.0 ± 30.9 167.7 ± 25.8 0.6557 Total K excreted, mg 223.5 ± 38.2 205.6 ± 33.1 0.7372 246.7 ± 31.7 235.8 ± 26.5 0.7965 *All the values in Table 4 are average values for ∼24-hour period or one egg laying cycle. S-smaller particle size calcium source; SSB-larger particle size calcium source. View Large Eggshell Traits Average hen day egg production for a period of 6 wk showed no difference (P > 0.05) between the 2 groups of broiler breeder hens fed 2 different particle sizes of limestone (Table 5). There were about 8 to 9 shell-less and double yolk eggs recorded during the 6-week experimental period in both groups. Three-day average egg weight, percent shell weight, and SWUSA did not change (P > 0.05) between the 2 groups. Feeding a larger particle size of limestone significantly (P < 0.05) increased the specific gravity of eggs as compared to feeding breeders smaller particle sized limestone (1.087 vs. 1.085). There was no change (P > 0.05) in the egg weight, percent shell weight, SWUSA, or specific gravity of eggs from the 2 groups of colostomized breeder hens fed larger and smaller particle sized limestone (Table 5). Table 5. Ca effects on egg production and shell quality traits for broiler breeder hens* fed small or large particle limestone. Non-colostomized birds Colostomized birds S SSB P > | t | S SSB P > | t | Average hen day egg number 36.5 ± 0.80 36.9 ± 0.80 0.7454 – – Egg weight, g 60.6 ± 0.40 59.5 ± 0.40 0.0612 60.8 ± 0.6 58.8 ± 0.6 0.1486 % Dry shell weight 9.10 ± 0.08 9.22 ± 0.08 0.3010 9.6 ± 0.2 9.8 ± 0.2 0.4226 SWUSA, mg/cm2 76.72 ± 0.72 77.31 ± 0.75 0.5652 80.1 ± 5.42 81.4 ± 5.42 0.8808 Specific Gravity 1.085 ± 0.0006 1.087 ± 0.0006 0.0382 1.086 ± 0.0032 1.087 ± 0.0029 0.8801 Non-colostomized birds Colostomized birds S SSB P > | t | S SSB P > | t | Average hen day egg number 36.5 ± 0.80 36.9 ± 0.80 0.7454 – – Egg weight, g 60.6 ± 0.40 59.5 ± 0.40 0.0612 60.8 ± 0.6 58.8 ± 0.6 0.1486 % Dry shell weight 9.10 ± 0.08 9.22 ± 0.08 0.3010 9.6 ± 0.2 9.8 ± 0.2 0.4226 SWUSA, mg/cm2 76.72 ± 0.72 77.31 ± 0.75 0.5652 80.1 ± 5.42 81.4 ± 5.42 0.8808 Specific Gravity 1.085 ± 0.0006 1.087 ± 0.0006 0.0382 1.086 ± 0.0032 1.087 ± 0.0029 0.8801 *n = 135 per treatment for non-colostomized birds; n = 6 per treatment for colostomized birds. S-smaller particle size calcium source; SSB-larger particle size calcium source. SWUSA-Shell weight per unit surface area. View Large Table 5. Ca effects on egg production and shell quality traits for broiler breeder hens* fed small or large particle limestone. Non-colostomized birds Colostomized birds S SSB P > | t | S SSB P > | t | Average hen day egg number 36.5 ± 0.80 36.9 ± 0.80 0.7454 – – Egg weight, g 60.6 ± 0.40 59.5 ± 0.40 0.0612 60.8 ± 0.6 58.8 ± 0.6 0.1486 % Dry shell weight 9.10 ± 0.08 9.22 ± 0.08 0.3010 9.6 ± 0.2 9.8 ± 0.2 0.4226 SWUSA, mg/cm2 76.72 ± 0.72 77.31 ± 0.75 0.5652 80.1 ± 5.42 81.4 ± 5.42 0.8808 Specific Gravity 1.085 ± 0.0006 1.087 ± 0.0006 0.0382 1.086 ± 0.0032 1.087 ± 0.0029 0.8801 Non-colostomized birds Colostomized birds S SSB P > | t | S SSB P > | t | Average hen day egg number 36.5 ± 0.80 36.9 ± 0.80 0.7454 – – Egg weight, g 60.6 ± 0.40 59.5 ± 0.40 0.0612 60.8 ± 0.6 58.8 ± 0.6 0.1486 % Dry shell weight 9.10 ± 0.08 9.22 ± 0.08 0.3010 9.6 ± 0.2 9.8 ± 0.2 0.4226 SWUSA, mg/cm2 76.72 ± 0.72 77.31 ± 0.75 0.5652 80.1 ± 5.42 81.4 ± 5.42 0.8808 Specific Gravity 1.085 ± 0.0006 1.087 ± 0.0006 0.0382 1.086 ± 0.0032 1.087 ± 0.0029 0.8801 *n = 135 per treatment for non-colostomized birds; n = 6 per treatment for colostomized birds. S-smaller particle size calcium source; SSB-larger particle size calcium source. SWUSA-Shell weight per unit surface area. View Large Plasma iP, tP, Ca++, and tCa Blood plasma at both 2 and 6 wk, at 5 different periods related to oviposition, revealed no significant (P > 0.05) change in the mean iP, tP, or tCa (Figures 1 and 2) when each period was compared between groups of breeders fed smaller and larger particle sized limestone. At 2 wk (Figure 1a), the 24 h plasma iP ranged from ∼2 to 6 mg/dL for both smaller and larger particle sized limestone fed groups, with a tendency of higher values before (at 20 to 24 h) and after (at 0 to 6 h) oviposition. At 6 wk (Figure 1b), the 24 h plasma iP range was ∼5 to 7 mg/dL with the peak value for breeders fed smaller particle sized limestone observed during 20 to 24 h post oviposition and larger particle sized limestone fed group during 18 to 20 h post oviposition. At 2 wk (Figure 2a), plasma Ca++ was significantly higher (P < 0.05) by 0.5 mg/dL for the larger particle sized limestone fed group compared to the smaller particle size limestone fed group during 20 to 24 h post oviposition (4 vs. 3. 5 mg/dL). At 6 wk (Figure 2b), plasma Ca++ was significantly higher (P < 0.05) by 1 mg/dL for the larger particle sized limestone fed group compared to the smaller particle sized limestone fed group during 18 to 20 h post oviposition. Figure 1. View largeDownload slide Plasma inorganic P (iP) and total P (tP) profile (with error bars for SEM) in broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; no significant differences were observed in mean iP or tP at 5 different periods when compared at each period between S and SSB fed groups. Figure 1. View largeDownload slide Plasma inorganic P (iP) and total P (tP) profile (with error bars for SEM) in broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; no significant differences were observed in mean iP or tP at 5 different periods when compared at each period between S and SSB fed groups. Figure 2. View largeDownload slide Plasma ionic Ca (Ca++) and total Ca (tCa) profile (with error bars for SEM) in broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; * = significant differences; differences were observed in mean plasma ionic Ca++ at 20 to 24 h (2-week feeding) and 18 to 20 h (6-week feeding) periods between S and SSB fed groups; no differences were observed in mean tCa at 5 different periods when compared at each period between smaller and larger particle size limestone fed groups. Figure 2. View largeDownload slide Plasma ionic Ca (Ca++) and total Ca (tCa) profile (with error bars for SEM) in broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; * = significant differences; differences were observed in mean plasma ionic Ca++ at 20 to 24 h (2-week feeding) and 18 to 20 h (6-week feeding) periods between S and SSB fed groups; no differences were observed in mean tCa at 5 different periods when compared at each period between smaller and larger particle size limestone fed groups. Urinary pH The total collection of urine, from the colostomized birds during all the 5 periods, at 2 and 6 wk, indicated higher urine pH of ∼7.5 to 8 and 6.5 to 7.5, respectively, during 0 to 11 h post oviposition for both the smaller and larger particle sized limestone fed groups (Figure 3a and 3b.). The urine pH levels showed a declining trend to a low of 5.5 during 11 to 20 h post oviposition followed by a shift towards increasing pH values during 20 to 24 h post oviposition, for both smaller and larger particle sized limestone fed groups. None of the pH values was significantly different (P > 0.05) at each period between the 2 groups fed different particle sizes of limestone, except during 6 to 11 h post oviposition where the larger particle sized limestone fed group showed an increase (by 0.99) in urine pH. Figure 3. View largeDownload slide Urine pH profile (with error bars for SEM) of broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; * = significant difference; no differences were observed in mean urine pH at 5 different periods when compared at each period between S and SSB fed groups except at 6 to 11 h at 6 wk feeding. Figure 3. View largeDownload slide Urine pH profile (with error bars for SEM) of broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; * = significant difference; no differences were observed in mean urine pH at 5 different periods when compared at each period between S and SSB fed groups except at 6 to 11 h at 6 wk feeding. Excretion Pattern of Urinary P and Ca The diurnal patterns of urinary tCa and tP excretion by broiler breeder hens are shown in Figure 4a and 4b. The tP excretion at both 2 and 6 wk collection remained low during 0 to 11 h (<3 mg/dL for both particle sizes). The P excretion was at maximum during 11 to 20 h post oviposition (43.90 mg/dL for breeders fed smaller sized particles and 57.4 mg/dL for breeders fed larger sized particles). This rise in P excretion was later followed by decreased excretion at 20 to 24 h (∼25 mg/dL for breeders fed large particles of limestone vs. ∼5 mg/dL or less for breeders fed smaller particle sizes of limestone) post oviposition. The tCa excretion was at maximum during 0 to 6 h post oviposition at 2 wk (125. 2 mg/dL for breeders fed smaller sized limestone particles and 76.4 mg/dL for breeders fed larger sized limestone particles) and during 0 to 11 h at 6 wk (average of ∼50 mg/dL for breeders fed both particle sizes of limestone). The excretion continued to decline during shell formation post 11 h sampling time for breeders fed both particle sizes of limestone. Figure 4. View largeDownload slide Diurnal pattern of urinary total P (tP) and total Ca (tCa) excretion (with error bars for SEM) for broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; * = significant difference; similar trend for tP and tCa excretion pattern was observed for both 2-week feeding and 6-week feedings at 5 different periods of urine sampled with no difference determined when compared within each period (except tCa at 2-week feeding) for S and SSB. Mean values for tP and tCa excretion between the periods were different after 2-week and 6-week feedings. Figure 4. View largeDownload slide Diurnal pattern of urinary total P (tP) and total Ca (tCa) excretion (with error bars for SEM) for broiler breeder hens fed 2 different particle sizes of Ca. a) 2-week feeding; b) 6-week feeding; S-smaller particle size calcium source; SSB-larger particle size calcium source; * = significant difference; similar trend for tP and tCa excretion pattern was observed for both 2-week feeding and 6-week feedings at 5 different periods of urine sampled with no difference determined when compared within each period (except tCa at 2-week feeding) for S and SSB. Mean values for tP and tCa excretion between the periods were different after 2-week and 6-week feedings. DISCUSSION Urinary and Excreta P, Ca, and K, and Shell Quality Though the quantity of urine excreted at 2 wk collection was about 2.5 times more compared to the quantity excreted at 6 wk (Table 4), the magnitude of change in Ca, P, and K excretion at 2 and 6 wk was not proportional to the quantity of urine excreted. The decreased urine water concentration at 6 wk (Table 4) indicates the birds’ ability to adapt in conserving water during the post-colostomy period. Previous reports (Hester et al., 1940; Colvin et al., 1966; Paulson, 1969) indicated polyurea or diuresis to occur for 3 d after operation. The surgical exteriorization of the colon (colostomy), however, did not affect feed consumption, egg size, or egg laying compared to normal birds. The trend in tP, tCa, and K excretion at both 2 and 6 wk (Table 4) suggests that the larger particle sized limestone results in less excretion of Ca, indicating more retention. The observation of increased Ca retention for breeders fed larger limestone particle sizes in the present study was similar to findings in a previous study by Ekmay and Coon (2010a) conducted with broiler breeder pure lines fed 2 different limestone particle sizes. The researchers observed 26.72% Ca retention for breeders fed a larger limestone particle size (3489.7 microns, 38.5% solubility) compared to 19.50% Ca retention for breeders fed a smaller limestone particle size (185.5 microns, 58.8% solubility). Further, they also mentioned that the retention percentages varied between the pure lines. Larger particle sized limestone has been shown to have higher in vivo solubility (Zhang and Coon, 1997), which would potentially lead to increased Ca retention. It could be difficult to detect an effect of limestone particle size on P retention in breeders, as breeder hens do not lay eggs as often (when compared to commercial laying hens) and would exert less pressure on dietary Ca for shell deposition. This would decrease the amount of bone P that would be mobilized with resultant loss of P in urine. Considering the egg laying biology of breeder hens, urine P may need to be evaluated over a 2- to 3-day period to see the dynamics of Ca and P losses in breeder hens as compared to the 24-hour period conducted in this study. Another factor that may have impacted the P retention in the present study was NPP levels in the diet. Ekmay and Coon (2011) conducted a balance study in 31 wk broiler breeders and found that there was not an increment in P retention above 30%, as the NPP levels were increased above 0.25% (to dietary NPP levels of 0.30, 0.35, or 0.40%). Inclusion of NPP levels above 0.25% resulted in a significant increase in P excretion. In the present study, NPP levels in the diets were analyzed at 0.39%, which would decrease the opportunity to see less P loss caused by breeders consuming large particle limestone. At 6 wk (Table 3), the tP retention increased by 2% and Ca retention by 3.69%, supporting the significant increase in tibia ash % and specific gravity of eggs for breeders fed larger particle sized limestone compared to breeder hens fed smaller particle sized limestone. The improved bone ash and egg specific gravity also was noticed for broiler breeders fed larger particle sized limestone in a previous breeder study by Ekmay and Coon (2010a). The P retention in colostomized birds did not show a trend similar to normal birds because the average values may have been impacted by variation differences from the small number of colostomized hens (10 normal birds vs. 3 colostomized birds) in the present study. Considering the recovery period of colostomized breeders, it is difficult to have a large number of viable colostomized hens. Comparing the % PP disappearance from the present study (Table 3) to % PP disappearance from the laying hens at peak egg production (Leske and Coon, 1999), about 35% more PP disappearance was obtained in the present study. This increase in PP disappearance in breeder hens could be due to relatively higher bone mobilization during egg production. This would lead to improved efficiency in hydrolyzing dietary PP to replenish depleted bone reserves (Ekmay and Coon, 2010b). When the excretion patterns of tCa and tP are analyzed (Figure 4a and 4b), it is very evident that the Ca excretion was at its peak during the first 11 h post oviposition, and at its lowest during 11 to 20 h post oviposition, in both larger and smaller particle sized limestone fed colostomized hens. During 20 to 24 h of post oviposition (only h before the next egg is laid), tCa excretion continued to decrease in hens fed the smaller particle sized limestone, whereas in hens fed the larger particle size, the Ca excretion started increasing at both 2 and 6 wk collection. The P excretion followed the trend that is expected in relation to Ca excretion. The P excretion was at its basal level during first 11 h post oviposition and suddenly increased to its maximum during 11 to 20 h post oviposition. During 20 to 24 h post oviposition, P excretion again declined; however, there was a tendency of higher urinary P loss during this period with the breeder hens fed small particle sized limestone at both 2 wk and 6 wk sampling periods. Breeders fed larger particle sized limestone, as discussed earlier, show increased dietary Ca retention, leading to reduced dependency on bone Ca for shell deposition. This consequently decreases quantity of P loss that is potentially generated from bone demineralization. Feeding 31 wk breeder hens with larger particle limestone (3489.7 microns, 38.5% solubility) decreased the daily P excretion by ∼50 mg compared to breeders fed with smaller particle sized limestone (185.5 microns, 58.8% solubility) (Ekmay and Coon, 2011). The overall higher urinary P excretion from 2.4 mg P/g DM to 4.1 mg P/g DM was reported in a 16-week feeding study with laying hens fed with small particle Ca size (0.5 to 0.8 mm) vs. larger Ca particle sized limestone (3.3 to 4.7 mm) at 0.128% P and 3.5% Ca basal dietary inclusion with NPP levels added from 0.128 to 0.33% (Coon et al., 2011). Wideman (1987) reported that Ca excretion increases while iP excretion decreases when the dietary Ca intake exceeds the rate of Ca utilization by the shell glands. The report was based on a study of Fussell (1960) that quantified total excretion of urinary Ca using colostomized laying hens fed diets containing 0.67% total P and 2% Ca for d 1 to 8, 0.75% Ca from d 9 to 17, and 5% Ca on d 18 to 27. In the present study, the larger particle size might have continued to provide the Ca for absorption from the gut during calcification, but the amount of Ca that was required for shell calcification could have been greater than the amount that could be absorbed during the rapid period of calcium deposition in eggshell. This rapid requirement could possibly have triggered the bone mobilization to release more Ca coupled with its counter ion P (Kerschnitzki et al., 2014; Taylor and Belanger, 1969). The increased Ca retention coupled with decreased average Ca excretion could be the reason for significant increase in bone ash concentration and specific gravity of eggs, and a tendency to increase percent shell weight and SWUSA for breeder hens fed larger particle sized limestone compared to that of smaller particle sized limestone fed birds. During shell calcification, decreased blood Ca++ results in increased parathyroid hormone (PTH) secretion from the parathyroid gland. PTH increases bone mineral mobilization, resulting in increased Ca and P concentration in the blood. The increased iP accumulation in the blood is prevented by stimulation of urinary iP excretion by PTH stimulation, and simultaneous decrease of Ca excretion due to reduced Ca filtration rates (Wideman, 1987). Plasma iP, tP, Ca++, and tCa The plasma iP, tP, and Ca++ for the smaller particle sized limestone fed group showed peak values during 20 to 24 h post oviposition (Figure 1a and 1b). Plasma tCa for the smaller particle sized limestone fed group showed peak values at 0 to 6 h post oviposition (Figure 2a), whereas the plasma iP, tP, Ca++, and tCa for the larger particle sized limestone fed group showed peak values during 18 to 20 h post oviposition (Figure 1a and 1b). The Miller et al. (1977) study conducted to measure changes in the serum P level of laying hens over a 24-hour period found similar results as in this study in which a predicted peak was at 3 to 4 h prior to oviposition in 63-week-old laying hens fed 3.25% Ca and 0.75% total P. The present study contrasts with Fienberg et al. (1937) and Peterson and Parrish (1939), who reported serum P levels peaked during 11.5 to 13 h of egg formation, and Ca was relatively constant during the various stages of egg formation in a single egg laying cycle. Fienberg et al. (1937) and Peterson and Parrish (1939) utilized different bleeding times that may have contributed to the differences. The early researchers reported bleeding times that were 1 to 2 h (time immediately following oviposition), 11.5 to 13 h, and 26 h (about time for the next oviposition) after oviposition. Paul and Snetsinger (1969) observed the highest plasma Ca++ 1 h post oviposition, falling slowly towards the later part of shell calcification, whereas the plasma P levels reached peak around 16 h post oviposition, in 60-week-old hens fed 2.5% Ca. Paul and Snetsinger (1969) reported bleeding times selected at 1, 9, 16, and 23 h post oviposition. Rao and Roland (1990) reported plasma peak iP at 14 h post oviposition, and plasma Ca++ peaks at 0 and 21 h post oviposition, in 30-week-old laying hens fed diets with 4% Ca and 0.6% total P for 3 d, with selected bleeding time periods of 0, 7, 14, and 21 h post oviposition. The Miller et al. (1977) report was based on 10 different time periods conducted in 3 trials during a 24-hour egg laying cycle, whereas Feinberg et al. (1937), Peterson and Parrish (1939), Paul and Snetsinger (1969), and Rao and Roland (1990) reports were based on 3, 3, 4, and 4 different time periods, respectively. The discrepancy in blood iP or Ca values during the egg formation cycle could be attributed to a seasonal factor (Peterson and Parrish, 1939), amount of dietary P and Ca, limestone solubility due to particle size, hen's Ca status (Rao and Roland, 1990), age factor, breed factor, and time of estimating blood P or Ca levels during egg laying cycle. Urine pH The pH of urine at both 2 and 6 wk (Figure 3a and 3b) was at its peak during 0 to 11 h post oviposition. The pH was lowest at 11 to 20 h post oviposition, and rose in 20 to 24 h collection. Rao and Roland (1990) reported pH peaked at 0 h (pH ∼ 7.7) and 7 h (pH ∼ 8.2) post oviposition. The decreased pH (lowest) was then noticed at 14 h (pH ∼ 4.5) post oviposition and again increased pH at 21 h (pH ∼ 6.4) post oviposition in 30-week-old white leghorn laying hens fed a diet containing 0.6% total P and 4% Ca for 3 days. The proportionate decrease in pH at 6 wk compared to 2 wk collection in the present study could be attributed to the reduced excretion of Ca, which is in support of the observation by Wideman et al. (1985) who reported increased urine pH with increased urinary Ca concentration. The pH during the first 11 h in both groups was higher and in proportion to increased urinary Ca concentration (Figure 3a and 3b). Later, during shell formation period, the concentration of urine started declining, and pH also started declining. The decline in urine pH also could be attributed to urinary acid buffering by phosphaturic response to PTH (Wolbach, 1955; Prashad and Edwards, 1973). During shell calcification, both hydrogen ions and carbonate ions are co-generated, creating metabolic acidosis. This metabolic acidosis is compensated by renal clearance of acid leading to decrease in urinary pH. In summary, feeding larger particle sized limestone compared to smaller particle sized limestone to 30-week-old normal broiler breeder hens for 6 wk resulted in a significant increase in tibia ash and specific gravity, whereas tP retention and tCa retention increased numerically, and excreta P decreased. Of the tP excreted from feces and urine for one egg laying cycle of 24 h, the urinary tP contribution for smaller and larger particle sized limestone fed colostomized broiler breeder hens was about 18%. Of the tCa excreted from both feces and urine for one egg laying cycle of 24 h, the urinary Ca contributions for smaller and larger particle sized limestone fed colostomized broiler breeder hens were similar at ∼9%. 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Poultry ScienceOxford University Press

Published: Jul 11, 2018

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