Consequences of varying dietary calcium and phosphorus levels on lipid profile, antioxidant and immunity parameters of growing Egyptian geese

Abstract

The background to determine the standard macro minerals requirement for geese is very important for growth, production and health. Until now, there are no clear standard mineral requirements for the local Egyptian geese. Consequently, the present experiment was aimed at estimating the influence of different dietary calcium (Ca) and phosphorus (P) concentrations on lipid profile, antioxidant and immunity parameters of the growing Egyptian geese. A total number of 120 Egyptian goslings (4 weeks old), with almost the same body weights were divided into 4 groups (30 goslings/group) randomly. All groups were subdivided into six replicates, each replicate contains five birds. A 2 × 2 factorial experiment was done with 2 dietary levels of Ca (0.70 and 0.85%) and P (0.35 and 0.45%) in the diets. Lipid parameters were not significantly changed except for low density lipoprotein (LDL) cholesterol and total cholesterol decreased significantly at dietary 0.85% Ca, and 0.45% P levels. The lipid parameters were not altered by the interaction between Ca and P levels except triglycerides and very low density lipoprotein (VLDL) cholesterol. Dietary supplementation of 0.85% Ca, and 0.45% P improved superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) and decreased the malondialdehyde (MDA) concentration. The most examined immune parameters were improved by the main and the interaction effect of P and Ca levels in geese diets. In conclusion, the present findings show that the best dietary Ca and P requirements for growing Egyptian geese are 0.85%, and 0.45% respectively, without any detrimental effects on health and immune status.

HIGHLIGHTS

• Calcium and phosphorus are very important nutrients in formulations of geese.

• Ca is essential for coagulation, eggshell formation, muscle and nerve function.

• Phosphorus has an important role in nervous system and is a significant component of eggshells, phospholipids and nucleic acids.

• The standard Ca and P requirement for growing geese are 0.85% and 0.45%, respectively.

Keywords:

• Antioxidant
• calcium
• geese
• immunity
• phosphorus

Introduction

Geese were the primary domesticated avian species in Egypt, for more than 4000 years ago. Moreover, the growth rate of Egyptian geese is the fastest of all qualified avian species (Farrell 2004; Ashour et al. 2020). Like most waterfowl species, geese will in general store fat in the body, and subsequently, a huge extent of their fast development happens in the skin, quills, and muscle to fat ratio. Thinking about the solid resistance and flexibility to roughage of geese, more affordable harvest results, for example, rice husk, buildup, Maize starch, full-fat rice wheat and cottonseed meal (CSM) are used by nutritionists in geese feed (Lu et al. 2011; Wang et al. 2014; Ding et al. 2016; Yu et al. 2019). Pellet and crumble feed forms are better than mash feed for Egyptian geese throughout the growing period, and optimal dietary CP requirements for Egyptian geese aged 1–7 and 8–12 weeks are 18% and 16%, respectively (Abou-Kassem et al. 2019). Due to the obscurity of feeding requirements for Egyptian geese, breeders have to rely on experience and feeding requirements to determine feeding allowances for geese (Arroyo et al. 2012). Generally, geese are fed with a concentrated diet, in addition to small quantities of forage. The growth rate depends on the ratios of concentrate and forage. Due to geese are reared under extensive conditions and provided with different quantities of green forage in most tropical countries, there are few studies on nutrient requirements of goose (Daghir 2008).

Calcium (Ca) and phosphorus (P) are very important nutrients in formulations for the diet of geese. Geese diets are developed in excess of Ca and P, which in metabolism are important; reflect levels of intestinal absorption, bone accretion, renal tubular reabsorption, glomerular filtration, and endogenous intestinal loss. Homeostasis Ca and P are primarily regulated by endocrines. Current commercial diets for both layers and broilers contain excess P and Ca, where their content could be reduced without affecting production (Li et al. 2017; Reda et al. 2020).

On the one hand, Ca improves heart power and smooth muscle movement, preserving the nervous and muscular tension. Ca is essential for coagulation, eggshell formation, muscle and nerve function (Dahifar et al. 2006; Ross et al. 2011). On the other hand, P has an important role in nerve function and is a significant component of eggshells, phospholipids and nucleic acids. It is a key mediator of energy metabolism through adenosine triphosphate (ATP) (Li et al. 2016).

Cereal grains have very low concentrations of Ca, but high P concentrations, most of which is bound as phytate and is considered not biologically available. However, it should be noted that plant feed ingredients, especially wheat, may contain significant endogenous phytase activity (Selle et al. 2003; Abd El-Hack et al. 2018), but it is lost during steam pelleting of diets (Jongbloed and Kemme 1990). Phytate also acts as an anti-nutritive factor by chelating divalent mineral cations and amino acids (Bryden et al. 2007). The most notable changes in diet formulation have been a move to essentially all plant diets, the adoption of digestibility values for amino acids and the widespread addition of feed enzymes, such as xylanase and phytase. Luigia and Agostino (2016) mentioned that increased serum calcium level in sample population correlates with deteriorating lipid profile.

Phosphorus plays an important role in improving immune system and has positive effects against pathogenic microorganisms. Phosphorus serves the activities of the immune system and promotes a healthy microbial environment in the gastrointestinal tract and acts as a buffer to possible pathogens (Charlotte et al. 2015). During the starter period of broiler chickens, body weight and gain as well as feed intake in the 30% available P deficient group were lower than that of the normal group (Imari et al. 2020). But, the levels of Ca, P and alkaline phosphatase in blood were not significantly affected by available P levels on days 10 and 42 (Imari et al. 2020). It has been concluded that the levels of available P in diets of modern broiler chickens can be reduced by 20% (Delezie et al. 2015).

Calcium considered lymphocyte second messenger. The remaining lymphocytes retain low calcium concentration. Antigen receptor activity causes calcium inflow from extracellular space by many methods; Ca2 + inflow occur and are introduced at specific levels of lymphocyte growth and maturation (Monika and Jean-Pierre 2009). The goal of this research was to determine the effect of dietary calcium and phosphorus different levels on lipid profile, antioxidant and immune parameters of growing Egyptian geese.

Materials and methods

All procedures of the experiment were performed with reference to the Committee of Local Experimental Animal Care and approved by ethics of our Poultry Department institutional committee, University of Zagazig, Egypt. This research was performed at the waterfowl Research Farm, Poultry Department, College of Agriculture, Zagazig University, Egypt. A total of 120 Egyptian goslings, 4 weeks old, with almost the same body weights (1300 ± 151) were randomly distributed to 4 groups, each group contains 30 birds, each group was then subdivided in to 6 replicates and each one has 5 birds. During the experimental period, the geese were kept in appropriate roosts, under the same hygienic conditions. In the first 3 days of the experiment, the animals were subjected to 24 h of light per day regime, then to 12 h of darkness and 12 h light per day for rest of the experimental period. Fresh water and feed were available during the entire time for all birds.

Design and diets

The research was carried out in a factorial configuration of 2 × 2. Two levels of calcium (0.85% and 0.70%) and two P levels (0.45% and 0.35%) were used during the experimental phase (4–12 weeks of age) to analyse their effect on the lipid profile, antioxidant and immune parameters of rising Egyptian geese. Diets were formulated according to recommendations of NRC (1994), as shown in Table 1.

Table 1. Composition and chemical analysis of the basal diets as fed.

Item*	Ca 0.70 %		Ca 0.85 %
	P 0.35 %	P 0.45 %	P 0.35%	P 0.45 %
Ingredients (g/kg diet)
Yellow Corn	66.00	66.00	66.2	66.00
Soybean meal (44%)	18.34	18.26	18.4	18.34
Gluten meal (60%)	4.50	4.40	4.49	4.55
Wheat bran	8.13	8.13	7.50	7.50
Di Calcium phosphate	1.25	1.8	1.25	1.80
Limestone	0.82	0.45	1.20	0.85
Vit-min Premix**	0.30	0.30	0.30	0.30
NaCl	0.30	0.30	0.30	0.30
DL Methionine	0.12	0.12	0.12	0.12
L-Lysine HCl	0.24	0.24	0.24	0.24
Total	100	100	100	100
Calculated analysis***
Crude protein %	18.10	18.00	18.04	18.03
ME kcal/kg diet	2906	2902	2907	2902
Calcium %	0.70	0.70	0.85	0.85
Phosphorous (Avai.)	0.35	0.45	0.35	0.45
Lysine %	1.00	1.00	1.00	1.00
Methionine + Cysteine %	0.75	0.75	0.75	0.75
Crude fibre %	3.68	3.66	3.63	3.62

*Ca: calcium and P: phosphorus

**Growth vitamin and Mineral premix Each 2.5 kg consists of : Vit A 12000, 000 IU; Vit D3, 2000, 000 IU; Vit. E. 10g; Vit k3 2 g; Vit B1, 1000 mg; Vit B2, 49g; Vit B6, 105 g; Vit B12, 10 mg; Pantothenic acid, 10 g; Niacin, 20 g, Folic acid, 1000 mg; Biotin, 50 g; Choline Chloride, 500 mg, Fe, 30 g; Mn, 40 g; Cu, 3 g; Co, 200 mg; Si, 100 mg and Zn, 45 g.

***Calculated according to NRC (1994).

Data collection

At the end of the experiment, 24 birds (six per group) were sampled for blood tests, and blood samples were randomly collected in heparinised tubes from five birds. Blood samples were centrifuged at (G force intensity = 2146.56 g) for 900 s with respect to biochemical parameters.

Lipid profile parameters

Using consumer kits (Bio diagnostic Company, Giza, Egypt), cholesterol (mg/dL), total cholesterol (TC; mg/dL), high density lipoprotein (HDL), triglyceride (TG; mg/dL) and low-density lipoprotein (LDL) rates were calculated spectrophotometrically.

Estimation of oxidative stress parameters

Based on commercial kits and spectrophotometer (Shimadzu, Tokyo, Japan), catalase (CAT) and superoxide dismutase (SOD) levels of malondialdehyde (MDA) and reduced glutathione (GSH) were estimated in plasma.

Immunological parameters estimation

Total and differential white blood cells (WBCS) count

Total WBCS, differential WBCs count and heterophil: lymphocyte (H: L) ratios were estimated according to Lauren et al. (2014). All blood smears were stained by using Wright–Giemsa Stain (EMD Chemicals Inc, Philadelphia, PA) evaluated microscopically. Descriptive analyses (standard deviation, mean, maximum and minimum) were calculated for estimated WBC count, WBC differential, and heterophil:lymphocyte (H:L) ratios.

Measurement of C reactive protein (CRP)

Level of CRP was determined by using enzyme-linked immunosorbent assay kit supplied by MyBiosource, San Diego, CA. MBS039949 ELISA kit is based on C reactive protein antibody and CRP antigen interactions to detect CRP antigen targets in serum samples.

Nitro blue tetrazolium (NBT) activity

NBT is yellow colour and reduced to produce formazan which is blue in colour, in these reaction oxygen radicals in neutrophil and monocyte were analysed by using of NBT. 0.1 mL of blood samples were placed in the microtiter plate wells, 0.2% of the NBT solution was added and incubated at room temperature for 30 minutes. Approximately 0.05 ml NBT blood cell suspension was applied to 1 mL N,N-dimethyl formamide, and centrifuged at 3000 xg for 5 min. Supernatant was tested using a 620 nm spectrophotometer in one ml of cuvettes (Siwicki et al. 1985).

Neutrophil adherence assays

Glass adherence is an indication of activated monocytes and neutrophils, this test was carried out to demonstrate activation and excess radical oxygen production. NBT glass adherent assays were conducted by inserting single drops (0.1 mL) of blood on two glass covers. At room temperature, coverslips were incubated for 30 min, and then softly washed by the phosphate buffer saline. Drops of 0.1 mL of 0.2% NBT were mounted on the microscope slide and a coverslip was placed on top so that the NBT solution could incubate the adherent cells at room temperature for 30 min. At ×400, activated neutrophils were counted under a microscope (Anderson et al. 1992).

Serum bactericidal test (SBT)

Bacterial colonies were centrifuged from each Salmonella typhimurium, and pellets were washed and suspended in phosphate buffer saline. The suspension's optical density was modified at 540 nm to 0.5%. Five times, the bacterial solution was mixed serially (1:10) with Phosphate buffer saline. The serum bacterial activity was measured by incubating at 37 °C for one hour two μL of bacterial suspension with 20 μL of serum in a micro-vial. Control group, saline phosphate buffer substitute’s serum. The number of viable bacteria is identified after incubation by counting colonies on a trypticase soya agar plate at 37 °C for 24 h after incubation (Rao et al. 2006).

Lysozyme activity

Lysozyme is an essential immune system parameter and functions as a bactericidal agent. Lysozyme expression was measured using a turbidity assay in which 0.2 mg mL⁻¹ lyophilised micrococcus lysodekticus was used as a substrate in a sodium phosphate buffer of 0.04 M (pH 5.75) (Parry et al. 1965). Fifty μl of serum was applied to 2 mL of bacterial suspension; with an absorbance reduction of 540 nm was measured at 22 °C after 0.5 and 4.5 min of incubation. One unit of action for lysozyme was acting as a 0.001 min⁻¹ reduction in absorbance.

Phagocytosis estimation

Phagocytosis was carried out by using the direct counting procedure according to Miller (1974) with phagocytic particle Enterobacter cloacae that was grown and enriched with brain heart agar and quantitated by pour plate count before killing with formalin. Heparinised (10 U/mL) whole blood from individual geese was grouped before the total phagocytes from total cells were calculated (Natt and Herrick 1952) and accounting differential leukocytes (Lucas and Jamroz 1961). Bacteria were added to 0.9 mL of blood in 0.1 mL of 0.85% NaCl to give the initial 10:1 ratio of bacteria to total phagocytes.

In a water bath shaker, the mixture was incubated at 41 °C (60 cycle/min). The blood smears were stained with Wright dye at spaced intervals from incubation mixture. Then, we counted 100 heterophils per slide to assess the proportion of phagocytising heterophils. Mean phagocytosis was estimated by counting the number of bacteria that engulfed per heterophil exhibiting phagocytosis (25 phagocytising heterophils per slide were counted).

Statistical analysis

The data collected were statistically evaluated on 2 × 2 factorial arrangements using the following model: $${\mathrm{Y}}_{\text{ijk}}=\mu +{\mathrm{A}}_{\mathrm{i}}+{\mathrm{S}}_{\mathrm{j}}+{\text{AS}}_{\text{ij}}+{\mathrm{e}}_{\text{ijk}},$$ where Y_ijk is an event, A_i is an effect of Ca level (I = 1–2), μ is the total mean, AS_ij is the relationship of two variables, S_j is an effect of P level (j = 1–2) and e_ijk is an experimental random error. Tukey’s post-hoc test was performed to determine variations between treatments. The variations were significant at p < .05.

Results and discussion

Lipid profile parameters

Adding various levels of calcium and phosphorus to Egyptian geese diet did not significantly affect (p > .05) triglycerides, HDL-cholesterol, and VLDL-cholesterol, whereas total cholesterol and LDL-cholesterol were significantly affected as clarified in Table 2. LDL cholesterol and total cholesterol (p ≤ .05) were significantly decreased by supplementation of 0.85% Ca, and 0.45% P levels. With respect to the interaction effects, the lipid profile parameters were not significantly altered (p > .05) by the interaction among calcium and phosphorus levels in Egyptian geese diet except triglycerides and VLDL cholesterol (p < .001) as clearly in Table 3. Plasma triglycerides and VLDL cholesterol concentration were the lowest at 0.70% Ca, 0.45% P followed by 0.85% Ca, 0.45% P interactions, respectively. Our results were partially agreement with that recorded by Ankra-Badu et al. (2010) and Lee et al. (2019) where the best results on lipid profile were obtained by supplementation 0.85%Ca and 0.45%P in the diets of white Roman geese.

Table 2. Lipid profile and antioxidant indices of growing geese as affected by the main effect of calcium and phosphorus levels.

Traits	Calcium levels (%)		P-value	Phosphorus levels (%)		P-value
	0.70 %	0.85 %		0.35 %	0.45 %
Lipid profile*
Triglycerides (mg/dL)	139.1 ± 18.1	135.6 ± 23.5	0.477	134.6 ± 22.6	140.1 ± 18.9	0.283
Total cholesterol (mg/dL)	186.4 ± 15.6	167.1 ± 18.0	0.004	163.0 ± 13.3	190.5 ± 12.5	<0.001
HDL cholesterol (mg/dL)	48.26 ± 1.56	46.49 ± 1.25	0.077	47.13 ± 2.07	47.62 ± 1.22	0.592
LDL cholesterol (mg/dL)	110.3 ± 19.6	93.49 ± 13.0	0.010	88.91 ± 9.49	114.9 ± 15.0	0.001
VLDL cholesterol (mg/dL)	27.82 ± 3.63	27.11 ± 4.69	0.477	26.92 ± 4.53	28.01 ± 3.78	0.283
Antioxidant parameters*
SOD (U/L)	149.0 ± 20.6	101.4 ± 19.0	<0.001	108.7 ± 26.8	141.6 ± 27.8	<0.001
CAT (U/L)	138.9 ± 15.8	91.32 ± 18.5	<0.001	101.3 ± 28.0	128.9 ± 26.6	0.001
MDA (nmol/ml)	3.650 ± 0.72	6.345 ± 0.88	<0.001	5.667 ± 1.59	4.329 ± 1.43	<0.001
GSH (ng/mL)	0.284 ± 0.04	0.187 ± 0.03	<0.001	0.206 ± 0.05	0.264 ± 0.06	<0.001

Means in the same row within each classification bearing different letters are significantly (P ≤ 0.05) different.

*HDL: high density lipoprotein, LDL: low-density lipoprotein, VLDL: very low-density lipoprotein, SOD: superoxide dismutase, CAT: catalase, MDA: malondialdehyde, GSH: reduced glutathione

Table 3. Lipid profile and antioxidant indices of growing geese as affected by the interaction among treatments.

Traits	0.70 % Ca		0.85 % Ca		P-value
	0.35 % P	0.45 % P	0.35 % P	0.45 % P
Lipid profile*
Triglycerides (mg/dL)	154.1 ± 7.86^a	124.1 ± 9.38^b	115.1 ± 8.95^b	156.0 ± 6.52^a	<0.001
Total cholesterol (mg/dL)	173.2 ± 6.96	199.5 ± 6.28	152.7 ± 8.93	181.5 ± 10.2	0.797
HDL cholesterol (mg/dL)	48.30 ± 2.32	48.21 ± 0.86	45.95 ± 1.07	47.02 ± 1.39	0.525
LDL cholesterol (mg/dL)	94.12 ± 10.3	126.5 ± 7.99	83.70 ± 6.12	103.3 ± 9.79	0.239
VLDL cholesterol (mg/dL)	30.81 ± 1.57^a	24.83 ± 1.88^b	23.02 ± 1.79^b	31.20 ± 1.30^a	<0.001
Antioxidant parameters*
SOD (U/L)	131.7 ± 7.27	166.3 ± 10.4	85.81 ± 13.1	117.0 ± 1.71	0.754
CAT (U/L)	125.1 ± 5.34	152.6 ± 5.21	77.53 ± 15.3	105.1 ± 7.00	0.999
MDA (nmol/ml)	4.260 ± 0.33	3.041 ± 0.23	7.073 ± 0.54	5.618 ± 0.27	0.588
GSH (ng/mL)	0.252 ± 0.01	0.315 ± 0.02	0.160 ± 0.02	0.213 ± 0.01	0.556

Means in the same row within each classification bearing different letters are significantly (P ≤ 0.05) different.

*HDL: high density lipoprotein, LDL: low-density lipoprotein, VLDL: very low-density lipoprotein, SOD: superoxide dismutase, CAT: catalase, MDA: malondialdehyde, GSH: reduced glutathione

Antioxidant parameters

Concentration of serum antioxidant parameters was slightly altered (p = .001) due to the supplementation of different levels of Ca and P in the diet of Egyptian geese as explained in data recorded in Table 2. Since, antioxidant enzymes (SOD, CAT and GSH), also increased their efficacy by using 0.85% Ca and 0.45% P supplement rates (p = .000). Also, MDA concentration as a lipid peroxidation marker decreased significantly (p < .001) at supplementation by these levels. Related to the interaction among Ca and P levels effects, antioxidant indices levels were not significantly changed due to Ca and P interaction levels (p > .05). These results were partially agreement with Zhang et al. (2020) who reported that serum concentrations of T-AOC and CAT were decreased by dietary Ca and P deficiencies without 25-OH-D₃ (Interaction, p < .05) and unaffected by dietary deficiencies of Ca and P with 25-OH-D₃ at 42 days of age. In the present study, antioxidant parameters (SOD, CAT and GSH) were elevated numerically at 0.85% Ca and 0.45% P interaction and 0.70% Ca and 0.45% P interaction. The deficiency in Ca leads to a significant decrease of antioxidant enzymes (Choi and Jung 2017). Chen et al. (2017) reported that the deficiency in P depressed antioxidant enzymes activities and GSH content and downregulated the mRNA levels of antioxidant enzymes. Also, MDA levels were numerically decreased at interaction supplementation of these Ca and P levels. Dietary supplementation of 25-OH-D₃ decreased (p < .05) serum concentration of MDA in broilers (Zhang et al. 2020). Oxidation of lipid is a fundamental indicator for poor meat quality and more rancidity%, moreover development of forbidden alterations in colours, flavour and odour (Gray et al. 1996).

The elevated levels of MDA are an indicator of oxidative damage in lipid peroxidation and in cells caused by ROS (reactive oxygen species) (Nielsen et al. 1997). Malondialdehyde is one of the final end products of peroxidation of polyunsaturated fatty acids in host cells. The expansion of the free radicals allows MDA to overproduce. Malondialdehyde level is commonly regarded as the predictor of oxidative status and the state of cell strengthening in damaging cells (Gaweł et al. 2004). In fact, MDA is an after-effect of compromised lipids and is commonly used to predict lipid peroxidation in host cells (Raharjo and Sofos 1993).

Immunity parameters

Total and differential WBC count was estimated to all collected samples, addition of different levels of calcium and phosphorous in diet of geese highly significantly effect on total WBCs count as illustrated in Table 4. Heterophile percentage, lymphocyte and eosinophil percentages were not significantly affected with supplementation of different levels of phosphorus and calcium in geese diets. The findings obtained were partly in line with those reported by Tian et al. (2013) who mentioned that optimal diet vitamin D3 supplementation could increase the metabolism of calcium and the immune function of Yangzhou goslings. On the other hand, the highest lymphocyte (H) and lowest blood heterophil (L) percentages and lowest ratio of H:L were recorded in birds fed with the ration containing a standard Ca–P (Askari et al. 2015).

Table 4. Immune parameters of growing geese as affected by the main effect of calcium and phosphorus levels.

Traits	Calcium levels (%)		P-value	Phosphorus levels (%)		P-value
	0.70 %	0.85 %		0.35 %	0.45 %
WBCS*	20.90 ± 7.22	33.73 ± 2.72	<0.001	23.71 ± 10.0	30.92 ± 4.34	<0.001
Heterophil %	17.26 ± 0.41	17.07 ± 0.31	0.067	17.01 ± 0.28	17.32 ± 0.40	0.004
lymphocyte%	56.74 ± 1.31	56.50 ± 0.91	0.533	56.89 ± 1.16	56.35 ± 1.03	0.155
Esinophile%	22.60 ± 1.30	22.52 ± 1.01	0.853	22.61 ± 1.33	22.51 ± 0.97	0.791
Monocyte%	1.572 ± 0.21	1.578 ± 0.19	0.935	1.556 ± 0.19	1.594 ± 0.21	0.572
Basophile %	1.600 ± 0.20	1.556 ± 0.22	0.532	1.544 ± 0.22	1.611 ± 0.20	0.350
NBT mg/ml	3.181 ± 0.62	4.388 ± 0.22	<0.001	3.468 ± 0.91	4.101 ± 0.40	<0.001
Neutrophil adherence Cell/field	9.827 ± 2.40	12.88 ± 0.48	<0.001	10.06 ± 2.64	12.64 ± 0.65	<0.001
Alpha-1 globulin (g/dL)	0.588 ± 0.05	0.543 ± 0.10	0.123	0.535 ± 0.09	0.597 ± 0.06	0.046
Alpha-2 globulin (g/dL)	0.835 ± 0.08	0.890 ± 0.11	0.405	0.872 ± 0.12	0.853 ± 0.08	0.777
Beta globulin (g/dL)	0.485 ± 0.08	0.833 ± 0.35	<0.001	0.813 ± 0.37	0.505 ± 0.07	0.001
Gamma globulin (g/dL)	0.842 ± 0.08	1.253 ± 0.47	<0.001	1.258 ± 0.47	0.837 ± 0.07	<0.001
bactericidal activity	105.9 ± 9.23	99.94 ± 2.39	<0.001	106.5 ± 8.69	99.39 ± 2.77	<0.001
Lysozyme activity	10.31 ± 2.99	12.81 ± 0.69	<0.001	9.903 ± 2.59	13.22 ± 0.48	<0.001
Phagocytic percentage (P%)	0.733 ± 0.16	0.921 ± 0.04	<0.001	0.750 ± 0.17	0.904 ± 0.04	<0.001
Phagocytic index (P.I.)	0.816 ± 0.12	0.911 ± 0.04	<0.001	0.809 ± 0.11	0.918 ± 0.04	<0.001
Serum CRP (mgLdl)	29.71 ± 1.61	31.67 ± 0.46	<0.001	29.79 ± 1.67	31.59 ± 0.58	<0.001

Means in the same row within each classification bearing different letters are significantly (P ≤ 0.05) different.

*WBCS: White blood cells, NBT: Nitoblue tetrazolium activity, CRP: C reactive protein

The supplementation of 0.45% phosphorus could significantly increase total WBCS count in compared with group that received 0.35% phosphorus and these results was agreed with Xu et al. (2014) who mentioned that phosphorus deficiency can lead to decreased numbers of the peripheral blood T-lymphocyte populations, affecting the cellular immune system in layers.

The percentage of monocytes increased with body weight in geese, while atypical lymphocyte cell morphology was observed in 4% of adult geese. The use of 0.85% calcium or 0.45% phosphorus in growing geese diets increased NBT activity. Regarding the interaction effect, NBT activity was significantly affected by the interaction among calcium and phosphorus levels. The highest value was recorded by birds fed diet contained 0.85% Ca and 0.45%P at the experimental period, as shown in Tables 4 and 5.

Table 5. Immune parameters of growing geese as affected by the interaction among treatments.

Traits	0.85 % Ca		0.70 % Ca		P-value
	0.35 % P	0.45 % P	0.35 % P	0.45 % P
WBCS*	14.27 ± 2.85^c	27.53 ± 1.96^b	33.15 ± 2.17^a	34.31 ± 3.20^a	<0.001
Heterophil %	16.96 ± 0.28^b	17.56 ± 0.27^a	17.05 ± 0.28^b	17.08 ± 0.36^b	0.008
lymphocyte%	57.07 ± 1.29	56.41 ± 1.31	56.72 ± 1.06	56.29 ± 0.72	0.766
Esinophile%	22.56 ± 1.56	22.64 ± 1.08	22.67 ± 1.16	22.38 ± 0.88	0.643
Monocyte%	1.556 ± 0.17	1.589 ± 0.24	1.556 ± 0.21	1.600 ± 0.18	0.935
Basophile %	1.600 ± 0.21	1.600 ± 0.21	1.489 ± 0.23	1.622 ± 0.20	0.350
NBT mg/ml	2.608 ± 0.21^c	3.753 ± 0.16^b	4.328 ± 0.22^a	4.449 ± 0.23^a	<0.001
Neutrophil adherence Cell/field	7.511 ± 0.25^d	12.14 ± 0.28^c	12.61 ± 0.30^b	13.14 ± 0.50^a	<0.001
Alpha-1 globulin (g/dL)	0.613 ± 0.04^a	0.563 ± 0.05^a	0.457 ± 0.04^b	0.630 ± 0.06^a	0.003
Alpha-2 globulin (g/dL)	0.830 ± 0.11	0.840 ± 0.08	0.913 ± 0.14	0.867 ± 0.10	0.663
Beta globulin (g/dL)	0.490 ± 0.12^b	0.480 ± 0.03^b	1.137 ± 0.12^a	0.530 ± 0.10^b	0.001
Gamma globulin (g/dL)	0.840 ± 0.09^b	0.843 ± 0.09^b	1.677 ± 0.13^a	0.830 ± 0.07^b	<0.001
bactericidal activity	113.6 ± 6.54^a	98.33 ± 2.83^b	99.44 ± 2.40^b	100.4 ± 2.40^b	<0.001
Lysozyme activity	7.449 ± 0.57^c	13.17 ± 0.53^a	12.36 ± 0.60^b	13.26 ± 0.45^a	<0.001
Phagocytic percentage (P %)	0.588 ± 0.06^c	0.879 ± 0.02^b	0.912 ± 0.04^ab	0.930 ± 0.03^a	<0.001
Phagocytic index (P.I.)	0.711 ± 0.04^b	0.920 ± 0.04^a	0.907 ± 0.04^a	0.916 ± 0.04^a	<0.001
Serum CRP (mgLdl)	28.21 ± 0.39^c	31.20 ± 0.55^b	31.37 ± 0.39^b	31.98 ± 0.28^a	<0.001

Means in the same row within each classification bearing different letters are significantly (P ≤ 0.05) different.

*WBCS: White blood cells, NBT: Nitoblue tetrazolium activity, CRP: C reactive protein

After feeding experiment, neutrophils adherence test showed significant increases in geese groups received diet contained 0.85% calcium and 0.45% phosphorus. Neutrophils adherence significantly affected by the interaction among calcium and phosphorus levels, as agreed with results obtained by Tian et al. (2013).

The viable Salmonella typhimurium count were significantly higher in group received 0.75% calcium and 0.35% phosphorus while count was decreased in geese group which received 0.85% calcium and 0.45% phosphorus. While, lysozyme activity showed a significant increase in all groups received calcium and phosphorous. No significant difference was observed due to different levels of calcium and phosphorous.

The phagocytic percentage (P%) of blood polymorphonuclear (PMN) cells was higher in group of geese which fed on 0.85% calcium and 0.45% phosphorous, as shown in the following (Tables 4 and 5), these results were agreed with Melendez (2013) who mentioned that calcium acts as a cofactor for activation of intracellular signalling proteins, which activate phagocytosis. Requirement of calcium for initial steps in phagocytosis, and phagosome maturation, depending on type of cell and receptor that is driving phagocytosis. Also, obtained results agreed with Kiersztejn et al. (1992) who estimated that phosphate depletion impairs the phagocytic ability of polymorphonuclear leukocytes (PMNL).

Conclusions

The obtained results were concluded that optimal dietary Ca and P requirements for growing Egyptian geese are 0.85% and 0.45%, respectively. Also, dietary Ca and P levels have a good effect on lipid profile indices, and increased the activity of antioxidant enzymes (SOD, CAT and GSH) and decreased MDA as a marker of oxidative status of polyunsaturated fatty acids. Also, the levels of 0.85% Ca and 0.45% P increased total WBCs count, phagocytic index and the most immune parameters of the growing geese. So, we can conclude that the standard Ca and P requirements for growing Egyptian geese are 0.85% and 0.45%, respectively.

Ethical approval

Animal care and maintenance were performed in accordance with the guidelines of the Egyptian Research Ethics Committee and the guidelines specified in the Guide for the Care and Use of Laboratory Animals (2011).

Acknowledgement

This project was supported financially by the Science and Technology Development Fund (STDF), Egypt, Grant no. 26193).

Disclosure statement

No potential conflicts of interest declared.

Additional information

Funding

This project was supported financially by the Science and Technology Development Fund (STDF), Egypt, Grant no. 26193).