Effect of Phytase Supplementation on Growth Performance, Mineral Digestibility, and Tibia Calcium and Phosphorous in Broilers Fed Low Phosphorus Diets

ABSTRACT The aim of present study was to investigate the effect of phytase supplementation on growth performance, mineral digestibility, and tibia calcium and phosphorous in broilers fed low phosphorus diets. Three hundred broiler chicks were allotted to six different treatments with five replicates each (10 chicks/replicate). T1: Control, 0.5% available phosphorus (Av.P); T2: 0.35P, 0.35% Av.P; T3: 0.20P, 0.20% Av.P; T4: 0.35P-1Phy, diet 0.35P + 1000FTU/kg phytase; T5: 0.20P-2Phy, diet 0.20P + 2000 ftu/kg phytase; T6: 0.20P-3Phy, diet 0.20P + 3000 ftu/kg phytase. Feed intake and weight gain were higher (p=0.001) in broilers fed phytase supplemented diets. Feed conversion ratio was better (p<0.01) in the 0.35P-1Phy than in the others. Calcium and P digestibility was higher (p<0.05) in the 0.35P-1Phy than the 0.35P, 0.20P and 0.20P-2Phy, groups. The highest (p<0.05) tibia ash was observed in the 0.35P and 0.35P-1Phy groups rather than the 0.20P at 21st day, while at 33rd day it was higher (p<0.05) in the control than in 0.20P. At 21st day, tibia Ca content was higher (p<0.05) in the 0.35P-1Phy group as compared to 0.20P and 0.35P, while at 33rd day, tibia Ca content in the control and 0.20P-2Phy groups was higher (p<0.05) than that of 0.20P and 0.35P. Furthermore, tibia P content was higher (p<0.05) in all phytase supplemented groups. It could be concluded that dietary available phosphorus at 0.35% with phytase addition at 1000 FTU/kg reduces the cost per unit weight gain by 9.17%, with positive effects on growth performance.


INTRODUCTION
Calcium (Ca) and phosphorus (P) play a significant role in bone development and chicken well-being, as well as providing compressive strength and rigidity to the bones (Tizziani et al., 2016).Most of the ingredients of plant origin used for poultry feed formulation contain P as a phytate complex, which affects the availability of P (Tahir et al., 2012).Wheat and corn both contain 0.18% phytate phosphorus, but the bioavailability of phosphorus from both sources is different, i.e. 61.5 and 47.06 %, respectively (Ahmed et al., 2003;Mossa et al., 2018).Phytate also binds many minerals like Ca, P, iron, zinc and makes them unavailable to birds, which results in weak bones and stunted growth (Hakami et al., 2022).Phosphorus deficiency causes bone abnormalities like osteopenia, osteoporosis, and osteodystrophy (Applegate et al., 2003;Manangi & Coon, 2008), leading to bone breakage or defects.To fulfill P requirements, inorganic phosphorus (mostly dicalcium phosphate "DCP") is supplemented in poultry feed, but it increases feeding costs (Makiyama et al., 2012).This problem could be resolved by supplementing phytase in poultry rations to increase the bioavailability of P already present as phytate complex in feed ingredients, which ultimately decreases the feeding cost (Gehring et al., 2013).Dietary

Effect of Phytase Supplementation on Growth Performance, Mineral Digestibility, and Tibia Calcium and Phosphorous in Broilers Fed Low Phosphorus Diets
supplementation of phytase enhances the bioavailability of P in broiler chicks.Phytase supplementation of 5000 FTU/kg hydrolyzes 99.45 % of the phosphorus present in cereals (Manangi & Coon, 2008) and could replace DCP in broiler feed (Scholey et al., 2018).The phytase enzyme releases phosphorus by breaking the phytic acid and helps in the synthesis of myoinositol, giving better results in growth and development (Cowieson et al., 2011;Baloch et al., 2021).Phytase catalyzes the inorganic phosphate that contains chelated minerals and improves their availability to birds (Al-Harthi et al., 2020).Keeping in view the importance of phytase in poultry diets, the present study was designed to investigate the effect of phytase supplementation on growth performance, mineral digestibility, and tibia calcium and phosphorous in broilers fed low phosphorus diets.It was hypothesized that phytase supplementation could increase the availability of phosphorus to broilers and dietary phosphorus levels could be reduced without compromising the growth performance in broilers.Therefore, this experiment was designed to study the effect of phytase supplementation on growth performance, mineral digestibility, and tibia calcium and phosphorous in broilers fed low phosphorus diets.

MATERIAL AND METHODS
This study was carried out after the animal experiment was approved by the Office of Graduate Studies, University of Agriculture, Faisalabad (Decision number: DGS/18981-84; Dated: 05/08/2020).
House preparation: Feeding trial was conducted at Research Farms of University of Agriculture, Faisalabad, Pakistan.Cleaning, washing, fumigating, and sealing the house prior to the arrival of chicks helped reduce the number of bacteria and other microorganisms in the environment.All proper sanitary procedures were followed during the experimental trial.The chickens were vaccinated on day 1, day 8, day 18, and day 25 with ND + IB, IBD, and IBD vaccines, respectively.
Experimental design and birds: Three hundred day-old broiler chicks (Ross-308) with average weight of 40.05 grams were divided into 6 different treatments, following a completely randomized design (Table 1).Treatments were as following: T1: Control, 0.5% available phosphorus (Av.P); T2: 0.35P, 0.35% Av.P; T3: 0.20P, 0.20% Av.P; T4: 0.35P-1Phy, diet 0.35P + 1000 FTU/kg phytase; T5: 0.20P-2Phy, diet 0.20P + 2000 FTU/ kg phytase; T6: 0.20P-3Phy, diet 0.20P + 3000 FTU/kg phytase, each for starter and finisher phase.The basal diet was formulated according to the breed-specific guide (Ross-308) as shown in Table 2 and 3. Faczyme-10HG (heat stable phytase with 10.000 U/g, Tangshan Finely Animal Care Co., Ltd.China) was used as a source of phytase.Birds were provided with free access to feed and water throughout the feeding trial and an optimal environment was provided.The experiment lasted for 33 days and was divided into two phases (starter phase from day 1 to 21 and finisher phase from day 22 to 33).During the first week, temperature was maintained at 35 °C, being reduced by 5 °C per week and maintained at 21 °C during the rest of the experimental period.Relative humidity was adjusted to 65%, and a stocking density of 0.06 m 2 per bird was used.

Samples collection and measurements
Growth Performance: Initial weight of chicks was recorded on arrival at the shed.The weight of feed offered and refusal were recorded to determine feed intake.Body weight of chicks was recorded weekly to determine the weight gain.Feed conversion ratio (FCR) was calculated from data on feed intake and weight gain,.
Carcass Characteristics: At the end of the feeding trial, two birds were collected randomly from each replicate.Those birds were weighted and slaughtered through halal neck cut method as explained by (Ali et al., 2011).After slaughtering, visceral organs and giblets were removed and carcass weight was measured to calculate dressing percentage.After evisceration, the absolute weight of various internal organs such as heart, liver and gizzard of the slaughtered birds were recorded.The weight of internal organs were expressed as a percentage of carcass weight.
Minerals Digestibility: Calcium and phosphorus digestibility were determined at the end of each phase by total collection method.Celite ® was used as an external marker and added in the last five days of feed at the rate of 1% of feed, and fecal samples were collected during the last two days.Composited samples for each replicate were dried at 65 °C and stored till further processing.Feed and fecal material were analyzed (AOAC, 2000) for Ca, P and celite present in it to calculate digestibility of Ca and P.

Effect of Phytase Supplementation on Growth Performance, Mineral Digestibility, and Tibia Calcium and Phosphorous in Broilers Fed Low Phosphorus Diets
Digestibility (%) = 100 -(100 × maker in feed (%) / marker in feces (%) × mineral in feces (%) / mineral in feed (%)) Tibia Ash Determination: The tibiae were dried at 110 °C for 12 hours, then extracted with ether, dried once more, and finally weighed.The dry, fatfree bones were ashed at 550 °C in a muffle furnace.The percentage of dry, fat-free bone mass that was transferred to ash weight was determined as explained by (Catalá-Gregori et al., 2006).
Tibia Calcium and Phosphorous: Tibia samples were also collected from slaughtered birds and analyzed for the concentration of Ca and P present in them (AOAC, 2000).

Complete Blood Count:
On the last day of trial, 2 birds from each replicate were randomly picked and blood was collected through slaughtering and stored in EDTA tubes for determination of complete blood count (Shoaib et al., 2021).
Economic analysis: The experiment was conducted in 2020, but the latest cost of feed ingredients, chick cost, as well as miscellaneous expenses has been used.Economic parameters of the flock were determined by calculating the cost of chicks, feed ingredients (including phytase enzyme), and miscellaneous costs for flock raising.
Statistical Analysis: The observed data were analyzed using a statistical software.Analysis of variance were conducted through a completely randomized design using Minitab 17, and means of treatments were compared using Tukey's Test (Steel et al., 1997;Minitab, 2010).

RESULTS
During the starter phase, feed intake and weight gain were higher in phytase supplemented groups than non-supplemented groups.Better FCR was recorded in birds that received 0.35P-1Phy than in other treatments.Similarly, during the finisher phase, birds receiving enzymes in a low phosphorus diet had higher feed intake and weight gain.Improved FCR was recorded in birds receiving the 0.35P-1Phy and 0.20P-3Phy treatments.During the overall period, feed intake and weight gain were higher in birds of phytase supplemented groups than non-supplemented groups.Better FCR was recorded in birds receiving the 0.35P-1Phy treatment than other groups (Table 4).
Data regarding carcass characteristics at day 21 and 33 are given in Table 5.At day 21, dressing percentage and relative organ weights were not affected (p>0.05) by different phosphorus levels and phytase supplementation.Similarly, at day 33, all parameters of carcass characteristics were similar (p>0.05),except A significant (p<0.05)effect of dietary treatments was found on Ca and P digestibility at the 21 st and 35 th days of experiment (Figure 1).At the 21 st day, Ca digestibility was higher (p<0.05) in the control, 0.35P-1Phy and 0.20P-3Phy groups.At the 33 rd day, higher (p<0.05)Ca digestibility was observed for the 0.35P-1Phy group as compared to other treatments.The highest (p<0.05)P digestibility was found in the control group and in birds of the 0.35P-1Phy group at the 21 st and 33 rd day, respectively.Tibia ash contents were also significantly (p<0.05)affected by dietary treatments (Figure 2).The highest ash content was found for 0.35P and 0.35P-1Phy on the 21st day.Meanwhile, on day 33, tibia ash content of the control was higher than other groups.

Effect of Phytase Supplementation on Growth Performance, Mineral Digestibility, and Tibia Calcium and Phosphorous in Broilers Fed Low Phosphorus Diets
Tibia Ca and P contents were also significantly (p<0.05)affected by dietary treatments (Figure 2).The highest tibia Ca content was found in the 0.35P-1Phy group at the 21 st day.At day 33, tibia Ca contents of the control and 0.20P-2Phy groups were higher than in other groups.On the other hand, tibia P content of all phytase supplemented groups was higher than other groups at day 21.At day 35, the tibia P content of the control and 0.35P-1Phy groups were higher than other groups.
Blood hematology parameters (WBC, RBC, HGB, HCT, MCV, MCH, MCHC and PLT) were not affected (p>0.05) by phytase addition in broilers fed a low phosphorus diet (Table 6).The economical appraisal of the experiment on the basis of dietary treatments is shown in (Table 7).The highest total cost of production (PKR 641.00) was observed in the 0.20P-3Phy group.Cost per kg weight gain was lower than the control in all phytase supplemented groups.Cost per unit weight gain in the 0.35P-1Phy group was PKR 38.45 less than that of control.

DISCUSSION
Results showed that boilers could grow better at low dietary available P with supplementation of phytase.They are supported by Hofmann et al. (2022), who showed that addition of phytase at 1,500 FTU/kg in reduced protein, amino acid and P diet had improved weight gain and FCR.Babatunde et al. (2022)  that birds on a low P diet had lower weight gain and feed efficiency, but these parameters were improved with addition of phytase.Zarghi et al. (2022) revealed that phytase addition in wheat-based diets improved weight gain.In addition, Zanu et al. (2020) proposed that phytase supplementation improved intestinal morphology, and broilers could grow better with a healthy intestine.Scholey et al. (2018) stated that phytase could be used to provide the required level of P to broilers during finisher phase without using inorganic P, and that it is also an economical approach to fulfilling the P requirement of broilers.Furthermore, inorganic P sources like DCP are more expensive than phytase, so supplementation of phytase as an alternative to DCP ultimately reduces feed cost and is also environmentally friendly.Phytase supplementation also reduced the fecal phytate P excretion to the environment and reduced pollution (Pieniazek et al., 2017).Truong et al. (2017) stated that phytase also enhanced the sodium reabsorption from the small intestine, which is indirectly linked to improving the absorption of nutrients.Phytate enhances the availability and efficiency of nutrients, which results in better production and lowers anti-nutritional effects in broilers (Beeson et al., 2017).Phytase supplementation has been reported to increase the digestibility of amino acids and proteins in broilers (Gehring et al., 2013), which might be the reason for improved growth performance.Phytase supplementation also regulates the endogenous enzyme secretions in broilers (Liu et al., 2009).Pirgozliev et al. (2008) stated that broiler diets containing phytase at the levels of 250, 500 and 2500 FTU/kg increase BWG to 39.6, 41.1 and 44.0 gm per day, respectively.
A significant effect of dietary treatments was found on Ca and P digestibility.Ca and P digestibility was improved by phytase supplementation.Phytic acid reduces the availability and absorption of amino acids and protein by making them into insoluble complexes and lowering their digestibility (De Sousa et al., 2015).Results are in line with Babatunde et al. (2022), who observed that Ca and P digestibility were improved with the addition of phytase.Zarghi et al. (2022) showed that phytase addition in a wheat-based diet improved dry matter digestibility.Supplementation of phytase is the best way for releasing chelated minerals from phytates.However, contrasting results were found by Mossa et al. (2018), who stated that supplemental phytase did not affect mineral digestibility.Dave & Modi (2019) reported a lower Ca digestibility for limestone when a purified assay diet was used and compared to a maize-based diet.Conflicting results might be due to different dosage rate of phytase, bird species, durations of the experiment, or source of phytase (fungal or bacterial) (Abd El-Hack et al., 2018).
Reduced Ca and P levels in broiler ash contents (p<0.05%DM) given the diet with phytase supplementation was expected, as phytase enhances the release of phosphorus and other minerals from feedstuffs.According to various scientists, bone ash content is the most effective way to determine the amount of phosphorus produced by phytase in diets including maize and soybean meal Pereira et al. (2012), Nelson and Walker (1964).
Tibia Ca and P deposition during the starter and finisher phases were significantly improved by dietary supplementation of phytase in broiler diets.Results supported by Hofmann et al. (2022) showed that the addition of phytase at 1500 FTU/kg in reduced protein, amino acid, and P diets had improved tibia ash weight.Yan et al. (2001) stated that P availability could be increased by maintaining the Ca and P balance through phytase supplementation.Leeson et al. (2000) reported that tibias rich in ash results in better bone mineralization.High tibia ash percentage relates to more Ca and P retention in the tibia.Rutherfurd et al. (2004) concluded that phytase supplementation at the level of 1000 and 2000 FTU/kg in low-P diets enhanced both tibial mineral concentration and density by 35% and 24%, respectively.Phytase supplementations improved Ca and P digestibility (Ravindran et al., 2006), which ultimately enhanced the availability of these minerals and might be the reason for enhanced Ca and P deposition in the tibia.Blood hematology parameters were not affected by phytase addition in broilers fed a low phosphorus diet.Results are in line with the findings of Baloch et al. (2021), who stated that different levels of phytase had no effect on blood hematology parameters.Al-Harthi et al. (2020) reported that phytase addition in broiler diets had no effect on blood hematology parameters.
Improvement in the economic appraisal was observed with the supplementation of phytase enzyme in broiler diets.These findings agree with those of Rezaei et al. (2007), who concluded that phytase supplementation could improve production efficiency by enhancing growth performance, and Ca and P retention, and reducing production costs.

CONCLUSION
It could be concluded that dietary available phosphorus could be reduced by up to 0.35% with supplementation of phytase at the level of 1000 FTU/ kg in broiler diets.Phytase supplementation improved calcium and phosphorus availability by increasing their digestibility, and also improved growth performance, and tibia calcium and phosphorus contents, without compromising carcass traits in broilers.Moreover, phytase supplementation at a reduced dietary available phosphorus level could reduce the cost per unit weight gain up to 9.17%.

Table 2 -
Ingredients and nutrient composition of basal diets (Starter phase).

Yaseen MA, Rahman MA, Bhatti SA, Rahman MS, Yaqoob MU, Ahmad F, Zahid MU, Shoaib M Effect of Phytase Supplementation on Growth Performance, Mineral Digestibility, and Tibia Calcium and Phosphorous in Broilers Fed Low Phosphorus Diets for
heart and gizzard weight.Birds in the control group had higher (p<0.05)relative heart and gizzard weight than other groups.

Table 4 -
Effect of phytase supplementation on growth performance of broilers fed low phosphorus diets.

Table 5 -
Effect of phytase supplementation on carcass characteristics of broilers fed low phosphorus diets.

Table 6 -
Effect of phytase supplementation on complete blood count of broilers fed low phosphorus diets.