Animal Science

The performance, haematological indices and serum chemistry of two breeds of broiler chickens fed Ficus exasperate leaf powder and Vitamin C supplemented diets

0000-0002-7806-6662C.O Osowe¹  , 0000-0001-9652-7250O.A Adu¹  , 0000-0002-2175-1490O.D Oloruntola^{2  *}  , 0000-0003-3471-7758C.A Chineke¹ 

¹Department of Animal Production and Health, The Federal University of Technology, Akure, Nigeria.

²Department of Animal Science, Adekunle Ajasin University, Akungba-Akoko, Nigeria.

ABSTRACT

A total of 240 Arbor acre (AB) and 240 Cobb 500 (CO) broiler chickens given Ficus exasperate leaf powder (1%FELP) and 200mg/kg Vitamin C (VC) supplemented diets were studied for performance and blood indices. Diets 1 and 2 (controls) were fed to AB and CO, respectively; Diets 3 and 4 (VC supplemented) were fed to AB and CO, respectively; Diets 5 and 6 (FELP supplemented) were fed to AB and CO, respectively; while diets 7 and 8 (VC+FELP supplemented) were fed to AB and CO, respectively. At 6 age weeks, body weight gain (BWG) of CO improved (P<0.05) over the AB birds, while VC improved BWG of CO over AB. VC improved the feed conversion ratio (FCR). VC and FEPL increased the packed cell volume and the red blood cell (RBC); while the RBC of CO was higher (P<0.05) than AB. The VC and FELP increased the haemoglobin concentration. The white blood cell count improved (P<0.05) by FELP. The VC and VC x FELP reduced (P<0.05) the serum aspartate aminotransferase concentration. VC and FELP reduced (P<0.05) the serum creatinine concentration. The FELP reduces (P<0.05) the serum cholesterol. Conclusively, CO and 200 mg/kg Vitamin C supplementation support optimal body weight gain. For better immunity and hypocholesterolemia, 1 % FELP supplementation is indicated; while 1 % FELP might be coupled with 200 mg/kg Vitamin C to enhance feed consumption, red blood cell count, packed cell volume, haemoglobin concentration; to prevent liver and tissue damage and protect renal cells from damage in broiler chickens.

Key words: avian; genetic make-up; phytogens; citric acid; stress

In the tropics, where in most places poultry species are reared outside their thermoneutral zone of 18 ºC-24 ºC, the negative effects of high ambient temperature on broiler chicken performance and health status are on the rise (^{Olanrewaju et al. 2010}), and this is cause for concern. This is owing to climate problems, which have resulted in widespread heat stress among livestock species (^{Kpomasse et al. 2021}). Previous reports indicate that supplementation of antioxidants ameliorated the effects of heat stress on poultry; and there are calls for the adjustment of birds’ diets to the climatic conditions of the regions where they are produced (^{Suganya et al. 2015}, ^{Adeyeye et al. 2020} and ^{Kpomasse et al. 2021}).

Supplements are included in a nutritionally balanced diet to elicit growth, antioxidant status, and feed conversion ratio improvement of the host (^{Dhama et al. 2014}). Antibiotics, prebiotics, antioxidants, probiotics, coccidiostats, exogenous enzymes, and phytogenics or phytochemicals are among the popular dietary supplements used in the broiler industry (^{Dhama et al. 2014}).

There is legislation against the use of antibiotics as supplements or growth promoters in animal production and the increasing awareness and demand of consumers for antibiotic-free animal protein (^{Gadde et al. 2017}). However, phytogenics or herbs and Vitamin C are increasingly being used as feed supplements in animal production to boost growth and improve the antioxidant status of the animals (^{Adeyeye et al. 2020}, ^{Adebayo et al. 2020} and ^{Kpomasse et al. 2021}).

Aromatic plants/phytogenics have been employed since ancient times for their therapeutic benefits as well as their ability to impart aroma and flavour to meals or food. Natural medicine products produced from herbs have been used as feed additives in chicken production to increase growth by combining active and complex molecules, such as those found in phytogens (^{Khan et al. 2012a},^b). Phytogenic food supplementation has been shown in trials to promote or maintain gut microbiota, which improves host nutrition, growth, and health through better nutrient utilization (^{Hashemi and Davoodi 2011}, ^{Oloruntola et al. 2020} and ^{Oloruntola et al. 2021}). The active components in the herbs have been shown to inhibit lipid peroxidation and reduce iron complex, as well as produce nitric oxide, scavenge superoxide radicals, and hydrogen peroxide from activated macrophages (^{Dhama et al. 2014}, ^{Oloruntola et al. 2020} and ^{Oloruntola et al. 2021}).

Vitamin C supplementation has been shown to improve growth vigour, immune system stimulation, feed conversion, and gut microbiota regulation. Furthermore, Vitamin C is important for improved feed utilisation and metabolism, as well as stress reduction. (^{Sahin et al. 2003} and ^{Dhama et al. 2014}).

The "Sandpaper Leaf Tree," Ficus exasperata Vahl. (Moraceae), is rapidly being used for several ailments, prompting a rise in research to back up its conservative claims (^{Bafor and Igbinuwen 2009}). The leaves of F. exasperata have anticonvulsant, antibacterial, hypoglycemic, antiulcer, hypotensive, hypolipidemic, anti-inflammatory, oxytocin inhibitory, antipyretic, and anxiolytic activities (^{Ahmed et al. 2012}). The earlier study of the composition and properties of Ficus exasperate by ^{Osowe et al. (2021)} reveals that the F. exasperate leaf has 11.41 % ash, 17.26 % crude fibre, 9.61 % crude fat, 15.01% crude protein; 85% DPPH, 8 mg/g Vitamin C, 165.28 mg/g flavonoids, 56.52 mg/g phenols, 3.33 mg/g, 80.72 mg/g saponins, and 81 mg/g alkaloids.

Broiler chicken development, blood indices, serum biochemical indices, and antioxidant status are influenced by phytogen, vitamin and mineral supplementation doses, genetic line, and age. (^{Al-Masad 2012}, ^{Oloruntola et al. 2018} and ^{Adebayo et al. 2020}). As a result, the objective of this study is to examine two breeds of broiler chickens (Arbor acre and Cobb 500), being raised under a typical tropical ambient temperature (27.77±0.4 degrees Celsius) and given 1 % Ficus exasperate leaf powder and 200 mg/kg vitamin C supplemented diets for performance, haematological indices, serum biochemistry indices, and antioxidant status.

Materials and Methods

Ethical approval. collection, processing, and analysis of phytogens. The experiment's standards and criteria for animal and animal protocol were accepted by the Research and Ethics Committee of the Department of Animal Production and Health, the Federal University of Technology, Akure (FUTA), Nigeria. The experiment took place in February and March of 2021, with an average ambient temperature of 27.77±0.4 degrees Celsius and relative humidity of 74.5 %. ^{Osowe et al. (2021)} described the procedure for gathering, preparing, and analyzing Ficus exasperata leaf powder (FELP).

Vitamin C and ingredients for experimental feed. Local markets in Akure, Nigeria, were used to obtain Vitamin C powder (Avondale Laboratories Limited, Banbury, England) and other feed ingredients such as maize, maize bran, rice bran, soybean meal, soy oil, fish meal, limestone, bone meal, common salt, broiler premix, methionine, and lysine.

Experimental diets, experimental design, and the birds’ living environment. To address the dietetic requirements of the experimental broiler starter and finisher chickens (crude protein: 22.18 % and 20.03 %; crude fibre: 3.51 % and 3.59 %; crude fat: 4.22 % and 2.39 %; metabolizable energy: 12.61 kJ/kg and 12.99.1 kJ/kg), a basic experimental diet was compounded for the starting phase (0 to 21 days) and the finishing phase (21-42 days) (table 1). The crude protein, crude fat, and crude fat content of the experimental diets were determined (^{AOAC 2016}).

Table 1 Composition of the experimental diets 

Ingredients (%)	Starter phase	Finisher phase
Maize	50.35	58.35
Maize bran	3.00	0.00
Rice bran	0.00	3.00
Soybean meal	38.00	30.00
Soy oil	1.00	1.00
Fish meal	3.00	3.00
Limestone	0.50	0.50
Bone meal	3.00	3.00
Salt	0.30	0.30
Premix	0.30	0.30
Methionine	0.30	0.30
Lysine	0.25	0.25
Nutrient composition (%)
*Crude protein	22.18	20.03
*Crude fibre	3.51	3.59
*Crude fat	4.22	2.39
Metabolizable energy (Kcal/kg)	3018.0	3108.10
Methionine	0.46	0.45
Lysine	1.12	1.09
Available phosphorus	0.47	0.42
Calcium	1.02	0.95

The baseline diet was divided into eight equal portions and called diets 1 to 8 for each phase. Diets 1 and 2 were fed to Arbor acre and Cobb 500, respectively, with no supplements; Diets 3 and 4 were fed to Arbor acre and Cobb 500, respectively, and were supplemented with 200 mg/kg Vitamin C powder; Diets 5 and 6 were fed to Arbor acre and Cobb 500, respectively, and were supplemented with 1 % FELP; while diets 7 and 8 were fed to Arbor acre and Cobb 500, respectively and were supplemented with 200 mg/kg Vitamin C and 1% FELP, respectively.

The feeding trial took place at the FUTA Teaching and Research Farm in Nigeria. For this experiment, a total of 480 broiler chickens (240 Arbor acre; 240 Cobb 500) were used. Specifically, 240 Arbor acre broiler chicks were randomly assigned to diets 1, 3, 5, and 7 in a completely randomised arrangement; while 240 one-day-old Cobb 500 were also allocated to diets 2, 4, 6, and 8 at random. Six times, each diet was repeated with ten birds each time (10 birds/repeat). Wood flakes were used to blanket the floor (2m x 1m) of the experimental pen that housed each replicate to a depth of 3 cm. To keep the chicks warm, heat lamps were positioned above the brooder. The temperature in the experimental house was maintained at 31±2 degrees Celsius for the earliest week, then dropped by 2 degrees Celsius each subsequent week until it reached 26±2 degrees Celsius. On the first day, the lights were left on for 24 hours, and on subsequent days, it was left on for 23 hours.

Performance characteristics. The body weight (BW) and feed intake (FI) of the experimental broiler birds were examined and measured every seven days. The average body weight gain (BWG) was calculated using the difference between the birds’ initial and final BW. The feed conversion ratio was computed by dividing the amount of feed consumed by the amount gained in weight.

Blood samples. On day 42 of the feeding study, 24 birds (3/replications) from each dietary group were randomly selected and bled using a syringe and needle via the wing vein. For serum proteins (total protein, albumin, globulin), biochemical components (creatinine, and cholesterol), enzyme activities (aspartate aminotransferase, and alanine transaminase) a portion of the blood sample (4 mL) was poured into a plain blood sample vial. The blood sample in each of the sample bottles was spun before analysis, and the serum was decanted into another plain bottle before freezing at -20 °CA Reflectron ^®Plus 8C79 (Roche Diagnostic, GombH Mannheim, Germany) and kits were used to detect serum proteins, biochemical components, and enzyme activity. For the measurement of haematological indices, the leftover blood (2 mL) was put into an Ethylenediaminetetraacetic acid blood sample collection tube. The standard procedures for determining haematological indices were followed (^{Cheesbrough 2000}).

Statistical data evaluation. The data was analysed using the SPSS version 20 General Linear Model procedure for a complete randomized design with 2x2x2 factorial arrangements. The data were tested for the main effects (breeds, Vitamin C, and FELP inclusion) and their interactions.

The P<0.05 was used to determine significance. Means were separated using Duncan multiple range test using SPSS.

Results

Performance. The growth performance response of different breeds of broiler chickens to Ficus exasperata leaf powder and Vitamin C dietary supplementation is shown in table 2. At the starter phase (1-3 weeks), FELP significantly (P<0.05) reduced the FI. This results in the reduced (P<0.05) FI recorded in treatments 5, 6, 7, and 8 compared to the control treatments (1 and 2). The feed conversion ratio (FCR) improved (P<0.05) by breed and FELP supplementation; consequently, the FCR was better (P<0.05) in the AB breed, compared to the CO breed, and in FELP supplemented treatment (5, 6, 7 and 8) compared to no FELP supplemented treatments (1, 2, 3, and 4).

Table 2 The growth performance response of different breeds of broiler chickens to Ficus exasperata, leaf powder and Vitamin C dietary supplementations 

TRT	BRD	VC, mg/kg	FELP, %	IW- g/b	BWG, 1-3 wks	FI, 1-3 wks	FCR, 1-3 wks	BWG, 4-6 wks	FI, 4-6 wks	FCR, 4-6 wks	BWG, 1-6 wks	FI, 1-6 wks	FCR, 1-6 wks
1	AB	0	0	37.65	845.36	1024.24^a	1.21^ab	1490.22^b	3269.07	2.26^ab	2335.58^b	4293.32	1.86^ab
2	CO	0	0	37.55	792.34	984.24^a	1.24^a	1887.54^a	2751.00	1.47^c	2679.88^a	3735.23	1.39^c
3	AB	200	0	37.32	812.60	988.37^a	1.22^ab	1604.74^b	2563.15	1.59^bc	2417.34^b	3551.52	1.46^bc
4	CO	200	0	37.54	807.01	1004.17^a	1.24^a	1915.37^a	2678.75	1.39^c	2722.38^a	3682.92	1.35^c
5	AB	0	1	37.59	838.44	894.84^bc	1.06^d	1451.00^b	3402.66	2.37^a	2289.45^b	4297.51	1.88^a
6	CO	0	1	37.51	777.79	853.88^c	1.09^cd	1486.68^b	3374.69	2.27^ab	2264.47^b	4228.58	1.86^ab
7	AB	200	1	37.64	797.96	869.13^c	1.08^d	1561.69^b	3106.75	1.98^abc	2359.63^b	3975.89	1.68^abc
8	CO	200	1	37.40	815.29	947.31^b	1.16^bc	1622.26^b	2876.20	1.77^abc	2437.56^b	3823.52	1.56^abc
SEM				0.04	8.45	14.63	0.01	39.86	90.05	0.09	37.67	89.01	0.05
P-value				0.59	0.55	0.01	0.01	0.01	0.07	0.02	0.01	0.19	0.03
	AB			37.55	823.59	944.15	1.14^b	1526.91^b	3085.41	2.05^a	2350.51^b	4029.56	1.72
	CO			37.50	798.11	947.40	1.18^a	1727.96^a	2920.16	1.73^b	2526.07^a	3867.56	1.54
	SEM			0.06	12.27	12.25	0.01	32.39	107.14	0.10	28.64	115.15	0.06
	P-value			0.61	0.16	0.85	0.01	0.01	0.29	0.04	0.01	0.33	0.06
		0		37.57	813.48	939.30	1.15	1578.86^b	3199.36^a	2.09^a	2392.35^b	4138.66^a	1.75^a
		200		37.47	808.21	952.25	1.17	1676.01^a	2806.21^b	1.68^b	2484.23^a	3758.46^b	1.51^b
		SEM		0.06	12.22	12.25	0.01	32.39	107.14	0.10	28.64	115.15	0.06
		P value		0.29	0.76	0.46	0.17	0.05	0.02	0.01	0.03	0.03	0.01
			0	37.51	814.33	1000.25^a	1.23^a	1724.46^a	2815.49^b	1.68^b	2538.79^a	3815.75	1.52^b
			1	37.53	807.37	891.29^b	1.10^b	1530.41^b	3190.07^a	2.10^a	2337.78^b	4081.37	1.75^a
			SEM	0.06	12.22	12.25	0.01	32.39	107.14	0.10	28.64	115.15	0.06
			P value	0.81	0.69	0.01	0.01	0.01	0.02	0.01	0.01	0.12	0.01
	Interactions P-value
	BRD x VC			0.21	3.28	6.36	0.45	0.11	0.50	0.64	0.15	0.86	0.52
	BRD x FELP			1.50	0.40	0.78	0.47	11.14	0.05	1.33	13.55	0.09	1.63
	VC x FELP			0.61	0.40	1.45	1.49	0.32	0.01	0.05	0.54	0.01	0.03
	BRD x VC x FELP			1.84	0.19	0.83	0.53	0.37	1.90	1.46	0.77	1.40	1.58

Means with a different superscript in the same column are significantly (P<0.05) different; TRT: Treatments; BRD: Breeds; VC: Vitamin C; FELP: Ficus exasperata IW: Initial weight; BWG: Body weight gain; FI: Feed intake; FCR: Feed conversion ratio; AB: Arbor acre; CO: Cobb 500; SEM: Standard error of the means.

At the finisher phase (4-6 weeks), the body weight gain (BWG), FI, and FCR were affected (P<0.05) by the dietary treatments. The BWG of CO was greater (P<0.05) than AB, the vitamin C supplementation improved (P<0.05) the BWG; while the FELP supplementation reduced (P<0.05) the BWG. Consequently, the best (P<0.05) BWG was recorded in treatments 2 and 4. The Breed x FELP and vitamin C x FELP were significant (P<0.05) for BWG. The vitamin C supplementation significantly (P<0.05) reduced the FI; while the FELP supplementation increased (P<0.05) the FI. The Vitamin C x FELP improved the FCR. The FCR was improved (P<0.05) by breed and Vitamin C supplementation; while FELP impaired the FCR.

At the overall phase (1-6 weeks), the breed and Vitamin C supplementation improved (P<0.05) the BWG; while the FELP retards the BWG. Consequently, the BWG recorded in treatments 2, and 4 were higher (P<0.05), compared to the rest of treatments 1, 3, 5, 6, 7, and 8. The Vitamin C x FELP was significant (P<0.05) for FI. Vitamin C supplementation reduced the FI. The Vitamin C x FELP was significant (P<0.05) for FCR. Vitamin C supplementation improved (P<0.05) FCR; while the FELP impaired the FCR. Accordingly, there was improved (P<0.05) FCR in treatments 2 and 4, compared to treatments 1, 3, 5, 6, 7, and 8.

Haematological indices. All the haematological indices in this study were significantly (P<0.05) affected by the treatment effects, except (P>0.05) for the mean cell haemoglobin concentration (MCHC), granulocytes, and monocytes (table 3). The packed cell volume (PCV) was significantly (P<0.05) affected by breed x Vitamin C, Vitamin C x FELP, and breed x Vitamin C x FELP. In addition, the Vitamin C and FELP supplementations improved (P<0.05) the PCV level. In consequence, the PCV was highest (P<0.05) in treatments 6 and 7, compared to treatments 1, 2, 3, 4, 5, and 8. The red blood cell (RBC) counts were significantly (P<0.05) affected by Vitamin C x FELP and breed x Vitamin C and FELP. The RBC count of CO was lower (P<0.05) than AB; while the Vitamin C and FELP independently improved (P<0.05) the RBC count. Thus, the RBC count of birds in treatment 7 was higher (P<0.05) compared to the rest treatments.

Table 3 The haematological indices response of different breeds of broiler chickens to Ficus exasperata leaf powder and Vitamin C dietary 

TRT	BRD	VC, mg/kg	FELP, %	PCV, %	RBC, x10^6/l	HB, g/dl	MCHC, g/dl	MCV, fl	MCH, Pg/cell	WBC, x10^9/l	GRA, x10^9/l	LYM, x10^9/l	MON, x10^9/l
1	AB	0	0	31.50^c	2.25^d	10.50^cd	33.34	137.51^b	40.84^b	2.65^d	0.79	1.64^bc	0.22
2	CO	0	0	30.50^de	2.02^d	10.16^ed	33.32	139.42^ab	44.47^ab	2.82^cd	0.78	1.29^c	0.74
3	AB	200	0	30.12^de	2.63^c	10.04^ed	33.33	114.76^c	38.25^c	2.75^d	0.92	1.71^b	0.12
4	CO	200	0	31.37^cd	2.75^c	10.46^cd	33.34	113.89^c	37.96^c	2.79^cd	1.06	1.46^bc	0.27
5	AB	0	1	32.50^b	2.00^d	9.83^e	33.33	137.66^b	42.22^b	3.15^abc	0.76	1.86^b	0.52
6	CO	0	1	33.94^ab	2.04^d	11.31^ab	33.33	143.51^a	46.83^a	3.32^a	0.93	1.81^b	0.58
7	AB	200	1	34.50^a	3.45^a	11.50^a	33.33	85.54^d	28.51^d	3.50^a	0.88	2.58^a	0.04
8	CO	200	1	32.72^b	3.21^b	10.90^bc	33.33	88.55^cd	34.07^cd	3.19^ab	0.96	1.75^b	0.47
SEM				0.48	0.14	0.16	0.00	5.71	2.05	0.7	0.03	0.08	0.07
P-value				0.01	0.01	0.01	0.57	0.01	0.01	0.01	0.23	0.01	0.21
	AB			31.41	2.73^a	10.46	33.30	122.12^b	40.70^b	3.01	0.83	1.95^a	0.23
	CO			32.13	2.50^b	10.71	33.30	133.76^a	44.58^a	3.03	0.94	1.58^b	0.52
	SEM			0.24	0.06	0.83	0.00	2.79	0.93	0.05	0.04	0.06	0.09
	P-value			0.06	0.01	0.06	1.00	0.01	0.01	0.81	0.13	0.01	0.06
		0		31.36^b	2.07^b	10.45^b	33.33	139.77^a	50.59^a	2.98	0.82	1.65^b	0.52
		200		32.18^a	3.16^a	10.72^a	33.32	104.11^b	34.70^b	3.06	0.96	1.87^a	0.23
		SEM		0.24	0.06	0.08	0.00	2.79	0.93	0.06	0.04	0.06	0.09
		P value		0.03	0.01	0.01	0.67	0.01	0.01	0.39	0.03	0.02	0.06
			0	30.87^b	2.41^b	10.29^b	33.33	130.15	43.38	2.75^b	0.89	1.52^b	0.33
			1	32.66^a	2.83^a	10.88^a	33.32	125.73	41.91	3.29^a	0.88	2.01^a	0.40
			SEM	0.24	0.06	0.08	0.00	2.79	0.93	0.06	0.04	0.06	0.09
			P value	0.01	0.01	0.01	0.67	0.28	0.28	0.01	0.92	0.01	0.64
	Interactions P-value
	BRD x VC			0.01	0.12	0.01	0.22	0.35	0.35	0.08	0.82	0.07	0.99
	BRD x FELP			0.10	0.06	0.11	0.67	0.11	0.11	0.31	0.64	0.42	0.75
	VC x FELP			0.01	0.01	0.01	0.41	0.01	0.01	0.69	0.32	0.25	0.95
	BRD x VC x FELP			0.01	0.01	0.01	0.11	0.59	0.59	0.29	0.36	0.02	0.19

Means with a different superscript in the same column are significantly (P<0.05) different; TRT: Treatments; BRD: Breeds; AB:Abore acre; CO: Cobb 500; VC: Vitamin C; FELP: Ficus exasperate leaf powder; PCV: Packed cell volume; RBC: Red blood cells; HBc: Haemoglobin conc.; MCHC: Mean cell haemoglobin concentration; MCV: Mean cell volume; MCH: Mean cell haemoglobin; WBC: White blood cells; GRA: Granulocytes; LYM: Lymphocytes; MON: Monocytes; SEM: Standard error of the means.

The haemoglobin concentration (HBc) was significantly (P<0.05) affected by breed x Vitamin C, Vitamin C x FELP, and breed x Vitamin C and FELP. The Vitamin C and FELP supplementation independently improved (P<0.05) the HBc of the broiler chickens. Hence, the significantly (P<0.05) highest HBc was recorded in treatment 7, compared to the controls, and the rest treatments. The MCV and MCH were affected (P<0.05) by the interaction of Vitamin C and FELP. Additionally, the MCV and MCH were improved (P<0.05) by the breed but impaired (P<0.05) by Vitamin C supplementation. Accordingly, the best (P<0.05) MCV and MCH values recorded in treatment 6 were similar (P>0.05) to treatment 2, but significantly (P<0.05) higher than treatments 1, 3, 4, 6, 7, and 8.

The white blood cell (WBC) counts were increased (P<0.05) by FELP supplementation. Consequently, the highest (P<0.05) WBC counts recorded in treatments 6, and 7 were similar (P>0.05) to treatments 5 and 8 but were higher (P<0.05) than in the rest treatments. The breed x Vitamin C and FELP were significant (P<0.05) for lymphocyte counts. The highest (P<0.05) lymphocyte counts were recorded in AB; while the Vitamin C and FELP supplementations independently improved the lymphocyte count. So, the lymphocyte counts were highest (P<0.05) in treatment 7, compared to the control and other treatments.

Serum Chemistry. The serum biochemical profiles of different breeds of broiler chicken fed Ficus exasperata leaf powder and Vitamin C supplemented diets are shown in table 4. The vitamin C x FELP supplements were significant (P<0.05) for serum aspartate aminotransferase (AST). The vitamin C supplementation reduced (P<0.05) the AST concentration, so the least (P<0.05) AST concentration recorded in treatments 3, 4, and 8 was similar (P>0.05) to 7, but was significantly (P<0.05) lower than treatments 1, 2, 5, and 6. The vitamin C and FELP independently reduced (P<0.05) the serum creatinine concentration. Consequently, the lowest creatinine concentration recorded in treatments 3, 4, 7, and 8 reported, though similar to treatments 5 and 6, was significantly higher (P<0.05) than in treatments 1 and 2. The FELP supplementation reduced (P<0.05) the serum cholesterol concentration. So, the serum cholesterol level was significantly (P<0.05) lower in treatments 5, 6, 7, and 8, compared to the rest treatments. Vitamin C supplementation increased the serum total protein and albumin concentration. The FELP improved (P<0.05) the globulin concentration, hence the highest (P<0.05) globulin concentration was recorded in treatments 5, 6, 7, and 8, compared to treatments 1, 2, 3, and 4.

Table 4 Serum biochemical profiles of different breeds of broiler chicken fed Ficus exasperata leaf powder and Vitamin C supplemented diets 

TRT	BRD	VC, mg/kg	FELP, %	AST, (IU/L)	ALT, (IU/L)	CREA, (µmol/L)	CHOL, (mmol/L)	TP, (g/L)	ALB, (g/L)	GLB, (g/L)
1	AB	0	0	160.03^a	22.80	82.91^ab	4.14^a	53.30	23.03	30.27^c
2	CO	0	0	155.24^ab	21.94	84.38^a	3.95^a	54.57	23.27	31.30^c
3	AB	200	0	115.65^d	20.74	68.37^c	3.69^a	64.23	31.65	32.58^bc
4	CO	200	0	114.80^d	21.83	68.26^c	3.94^a	64.86	34.05	30.81^c
5	AB	0	1	140.80^bc	19.21	73.07^bc	2.51^b	62.45	26.19	36.26^a
6	CO	0	1	138.80^c	20.51	73.41^bc	2.65^b	58.72	24.14	34.57^ab
7	AB	200	1	126.93^cd	20.15	65.92^c	2.94^b	67.31	31.53	35.78^a
8	CO	200	1	116.75^d	20.51	66.23^c	2.67^b	69.42	34.66	34.75^ab
SEM		3.8	0.62	1.71	0.14	1.57	1.41	0.51
P-value		0.01	0.92	0.01	0.01	0.06	0.12	0.01
	AB			135.85	20.72	72.57	3.32	61.82	28.10	33.72
	CO			131.40	21.20	73.07	3.30	61.89	29.03	32.86
	SEM		2.55	0.98	1.65	0.08	1.85	1.75	0.38
	P-value		0.23	0.73	0.83	0.87	0.97	0.71	0.13
		0		148.72^a	21.11	78.44^a	3.31	57.26^b	24.16^b	33.10
		200		118.53^b	20.80	67.19^b	3.31	66.45^a	32.97^a	33.48
		SEM		2.55	0.98	1.65	0.08	1.85	1.75	0.38
		P value	0.01	0.82	0.01	0.99	0.01	0.01	0.49
			0	136.43	21.82	75.98^a	3.93^a	59.24	28.00	31.24^b
			1	130.82	20.09	69.65^b	2.69^b	64.47	29.13	35.34^a
			SEM	2.55	0.98	1.65	0.08	1.85	1.75	0.38
			P value	0.13	0.23	0.01	0.01	0.06	0.65	0.01
	Interactions P-value
	BRD x VC			0.77	0.85	0.86	0.95	0.62	0.47	0.33
	BRD x FELP			0.65	0.80	0.94	0.68	0.74	0.87	0.34
	VC x FELP			0.01	0.58	0.10	0.07	0.59	0.72	0.34
	BRD x VC x FELP			0.41	0.60	0.87	0.08	0.54	0.76	0.13

Means with a different superscript in the same column are significantly (P<0.05) different; TRT: Treatments; BRD: Breeds; AB: Arbor acre; CO: Cobb 500; VC: Vitamin C; FELP: Ficus exasperata leaf powder; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; CREA: Creatine; CHOL: Cholesterol; TP: Total Protein; ALB: Albumin; GLB: Globulin; SEM: Standard error of the means.

Discussion

When birds are grown outside of the thermo-neutral zone (18 ºC -24 ºC), they perform poorly, so they re-organize their energy and protein supplies to cope with heat stress, but at the cost of reduced reproductive efficiency and growth (^{Park and Kim 2017}). The observed variation in the performance characteristics response of the broiler chickens to the dietary treatments at the starter and finisher phase of production could be due to differences in the management, physiological status and nutritional factors between the starter and finisher phase (^{Oloruntola et al. 2018}). The improved BWG in CO breed fed the non-supplemented diets (treatment 2) and 200 mg/kg Vitamin C supplemented diet (treatment 4) in this study during the finisher and overall phases, depict the effects of breed, FELP, and vitamin C supplementation on the body weight gain of broiler chickens. Improved growth performance in broiler chickens has been attributed to genetics and breeding, nutrition, and management practices (^{Tavarez and Solis de los Santos, 2016}).

During the finisher and overall stages of this study, the superior BWG reported in CO breed compared to AB breed illustrates the variation in incremental improvement by genetic selection for growth rate across the two broiler chicken breeds under consideration. This finding demonstrates that genetics is one of the most important factors influencing broiler growth (^{Rance et al. 2002} and ^{Tavarez and Solis de los Santos 2016}). In addition, the observed improved BWG by Vitamin C supplementation supports the earlier report of ^{Al-Masad (2012)}, who recorded higher body weight in broilers fed diets supplemented with Vitamin C and reared under high ambient temperature, compared to those fed un-supplemented diets.

Since heat exposure produces oxidative stress in broilers, which reduces their growth performance (^{Shakeri et al. 2019}), the possible causes of better BWG documented in the experimental birds could be Vitamin C’s activity as an antioxidant that can inhibit growth performance decline owing to heat stress (^{Al-Masad 2012}). In biological systems, ascorbic acid acts as a free radical scavenger and reducer, scavenging oxidising free radicals and harmful oxygen-derived species such as hydrogen peroxide, hydroxyl radicals, and singlet oxygen (^{Hacışevki, 2009}). The lower BWG in the experimental birds due to FELP supplementation in this study suggests a possible compromise of nutritive quality and impairment of feed acceptability by the birds, because some phytochemicals and phytogenics may interfere with nutrient bioavailability in the birds, especially when present at levels above the tolerable concentration in their diets (^{Dhama et al. 2014} and ^{Oloruntola, 2021}).

Multiple studies have demonstrated a decline in feed consumption due to the high inclusion level (>1500 mg/kg) of phytochemical feed supplements and the inherent properties of some compounds, such as a strong flavour and odour (^{Yan et al. 2011}, ^{Oloruntola et al. 2016 a}, ^b and ^{Valenzuela-Grijalva et al. 2017}). Although FELP was not consumed at a high dietary level in this trial, its bioactive components may have contributed to the reduced feed intake at the starter phase by activating the release of satiety hormone (cholecystokinin) by the cells of the upper small intestine, delaying stomach emptying, and increasing feelings of satiety and anorexia (^{Tucci 2010}). Furthermore, the usage of feed supplements can affect an animal’s willingness to eat a given feed (^{Valenzuela-Grijalva et al. 2017}). This is in tandem with the interactive effects of the breed with FELP (at the finisher phase) and vitamin C with FELP (at the overall phase) in this study. Previous studies reported improved FI in broiler chickens as a result of interaction between thyme and cinnamon (^{Al-Kassier 2009}) and CrPic and vitamin C (^{Adebayo et al. 2022}). However, during the finisher and overall periods of the experimental birds, the FI was consistent across treatment groups. This observation suggests that the birds’ FI intake capacity changes or improves as they grow older and is consistent with increased FI in broiler chickens on day 22 compared to day 14, being reported by ^{Babatunde et al (2019)}. The reduction in the FI recorded in the FELP treated groups (5, 6, 7, and 8) and the significant effect of FELP supplementation in this study suggest the negative effects of the FELP on the feed acceptability and consumption by the birds during the early stage of their life (the starter phase). The reduced FI in the affected treatment groups could be due to the phytoconstituents of the FELP. For instance, tannin, when present at high levels in diets reduces voluntary feed intake and nutrient digestibility (^{Hassan et al. 2020} and ^{Osowe et al. 2021}).

The feed conversion ratio (FCR) is a proxy for efficiency in terms of performance and shows the amount of feed required to gain one unit of body weight on a live bird over a given period. As revealed in this study, breed, Vitamin C and FELP supplementation have varying noticeable effects on the FCR of the broiler chickens. The effects of the interaction of vitamin C and FELP on FCR in this study agreed with earlier studies that a blend of feed supplements could synergise and complement each other to produce noticeable effects on performance characteristics such as FCR (^{Al-Kassier 2009} and ^{Adebayo et al. 2022}).

The improved FCR recorded in the CO breed over the AB breed during the starter phase (1-3 weeks) of this study shows that improved genetics and breeding are among the factors behind the improved performance and in particular the feed efficiency in broiler chickens production (^{Tavarez and Solis de Los Santos 2016}). In actuality, broiler chicken production performance is always changing as a function of genetics and breeding (^{Zuidhof et al. 2014}). In addition, the improved FCR recorded in the broiler chickens as a result of vitamin C supplementation in this study negates the earlier report of ^{Abudabos et al (2018)}, who recorded similar FCR in broiler chickens fed the control and vitamin C supplemented diets. However, the addition of 250 mg/kg of Vitamin C to broiler chicken feed has been demonstrated to improve body weight gain and feed efficiency. Increased performance, such as FCR, may be attributable to thyroid activity and increased oxygen consumption in the presence of ascorbic acid, especially when the ambient temperature is high (^{Kassim and Norziha 1995} and ^{Khan et al. (2012a)}. The negative effects of FELP on FCR in this study suggest the phytogens have some phytochemicals at concentrations above the tolerable level for the birds (^{Valenzuela-Grijalva et al. 2017}).

Haematological measures can be used to identify an animal’s pathogenic, physiological, and nutritional status (^{Adeyeye et al. 2020}). Blood has an important function in thermoregulation in birds in a typical tropical climate. The wattles, comb, and skin’s blood vessels dilate to transport internal body heat to the skin surface, causing convective, radiative, and conductive heat loss (^{Kpomasse et al. 2021}). The fact that factors such as vitamin C, FELP, and breed; and the interactions: breed x vitamin C; vitamin C x FELP and breed x vitamin C x FELP increased PCV, RBC, and HB in this study suggests that these nutrients are important in increasing the proportion of total erythrocyte mass to total blood volume, the number of erythrocytes, and the concentration of protein molecules in the red blood cells that carry oxygen to the lungs.

Therefore, leveraging on this finding, broiler chickens with anaemia could be treated by adding vitamin C and FELP to their diets, either independently or combined. The improved PCV, RBC, and HB by the vitamin C, FELP, breed x vitamin C x FELP could be the antioxidant activity or properties of these supplements synagising with the breed factor (^{Yadav et al. 2016} and ^{Osowe et al. 2021}). Furthermore, FELP’s contribution to the broiler diet’s essential mineral profile (^{Osowe et al. 2021}), as well as the possible complementary role of supplemental vitamin C in assisting the breakdown of amino acids and aiding in the absorption of minerals, particularly iron, by keeping them in a reduced ferrous state, could be responsible for the improved erythrogram caused by these supplements (^{Yadav et al. 2016}). The higher RBC counts in the AB breed compared to the CO breed suggest that genetic variables may influence erythrocyte count. Previous research has shown that genetic variables influence haematological parameters such as PCV, RBC, HBC, mean cell haemoglobin, and mean cell haemoglobin concentration, among others (^{Chineke et al. 2006} and ^{Etim et al. 2014}). The PCV, RBC, and HB values in this study are all within normal ranges (PCV: 22-35 %, RBC: 2.5-3.5 x 106/L, and HB: 7-13 g/dL) reported by ^{Bounous and Stedman (2000)}.

The interaction of vitamin C and FELP producing reduction of the MCV and MCH indicates the presence of microcytic anaemia, which is frequently associated with the iron shortage. Furthermore, the increased MCV and MCH levels in CO breeds indicate macrocytic anaemia, which is defined by excessively large red blood cells and implies that red blood cells are fewer and transport less haemoglobin, meaning that the blood is not as oxygen-rich as it should be (^{Aslinia et al. 2006}). However, the levels observed in this study are within the normal range (MCV: 90 - 140 fl; MCH: 33-47 pg/cell) as reported by (^{Bounous and Stedman 2000}); therefore the raised MCV values induced by breeds or reduced MCV values produced by Vitamin C supplementation may not be of health concern (^{Bounous and Stedman 2000}).

The body is protected by leukocytes and its differentials from harmful or foreign organism infiltration, as well as antibody production and dispersion (^{Oloruntola et al. 2016a}). Furthermore, several phytogens have immunomodulatory properties (^{Dashputre and Naikwade 2010} and ^{Oloruntola et al. 2016 a},^b). This explains the increased WBC counts as a result of FELP supplementation, compared to the control in this study The immunomodulatory properties of phytogens could be linked to dietary substances like phytoestrogens, which, when ingested, can mimic the effects of the hormone oestrogen, (^{Cady et al. 2020}) oestrogen controls immune response by suppressing the negative selection of high-affinity auto-reactive B cells, influencing B cell function, and triggering the Th2 response (^{Taneja 2018}). The increased lymphocyte counts observed in this study as a result of interactions (breed x vitamin C x FELP), vitamin C supplementation, and FELP supplementation highlight the potential impact of vitamin C (^{Shojadoost et al. 2021}), FELP (^{Dashputre and Naikwade 2010} and ^{Oloruntola et al. 2016 a},^b) and their interactions with breed factors (^{Zahoor et al. 2018}) on the immunological condition of birds. Additionally, the higher lymphocyte count in the AB breed compared to the CO breed in this study suggests a difference in the birds’ immune response capabilities and the impact of genetic makeup on the experimental birds’ immune response (^{Zahoor et al. 2018}).

The transaminase enzyme, aspartate aminotransferase (AST) catalyzes the transformation of aspartate and alpha-ketoglutarate to oxaloacetate and glutamate. Except for bone, AST may be detected in all tissues, with the highest amounts in skeletal muscle and the liver. The concentration of AST rises after injury, bruising, necrosis, neoplasm, or infection of the liver or muscle (^{Washington and Van Hoosier, 2012}). Therefore, the reduction of the serum AST concentration by interaction (vitamin C x FELP) and vitamin C concentration in this study shows the antioxidative properties and protective role of vitamin C and the interaction effects of vitamin C with the phytogenic supplement (FELP) against liver or muscle damage. Vitamin C protects proteins from alkylation by electrophilic lipid peroxidation products, oxidative stress-induced cellular damage by rummaging reactive oxygen species, and vitamin E-dependent neutralisation of lipid hydroperoxyl radicals (^{Traber and Stevens, 2011}). In addition, Vitamin C helps endothelial nitric oxide synthase (eNOS) functions by recycling the eNOS cofactor tetrahydrobiopterin, which is important for arterial elasticity and blood pressure management (^{Miranda et al. 2009}, ^{Chavez et al. 2010} and ^{Traber and Stevens 2011}).

Creatinine is a nitrogen waste product that is not protein-based, and its measurement can be used to assess renal function (^{Salazar 2014}). The reduction of serum creatinine concentration by the Vitamin C and FELP supplementation as recorded in this study also elucidates the health benefits of Vitamin C and FELP dietary supplementation in preserving the physiological and anatomical functions of the kidney, which may owe to the antioxidant properties of Vitamin C (^{Traber and Stevens 2011}) and FELP (^{Osowe et al. 2021}).

Serum cholesterol is produced in the liver and is derived from the diet. A high cholesterol level is a sign of heart disease. Reduced serum cholesterol levels can be caused by a low-fat diet, a decrease in cholesterol uptake, or intestinal mal-absorption (^{Adegbeye et al. 2020}). In this study, the phytogens’ hypocholesterolemic capabilities are revealed by the broiler chickens’ reduced blood cholesterol levels after being fed FELP supplemented diets. The hypocholesterolemic effect reported may be owing to the phytosterols in FELP limiting cholesterol absorption in the intestine due to the structural similarities of cholesterol and phytosterol (^{Adegbeye et al. 2020} and ^{Ayodele et al. 2021}). Furthermore, one of the phytochemicals in FELP, saponins, is known to modulate blood lipids (^{Osowe et al. 2021}).

Haemo-concentration, increased globulin production, lipaemia, and haemolysis are the most common causes of elevated serum total protein. However, the observed increase in the serum total protein and albumin, due to Vitamin C supplementation may not be pathological because, the serum total protein levels (53.30 -69.42 g/L) and albumin level (23.03-34.05 g/L) recorded in this study are within the normal range for total protein (52-69 g/L) and albumin (21 - 34.5 g/L) reported by ^{Mitruka and Rawnsley (1977)} and ^{Oloruntola et al. (2016a)} for chickens. Vitamin C supplementation has been shown to raise blood protein levels (^{Karakilcik et al. 2004} and ^{Nayila 2020}). Globulins are a diverse collection of large serum proteins that do not include albumin (^{Odunitan-Wayas et al. 2018}). In addition to the aforementioned variables that cause an increase in total protein, an increase in alpha, beta, or gamma globulin can cause an increase in serum globulin. By implication, the increased globulin concentration observed in this study because of FELP supplementation suggests the phytogens stimulated the increased globulin production in the serum. This agrees with ^{Ghazalah and Ali (2008)}, who recorded increased serum protein and globulin concentration in broiler chickens fed a 5g/kg rosemary leaves supplemented diet. Phytogenics can stimulate the intestinal walls, increasing the secretion of digestive enzymes, as well as enhancing the absorption of more nutrients and, as a result, an improved protein profile (^{Abudabos et al. 2016}).

Conclusions

In conclusion, to achieve optimal body weight gain in broiler chickens, breed selection (CO) and 200 mg/kg Vitamin C supplementation are recommended, especially in a typical tropical environment. However, for better immunity and hypocholesterolemia in the birds, 1 % FELP dietary supplementation is indicated. As a nutritional supplement, 1 % FELP might be coupled with 200 mg/kg of vitamin C to enhance feed consumption, red blood cell count, packed cell volume, and haemoglobin concentration; to prevent liver and tissue damage and protect renal cells from damage in broiler chickens.

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Received: April 02, 2022; Accepted: June 29, 2022

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We declare that there is no conflict of interest on the part of the authors of this study

Author’s contribution: C.O Osowe: Conceptualization, investigation, data curation, writing - original draft. O.A Adu: Conceptualization, investigation, data curation, writing - original draft, writing - review & editing. O.D. Oloruntola: Investigation, data curation, writing - original draft, writing - review & editing. C.A Chineke: Conceptualization, investigation, writing - original draft, writing - review & editing