Relative bioavailability determination of highly concentrated cholecalciferol (vitamin D3) sources employing a broiler chick bioassay

Relative bioavailability determination of highly concentrated cholecalciferol (vitamin D3)... Abstract Cholecalciferol (D3) deficiency in animals and humans is associated with skeletal deformities and retarded growth. Hence, the evaluation of D3 sources is useful to ensure adequate biological activity. For this experiment, two sources of D3 were used to compare both biological activities using a chick bioassay. Newly hatched broiler chickens were caged in 2 battery brooders for a 17-day trial period. Experimental treatments were created from a common basal D3-deficient corn-soy broiler starter diet. The basal diet was supplemented with 0 (NC), 62.5, 125, 250, 500, or 1,000 IU D3/kg of feed. All birds were fed the basal diet devoid of D3 (NC) over the first 9 d to deplete the maternal D3, followed by a 12-hour fasting period. On d 10, the test diets were offered ad libitum for 7 days. Feed consumption, body weight (BW), and mortality were recorded to evaluate weight gain (WG) and feed efficiency (FE). Additionally, bone mineral content (BMC), bone mineral density (BMD), percent fat-free dried tibia ash (TBA), and tibia breaking strength (TBS) were used to evaluate bone mineralization. Relative bioavailability (RBV) of D3 was determined by the slope-ratio method using the powdered D3 as the reference standard source. Estimated RBV of the D3 beadlets source was 0.74, 0.75, 0.79, 0.80, 0.65, and 0.63 when BW, WG, TBA, TBS, BMC, and BMD were used as response criteria, respectively. In conclusion, this protocol was able to detect differences between highly concentrated D3 sources and may be employed to evaluate D3 biological activity. DESCRIPTION OF PROBLEM Cholecalciferol or vitamin D3 (D3) is obtained from the diet or produced in vivo by irradiation of the D3 provitamin 7-dehydrocholesterol found in the epidermal layers of the skin, where by exposure to the ultraviolet light, it is synthesized to D3 [1]. Dietary or endogenous synthesized D3 is transported via the blood to the liver where it is converted to 25-hydroxycholecalciferol (25-OH-D3), the major circulating form of vitamin D3. The newly formed 25-OH-D3 must then be transported to the kidneys for conversion into 1,25-dihydroxycholecalciferol (1,25(OH)2 D3) which is the fully active hormonal D3 metabolite [2]. The nutritional importance of D3 is to participate in the regulation of calcium and phosphorous homeostasis to assure proper metabolic functions and promote bone development [3]. Under natural conditions, vitamin D3 deficiency is not likely to occur since poultry are able to synthesize their own D3 from the sun. However, under intensive commercial poultry conditions where birds are raised in closed facilities and access to sunlight is limited, the risk of having skeletal disorders such as tibia dyschondroplasia or rickets [4] due to D3 deficiency is one of the most common problems in live production operations. Sullivan [5] estimated annual losses in the United States due to skeletal problems in broiler production of around $80 to $120 million USD, which adjusted to the annual inflation [6] would represent $134 to $200 million USD in 2017. For this reason, D3 supplements are widely used to formulate poultry feeds. Despite the number of dietary D3 sources currently available, not all of them will have the same biological potency or will be equally bioavailable. Yang et al. [7] reported very low correlations (below 60%) between the chemical and biological assay potency of vitamin D3 sources supplemented to turkey diets. Bioavailability determination of nutrients is extremely important to livestock nutrition. Estimating the bioavailability of nutrient sources allows nutritionists to formulate diets that precisely meet the animal's nutritional requirements [8, 9]. Comparison of biological activity between nutrient sources is usually expressed as relative bioavailability (RBV). This term is defined as “the ratio between the amount of the standard and testing source required to produce equivalent responses” [10, 9]. Bioassay protocols used to evaluate RBV of nutrients are based on dose-response data [11] in which a nutrient source is fed at different levels and then compared to a reference standard based on a biological response such as growth, bone mineralization, nutrient retention, etc. Bioavailability assays are influenced by many factors [9]. When D3 is used in poultry research, the maternal D3 carryover effect appears to play a key role in the response of the progeny to D3 supplementation. Moran [12] studied the effect of maternal deposition of nutrients in the egg yolk and found that any nutritional inadequacies in the parental flock will have a negative impact on the performance of the resulting chicks. Coto et al. [13] reported the presence of a distinct carryover effect from broiler breeders fed different combinations of D3 (0, 300, 600, 1,200, and 2,400 IU D3/kg) and 25-OH-D3 (0, 68 μg/kg), which influenced the performance, bone development, and incidence of tibia dyschondroplasia in newly hatched chicks. In a different experiment, Coto et al. [14] reported that dietary vitamin D3 in the breeder flock will affect egg shell thickness, egg production, and egg mass, which ultimately will affect performance of the progeny. Additionally, Saunders-Blades and Korver [15] reported that progeny performance was inconsistently influenced by supplemental D3 (3,000 IU/kg) in the diet or a combination of dietary D3 (3,000 IU/kg) and 25-OH-D3 (34.5 μg/Liter) supplemented to broiler breeders through drinking water. However, supplemental 25-OH-D3 reduced embryonic mortality and improved hatchability. In agreement with previous literature reports, research done in our laboratory has observed considerable variability in response to dietary vitamin D3. In some cases, it can be difficult to find a significant decrease in performance or bone mineralization as indicators of vitamin D3 biological activity in young broilers. Therefore, the present study was conducted with the objective of establishing an appropriate protocol that would reduce the effect of maternal D3 and would increase the sensitivity of our response variables to supplemental D3, allowing to better evaluate the RBV of highly concentrated dietary D3 sources in growing broiler chickens. MATERIALS AND METHODS Birds and General Management A total of 300 straight run Ross-308 newly hatched broiler chickens was purchased from a local commercial hatchery. At the time of arrival to the experimental facilities, birds were allocated to 2 Petersime battery brooders (≈7 birds per cage) inside an environmentally controlled rearing room. A basal D3-deficient corn-soy broiler starter diet (Table 1) was fed ad libitum for the first 9 d of the study to deplete the maternal stores of D3 followed by a 12-hour fasting period. On d 10 of the trial, birds were weighed in groups of 20 and average body weight (BW) was calculated. The average BW was used to create 48 groups of 5 chickens with close to “identical” BW and variance. All birds that showed abnormalities were discarded. The remaining birds (n = 240) were then randomly reallocated to the battery brooders and distributed into 11 dietary treatments (5 birds per pen and 4 pen replicates per treatment). Treatment diets were offered ad libitum for 7 days. Fluorescent 48-inch tube lamps covered with red shields were used to provide 24-hour constant light. The complete absence of UV light inside the rearing rooms has been previously verified by the Texas A&M Environmental Health and Safety Office [16] to prevent endogenous synthesis of D3. Water was offered ad libitum during the whole trial. Birds were monitored daily with regard to general flock condition, temperature, lighting, water, feed, and any unanticipated events for the rearing facility. All procedures in this experiment were approved by the Animal and Care Committee of Texas A&M University (IACUC 2017–0072). Table 1. Basal vitamin D3 deficient broiler starter diet. Ingredient  Basal diet1 (%)  Yellow corn  62.7  Dehulled soybean meal (48% CP)  32.1  DL-Methionine  0.15  Lysine-HCl  0.01  Corn oil  1.83  Limestone  1.06  Monoclacium phosphate  1.19  Sodium chloride (salt)  0.46  Customized vitamin-mineral premix2  0.50  Ingredient  Basal diet1 (%)  Yellow corn  62.7  Dehulled soybean meal (48% CP)  32.1  DL-Methionine  0.15  Lysine-HCl  0.01  Corn oil  1.83  Limestone  1.06  Monoclacium phosphate  1.19  Sodium chloride (salt)  0.46  Customized vitamin-mineral premix2  0.50  1Calculated nutritional content was as follows: 22% crude protein, 3,050 kcal/kg metabolizable energy, 0.75% calcium, 0.375% available phosphorous, 0.48% methionine, 0.85% methionine+cystine, 1.2% lysine, 0.26% tryptophan, 0.82% threonine, 1.46% arginine, 3.7% crude fat, 2.17% crude fiber, 0.2% sodium, 0.93% potassium, 0.3% chloride. 2Vitamin-mineral premix added at this rate yields per kg of diet: 10 mg copper, 2 mg iodine, 20 mg iron, 125 mg manganese, 125 mg zinc, 0.2 mg selenium, 8,000 IU vitamin A, 40 IU vitamin E, 2 mg menadione, 4 mg thiamine, 8 mg riboflavin, 60 mg niacin, 15 mg pantothenic acid, 4 mg pyridoxine, 0.18 mg biotin, 2 mg folic acid, 0.02 mg vitamin B12, 600 mg choline. View Large Table 1. Basal vitamin D3 deficient broiler starter diet. Ingredient  Basal diet1 (%)  Yellow corn  62.7  Dehulled soybean meal (48% CP)  32.1  DL-Methionine  0.15  Lysine-HCl  0.01  Corn oil  1.83  Limestone  1.06  Monoclacium phosphate  1.19  Sodium chloride (salt)  0.46  Customized vitamin-mineral premix2  0.50  Ingredient  Basal diet1 (%)  Yellow corn  62.7  Dehulled soybean meal (48% CP)  32.1  DL-Methionine  0.15  Lysine-HCl  0.01  Corn oil  1.83  Limestone  1.06  Monoclacium phosphate  1.19  Sodium chloride (salt)  0.46  Customized vitamin-mineral premix2  0.50  1Calculated nutritional content was as follows: 22% crude protein, 3,050 kcal/kg metabolizable energy, 0.75% calcium, 0.375% available phosphorous, 0.48% methionine, 0.85% methionine+cystine, 1.2% lysine, 0.26% tryptophan, 0.82% threonine, 1.46% arginine, 3.7% crude fat, 2.17% crude fiber, 0.2% sodium, 0.93% potassium, 0.3% chloride. 2Vitamin-mineral premix added at this rate yields per kg of diet: 10 mg copper, 2 mg iodine, 20 mg iron, 125 mg manganese, 125 mg zinc, 0.2 mg selenium, 8,000 IU vitamin A, 40 IU vitamin E, 2 mg menadione, 4 mg thiamine, 8 mg riboflavin, 60 mg niacin, 15 mg pantothenic acid, 4 mg pyridoxine, 0.18 mg biotin, 2 mg folic acid, 0.02 mg vitamin B12, 600 mg choline. View Large Dietary Treatments and Vitamin D3 Sources A basal mash corn-soy broiler starter diet devoid of D3 was formulated using a customized vitamin/mineral premix containing no D3 and corn oil as the fat source. To increase the sensitivity of our response variables to the experimental treatments, the basal diet was formulated with a marginal concentration of calcium (0.75%) and available phosphorous (0.37%). The basal diet was subdivided into 11 equally sized batches and supplemented with 0, 62.5, 125, 250, 500, or 1,000 IU D3/kg of diet based on the labeled concentration of 2 sources of D3. A spray-dried D3 powder (500,000 IU/g) and a beadlet type (500,000 IU/g) D3 source were used. To create the treatment diets, a specific amount of D3 from each source was weighed and directly mixed with the basal diet for 10 min using a stainless steel mixer [17]. The 0 IU D3/kg of diet treatment served as the common negative control (NC) group for both sources. Performance Evaluation Average BW and feed intake (FI) per pen were recorded on d 10 and 17 to calculate average weight gain (WG) and feed efficiency (FE). Mortality was recorded daily and used to adjust FE. Bone Mineralization Analysis On d 17 of the trial, all birds were euthanized using CO2, labeled, and immediately transported to the Texas A&M Applied Exercise Science Laboratory to perform a whole body analysis using a Prodigy Dual X-ray absorptiometry (DXA) scan [18]. Chickens were placed in prone position with their wings and legs at the sides of the body. Date were analyzed using the small animal software [18], which is specifically designed for animals <20 kg. Average total bone mineral content (BMC) and bone mineral density (BMD) per pen were calculated. After the DXA scan, both tibiae were removed, labeled, and stored in a freezer (–20°C) until further analysis. Right tibiae were gently boiled for 2 h and defatted in 4 L of petroleum ether for 48 hours. Defatted bones were then dried in a forced draft oven (95°C) until constant weight. Finally, the dried bones were ashed at 650°C for 23 hours. Percent tibia bone ash (TBA) was calculated based on starting dry bone weight and remaining ash. The left tibiae were cleaned from any adhering tissue and used to assay breaking strength (TBS) using a texture analyzer [19] charged with a 50 kg load cell, and a crosshead speed of 100 mm/min with the tibia supported on a 3-point bending ring and a 2.5 cm constant span. Statistical Analysis Data were analyzed as (2 × 5) 2-way ANOVA using the GLM procedure of SPSS [20]. Source, level, and source*level interaction were used as fixed factors in the model. Level main effects were analyzed using Duncan's multiple range tests. If a significant interaction was detected, means were re-analyzed as one-way ANOVA and then separated by Duncan's multiple range tests. Significance was accepted at P ≤ 0.05. Slope-ratio regression analysis was performed following the Littell et al. [21] procedure to determine RBV. The powdered D3 was used as the reference standard source (set at 100%) and the beadlet type as the test source. The 0 IU/kg D3 treatment served as a common negative control reference treatment. For this experiment, FI was not different (P > 0.05) across treatment diets. Therefore, RBV analysis was done using D3 concentration as the independent variable. To linearize the response function, D3 level was subjected to log10 transformation before checking validity of slope-ratio assumptions of linearity and equality of intercepts. If response variables failed to meet these assumptions, they were not used in RBV determination. RESULTS AND DISCUSSION Modern broiler strains are the result of yr of intensive genetic selection towards maximum FE and growth [22, 23]. This leads to the idea that older broiler strains have altered their physiology and capacity to respond to dietary nutrients. As detailed by Applegate and Angel [24], a big part of the nutrition research used to establish nutrient requirements can date as early as 1947. Therefore, it is necessary to re-evaluate and adapt strategies that allow nutritionists to precisely evaluate the ability of modern poultry strains to respond to different nutrient sources. Bioavailability determination of D3 supplements has historically relied on the AOAC (932.16) chick bioassay [25]. However, based on current feeding practices and previously mentioned changes in the nutritional response of modern broiler strains, the present protocol was designed with the objective of reducing external sources of variation and increasing the sensitivity of the response variables to highly concentrated D3 sources commonly used in formulation of poultry diets. For this purpose, we focused on reducing the effect of maternal D3 by establishing a 9-day depletion period and a 12-hour fasting. Previous research done in our laboratory suggests that broiler chicks will deplete maternal D3 stores at around 8 d post hatched (H. Leyva-Jimenez, unpublished data). The fasting period cleans the digestive tract and tends to standardize FI of treatment diets. Moreover, in agreement with previous literature reports [26–32], dietary calcium and available phosphorous concentration was lowered in reference to the NRC [33] nutrient recommendations to increase the sensibility of the response variables to supplemental D3, but an optimal 2:1 calcium ratio to available phosphorous was kept to promote normal bone development. On the other hand, following AOAC recommendations, chicks were caged in an environmentally controlled rearing room away from sunlight, and newly hatched chicks were used in this experiment. Additionally, corn oil was used as the fat source to formulate the D3-deficient basal diet to avoid potential contamination risk. No interaction (P > 0.05) or main effects were observed between source and level for any of the performance variables. However, an increasing trend in BW, WG, and FE was found with increasing concentration of D3 in the diet and when compared to the NC control group (Table 2). Mortality was not influenced by vitamin D3 supplementation. For bone mineralization results, no interaction (P > 0.05) was found between source and level to supplemental D3. But main effect differences (P < 0.05) were observed for TBA, TBS, and BMC (Table 3). Additionally, main level effects were observed for TBA, for which higher levels of D3 supplementation appear to improve bone mineralization. The supplementation of vitamin D3 to the broiler diets improved bone mineralization for both sources when compared to the NC group. No differences were found in BMD across dietary D3 supplementation. The results of this experiment show a decrease in performance and bone mineralization in the NC group when compared to the treatment diets for both D3 sources. This suggests that the 9-day depletion period was effective in reducing the carryover effect of maternal D3. Table 2. Effect of vitamin D3 supplementation on performance of 17-day-old broiler chickens. IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Body weight (g/bird)  STD  430  456  448  457  455  459  456  3  TEST    443  449  452  457  441  450    Total    449  448  454  457  450  452    PSEM    5      Weight gain (g/bird)  STD  266  295  288  294  294  296  293  3  TEST    285  284  290  291  283  287    Total    289  286  292  292  289  290    PSEM    5    Feed efficiency (g/g)  STD  0.68  0.70  0.71  0.73  0.73  0.73  0.72  0.003  TEST    0.71  0.72  0.71  0.71  0.70  0.71    Total    0.70  0.71  0.72  0.72  0.71  0.71    PSEM    0.007    Feed intake (g/bird)  STD  387  427  394  402  404  400  404  9  TEST    399  396  409  409  404  403    Total    411  395  405  406  402  404    PSEM    14    Mortality (%)  STD  0  5  0  0  0  0  0.83  1  TEST    0  0  0  0  0  0    Total    3  0  0  0  0  0.42    PSEM    1    IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Body weight (g/bird)  STD  430  456  448  457  455  459  456  3  TEST    443  449  452  457  441  450    Total    449  448  454  457  450  452    PSEM    5      Weight gain (g/bird)  STD  266  295  288  294  294  296  293  3  TEST    285  284  290  291  283  287    Total    289  286  292  292  289  290    PSEM    5    Feed efficiency (g/g)  STD  0.68  0.70  0.71  0.73  0.73  0.73  0.72  0.003  TEST    0.71  0.72  0.71  0.71  0.70  0.71    Total    0.70  0.71  0.72  0.72  0.71  0.71    PSEM    0.007    Feed intake (g/bird)  STD  387  427  394  402  404  400  404  9  TEST    399  396  409  409  404  403    Total    411  395  405  406  402  404    PSEM    14    Mortality (%)  STD  0  5  0  0  0  0  0.83  1  TEST    0  0  0  0  0  0    Total    3  0  0  0  0  0.42    PSEM    1    1Pooled standard error of the mean (PSEM). n = 8 (main level effects) and n = 20 (main source effects). ¥Reference standard (STD) and test source (TEST). View Large Table 2. Effect of vitamin D3 supplementation on performance of 17-day-old broiler chickens. IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Body weight (g/bird)  STD  430  456  448  457  455  459  456  3  TEST    443  449  452  457  441  450    Total    449  448  454  457  450  452    PSEM    5      Weight gain (g/bird)  STD  266  295  288  294  294  296  293  3  TEST    285  284  290  291  283  287    Total    289  286  292  292  289  290    PSEM    5    Feed efficiency (g/g)  STD  0.68  0.70  0.71  0.73  0.73  0.73  0.72  0.003  TEST    0.71  0.72  0.71  0.71  0.70  0.71    Total    0.70  0.71  0.72  0.72  0.71  0.71    PSEM    0.007    Feed intake (g/bird)  STD  387  427  394  402  404  400  404  9  TEST    399  396  409  409  404  403    Total    411  395  405  406  402  404    PSEM    14    Mortality (%)  STD  0  5  0  0  0  0  0.83  1  TEST    0  0  0  0  0  0    Total    3  0  0  0  0  0.42    PSEM    1    IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Body weight (g/bird)  STD  430  456  448  457  455  459  456  3  TEST    443  449  452  457  441  450    Total    449  448  454  457  450  452    PSEM    5      Weight gain (g/bird)  STD  266  295  288  294  294  296  293  3  TEST    285  284  290  291  283  287    Total    289  286  292  292  289  290    PSEM    5    Feed efficiency (g/g)  STD  0.68  0.70  0.71  0.73  0.73  0.73  0.72  0.003  TEST    0.71  0.72  0.71  0.71  0.70  0.71    Total    0.70  0.71  0.72  0.72  0.71  0.71    PSEM    0.007    Feed intake (g/bird)  STD  387  427  394  402  404  400  404  9  TEST    399  396  409  409  404  403    Total    411  395  405  406  402  404    PSEM    14    Mortality (%)  STD  0  5  0  0  0  0  0.83  1  TEST    0  0  0  0  0  0    Total    3  0  0  0  0  0.42    PSEM    1    1Pooled standard error of the mean (PSEM). n = 8 (main level effects) and n = 20 (main source effects). ¥Reference standard (STD) and test source (TEST). View Large Table 3. Effect of vitamin D3 supplementation on bone mineralization of 17-day-old broiler chickens. IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Tibia bone ash (%)  STD  43  47.4  47.3  48.3  48.1  48.8  48.0a  0.29  TEST    47.0  45.3  46.9  47.7  47.8  47.0b    Total    47a,b  46.5b  47.6a,b  47.9a,b  48.3a  47.5    PSEM    0.48      Tibia breaking strength (kg)  STD  6.1  7.8  7.5  7.6  7.9  8.4  7.9a  0.13  TEST    8.0  7.2  7.7  8.0  7.4  7.5b    Total    7.4  7.3  7.7  8.0  7.9  7.7    PSEM    0.21    Bone mineral content (g)  STD  1.7  2.45  2.11  2.11  2.47  2.43  2.30a  0.074  TEST    2.04  1.87  2.20  2.19  2.03  2.06b    Total    2.20  2.00  2.15  2.33  2.23  2.20    PSEM    0.12    Bone mineral density (g/cm2)  STD  0.044  0.060  0.053  0.050  0.060  0.060  0.056  0.002  TEST    0.053  0.047  0.053  0.053  0.052  0.051    Total    0.056  0.050  0.051  0.056  0.056  0.054    PSEM    0.003    IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Tibia bone ash (%)  STD  43  47.4  47.3  48.3  48.1  48.8  48.0a  0.29  TEST    47.0  45.3  46.9  47.7  47.8  47.0b    Total    47a,b  46.5b  47.6a,b  47.9a,b  48.3a  47.5    PSEM    0.48      Tibia breaking strength (kg)  STD  6.1  7.8  7.5  7.6  7.9  8.4  7.9a  0.13  TEST    8.0  7.2  7.7  8.0  7.4  7.5b    Total    7.4  7.3  7.7  8.0  7.9  7.7    PSEM    0.21    Bone mineral content (g)  STD  1.7  2.45  2.11  2.11  2.47  2.43  2.30a  0.074  TEST    2.04  1.87  2.20  2.19  2.03  2.06b    Total    2.20  2.00  2.15  2.33  2.23  2.20    PSEM    0.12    Bone mineral density (g/cm2)  STD  0.044  0.060  0.053  0.050  0.060  0.060  0.056  0.002  TEST    0.053  0.047  0.053  0.053  0.052  0.051    Total    0.056  0.050  0.051  0.056  0.056  0.054    PSEM    0.003    1Pooled standard error of the mean (PSEM). n = 8 (main level effects) and n = 20 (main source effects). a,bMeans within a row or column with no common superscript differ significantly (P < 0.05). ¥Reference standard (STD) and test source (TEST). View Large Table 3. Effect of vitamin D3 supplementation on bone mineralization of 17-day-old broiler chickens. IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Tibia bone ash (%)  STD  43  47.4  47.3  48.3  48.1  48.8  48.0a  0.29  TEST    47.0  45.3  46.9  47.7  47.8  47.0b    Total    47a,b  46.5b  47.6a,b  47.9a,b  48.3a  47.5    PSEM    0.48      Tibia breaking strength (kg)  STD  6.1  7.8  7.5  7.6  7.9  8.4  7.9a  0.13  TEST    8.0  7.2  7.7  8.0  7.4  7.5b    Total    7.4  7.3  7.7  8.0  7.9  7.7    PSEM    0.21    Bone mineral content (g)  STD  1.7  2.45  2.11  2.11  2.47  2.43  2.30a  0.074  TEST    2.04  1.87  2.20  2.19  2.03  2.06b    Total    2.20  2.00  2.15  2.33  2.23  2.20    PSEM    0.12    Bone mineral density (g/cm2)  STD  0.044  0.060  0.053  0.050  0.060  0.060  0.056  0.002  TEST    0.053  0.047  0.053  0.053  0.052  0.051    Total    0.056  0.050  0.051  0.056  0.056  0.054    PSEM    0.003    IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Tibia bone ash (%)  STD  43  47.4  47.3  48.3  48.1  48.8  48.0a  0.29  TEST    47.0  45.3  46.9  47.7  47.8  47.0b    Total    47a,b  46.5b  47.6a,b  47.9a,b  48.3a  47.5    PSEM    0.48      Tibia breaking strength (kg)  STD  6.1  7.8  7.5  7.6  7.9  8.4  7.9a  0.13  TEST    8.0  7.2  7.7  8.0  7.4  7.5b    Total    7.4  7.3  7.7  8.0  7.9  7.7    PSEM    0.21    Bone mineral content (g)  STD  1.7  2.45  2.11  2.11  2.47  2.43  2.30a  0.074  TEST    2.04  1.87  2.20  2.19  2.03  2.06b    Total    2.20  2.00  2.15  2.33  2.23  2.20    PSEM    0.12    Bone mineral density (g/cm2)  STD  0.044  0.060  0.053  0.050  0.060  0.060  0.056  0.002  TEST    0.053  0.047  0.053  0.053  0.052  0.051    Total    0.056  0.050  0.051  0.056  0.056  0.054    PSEM    0.003    1Pooled standard error of the mean (PSEM). n = 8 (main level effects) and n = 20 (main source effects). a,bMeans within a row or column with no common superscript differ significantly (P < 0.05). ¥Reference standard (STD) and test source (TEST). View Large Slope-ratio validation tests for performance and bone mineralization response variables are presented in Table 4. All variables except FE resulted in a linear relationship with equal slopes. Therefore, regression analysis was not performed using FE as a response criterion to estimate D3 biological activity. Relative bioavailability of the beadlet source to the reference standard was estimated to be 0.74, 0.75, 0.79, 0.80, 0.65, and 0.63 when BW, WG, TBA, TBS, BMC, and BMD were used as criteria to estimate D3 biological activity, respectively (Table 5). The average RBV of the test beadlet D3 was found to be 0.73 or 73% of the reference standard. Table 4. Slope-ratio assumption validation test.   Test (P-value)  Response  Linearity  Equality of intercepts  BW  0.826  0.946  WG  0.962  0.826  FE  0.753  0.013  TBA  0.630  0.556  TBS  0.281  0.570  BMC  0.445  0.985  BMD  0.440  0.977    Test (P-value)  Response  Linearity  Equality of intercepts  BW  0.826  0.946  WG  0.962  0.826  FE  0.753  0.013  TBA  0.630  0.556  TBS  0.281  0.570  BMC  0.445  0.985  BMD  0.440  0.977  Validation test was performed on body weight (BW), weight gain (WG), feed efficiency (FE), percent tibia bone ash (TBA), tibia breaking strength (TBS), bone mineral content (BMC), and bone mineral density (BMD). Failure to meet assumptions (P ≤ 0.05) indicates that model is not linear or has unequal intercepts and therefore slope-ratio analysis should not proceed. View Large Table 4. Slope-ratio assumption validation test.   Test (P-value)  Response  Linearity  Equality of intercepts  BW  0.826  0.946  WG  0.962  0.826  FE  0.753  0.013  TBA  0.630  0.556  TBS  0.281  0.570  BMC  0.445  0.985  BMD  0.440  0.977    Test (P-value)  Response  Linearity  Equality of intercepts  BW  0.826  0.946  WG  0.962  0.826  FE  0.753  0.013  TBA  0.630  0.556  TBS  0.281  0.570  BMC  0.445  0.985  BMD  0.440  0.977  Validation test was performed on body weight (BW), weight gain (WG), feed efficiency (FE), percent tibia bone ash (TBA), tibia breaking strength (TBS), bone mineral content (BMC), and bone mineral density (BMD). Failure to meet assumptions (P ≤ 0.05) indicates that model is not linear or has unequal intercepts and therefore slope-ratio analysis should not proceed. View Large Table 5. Relative bioavailability of beadlet type D3 to reference standard D3 based on D3 concentration (IU/kg diet) on 17-day-old broiler chickens.     Slope ± PSEM1        Response  Intercept  ¥STD  TEST  P-value  R2  *RBV  BW  431  9.882 ± 2.57  7.365 ± 2.57  0.002  0.27  0.74  WG  268  10.241 ± 2.30  7.670 ± 2.30  <0.01  0.33  0.75  TBA  42.2  1.952 ± 0.21  1.540 ± 0.21  <0.01  0.67  0.79  TBS  6.2  0.704 ± 0.10  0.512 ± .010  <0.01  0.53  0.80  BMC  1.698  0.248 ± 0.05  0.160 ± 0.05  <0.01  0.36  0.65  BMD  0.045  0.005 ± 0.01  0.003 ± 0.01  <0.01  0.26  0.63      Slope ± PSEM1        Response  Intercept  ¥STD  TEST  P-value  R2  *RBV  BW  431  9.882 ± 2.57  7.365 ± 2.57  0.002  0.27  0.74  WG  268  10.241 ± 2.30  7.670 ± 2.30  <0.01  0.33  0.75  TBA  42.2  1.952 ± 0.21  1.540 ± 0.21  <0.01  0.67  0.79  TBS  6.2  0.704 ± 0.10  0.512 ± .010  <0.01  0.53  0.80  BMC  1.698  0.248 ± 0.05  0.160 ± 0.05  <0.01  0.36  0.65  BMD  0.045  0.005 ± 0.01  0.003 ± 0.01  <0.01  0.26  0.63  Slope-ratio analysis was performed on body weight (BW), weight gain (WG), percent tibia bone ash (TBA), tibia breaking strength (TBS), bone mineral content (BMC), and bone mineral density (BMD) as criteria to determine relative bioavailability of supplemental D3 sources. *RBV = slope (TEST)/slope (STD). ¥Powdered reference standard (STD) and beadlet test source (TEST). 1Pooled standard error of the mean (PSEM). View Large Table 5. Relative bioavailability of beadlet type D3 to reference standard D3 based on D3 concentration (IU/kg diet) on 17-day-old broiler chickens.     Slope ± PSEM1        Response  Intercept  ¥STD  TEST  P-value  R2  *RBV  BW  431  9.882 ± 2.57  7.365 ± 2.57  0.002  0.27  0.74  WG  268  10.241 ± 2.30  7.670 ± 2.30  <0.01  0.33  0.75  TBA  42.2  1.952 ± 0.21  1.540 ± 0.21  <0.01  0.67  0.79  TBS  6.2  0.704 ± 0.10  0.512 ± .010  <0.01  0.53  0.80  BMC  1.698  0.248 ± 0.05  0.160 ± 0.05  <0.01  0.36  0.65  BMD  0.045  0.005 ± 0.01  0.003 ± 0.01  <0.01  0.26  0.63      Slope ± PSEM1        Response  Intercept  ¥STD  TEST  P-value  R2  *RBV  BW  431  9.882 ± 2.57  7.365 ± 2.57  0.002  0.27  0.74  WG  268  10.241 ± 2.30  7.670 ± 2.30  <0.01  0.33  0.75  TBA  42.2  1.952 ± 0.21  1.540 ± 0.21  <0.01  0.67  0.79  TBS  6.2  0.704 ± 0.10  0.512 ± .010  <0.01  0.53  0.80  BMC  1.698  0.248 ± 0.05  0.160 ± 0.05  <0.01  0.36  0.65  BMD  0.045  0.005 ± 0.01  0.003 ± 0.01  <0.01  0.26  0.63  Slope-ratio analysis was performed on body weight (BW), weight gain (WG), percent tibia bone ash (TBA), tibia breaking strength (TBS), bone mineral content (BMC), and bone mineral density (BMD) as criteria to determine relative bioavailability of supplemental D3 sources. *RBV = slope (TEST)/slope (STD). ¥Powdered reference standard (STD) and beadlet test source (TEST). 1Pooled standard error of the mean (PSEM). View Large In this D3 experiment, biological activity was evaluated using performance and bone mineralization measurements. However, TBA and TBS had the best coefficients of determination (R2), 0.67 and 0.53, respectively, and this suggests that TBA and TBS are better indicators of D3 biological activity when compared to performance. This is in agreement with previous studies that found measurements of bone quality, such as bone ash content or incidence of rickets, to be more sensitive indicators of D3 activity than performance responses [34–36]. On the other hand, bone density responses (BMC and BMD) had low R2 when compared with TBA and TBS. Although bone densitometers such as the DXA scan have been used to evaluate bone mineralization in poultry and have found high correlation with chemical and mechanical assays [37–41], in this particular case, age and weight of the birds might be influencing the DXA measurements, as suggested by Mitchell et al. [42] who found that agreement between DXA body scans and chemical assays was reduced with smaller birds (<2 kg). Despite low R2 registered for DXA responses, we believe DXA is a good non-invasive procedure that allows continuous assessment of bone development in young birds, but further research is needed to refine the scan protocol and increase the response accuracy to supplemental D3 sources. Relative bioavailability differences between D3 sources could be due to differences in particle size [43] that caused segregation during the mixing process, coating material of the beadlet [44, 45] that could potentially prevent D3 digestion and absorption, or lower biological activity [7] from the chemical activity reported by the manufacturer. CONCLUSIONS AND APPLICATIONS Estimated RBV of D3 beadlets source was 0.74, 0.75, 0.79, 0.80, 0.65, and 0.63 when BW, WG, TBA, TBS, BMC, and BMD were used as response criteria, respectively. 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Relative bioavailability determination of highly concentrated cholecalciferol (vitamin D3) sources employing a broiler chick bioassay

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Applied Poultry Science, Inc.
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© 2018 Poultry Science Association Inc.
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1056-6171
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1537-0437
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10.3382/japr/pfy007
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Abstract

Abstract Cholecalciferol (D3) deficiency in animals and humans is associated with skeletal deformities and retarded growth. Hence, the evaluation of D3 sources is useful to ensure adequate biological activity. For this experiment, two sources of D3 were used to compare both biological activities using a chick bioassay. Newly hatched broiler chickens were caged in 2 battery brooders for a 17-day trial period. Experimental treatments were created from a common basal D3-deficient corn-soy broiler starter diet. The basal diet was supplemented with 0 (NC), 62.5, 125, 250, 500, or 1,000 IU D3/kg of feed. All birds were fed the basal diet devoid of D3 (NC) over the first 9 d to deplete the maternal D3, followed by a 12-hour fasting period. On d 10, the test diets were offered ad libitum for 7 days. Feed consumption, body weight (BW), and mortality were recorded to evaluate weight gain (WG) and feed efficiency (FE). Additionally, bone mineral content (BMC), bone mineral density (BMD), percent fat-free dried tibia ash (TBA), and tibia breaking strength (TBS) were used to evaluate bone mineralization. Relative bioavailability (RBV) of D3 was determined by the slope-ratio method using the powdered D3 as the reference standard source. Estimated RBV of the D3 beadlets source was 0.74, 0.75, 0.79, 0.80, 0.65, and 0.63 when BW, WG, TBA, TBS, BMC, and BMD were used as response criteria, respectively. In conclusion, this protocol was able to detect differences between highly concentrated D3 sources and may be employed to evaluate D3 biological activity. DESCRIPTION OF PROBLEM Cholecalciferol or vitamin D3 (D3) is obtained from the diet or produced in vivo by irradiation of the D3 provitamin 7-dehydrocholesterol found in the epidermal layers of the skin, where by exposure to the ultraviolet light, it is synthesized to D3 [1]. Dietary or endogenous synthesized D3 is transported via the blood to the liver where it is converted to 25-hydroxycholecalciferol (25-OH-D3), the major circulating form of vitamin D3. The newly formed 25-OH-D3 must then be transported to the kidneys for conversion into 1,25-dihydroxycholecalciferol (1,25(OH)2 D3) which is the fully active hormonal D3 metabolite [2]. The nutritional importance of D3 is to participate in the regulation of calcium and phosphorous homeostasis to assure proper metabolic functions and promote bone development [3]. Under natural conditions, vitamin D3 deficiency is not likely to occur since poultry are able to synthesize their own D3 from the sun. However, under intensive commercial poultry conditions where birds are raised in closed facilities and access to sunlight is limited, the risk of having skeletal disorders such as tibia dyschondroplasia or rickets [4] due to D3 deficiency is one of the most common problems in live production operations. Sullivan [5] estimated annual losses in the United States due to skeletal problems in broiler production of around $80 to $120 million USD, which adjusted to the annual inflation [6] would represent $134 to $200 million USD in 2017. For this reason, D3 supplements are widely used to formulate poultry feeds. Despite the number of dietary D3 sources currently available, not all of them will have the same biological potency or will be equally bioavailable. Yang et al. [7] reported very low correlations (below 60%) between the chemical and biological assay potency of vitamin D3 sources supplemented to turkey diets. Bioavailability determination of nutrients is extremely important to livestock nutrition. Estimating the bioavailability of nutrient sources allows nutritionists to formulate diets that precisely meet the animal's nutritional requirements [8, 9]. Comparison of biological activity between nutrient sources is usually expressed as relative bioavailability (RBV). This term is defined as “the ratio between the amount of the standard and testing source required to produce equivalent responses” [10, 9]. Bioassay protocols used to evaluate RBV of nutrients are based on dose-response data [11] in which a nutrient source is fed at different levels and then compared to a reference standard based on a biological response such as growth, bone mineralization, nutrient retention, etc. Bioavailability assays are influenced by many factors [9]. When D3 is used in poultry research, the maternal D3 carryover effect appears to play a key role in the response of the progeny to D3 supplementation. Moran [12] studied the effect of maternal deposition of nutrients in the egg yolk and found that any nutritional inadequacies in the parental flock will have a negative impact on the performance of the resulting chicks. Coto et al. [13] reported the presence of a distinct carryover effect from broiler breeders fed different combinations of D3 (0, 300, 600, 1,200, and 2,400 IU D3/kg) and 25-OH-D3 (0, 68 μg/kg), which influenced the performance, bone development, and incidence of tibia dyschondroplasia in newly hatched chicks. In a different experiment, Coto et al. [14] reported that dietary vitamin D3 in the breeder flock will affect egg shell thickness, egg production, and egg mass, which ultimately will affect performance of the progeny. Additionally, Saunders-Blades and Korver [15] reported that progeny performance was inconsistently influenced by supplemental D3 (3,000 IU/kg) in the diet or a combination of dietary D3 (3,000 IU/kg) and 25-OH-D3 (34.5 μg/Liter) supplemented to broiler breeders through drinking water. However, supplemental 25-OH-D3 reduced embryonic mortality and improved hatchability. In agreement with previous literature reports, research done in our laboratory has observed considerable variability in response to dietary vitamin D3. In some cases, it can be difficult to find a significant decrease in performance or bone mineralization as indicators of vitamin D3 biological activity in young broilers. Therefore, the present study was conducted with the objective of establishing an appropriate protocol that would reduce the effect of maternal D3 and would increase the sensitivity of our response variables to supplemental D3, allowing to better evaluate the RBV of highly concentrated dietary D3 sources in growing broiler chickens. MATERIALS AND METHODS Birds and General Management A total of 300 straight run Ross-308 newly hatched broiler chickens was purchased from a local commercial hatchery. At the time of arrival to the experimental facilities, birds were allocated to 2 Petersime battery brooders (≈7 birds per cage) inside an environmentally controlled rearing room. A basal D3-deficient corn-soy broiler starter diet (Table 1) was fed ad libitum for the first 9 d of the study to deplete the maternal stores of D3 followed by a 12-hour fasting period. On d 10 of the trial, birds were weighed in groups of 20 and average body weight (BW) was calculated. The average BW was used to create 48 groups of 5 chickens with close to “identical” BW and variance. All birds that showed abnormalities were discarded. The remaining birds (n = 240) were then randomly reallocated to the battery brooders and distributed into 11 dietary treatments (5 birds per pen and 4 pen replicates per treatment). Treatment diets were offered ad libitum for 7 days. Fluorescent 48-inch tube lamps covered with red shields were used to provide 24-hour constant light. The complete absence of UV light inside the rearing rooms has been previously verified by the Texas A&M Environmental Health and Safety Office [16] to prevent endogenous synthesis of D3. Water was offered ad libitum during the whole trial. Birds were monitored daily with regard to general flock condition, temperature, lighting, water, feed, and any unanticipated events for the rearing facility. All procedures in this experiment were approved by the Animal and Care Committee of Texas A&M University (IACUC 2017–0072). Table 1. Basal vitamin D3 deficient broiler starter diet. Ingredient  Basal diet1 (%)  Yellow corn  62.7  Dehulled soybean meal (48% CP)  32.1  DL-Methionine  0.15  Lysine-HCl  0.01  Corn oil  1.83  Limestone  1.06  Monoclacium phosphate  1.19  Sodium chloride (salt)  0.46  Customized vitamin-mineral premix2  0.50  Ingredient  Basal diet1 (%)  Yellow corn  62.7  Dehulled soybean meal (48% CP)  32.1  DL-Methionine  0.15  Lysine-HCl  0.01  Corn oil  1.83  Limestone  1.06  Monoclacium phosphate  1.19  Sodium chloride (salt)  0.46  Customized vitamin-mineral premix2  0.50  1Calculated nutritional content was as follows: 22% crude protein, 3,050 kcal/kg metabolizable energy, 0.75% calcium, 0.375% available phosphorous, 0.48% methionine, 0.85% methionine+cystine, 1.2% lysine, 0.26% tryptophan, 0.82% threonine, 1.46% arginine, 3.7% crude fat, 2.17% crude fiber, 0.2% sodium, 0.93% potassium, 0.3% chloride. 2Vitamin-mineral premix added at this rate yields per kg of diet: 10 mg copper, 2 mg iodine, 20 mg iron, 125 mg manganese, 125 mg zinc, 0.2 mg selenium, 8,000 IU vitamin A, 40 IU vitamin E, 2 mg menadione, 4 mg thiamine, 8 mg riboflavin, 60 mg niacin, 15 mg pantothenic acid, 4 mg pyridoxine, 0.18 mg biotin, 2 mg folic acid, 0.02 mg vitamin B12, 600 mg choline. View Large Table 1. Basal vitamin D3 deficient broiler starter diet. Ingredient  Basal diet1 (%)  Yellow corn  62.7  Dehulled soybean meal (48% CP)  32.1  DL-Methionine  0.15  Lysine-HCl  0.01  Corn oil  1.83  Limestone  1.06  Monoclacium phosphate  1.19  Sodium chloride (salt)  0.46  Customized vitamin-mineral premix2  0.50  Ingredient  Basal diet1 (%)  Yellow corn  62.7  Dehulled soybean meal (48% CP)  32.1  DL-Methionine  0.15  Lysine-HCl  0.01  Corn oil  1.83  Limestone  1.06  Monoclacium phosphate  1.19  Sodium chloride (salt)  0.46  Customized vitamin-mineral premix2  0.50  1Calculated nutritional content was as follows: 22% crude protein, 3,050 kcal/kg metabolizable energy, 0.75% calcium, 0.375% available phosphorous, 0.48% methionine, 0.85% methionine+cystine, 1.2% lysine, 0.26% tryptophan, 0.82% threonine, 1.46% arginine, 3.7% crude fat, 2.17% crude fiber, 0.2% sodium, 0.93% potassium, 0.3% chloride. 2Vitamin-mineral premix added at this rate yields per kg of diet: 10 mg copper, 2 mg iodine, 20 mg iron, 125 mg manganese, 125 mg zinc, 0.2 mg selenium, 8,000 IU vitamin A, 40 IU vitamin E, 2 mg menadione, 4 mg thiamine, 8 mg riboflavin, 60 mg niacin, 15 mg pantothenic acid, 4 mg pyridoxine, 0.18 mg biotin, 2 mg folic acid, 0.02 mg vitamin B12, 600 mg choline. View Large Dietary Treatments and Vitamin D3 Sources A basal mash corn-soy broiler starter diet devoid of D3 was formulated using a customized vitamin/mineral premix containing no D3 and corn oil as the fat source. To increase the sensitivity of our response variables to the experimental treatments, the basal diet was formulated with a marginal concentration of calcium (0.75%) and available phosphorous (0.37%). The basal diet was subdivided into 11 equally sized batches and supplemented with 0, 62.5, 125, 250, 500, or 1,000 IU D3/kg of diet based on the labeled concentration of 2 sources of D3. A spray-dried D3 powder (500,000 IU/g) and a beadlet type (500,000 IU/g) D3 source were used. To create the treatment diets, a specific amount of D3 from each source was weighed and directly mixed with the basal diet for 10 min using a stainless steel mixer [17]. The 0 IU D3/kg of diet treatment served as the common negative control (NC) group for both sources. Performance Evaluation Average BW and feed intake (FI) per pen were recorded on d 10 and 17 to calculate average weight gain (WG) and feed efficiency (FE). Mortality was recorded daily and used to adjust FE. Bone Mineralization Analysis On d 17 of the trial, all birds were euthanized using CO2, labeled, and immediately transported to the Texas A&M Applied Exercise Science Laboratory to perform a whole body analysis using a Prodigy Dual X-ray absorptiometry (DXA) scan [18]. Chickens were placed in prone position with their wings and legs at the sides of the body. Date were analyzed using the small animal software [18], which is specifically designed for animals <20 kg. Average total bone mineral content (BMC) and bone mineral density (BMD) per pen were calculated. After the DXA scan, both tibiae were removed, labeled, and stored in a freezer (–20°C) until further analysis. Right tibiae were gently boiled for 2 h and defatted in 4 L of petroleum ether for 48 hours. Defatted bones were then dried in a forced draft oven (95°C) until constant weight. Finally, the dried bones were ashed at 650°C for 23 hours. Percent tibia bone ash (TBA) was calculated based on starting dry bone weight and remaining ash. The left tibiae were cleaned from any adhering tissue and used to assay breaking strength (TBS) using a texture analyzer [19] charged with a 50 kg load cell, and a crosshead speed of 100 mm/min with the tibia supported on a 3-point bending ring and a 2.5 cm constant span. Statistical Analysis Data were analyzed as (2 × 5) 2-way ANOVA using the GLM procedure of SPSS [20]. Source, level, and source*level interaction were used as fixed factors in the model. Level main effects were analyzed using Duncan's multiple range tests. If a significant interaction was detected, means were re-analyzed as one-way ANOVA and then separated by Duncan's multiple range tests. Significance was accepted at P ≤ 0.05. Slope-ratio regression analysis was performed following the Littell et al. [21] procedure to determine RBV. The powdered D3 was used as the reference standard source (set at 100%) and the beadlet type as the test source. The 0 IU/kg D3 treatment served as a common negative control reference treatment. For this experiment, FI was not different (P > 0.05) across treatment diets. Therefore, RBV analysis was done using D3 concentration as the independent variable. To linearize the response function, D3 level was subjected to log10 transformation before checking validity of slope-ratio assumptions of linearity and equality of intercepts. If response variables failed to meet these assumptions, they were not used in RBV determination. RESULTS AND DISCUSSION Modern broiler strains are the result of yr of intensive genetic selection towards maximum FE and growth [22, 23]. This leads to the idea that older broiler strains have altered their physiology and capacity to respond to dietary nutrients. As detailed by Applegate and Angel [24], a big part of the nutrition research used to establish nutrient requirements can date as early as 1947. Therefore, it is necessary to re-evaluate and adapt strategies that allow nutritionists to precisely evaluate the ability of modern poultry strains to respond to different nutrient sources. Bioavailability determination of D3 supplements has historically relied on the AOAC (932.16) chick bioassay [25]. However, based on current feeding practices and previously mentioned changes in the nutritional response of modern broiler strains, the present protocol was designed with the objective of reducing external sources of variation and increasing the sensitivity of the response variables to highly concentrated D3 sources commonly used in formulation of poultry diets. For this purpose, we focused on reducing the effect of maternal D3 by establishing a 9-day depletion period and a 12-hour fasting. Previous research done in our laboratory suggests that broiler chicks will deplete maternal D3 stores at around 8 d post hatched (H. Leyva-Jimenez, unpublished data). The fasting period cleans the digestive tract and tends to standardize FI of treatment diets. Moreover, in agreement with previous literature reports [26–32], dietary calcium and available phosphorous concentration was lowered in reference to the NRC [33] nutrient recommendations to increase the sensibility of the response variables to supplemental D3, but an optimal 2:1 calcium ratio to available phosphorous was kept to promote normal bone development. On the other hand, following AOAC recommendations, chicks were caged in an environmentally controlled rearing room away from sunlight, and newly hatched chicks were used in this experiment. Additionally, corn oil was used as the fat source to formulate the D3-deficient basal diet to avoid potential contamination risk. No interaction (P > 0.05) or main effects were observed between source and level for any of the performance variables. However, an increasing trend in BW, WG, and FE was found with increasing concentration of D3 in the diet and when compared to the NC control group (Table 2). Mortality was not influenced by vitamin D3 supplementation. For bone mineralization results, no interaction (P > 0.05) was found between source and level to supplemental D3. But main effect differences (P < 0.05) were observed for TBA, TBS, and BMC (Table 3). Additionally, main level effects were observed for TBA, for which higher levels of D3 supplementation appear to improve bone mineralization. The supplementation of vitamin D3 to the broiler diets improved bone mineralization for both sources when compared to the NC group. No differences were found in BMD across dietary D3 supplementation. The results of this experiment show a decrease in performance and bone mineralization in the NC group when compared to the treatment diets for both D3 sources. This suggests that the 9-day depletion period was effective in reducing the carryover effect of maternal D3. Table 2. Effect of vitamin D3 supplementation on performance of 17-day-old broiler chickens. IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Body weight (g/bird)  STD  430  456  448  457  455  459  456  3  TEST    443  449  452  457  441  450    Total    449  448  454  457  450  452    PSEM    5      Weight gain (g/bird)  STD  266  295  288  294  294  296  293  3  TEST    285  284  290  291  283  287    Total    289  286  292  292  289  290    PSEM    5    Feed efficiency (g/g)  STD  0.68  0.70  0.71  0.73  0.73  0.73  0.72  0.003  TEST    0.71  0.72  0.71  0.71  0.70  0.71    Total    0.70  0.71  0.72  0.72  0.71  0.71    PSEM    0.007    Feed intake (g/bird)  STD  387  427  394  402  404  400  404  9  TEST    399  396  409  409  404  403    Total    411  395  405  406  402  404    PSEM    14    Mortality (%)  STD  0  5  0  0  0  0  0.83  1  TEST    0  0  0  0  0  0    Total    3  0  0  0  0  0.42    PSEM    1    IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Body weight (g/bird)  STD  430  456  448  457  455  459  456  3  TEST    443  449  452  457  441  450    Total    449  448  454  457  450  452    PSEM    5      Weight gain (g/bird)  STD  266  295  288  294  294  296  293  3  TEST    285  284  290  291  283  287    Total    289  286  292  292  289  290    PSEM    5    Feed efficiency (g/g)  STD  0.68  0.70  0.71  0.73  0.73  0.73  0.72  0.003  TEST    0.71  0.72  0.71  0.71  0.70  0.71    Total    0.70  0.71  0.72  0.72  0.71  0.71    PSEM    0.007    Feed intake (g/bird)  STD  387  427  394  402  404  400  404  9  TEST    399  396  409  409  404  403    Total    411  395  405  406  402  404    PSEM    14    Mortality (%)  STD  0  5  0  0  0  0  0.83  1  TEST    0  0  0  0  0  0    Total    3  0  0  0  0  0.42    PSEM    1    1Pooled standard error of the mean (PSEM). n = 8 (main level effects) and n = 20 (main source effects). ¥Reference standard (STD) and test source (TEST). View Large Table 2. Effect of vitamin D3 supplementation on performance of 17-day-old broiler chickens. IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Body weight (g/bird)  STD  430  456  448  457  455  459  456  3  TEST    443  449  452  457  441  450    Total    449  448  454  457  450  452    PSEM    5      Weight gain (g/bird)  STD  266  295  288  294  294  296  293  3  TEST    285  284  290  291  283  287    Total    289  286  292  292  289  290    PSEM    5    Feed efficiency (g/g)  STD  0.68  0.70  0.71  0.73  0.73  0.73  0.72  0.003  TEST    0.71  0.72  0.71  0.71  0.70  0.71    Total    0.70  0.71  0.72  0.72  0.71  0.71    PSEM    0.007    Feed intake (g/bird)  STD  387  427  394  402  404  400  404  9  TEST    399  396  409  409  404  403    Total    411  395  405  406  402  404    PSEM    14    Mortality (%)  STD  0  5  0  0  0  0  0.83  1  TEST    0  0  0  0  0  0    Total    3  0  0  0  0  0.42    PSEM    1    IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Body weight (g/bird)  STD  430  456  448  457  455  459  456  3  TEST    443  449  452  457  441  450    Total    449  448  454  457  450  452    PSEM    5      Weight gain (g/bird)  STD  266  295  288  294  294  296  293  3  TEST    285  284  290  291  283  287    Total    289  286  292  292  289  290    PSEM    5    Feed efficiency (g/g)  STD  0.68  0.70  0.71  0.73  0.73  0.73  0.72  0.003  TEST    0.71  0.72  0.71  0.71  0.70  0.71    Total    0.70  0.71  0.72  0.72  0.71  0.71    PSEM    0.007    Feed intake (g/bird)  STD  387  427  394  402  404  400  404  9  TEST    399  396  409  409  404  403    Total    411  395  405  406  402  404    PSEM    14    Mortality (%)  STD  0  5  0  0  0  0  0.83  1  TEST    0  0  0  0  0  0    Total    3  0  0  0  0  0.42    PSEM    1    1Pooled standard error of the mean (PSEM). n = 8 (main level effects) and n = 20 (main source effects). ¥Reference standard (STD) and test source (TEST). View Large Table 3. Effect of vitamin D3 supplementation on bone mineralization of 17-day-old broiler chickens. IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Tibia bone ash (%)  STD  43  47.4  47.3  48.3  48.1  48.8  48.0a  0.29  TEST    47.0  45.3  46.9  47.7  47.8  47.0b    Total    47a,b  46.5b  47.6a,b  47.9a,b  48.3a  47.5    PSEM    0.48      Tibia breaking strength (kg)  STD  6.1  7.8  7.5  7.6  7.9  8.4  7.9a  0.13  TEST    8.0  7.2  7.7  8.0  7.4  7.5b    Total    7.4  7.3  7.7  8.0  7.9  7.7    PSEM    0.21    Bone mineral content (g)  STD  1.7  2.45  2.11  2.11  2.47  2.43  2.30a  0.074  TEST    2.04  1.87  2.20  2.19  2.03  2.06b    Total    2.20  2.00  2.15  2.33  2.23  2.20    PSEM    0.12    Bone mineral density (g/cm2)  STD  0.044  0.060  0.053  0.050  0.060  0.060  0.056  0.002  TEST    0.053  0.047  0.053  0.053  0.052  0.051    Total    0.056  0.050  0.051  0.056  0.056  0.054    PSEM    0.003    IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Tibia bone ash (%)  STD  43  47.4  47.3  48.3  48.1  48.8  48.0a  0.29  TEST    47.0  45.3  46.9  47.7  47.8  47.0b    Total    47a,b  46.5b  47.6a,b  47.9a,b  48.3a  47.5    PSEM    0.48      Tibia breaking strength (kg)  STD  6.1  7.8  7.5  7.6  7.9  8.4  7.9a  0.13  TEST    8.0  7.2  7.7  8.0  7.4  7.5b    Total    7.4  7.3  7.7  8.0  7.9  7.7    PSEM    0.21    Bone mineral content (g)  STD  1.7  2.45  2.11  2.11  2.47  2.43  2.30a  0.074  TEST    2.04  1.87  2.20  2.19  2.03  2.06b    Total    2.20  2.00  2.15  2.33  2.23  2.20    PSEM    0.12    Bone mineral density (g/cm2)  STD  0.044  0.060  0.053  0.050  0.060  0.060  0.056  0.002  TEST    0.053  0.047  0.053  0.053  0.052  0.051    Total    0.056  0.050  0.051  0.056  0.056  0.054    PSEM    0.003    1Pooled standard error of the mean (PSEM). n = 8 (main level effects) and n = 20 (main source effects). a,bMeans within a row or column with no common superscript differ significantly (P < 0.05). ¥Reference standard (STD) and test source (TEST). View Large Table 3. Effect of vitamin D3 supplementation on bone mineralization of 17-day-old broiler chickens. IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Tibia bone ash (%)  STD  43  47.4  47.3  48.3  48.1  48.8  48.0a  0.29  TEST    47.0  45.3  46.9  47.7  47.8  47.0b    Total    47a,b  46.5b  47.6a,b  47.9a,b  48.3a  47.5    PSEM    0.48      Tibia breaking strength (kg)  STD  6.1  7.8  7.5  7.6  7.9  8.4  7.9a  0.13  TEST    8.0  7.2  7.7  8.0  7.4  7.5b    Total    7.4  7.3  7.7  8.0  7.9  7.7    PSEM    0.21    Bone mineral content (g)  STD  1.7  2.45  2.11  2.11  2.47  2.43  2.30a  0.074  TEST    2.04  1.87  2.20  2.19  2.03  2.06b    Total    2.20  2.00  2.15  2.33  2.23  2.20    PSEM    0.12    Bone mineral density (g/cm2)  STD  0.044  0.060  0.053  0.050  0.060  0.060  0.056  0.002  TEST    0.053  0.047  0.053  0.053  0.052  0.051    Total    0.056  0.050  0.051  0.056  0.056  0.054    PSEM    0.003    IU D3/kg of feed  Source¥  0  62.5  125  250  500  1,000  Total  PSEM1  Tibia bone ash (%)  STD  43  47.4  47.3  48.3  48.1  48.8  48.0a  0.29  TEST    47.0  45.3  46.9  47.7  47.8  47.0b    Total    47a,b  46.5b  47.6a,b  47.9a,b  48.3a  47.5    PSEM    0.48      Tibia breaking strength (kg)  STD  6.1  7.8  7.5  7.6  7.9  8.4  7.9a  0.13  TEST    8.0  7.2  7.7  8.0  7.4  7.5b    Total    7.4  7.3  7.7  8.0  7.9  7.7    PSEM    0.21    Bone mineral content (g)  STD  1.7  2.45  2.11  2.11  2.47  2.43  2.30a  0.074  TEST    2.04  1.87  2.20  2.19  2.03  2.06b    Total    2.20  2.00  2.15  2.33  2.23  2.20    PSEM    0.12    Bone mineral density (g/cm2)  STD  0.044  0.060  0.053  0.050  0.060  0.060  0.056  0.002  TEST    0.053  0.047  0.053  0.053  0.052  0.051    Total    0.056  0.050  0.051  0.056  0.056  0.054    PSEM    0.003    1Pooled standard error of the mean (PSEM). n = 8 (main level effects) and n = 20 (main source effects). a,bMeans within a row or column with no common superscript differ significantly (P < 0.05). ¥Reference standard (STD) and test source (TEST). View Large Slope-ratio validation tests for performance and bone mineralization response variables are presented in Table 4. All variables except FE resulted in a linear relationship with equal slopes. Therefore, regression analysis was not performed using FE as a response criterion to estimate D3 biological activity. Relative bioavailability of the beadlet source to the reference standard was estimated to be 0.74, 0.75, 0.79, 0.80, 0.65, and 0.63 when BW, WG, TBA, TBS, BMC, and BMD were used as criteria to estimate D3 biological activity, respectively (Table 5). The average RBV of the test beadlet D3 was found to be 0.73 or 73% of the reference standard. Table 4. Slope-ratio assumption validation test.   Test (P-value)  Response  Linearity  Equality of intercepts  BW  0.826  0.946  WG  0.962  0.826  FE  0.753  0.013  TBA  0.630  0.556  TBS  0.281  0.570  BMC  0.445  0.985  BMD  0.440  0.977    Test (P-value)  Response  Linearity  Equality of intercepts  BW  0.826  0.946  WG  0.962  0.826  FE  0.753  0.013  TBA  0.630  0.556  TBS  0.281  0.570  BMC  0.445  0.985  BMD  0.440  0.977  Validation test was performed on body weight (BW), weight gain (WG), feed efficiency (FE), percent tibia bone ash (TBA), tibia breaking strength (TBS), bone mineral content (BMC), and bone mineral density (BMD). Failure to meet assumptions (P ≤ 0.05) indicates that model is not linear or has unequal intercepts and therefore slope-ratio analysis should not proceed. View Large Table 4. Slope-ratio assumption validation test.   Test (P-value)  Response  Linearity  Equality of intercepts  BW  0.826  0.946  WG  0.962  0.826  FE  0.753  0.013  TBA  0.630  0.556  TBS  0.281  0.570  BMC  0.445  0.985  BMD  0.440  0.977    Test (P-value)  Response  Linearity  Equality of intercepts  BW  0.826  0.946  WG  0.962  0.826  FE  0.753  0.013  TBA  0.630  0.556  TBS  0.281  0.570  BMC  0.445  0.985  BMD  0.440  0.977  Validation test was performed on body weight (BW), weight gain (WG), feed efficiency (FE), percent tibia bone ash (TBA), tibia breaking strength (TBS), bone mineral content (BMC), and bone mineral density (BMD). Failure to meet assumptions (P ≤ 0.05) indicates that model is not linear or has unequal intercepts and therefore slope-ratio analysis should not proceed. View Large Table 5. Relative bioavailability of beadlet type D3 to reference standard D3 based on D3 concentration (IU/kg diet) on 17-day-old broiler chickens.     Slope ± PSEM1        Response  Intercept  ¥STD  TEST  P-value  R2  *RBV  BW  431  9.882 ± 2.57  7.365 ± 2.57  0.002  0.27  0.74  WG  268  10.241 ± 2.30  7.670 ± 2.30  <0.01  0.33  0.75  TBA  42.2  1.952 ± 0.21  1.540 ± 0.21  <0.01  0.67  0.79  TBS  6.2  0.704 ± 0.10  0.512 ± .010  <0.01  0.53  0.80  BMC  1.698  0.248 ± 0.05  0.160 ± 0.05  <0.01  0.36  0.65  BMD  0.045  0.005 ± 0.01  0.003 ± 0.01  <0.01  0.26  0.63      Slope ± PSEM1        Response  Intercept  ¥STD  TEST  P-value  R2  *RBV  BW  431  9.882 ± 2.57  7.365 ± 2.57  0.002  0.27  0.74  WG  268  10.241 ± 2.30  7.670 ± 2.30  <0.01  0.33  0.75  TBA  42.2  1.952 ± 0.21  1.540 ± 0.21  <0.01  0.67  0.79  TBS  6.2  0.704 ± 0.10  0.512 ± .010  <0.01  0.53  0.80  BMC  1.698  0.248 ± 0.05  0.160 ± 0.05  <0.01  0.36  0.65  BMD  0.045  0.005 ± 0.01  0.003 ± 0.01  <0.01  0.26  0.63  Slope-ratio analysis was performed on body weight (BW), weight gain (WG), percent tibia bone ash (TBA), tibia breaking strength (TBS), bone mineral content (BMC), and bone mineral density (BMD) as criteria to determine relative bioavailability of supplemental D3 sources. *RBV = slope (TEST)/slope (STD). ¥Powdered reference standard (STD) and beadlet test source (TEST). 1Pooled standard error of the mean (PSEM). View Large Table 5. Relative bioavailability of beadlet type D3 to reference standard D3 based on D3 concentration (IU/kg diet) on 17-day-old broiler chickens.     Slope ± PSEM1        Response  Intercept  ¥STD  TEST  P-value  R2  *RBV  BW  431  9.882 ± 2.57  7.365 ± 2.57  0.002  0.27  0.74  WG  268  10.241 ± 2.30  7.670 ± 2.30  <0.01  0.33  0.75  TBA  42.2  1.952 ± 0.21  1.540 ± 0.21  <0.01  0.67  0.79  TBS  6.2  0.704 ± 0.10  0.512 ± .010  <0.01  0.53  0.80  BMC  1.698  0.248 ± 0.05  0.160 ± 0.05  <0.01  0.36  0.65  BMD  0.045  0.005 ± 0.01  0.003 ± 0.01  <0.01  0.26  0.63      Slope ± PSEM1        Response  Intercept  ¥STD  TEST  P-value  R2  *RBV  BW  431  9.882 ± 2.57  7.365 ± 2.57  0.002  0.27  0.74  WG  268  10.241 ± 2.30  7.670 ± 2.30  <0.01  0.33  0.75  TBA  42.2  1.952 ± 0.21  1.540 ± 0.21  <0.01  0.67  0.79  TBS  6.2  0.704 ± 0.10  0.512 ± .010  <0.01  0.53  0.80  BMC  1.698  0.248 ± 0.05  0.160 ± 0.05  <0.01  0.36  0.65  BMD  0.045  0.005 ± 0.01  0.003 ± 0.01  <0.01  0.26  0.63  Slope-ratio analysis was performed on body weight (BW), weight gain (WG), percent tibia bone ash (TBA), tibia breaking strength (TBS), bone mineral content (BMC), and bone mineral density (BMD) as criteria to determine relative bioavailability of supplemental D3 sources. *RBV = slope (TEST)/slope (STD). ¥Powdered reference standard (STD) and beadlet test source (TEST). 1Pooled standard error of the mean (PSEM). View Large In this D3 experiment, biological activity was evaluated using performance and bone mineralization measurements. However, TBA and TBS had the best coefficients of determination (R2), 0.67 and 0.53, respectively, and this suggests that TBA and TBS are better indicators of D3 biological activity when compared to performance. This is in agreement with previous studies that found measurements of bone quality, such as bone ash content or incidence of rickets, to be more sensitive indicators of D3 activity than performance responses [34–36]. On the other hand, bone density responses (BMC and BMD) had low R2 when compared with TBA and TBS. Although bone densitometers such as the DXA scan have been used to evaluate bone mineralization in poultry and have found high correlation with chemical and mechanical assays [37–41], in this particular case, age and weight of the birds might be influencing the DXA measurements, as suggested by Mitchell et al. [42] who found that agreement between DXA body scans and chemical assays was reduced with smaller birds (<2 kg). Despite low R2 registered for DXA responses, we believe DXA is a good non-invasive procedure that allows continuous assessment of bone development in young birds, but further research is needed to refine the scan protocol and increase the response accuracy to supplemental D3 sources. Relative bioavailability differences between D3 sources could be due to differences in particle size [43] that caused segregation during the mixing process, coating material of the beadlet [44, 45] that could potentially prevent D3 digestion and absorption, or lower biological activity [7] from the chemical activity reported by the manufacturer. CONCLUSIONS AND APPLICATIONS Estimated RBV of D3 beadlets source was 0.74, 0.75, 0.79, 0.80, 0.65, and 0.63 when BW, WG, TBA, TBS, BMC, and BMD were used as response criteria, respectively. 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Journal of Applied Poultry ResearchOxford University Press

Published: Mar 5, 2018

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