Effect of Ginger Derived Phyto-Protease on Production Performance, Serum Biochemistry, Nutrient Digestibility, Gut Morphometry and Immunity of Broilers Fed High Level of Poultry By Product Meal-Based Diet

An experiment was conducted to assess the potential of ginger-derived phyto-protease on production performance, gut health


INTRODUCTION
T he feed efficiency of poultry needs to be improved significantly because feed is the commodity with the highest cost about 70% of the total production (Deek et al., 2020). Poultry production at the commercial level is facing a big challenge regarding quality feed availability at stable prices on a regular basis. The poultry industry relies on soybean meal as the primary source of protein which fulfills up to 77% of its protein requirement (Ogunji, 2004).
Many countries including Pakistan that imports full-fat soybean or meal face high price fluctuations and supply problems most of the time. According to USDA (2021) report, Pakistan imports 2.4 MMT of soybean costing 842 million US$ annually. Therefore, alternative indigenously available cheap protein sources will not only add versatility to feed ingredients for poultry but will also minimize soybean dependency to a certain extet (Shuaib et al., 2022). A locally manufactured poultry by-product meal (PBM) can potentially replace the plant protein source. PBM meal is produced by processing the inedible parts like viscera, feet, feathers, and heads of poultry carcass (Senkoylu et al., 2005) and is used as a protein source in the mono-gastric animal diet in many countries. Approximately 37% of the live weight of a broiler is considered slaughter house waste (Meeker and Hamilton, 2006) while non-edible parts account for approximately 28% of the live weight percentage of a broiler. In Pakistan, since there were 1407 million broilers in 2021 (Pakistan Economic Survey, 2021) with an average weight of 2kg and 28% non-edible parts they would produce O n l i n e

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787.92 million kg of waste product annually which needs to be managed efficiently to ensure environmental safety and reduce pollution. The use of this waste product as PBM in poultry ration will not only minimize this waste burden but will also minimize the cost of soybean meal and will act as a partial replacement for soybean. The CP value of PBM ranges from 45-65% (Karapanagiotidis et al., 2019) but aside from its benefits, there are some barriers to the incorporation of poultry by-product meal in broilers' diets which are its poor digestibility, quality of raw materials, processing conditions, variation in nutritive composition and inappropriate inclusion levels in poultry feed (Jafari et al., 2022). Several researchers suggest different levels of PBM replacement with soybean meal, some suggest a 10% inclusion level however, Hassanabadi et al. (2008) reported a 5% inclusion level without adverse effects on broilers' performance while Jafari et al. (2022) reported reduced feed efficiency and growth rate beyond 5% inclusion level. To overcome these inherent problems of poultry by-product meal and make it suitable for use in broilers' diets several processing methods like fermentation or bioprocession, rendering, and enzymatic valorization can be effectively applied (Barnes et al., 2015;Lewis et al., 2019).
Enzymatic valorization with exogenous protease extracted from ginger might improve the feeding value of PBM by complementing the endogenous enzyme system, improving the breakdown of certain proteins, and enhancing digestibility (Classen et al., 1991). These inconsistent results about PBM inclusion level in broiler diets urge further studies to figure out the correct inclusion level without compromising broilers' health. Therefore, this study was conducted to investigate the effect of soybean meal replacement by 6.5% PBM on broilers' performance, nutrient digestibility, serum biochemistry and gut morphology of broilers supplemented with ginger derived phyto-protease enzyme at different concentrations.

Experimental layout and bird's husbandry
This study was approved by the ethical committee of the Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan and was conducted in the poultry farm of the university. A total of 320-day-old broilers (ROSS-308) were purchased from a local hatchery and randomly distributed in four treatment groups in a completely randomized design. Each group had 4-replicates with 20 birds per replicate. Birds in all the treatment groups were presented with starter (0-21 days) and finisher (22-35 days) diets Table I as per   Table I

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(ROSS-308) requirements using Brill Formulation ® software. At different stages of rearing optimum environmental conditions of temperature, humidity, ventilation, and light were maintained as required. Ginger was purchased from the local market, washed, and sliced into small pieces. 200mL potassium phosphate buffer 100mM (pH 7.0) and 100g ginger were homogenized, then filtered through cheesecloth and centrifuged at 10,500x for 30 min. The supernatant was collected and used as a ginger protease (GP) with some modifications as described by Nafi et al. (2013). There were four dietary treatments (CON group; N-CON group, GPP1 group and GPP2 group). CON group was a normal corn-soybean meal diet, N-CON was a diet in which soybean meal was partially replaced with 6.5% PBM, GPP1 and GPP2 were the same 6.5%PBM (N-CON) based diets but added with ginger derived Phytoprotease enzyme at 50mg/kg and 100mg/kg.

Performance parameters
Feed intake was calculated by the formula as feed offered minus feed rejected. The final bird weight was subtracted from the initial weight to determine body weight gain. For each experimental unit, the feed conversion ratio (FCR) was calculated on weekly basis by dividing the feed consumed by the body weight of the birds.
To obtain dressing percentage carcass weight was divided by live weight (Badar et al., 2021). The relative weight of lymphoid organs (spleen, bursa, thymus) was calculated as organs weight as percent of BW= weight of an organ in g/ body weight in g × 100.

Serum biochemistry
Three birds per replicate were randomly selected for blood collection from wing veins in EDTA tubes. To separate serum, the samples were centrifuged at 3000 RPM for 15 min and then stored at -20 o C till further analysis. Serum was analyzed by using commercial kits through standard procedures and protocols after thawing for determination of total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), and serum triglycerides (Asghar et al., 2021).

Hemagglutination inhibition (HI)
In this trial, birds were vaccinated through drinking water against New Castle Disease Virus (NDV) on days 7 th and 17 th . On day 7 th birds were vaccinated against infectious bronchitis (IB). To determine antibody titer against NDV and IB three birds per replicate were randomly slaughtered for blood collection on day 35. A commercially available laboratory kit for HI was used for the determination of NDV antibody titer as described by Asghar et al. (2021). Antibody titer against IB was determined through ELISA.

Intestinal histomorphology
To evaluate intestinal histomorphology, 3cm duodenal tissue samples were taken from the duodenum center of three birds per replicate on day 35 th . The digesta was gently removed and the tissue was flushed with normal saline and then preserved in buffer formalin (10%) for 48 h. In the histopathology lab, the tissue samples were washed with tap water and then treated with an alcohol solution. Using cassettes, a properly cut tissue section was embedded in paraffin wax. Microtome was used to cut tissue sections into 4µm, then mounting those tissue sections on slides and staining with hematoxylin and eosin (H and E). Under a computer-aided light microscope using image analysis (Leica Camera AG, Solms, Germany) histological indices were analyzed as described by Laudadio et al. (2012).

Nutrients digestibility
Five birds per replicate were shifted to metabolic cages on day 36 for total fecal collection for the last four days till day 42. A weighed amount of feed was offered daily and feces were collected early the next morning for four days and weighed. A representative sample (20%) of feces was placed in plastic bags and stored at -20 o C till further analysis. For the determination of ileal nutrient digestibility 0.2% Cr 2 O 3 was used as an indigestible marker. The fecal samples within the pen were pooled and dried in an oven at 65 o C. The samples were ground to pass through a 0.5mm sieve. Similarly, digesta from the ileum portion of the small intestine of all the birds within the cage were collected and then frozen immediately. After drying, the samples were ground to pass through a 0.5mm sieve. The samples of diet, excreta and ileal digesta were analyzed for proximate analysis as described by AOAC (2005), and for calcium and phosphorus analysis by AOAC (2000). The concentration of chromium was determined with a UV absorption spectrophotometer (Hitachi Z-5000 Automatic Absorption Spectrophotometer, Tokyo, Japan) using the method of Pan et al. (2016). Adiabatic bomb calorimeter (Par instrument USA) was used to determine the gross energy of feed and fecal samples and then AME and nutrient digestibility were calculated using the formula of Stefanello et al. (2020) shown below.
Where GEi is gross energy (kcal/kg) of diet; GEo is gross energy (kcal/kg) of excreta. Ci and Co are concentrations of marker in the diet and digesta or excreta.
Where; Ci and Co are concentrations of marker in the diet and digesta or excreta (%), respectively; Ni and No are

Economics of the study (cost analysis)
Per chick cost was calculated for all the groups. The market rate of live birds per kg was used to calculate the mean gross return per chick.

Statistical analysis
The collected data was analyzed by the standard procedure of (ANOVA) analysis of variance using a completely randomized design (CRD) as suggested by Steel and Torrie (1981) and the Tukey test was used for significance among means with a level of significance set at (P<0.05).

Growth performance
The growth performance results of the birds are shown in Table II. Feed intake was significantly (P>0.05) lower in the N-CON group in both starter (1-21 day) and finisher phases (21-35 day), while the feed consumption of the same N-CON feed supplemented with ginger-derived Phyto-protease enzyme in GPP1 and GPP2 groups was comparable with CON group. Ginger derived Phytoprotease enzyme supplementation significantly improved the body weight gain of broilers in both phases. Feed efficiency was improved (P<0.05) by ginger protease enzyme in GPP1 and GPP2 groups compared to CON and N-CON groups.

Carcass traits, lymphoid organs weight, and serum biochemistry
Results related to carcass traits, lymphoid organs weight, and serum biochemistry are shown in Table III. The carcass yield of broilers was significantly affected by ginger protease enzyme in GPP1 and GPP2 groups compared to N-CON. Carcass yield was highest in GPP2 and lowest in the N-CON group. Ginger derived phytoprotease enzyme significantly affected dressing percentage. The dressing percentage of N-CON was the lowest and GPP2 was the highest. The percent relative weight of lymphoid organs (spleen and bursa) was significantly affected by ginger protease. Maximum weight of the spleen and bursa was noticed in the GPP2 group and minimum in the N-CON group. No significant effect of ginger protease was observed on the thymus. Total cholesterol, LDL, and triglycerides were (P<0.05) lowered, and HDL levels were raised in ginger protease-supplemented groups compared to control and negative control groups, the maximum effect of ginger protease was observed in the GPP2 group. The lowest values of total cholesterol, LDL, and triglycerides were observed in ginger protease-supplemented groups and highest in the N-CON group. HDL level was improved and highest in the GPP2 group and lowest in the N-CON group. CON, Normal commercial ration; N-CON, 6.5% of PBM inclusion; GPP1, 6.5%PBM +50mgkg -1 phyto-protease; GPP2, 6.5% PBM+100mgkg -1 phytoprotease. Means within the same rows bearing different superscripts differ significantly (p ≤ 0.05), FI, Feed intake; BWG, body weight gain; FCR, Feed conversion ratio.

Immunity and gut morphometry
The effect of soybean meal replacement with PBM at a 6.5% level added with or without ginger derived phyto-protease on broilers' immune response, livability, and gut morphology is shown in Table IV. On day 35 th the antibody titer against NDV was significant and highest in the GPP1 group while lowest in the N-CON group. The livability of broilers was significantly high in the GPP2 group compared to other treatment groups. The highest livability was observed in the GPP2 group and the lowest in the N-CON group. The antibody titer against infectious bronchitis was significantly affected by ginger protease supplementation. The highest titer was observed for the GPP2 group and the lowest in the N-CON group.
Gut morphometry of broilers was significantly affected and the GPP2 group observed significantly the highest villi length, width, and crypt depth compared to all other groups while the N-CON group showed the lowest villi length, width, and crypt depth. Villus surface area was significantly high in the GPP2 group compared to the N-CON group.

Nutrients digestibility
Results regarding nutrient digestibility and economics of broilers supplemented with ginger-protease enzyme are shown in Table V. Mean percent digestibility of dry matter, crude protein, crude fiber, calcium, and phosphorus improved significantly high in the GPP2 group and lowest in the N-CON group. The AME was significantly higher in the CON group followed by GPP2, and GPP1, and lowest in the N-CON group.

Economics
The results of economic parameters indicated that feed cost per chick was significantly low in the N-CON group but the gross return was significantly high in GPP2 and GPP1 groups compared to CON and N-CON groups. Despite the same feed cost GPP2 gross return and profit over feed cost were higher than the CON group.

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U. Ahmad et al.

DISCUSSION
This study was conducted to assess the effect of soybean meal replacement with poultry by-product meal at a 6.5% level of incorporation on broiler performance supplemented with or without ginger-protease enzyme. Feed intake, weight gain (WG), and FCR were improved with ginger protease supplementation in PBM-based diets. Depression in the growth performance of birds was obvious when fed PBM-based diets only, however with the supplementation of ginger-protease enzyme the growth performance improved significantly, this indicated that ginger-protease was responsible for complementing the endogenous secretion of proteolytic enzymes resulting in improved performance. The overall performance of birds was improved by enzyme supplementation which agrees with the previous findings of Cowieson et al. (2006), Mahmood et al. (2018), andSaaci et al. (2018) who noticed a positive correlation of aqueous ginger extract on body weight gain and feed intake of broilers. Mahmood et al. (2018) also reported improved feed intake with protease enzyme in PBM-based diets. Similarly, a positive effect of protease enzyme was noticed on carcass traits of broilers by Mahmood et al. (2018), while, Barazesh et al. (2013) reported that carcass characteristics were not affected by different concentrations of ginger opposing our observations. Similarly, broilers provided with protease in low-protein diets showed improvement in carcass yield (Ajayi, 2015). Enhanced utilization and deposition of protein might be responsible for improved carcass yield in phyto-protease-supplemented groups. Improvement in the percent weight of bursa and spleen was observed for ginger protease-supplemented groups (Raeesi et al., 2010) approving our observations. The per cent weight of the thymus was not affected by dietary ginger protease supplementation (Bondona et al., 2019) and these results agreed with our findings of a non-significant effect on thymus per cent weight. The non-significant effect of ginger protease on thymus weight was also confirmed by other researchers (Khushdil et al., 2012).
Serum cholesterol, triglycerides, and LDL level was significantly lowered while HDL level was improved in ginger protease-supplemented groups compared to CON and N-CON groups. Reduction in cholesterol and triglycerides were reported by feeding 0.2% ginger to broilers (Mohamed et al., 2012). The presence of HMG-COA (hydroxyl methyl glutaryl coenzyme A reductase) in ginger which is the rate-limiting step in cholesterol synthesis might be responsible for its hypolipidemic effect (Asghar et al., 2021). Cholesterol-lowering effects of the aqueous ginger extract were previously reported by Oleforuh et al. (2015). The antibody titer against Newcastle disease was significantly high among ginger derived phyto-protease supplemented groups on the 35 th day. Deniz Azhir et al. (2012) also reported the immuneboosting effect of ginger on broilers' immune systems. The increase in amino acid digestion by ginger protease might be responsible for the increase in antibody titer (Li et al., 2007) as dietary protein deficiency has long been known to impair immune functions and make humans and animals more susceptible to infectious diseases. The antioxidant effect of ginger and the presence of natural aromatic compounds like shogoals and gingerol might be another possible reason for the immune boosting effect of ginger O n l i n e

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Effect of Ginger Derived Phyto-Protease on Production Performance protease . Dry matter, crude protein, crude fiber, calcium, and phosphorus were significantly affected by ginger-protease enzyme in PBM-based diets. Our results were consistent with the observations of Kamel et al. (2015) and Duwa et al. (2020). However, Saaci et al. (2018) did not detect any significant effect of ginger extract on the DM and CP digestibility of broilers. This may be due to different types of feed, the duration of the experiment, and crude ginger extract. Digestibility of CP was improved by protease supplementation in broilers' diet (Vieira et al., 2013). The AME was enhanced in ginger protease-supplemented groups which may be attributed to the extra-proteinaceous effect of protease (Cowieson and Roos, 2014) that aid in the digestibility of fats apart from proteins thus improving AME. The significant changes among treatment groups for nutrient digestibility may be attributed to ginger derived phyto-protease enzymes contributing positively to digestive functions by stimulating the endogenous enzyme system. Broiler health and performance are affected by the development of the small intestine which is an important digestive organ involved in nutrient absorption (Kawalilak et al., 2010). To evaluate the effects of nutrients on gastrointestinal physiology a common measurement tool is villus structure and anatomy. Moreover, crypt depth and villus height have been documented to show significant correlations regarding performance improvement. By increasing the crypt depth and absorptive surface area of the small intestine of broilers, ginger protease improved the digestive and absorptive capacity which resulted in faster growth rates (Prakash and Srinivasan, 2012). The reduction in villus length, width, crypt depth, and surface area may be due to poor absorption of nutrients in PBM-based diets, however, the gut morphometry was significantly affected and villi length, villi width, and crypt depth were significantly improved by ginger derived phyto-protease supplementation in a PBM-based diet. Our results agreed with the reports of Karangiya et al. (2016), Shewita and Taha (2018) and Asghar et al. (2021) that there were improvements in the villus height, width, and surface area of the birds' duodenum. This increase in villi length and width causes an increase in the surface area of villi increasing the absorption of nutrients and resulting in improved growth and performance (Oladele et al., 2012). The electrolytes are produced through the excretory activity of crypt cells into the intestinal lumen ultimately boosting up digestive activities of the gut (Bowen, 2011).

CONCLUSION
The results of the current study clearly indicated that the negative effects of PBM on broilers' growth performance can be successfully alleviated through the addition of ginger derived Phyto-protease. This significantly improved broilers' performance, gut health, immunity, and nutrient digestibility in PBM-based diets. PBM may be used successfully as a partial replacement of soybean meal at 6.5% level of inclusion with the supplementation of ginger derived phyto-protease at (100 mg kg -1 feed) without any antagonistic effects on broilers health.