Influence of dietary 1 α lpha-hydroxycholecalciferol, individually or in combination with microbial phytase in calcium and phosphorus deficient diets on growth performance and tibia parameter of Japanese quails ( Coturnix japonica )

. The effect of 1 α -OH-D 3 in calcium-phosphorus (Ca-P) deficient diets on Japanese quail growth performance and tibia parameters was investigated. Eight-day-old ( n = 160) newly hatched quails were weighed and randomly allocated to 20 groups, each with 4 replicate pens of 8 birds. Treatments were as follows: T 1 , Ca-P-adequate; T 2 , Ca-P-deficient; T 3 , Ca-P-deficient + 500 FTU kg -1 of phytase (Ph); T 4 , Ca-P-deficient diet + 5 µg kg -1 of 1 α -OH-D 3 ; T 5 , Ca-P-deficient + Ph + 5 µg kg -1 of 1 α -OH-D 3 . Results showed that quails fed Ca-P-adequate had significantly higher body weight compared with quails fed Ca-P-deficient, Ca-P deficient supplemented with 1 α -OH-D 3 and Ca-P-deficient supplemented with 1 α -OH- D 3 and phytase, but did not differ from Ca-P-deficient diet supplemented with phytase. Quails fed Ca-P deficient were unable to achieve FCR comparable to quails fed Ca-P-adequate (p < 0.05). The percentage of bone ash data indicated that quails fed Ca-P-adequate had higher tibia ash compared with other groups except for quails fed Ca-P deficient diet supplemented with combination of 1 α -OH-D 3 and phytase. Quails fed Ca-P-adequate had higher tibia P compared with quails fed Ca-P-deficient. In conclusion, these results indicated that quails fed Ca-P-deficient supplemented with 5 µg kg -1 of 1 α -OH-D 3 in combination of 500 FTU kg -1 of phytase were able to achieve the same tibia ash and Ca compared with quails fed Ca-P-adequate.


Introduction
Approximately less than one-third of the phosphorus (P) in feedstuffs of plant origin is biologically accessible to poultry [National Research Council (NRC), 1994] because they do not have the digestive enzyme phytase to hydrolyze the phytate phosphorus (Nelson, McGillivray, Shieh, Wodzinski, & Ware, 1968).As a result, inorganic sources of P are incorporated to diets to meet P requirements of poultry (Selle & Ravindran, 2007).Growing pressure on farmers to decrease environmental pollution with poultry manure has stimulated research into ways to increase the accessibility of phytate phosphorus (PP) in feedstuffs (Landy & Toghyani, 2018).The addition of phytase enzyme is one such method to reduce environmental pollution (Edwards Junior, Shirley, Escoe, & Pesti, 2002;Jiang et al., 2013;Nelson, Shieh, Wodzinski, & Ware, 1971;Simons et al., 1990).Edwards Junior and Veltmann Juniorr (1983) indicated that enhancing dietary calcium (Ca) level will reduce PP usage by poultry due to the formation of insoluble Ca phytate.Similarly, Applegate, Angel, and Classen (2003) reported that intestinal mucosa phytase activity enhances and more PP is hydrolyzed at lower levels of dietary Ca compared with higher levels of Ca in broiler chickens.
Cholecalciferol (Vitamin D 3 ) is generally and widely used in poultry production.As an analog of vitamin D, 25-OH-D 3 is used as feed additive for the vitamin D requirement of poultry (Ghasemi et al., 2018).The metabolite 1α-hydroxycholecalciferol (1α-OH-D 3 ) is the derivative of cholecalciferol.Edwards Junior et al. (2002) reported that the 1 α-OH-D 3 is approximately five to eight times as effective as cholecalciferol in promoting growth and tibia quality.The results of a trial conducted by Landy and Toghyani (2014) indicated the ability of 1α-OH-D 3 to substitute for cholecalciferol in broiler chickens.Snow, Baker, and Parsons (2004) investigated efficiency of 1α-OH-D 3 and phytase in dietary containing low level of non-phytate phosphorus (NPP), the results indicated that interaction between 1α-OH-D 3 and phytase had affirmative effects on PP release of young broilers.Driver et al. (2005) also reported that combination of phytase and 1α-OH-D 3 could improve the percentage of tibia ash in broilers fed low level of Ca and P. Furthermore, Han et al. (2012a) reported that 1α-OH-D 3 had higher activity at low levels of dietary Ca in comparison to high levels of Ca.Han et al. (2015) reported that the supplementation of 5 μg of 1alpha-OH-D 3 per kg of diet could improve growth performance and tibia mineralization when dietary non-phytate phosphorus levels reached 0.30% in 1-to 21-d-old broilers.Landy, Toghyani, Bahadoran, and Eghbalsaied (2015) reported that supplementation of 1α-OH-D 3 in Ca-P deficient diet could improve tibia ash, Ca and P of broilers.
Despite these findings, there has been a dearth of information on the possible effects of 1α-OH-D 3 on Japanese quails.The aim of the experiment reported here was to investigate the effects of dietary1α-OH-D 3 , individually or in combination of microbial phytase on phytate P utilization in Ca-P deficient diet of growing Japanese quails.

Ethical approval
The birds were handled in compliance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals.Also, all procedures complied with the ethical guidelines of the Shahrekord University's Ethical Committee (approval ref No. 2017-03).

Animals and dietary treatments
Eight-day-old (n =160) as-hatched Japanese quails (Coturnix coturnix japonica) were individually weighed, selected, and divided into 20 groups of 8 chicks.The dietary treatments were as follows: T 1 , Ca-P-adequate (0.80% Ca, 0.59% total phosphorus (tP)); T 2 , Ca-P-deficient (0.60% Ca, 0.44% tP); T 3 , Ca-P-deficient + 500 FTU kg -1 of Phyzyme XP 5000 unique bacterial phytase developed by Danisco Animal Nutrition; T 4 , Ca-Pdeficient + 5 µgvkg -1 of 1α-OH-D 3 (Vitamin Derivatives Inc., Georgia, USA); T 5 , Ca-P-deficient + 500 FTU kg -1 of phytase + 5 µg kg -1 of 1α-OH-D 3 .The basal diet was formulated to meet or exceed the nutritional requirements of Japanese quails (NRC, 1994) except for Ca and P (Table 1).Chicks were placed in wire cages and had free access to feed and water throughout the whole trial.The chick house was completely enclosed and light was on during d 1 to 3, which was reduced to 14 h on d 16 and kept constant thereafter.The ambient temperature was controlled at 37 o C during the first d and then gradually reduced to 24 o C by the end of third wk and then was maintained constant.

Feed analyses
Dry matter (DM), Ca and tP content of formulated experimental diets were assayed for each experimental diet.Feed, were dried by using Oven for DM determinations.Ca and tP contents of experimental diets were evaluated by the ICPOES method 2011.14 (Association Official Analytical Chemist [AOAC], 1990).

Performance parameters
Average body weights (BW, g) of pens were recorded at day 8 and at the end of trial.Feed intake of each group was determined at 42 d of age.Growth performance was evaluated in terms of BW, average daily feed intake (DFI, g), and feed conversion ratio (FCR, Feed (g): Gain (g)).These data, determined on pen basis, and then averaged by treatment.

Chemical analysis
At the end of trial, 8 male quails/treatment were selected, sacrificed, and the left tibia of each quail was removed for determination of percentage of bone ash on a fat-free dry-weight basis, according to AOAC (1995; method 22.10).The percentages of Ca and P in tibia ash were determined via the ICPOES method 2011.14 (AOAC, 1990).

Statistical analysis
All of data were analyzed using the General Linear Model procedures of SAS (2012).Differences between means were tested using Tukey's HSD (Honestly Significant Difference).Values were considered statistically different at p ≤ 0.05.

Growth performance
Treatments failed to induce any significant effect on DFI (Table 2), though it tended to enhance in quails fed Ca-P-deficient diets supplemented with 1α-OH-D 3 (p > 0.05).At the end of trial, quails fed Ca-P-adequate diet had significantly higher BW (189 g) compared with quails fed Ca-P-deficient diet (163 g), Ca-P-deficient diet supplemented with 1α-OH-D 3 (168 g), and Ca-P-deficient diet supplemented with 1α-OH-D 3 and phytase (169 g), but did not differ from the quails fed Ca-P-deficient diet supplemented with phytase (174 g) that was intermediate.Supplementation of phytase to Ca-P-deficient diet could improve BW at 42 d of age, whereas the results were not statistically significant (p > 0.05).Quails that received the Ca-P-adequate diet during the trial were the most feed efficient (p < 0.05).The birds fed the Ca-Pdeficient diets throughout the trial were unable to achieve FCR index comparable to the quails fed Ca-Padequate diet, although it tended to improve in birds fed Ca-P-deficient diet supplemented with phytase and 1α-OH-D 3 .
In the present trial supplementation of 1α-OH-D 3 and phytase to Ca-P-deficient diet couldn't maximize growth performance of quails, although, supplementation of Ca-P-deficient diet with phytase could improve BW of quails at 42 d of age.Similarly, Attia et al. (2012) indicated that addition of phytase to broilers diet enhanced BW and this may be due to the increment in the availability and absorption of nutrients.In agreement with our results, Edwards Junior et al. (2002) reported that supplementation of 1α-OH-D 3 to dietary containing 0.30% of NPP could not improve FCR and BW in 1-to 16-d-old broilers but later in another trial Snow et al. (2004) indicated that addition of 1α-OH-D 3 and phytase to dietary containing 0.13% of NPP could improve the growth of broilers, although the growth was lower than those fed adequate levels of NPP.Our data in the current trial suggest that interaction for growth performance indices between phytase and 1α-OH D 3 might exist at lower level of NPP.
In the current trial the dietary treatments had no effect on tibia weight, length and diameter.In contrast with obtained results in our trial, Han et al. (2012b) reported that addition of 5 or 10 μg kg -1 of 1α-OH-D 3 to Ca-P-deficient diet could increase tibia length and weight of broilers, but it had not any effects on tibia width.Our data in the present trial showed that addition of phytase or 1α-OH-D 3 alone to Ca-P deficient diet had not any effect on tibia ash but, addition of phytase and 1α-OH-D 3 could promote tibia ash of quails.In contrast with our results, Edwards Junior et al. (2002) indicated that supplementation of 1α-OH-D 3 to P deficient diet could improve tibia ash and P utilization of broilers but later in another experiment Driver, Pesti, Bakalli, and Edwards Junior (2005) reported that supplementation of phytase and 1α-OH-D 3 could improve the percentage of tibia ash in broilers.Snow et al. (2004) indicated that interaction between phytase and 1α-OH-D 3 had beneficial effects on PP release of young broilers.It seems that interaction exist between 1α-OH-D 3 and phytase on bone mineralization.
In the current trial 1α-OH-D 3 in combination with phytase could improve tibia ash and Ca.The 1α-OH-D 3 improves PP utilization by facilitating Ca absorption and reducing the restriction of Ca on endogenous phytase.Han et al. (2009) reported that 1α-OH-D 3 facilitate intestinal P absorption due to stimulating small intestinal NaPi-IIb cotransporter gene expression.In addition, these findings show that 1α-OH-D 3 can regulate NaPi-IIb cotransporter gene transcription, and increase absorption of phosphate.a,b Values in the same column not sharing a common superscript differ (p < 0.05). 1 Ph represent 500 phytase units kg -1 , 1 α represent 5 µg kg -1 of 1 α-OH cholecalciferol and P+1 α represent 500 phytase units kg -1 plus 5 µg kg -1 of 1 α-OH cholecalciferol.a,b Values in the same column not sharing a common superscript differ (p < 0.05). 1 Ph represent 500 phytase units kg -1 , 1 α represent 5 µg kg -1 of 1 α-OH cholecalciferol and P+1 α represent 500 phytase units kg -1 plus 5 µg kg -1 of 1 α-OH cholecalciferol.

Conclusion
In conclusion, in the current study quails fed Ca-P-deficient diet supplemented with 5 µg kg -1 of 1α-OH-D 3 in combination of 500 FTU kg -1 of phytase were able to achieve the same tibia ash and Ca compared with quails fed Ca-P-adequate when dietary nonphytate phosphorus and Ca levels were 0.15% and 0.6%, respectively, whereas addition of 1α-OH-D 3 alone or in combination with phytase could not maximize growth performance of quails.

Table 1 .
The ingredients, calculated and determined composition of experimental diets.

Table 3 .
Effect SEM = standard error of the mean.