Arginine: lysine ratio influences on performance, egg quality, haematology, biochemistry, antioxidant status and immunity of dual-purpose breeding hens exposed to cyclic heat stress

Abstract

The aim of this study was to investigate the effects of arginine/lysine ratio (Arg/Lys) on the productive and reproductive performance, egg quality, immune and physiological parameters of hens reared under cyclic heat stress condition (CHS). A total of 140 females, and 20 males of 32 weeks of age, were randomly assigned to 4 treatment groups. The first group (positive control, PC) fed the basal corn-soybean meal diet with an Arg/Lys ratio of 1.25 and was kept at 22–24°C and 45–55% relative humidity. The other 3 treatments fed basal diet in which the Arg/Lys was set at 1.25, 1.37 and 1.50, respectively and were submitted to cyclic heat stress conditions (CHS, 38 °C ± 1, 55–65% RH) for three successive days a week from 10:00 am until 14:00. The results showed a reduction h-day egg production (EP), feed conversion ratio (FCR), shell weight, fertility and hatchability, hemoglobulin (Hgb), hydrogen power (pH) and plasma calcium and phosphorus concentrations for the negative control (NC) group compared to the positive control group. However, changing Arginine (Arg)/lysine (Lys) ratio in the basal diets for the groups under CHS from 1.25 to 1.37 and 1.50 improved EP, egg weight, egg mass and FCR when compared to NC group. Total lipid, total cholesterol, low-density lipoprotein, triglycerides, and malondialdehyde activity for the NC group significantly increased, while increasing the ratio of Arg/Lys to 1.37 and 1.50 improved the mentioned traits as to PC group. Our findings indicated that diets with an Arg/Lys ratio of 1.37 administered to laying hens farmed under cyclic heat stress conditions, are able to improve the laying performance, egg quality, fertility, hatchability, blood hematological and biochemical constituents, antioxidants, and immunity indices in comparison to the other groups, including the PC one. Thus, diets with an Arg/Lys ratio of 1.37 can be used to recover the adverse effect of CHS.

HIGHLIGHTS

• The diets with an Arg/Lys of 1.37 can be administered to laying hens to recover the adverse effect of cyclic heat stress.

• The arginine to lysine ratio can improve egg quality, fertility, hatchability, blood hematological and biochemical.

Keywords:

• Amino acids ratio
• thermal stress
• laying hens
• poultry
• animal health and production

Introduction

Global warming is a serious challenge that has negative impacts on immune status and health of animals worldwide and solution to this problem must be studied. High temperatures are one of the most serious environmental stressors, especially in hotter parts of the world (Renaudeau et al. 2011; Attia and Hassan 2017; Munonye et al. 2023). It’s likely that the bird’s amino acid requirements vary during hyperthermia, given the progressive changes in amino acid metabolism and protein pools that occur during heat stress (Temim et al. 2000). Thermal stress causes cell injury, changes in enzyme activity, and metabolic disturbances (Sahin et al. 2002a; Landy and Kavyani 2013), reduces thyroid activity, depresses blood total and ionised calcium, and the egg yolk precursor (Sahin et al. 2002b), impairs antioxidant capacity and initiates lipid peroxidation (Sahin and Kucuk 2001).

Amino acids are involved in the synthesis of immune-related proteins as well as the control of major immune-related signalling pathways, implying that they play an important role in defensive mechanisms (Andersen et al. 2016; Lee et al. 2023). During moderate inflammation, mammals can maintain a balance between arginine anabolism and catabolism, but this balance can be disrupted by the overriding of arginine catabolism during severe inflammation (Bansal and Ochoa 2003). Different research highlighted that supplementing AA in hens diet improves performance (Saeed et al. 2018), and heat stress has negative impacts on feed intake, growth, and feed conversion ratio of chickens (Li et al. 2015; Attia et al. 2016). Reduced nutrient digestibility, higher heat output, and reduced protein retention may all contribute to this decline in performance (Fouad et al. 2016; Orhan et al. 2018). Castro et al. (2016) reported that heat stress has been shown to affect physiological responses, impede nutritional digestion, and impair cell memory.

In poultry, Arg is an essential amino acid (AA) involved in a variety of metabolic pathways, including protein synthesis and immunology. At the macrophage level, arginine is transformed to nitric oxide (Jahanian 2009) which is a mediating nutrient for vasodilation and increased peripheral blood flow, which is a crucial thermoregulatory response to heat stress (Liu et al. 2019). Chickens have low activity of essential enzymes involved in endogenous Arg synthesis (Sung et al. 1991), thus, they have minimal Arg de novo synthesis and are heavily reliant on food Arg supplies. Because of a putative antagonism between lysine and arginine, dietary supplementation with synthetic lysine may influence the consumption and metabolism of arginine (Zhou et al. 2011). Arginine regulates protein synthesis by activating the target of rapamycin (TOR) signalling pathway, which influences nutrient metabolism, insulin release, and insulin-like growth factor-I (IGF-I) levels. It is also involved in non-specific immune responses and antioxidant responses, elevates disease resistance, and influences nutrient metabolism (Wang et al. 2015; Kheiri and Landy 2020). Recently, Brugaletta et al. (2023) observed that arginine supplementation counterbalances the effects of heat stress on energy homeostasis by increasing creatine levels and attenuating the increase in adenosine monophosphate levels, particularly in Pectoralis major muscle. According to (NRC) National Research Counil (1994), the Arginine: lysine ratio for laying hens is 1.014 with 700 mg of arginine and 690 mg lysine intake per day at 100 g of daily feed intake in 15% crude protein diet. Similar ratio was cited in the (NRC) National Research Council (1994), for diet contained 16.5% crude protein diet.

Due to absence of adequate studies evaluating the relationship between heat stress and essential amino acids ratio on laying hens, this study aimed to examine the effect of arginine/lysine ratio (Arg/Lys) on the productive and reproductive performance, egg quality, immune responses, some physiological parameters of the Mandara breeding hens exposed to cyclic heat stress.

Materials and methods

The study was conducted at the Animal Production Research Institute’s El-Sabahia Poultry Research Station in Alexandria Governorate under approval protocol number 01-10-003-37 and record no. 1563. In this experiment, the Mandara local strain, a hybrid between Alex and Dokki-4 (Abd-El-Gawad 1981), was used for 12 weeks, from 32 to 44 weeks of age. Native to Egypt, Mandara hens exhibit an average egg-laying capacity of 280 eggs per year. The eggs are white or cream in colour and weigh approximately 60 g.

One hundred and forty females, and twenty male (32 weeks old) were randomly assigned to four treatment groups and housed in 20 floor pens (2.0 m × 1.2 m × 2.0 m) in an environmentally controlled light-proof house (each sector has 40 floor pens). Each area can be separately managed in terms of environmental conditions and is furnished with wheat straw. Five replicates (7 hens + 1 cock) were used for each treatment. The first treatment was kept in the first sector at 22-24 °C and 45-55% relative humidity (RH) and fed the basal diet (corn meal diet) in mash form with a dietary Arg/Lys ratio of 1.25 and was used as a positive control (PC). The other treatments, kept in the second sector were submitted to cyclic heat stress conditions (CHS, 38 °C ± 1, 55-65% RH) for three successive days a week from 10:00 am until 14:00. The first CHS group was fed the basal diet with Arg/Lys 1.25 and used as a negative control (NC), the second and the third groups were the fed basal diet with Arg/Lys of 1.37 and 1.50, respectively (Table 1). The trial was run during weeks 32–44 of age, and feed and water were provided ad libitum. Vaccination and medical program were done according to common veterinarian care practice. Birds were exposed to photoperiod regimen 16 light − 8 dark cycles. The temperature- humidity index was calculated according to Berman (2016).

Table 1. Ingredient and chemical composition of the experimental diets.

Treatments	Tr1 + 2	Tr3	Tr4
Diets	Diet 1	Diet 2	Diet 3
Ingredients, (kg/ton)
Yellow corn 8.30% (CP)	630	630	630
Soybean meal 44% (CP)	260	260	260
Vit-Min Premix^a	3	3	3
NaHCO3	0	0	0
KCO3	0.23	0.23	0.23
KCl	0	0	0
NaCl	3.9	3.80	3.8
Dicalcium phosphate	19.4	19.4	19.4
Limestone	82.47	81.59	80.47
DL-Methionine	1.0	1.0	1.0
Arginine	0	0.98	2.1
Total	1000	1000	1000
Calculated composition,
CP, (g/kg)	165.4	165.4	165.4
ME, (kcal/Kg)	2706	2706	2706
Calcium, (g/kg)	32.7	32.4	3.20
Av. Phos, (g/kg)	4.90	4.90	4.90
Argnine (g/kg)	10.5	11.5	12.6
Lysine, (g/kg)	8.4	8.4	8.4
Arg/Ly	1.25	1.37	1.50
Methionine, (g/kg)	3.5	3.5	3.5
TSAA, (g/kg)	6.3	6.3	6.3
Na+, (g/kg)	1.70	1.65	1.65
K+, (g/kg)	7.20	7.20	7.20
Cl-, (g/kg)	2.80	2.70	2.70
DEB, (mEq)^b	180	180	180
Ether extract, (g/kg)	26.4	26.9	26.4
Crude fibre, (g/kg)	28.2	28.2	28.2
Chemical analysis,
CP, (g/kg)	161.3	163.3	163.1
Lysine, (g/kg)	8.2	8.2	8.2
Arginine, (g/kg)	10.3	11.4	12.2
Ether extract, (g/kg)	25.0	25.8	26.9
Crude fibre, (g/kg)	29.0	27.4	28.2
Ash, (g/kg)	57.8	62.6	63.1

Tr1 + 2: diets with treatment Arg/Ly 1.25; Tr3: diet with treatment Arg/Ly 1.37; Tr4: diet with treatment Arg/Ly 1.50.

^a Vit-Min premix provides per kilogram of diet: vitamin A, 12000 IU, vitamin E, 10 IU, menadione, 3 mg, Vit. D3, 2200 ICU, riboflavin, 10 mg, Ca pantothenate, 10 mg, nicotinic acid, 20 mg, choline chloride, 500 mg, vitamin B12, 10 µg, vitamin B6, 1.5 mg, vitamin B1, 2.2 mg, folic acid, 1 mg, biotin, 50 µg. Trace mineral (milligrams per kilogram of diet): Mn, 55, Zn, 50, Fe, 30, Cu, 10, Se, 0.10, Anti oxidant, 3 mg.

^b Dietary electrolyte balance (DEP): (Na++K+–Cl-).

CP: Crude Protein.

Body weight gain (BWG), feed conversion ratio (FCR), and egg mass (EM) were estimated after measuring feed intake (FI) and live body weight (LBW), mortality rate, egg production (EP percent), and egg weight (EW) in replicates at weekly intervals.

A total number of 15 eggs from each replicate were randomly chosen subjected to egg quality investigation. In particular, egg shape index, shell percent, thickness, and weight per unit of surface area (SWUSA, mg/cm²), were estimated according to Carter (1975) and Attia et al. (1995). While yolk index and Haugh units, were according to Funk (1948) and Haugh (1937), respectively.

At 44 weeks of age, 25 eggs from each replicate of each treatment were randomly collected to determine fertility and hatchability. On the day of hatch, hatching chicks were counted and weighed to the closest gram, and the relative weight was estimated by dividing the chick weight by the egg weight and multiplying by 100. The measurements were according to Attia et al. (1994, 1995).

In two blood tubes with and without heparin, blood samples were taken from the wing veins of five hens at the end of the experimental period of each treatment representing all treatments replicates. Blood samples were centrifuged for 20 min at 3,000 xg to separate plasma and serum, which were stored at 20 °C until analysis. Blood samples were obtained at random from each treatment group 2 h before the end of the heat stress cycle, to determine the hematological composition.

Serum glucose, total protein, albumin, globulin, triglycerides (Trig), total cholesterol, and high-density lipoprotein-cholesterol (HDL) were determined. Plasma calcium (Ca), phosphorus (P), total antioxidant capacity (TAC), malondialdehyde (MDA), and catalase (CAT) were measured. Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), alkaline phosphate (ALP), while globulin, estimated by difference. The immunoglobulin IgG, IgM, IgA, triiodothyronine (T₃) Thyroxin (T₄) were also determined. Biochemical constituents were determined using commercial kits produced by Diamond Diagnostics Company (29 Tahreer St. Dokki Giza Egypt).

The hematological parameters of the blood, such as haemoglobin (Hgb) are determined according to Drubkin (1964). The red blood cells (RBCs), White blood cells (WBCs) and WBC fractions were determined according to Hawkey and Dennett (1989; Schalm 1986). In addition, the pH value was evaluated using a digital electric pH metre immediately after the samples were collected.

During the experimental period, the interior temperature (^oC) and RH were recorded. The temperature-humidity index (THI) was calculated using Berman (2016) formula.

Apparent digestibility of nutrients

Five cocks of each treatment as one bird of each replicate per treatment were selected at 48 weeks of the age and used to determine the digestibility coefficient values of dry and organic matter (DM and OM), crude protein (CP), ether extract (EE) and natural fibre (CF) as affected by heat stress and dietary treatments, using the total collection method proposed by Attia et al. (2016). The crude protein (CP), lipid, ash and crude fibre were determined according to AOAC. (2007) using methods no. 934.01, 954.01, and 942.05, 978.10, respectively.

Arginine and lysine were analysed using freeze-dried samples, ground through 1-mm mesh screen. Samples were hydrolysed for 24 h with 6NHCl at 110 °C for the determination of AA by High-Speed Amino Acid Analyser (Hitachi LA8080 AminoSAAYA). Nitrogen analysis was carried out with a Leco N analyser. Pump (1) Buffer solutions and RG, and ammonia filtration at 20–80 °C, TDE3 reactor at 125–140 °C, using a detector at 440–570 nm (Pump 1). While Pump (2) contained ninhydrin reagent and washing solution. All samples were assayed in 3 duplicates.

Statistical analysis

Data were tested for normality and statistically analysed using the one-way ANOVA of GLM procedure (SAS 2009). Variables having significant differences were compared using Tukey test (SAS 2009). The statistical model used was as follows: $$\text{Yij}=\mathrm{\mu }+\text{Ti}+\text{eij},$$

Where, Yij = the dependent variable; μ= the overall meaning; Ti = the effect of treatments; eij = the random error. The replicate was the experimental unit. The results were expressed as average value, and the significance level was set at (p < 0.05).

Results and discussion

The high ambient temperature in the summer months, especially in tropical and sub-tropical areas, severely impacts feed intake, laying performance, reproductive performance, and physiological features of laying hens (Attia et al. 2006, 2009, 2011; Ajakaiye et al. 2011; Yoshida et al. 2011). The optimal temperature for laying hens is between 20 and 25 °C (Daghir 2008; Tumová and Gous 2012) and heat stress (HS) occurs when the ambient temperature rises above 27 °C (Bollengier-Lee et al. 1999; Attia et al. 2006). In recent years, the climate change and, in particular, the global warming represents a new important challenge for poultry breeders (Attia et al. 2009, 2012; Munonye et al. 2023) as can be considered responsible of an increase in temperature/humidity index inside the poultry house.

The average values of temperature and humidity recorded in our study during the application of cyclic heat stress confirmed that layers were subjected to high environmental temperatures and relative humidity percentages (Table 2). The average Temperature-Humidity Index (THI) of 91.5 indicated that layers raised in the CHS sector were exposed to emergency stress throughout testing periods According to the Weather Safety Index (Gross and Siegel 1983).

Table 2. The temperature degrees (Tdb^oC) and relative humidity (RH, %) during the experimental periods.

	Before CHS			During CHS
	Tdb	RH	THI	Tdb	RH	THI
January	19.67	48.83	61.37	37.17	35.67	89.73
February	22.00	50.25	65.77	37.42	41.58	91.92
March	23.00	48.75	67.56	38.08	40.83	92.89
mean ± SE	21.6 ± 1.71	49.3 ± 0.84	64.9 ± 3.18	37.6 ± 0.47	39.4 ± 3.22	91.5 ± 1.62

THI: temperature-humidity index Mild (72 to 79 THI) Moderate (80 to 89 THI) Severe (90 THI or greater); Tdb: dry bulb temperature; RH: relative humidity calculated according to Berman (2016); CHS: Cyclic Heat Stress. 

The average ambient temperature in our study was outside the accepted thermoneutral zones of 12–24 °C for chicken species maintained in temperate countries (Plyaschenko and Sidorov 1987) and of 20.9–28.5 °C for poultry species reared in tropical regions of the world (Plyaschenko and Sidorov 1987; Prinzinger et al. 1991). This conclusion supports the early findings that showed animals raised in environments with a temperature 32 °C and humidity index 76%, suffer from heat stress (Marai et al. 2002; Abdalla et al. 2018). The CHS had no influence on the final BW or BW change (Table 3) of hens: these findings could be explained by the ability of local hens’ strains to adapt to different environmental conditions (Moraa et al. 2022). However, our results disagree with those of Chand et al. (2016) who indicate that CHS decreased body weight of laying hens. Other researchers showed that BW and BW changes were dramatically reduced in hens reared under CHS (Attia et al. 2006, 2009, 2011; Aswathi et al. 2019). The findings presented here are consistent with the evidence that dietary adjustment may help to mitigate the negative effects of CHS through an improvement of oxidative stress status and performance of chickens (Syafwan et al. 2011; Singh et al. 2012; Attia et al. 2018). According to Syafwan et al. (2011), diets deficient in amino acids may enhance heat generation. Heat stress increases disease susceptibility and mortality because of the body’s immune system’s weakening (Sosnówka-Czajka et al. 2006; Maini et al. 2007). As laying hens were exposed to CHS, the mortality rate of breeding hens in the NC group (2.50%) increased when compared to the hens in the PC group (0.0%). However, increasing Agr/Lys ratio for the groups under CHS from 1.25 to 1.50 decreased the mortality rate from 2.5% (recorded for NC group) to 0.0 for the other experimental groups under CHS (Table 4). Ayo et al. (2010) indicated that heat stress in poultry is responsible of stressful behavioural responses such as panting, higher respiratory rate, and dehydration, which can lead to death due to heatstroke: the authors recorded 3.7% of death rate in laying birds during the hot-dry season in Nigeria. At the same time, Abd-Ellah (1995) observed a 28% rise in mortality in Egypt’s arid weather. The disparity between the two figures appears to be attributable to the severity of heat stress in Egypt’s desert climate, where the ambient temperature is around 43 °C. In addition, Attia et al. (2016) discovered that the survival rate of laying hens under heat stress (38 °C) was considerably lower (1.5%) in the experimental group than in the control group.

Table 3. Effect of dietary arginine/lysine ratio (Arg/lys) on final and change body weight (BW) and egg production, egg weight and egg mass of Mandara breeding hens under cyclic heat stress.

Arg /lys ratio		Body weight (BW)			Egg production (%)	Egg weight (g)	Egg mass (g/day)
		Initial BW (g)	Final BW (g)	BW Change (g)
1.25 (Control+)		1519.4	1630.8	111.4	50.90^c	55.81^a	26.32^b
Cyclic heat stress	1.25	1566.1	1693.3	127.2	43.86^d	51.70^b	24.5^b
	1.37	1507.3	1632.6	125.3	65.18^a	53.46^ab	34.82^a
	1.50	1499.6	1614.7	115.1	61.40^b	53.65^ab	32.94^a
SEM		110.35	120.15	33.25	1.18	1.17	0.91
p Value		0.1618	0.0945	0.5561	0.0001	0.0381	0.0001

^a,b,c,dmeans having different superscripts in the same columns are significantly different (p < 0.05).

SEM: standard error of means; P value: probability level; Arg/Lys = arginine/lysine ratio.

Table 4. Effect of dietary arginine/lysine ratio (Arg/lys) on feed intake, feed conversion ratio, fertility and hatchability of total and fertile eggs and survival rate of Mandara breeding hens under cyclic heat stress condition.

Arg /lys ratio		FI (g/day)	FCR (g feed/g egg)	Fertility%	Hatchability of total eggs, %	Hatchability of fertile eggs, %	Survival rate
							No	%
1.25 (Control+)		120^a	4.34^b	91.3^ab	89.2^a	97.6^a	40/0	0
Cyclic heat stress	1.25	120^a	5.12^a	89.4^b	76.1^b	85.0^c	40/2	2.5
	1.37	113^c	3.36^c	95.4^a	90.0^a	94.4^a	40/0	0
	1.50	116^b	3.42^c	86.94^c	79.17^b	90.99^b	40/0	0
SEM		0.80	0.17	5.90	4.60	3.56	ND	ND
p Value		0.0001	0.0001	0.0241	0.0364	0.0421	ND	ND

^a,b,cmeans having different superscripts in the same columns are significantly different (p < 0.05). SEM:standard error of means; p value: probability level; FI: feed intake; FCR: feed conversion ratio; Arg/Lys = arginine/lysine ratio; ND: not done.

The EP of the NC group was reduced by 14.34% for the entire trial period (33-44 weeks of age), according to the results of this study (Table 3). However, the results showed that increasing the Arg/Lys ratio in the diets of laying hens exposed to CHS, entirely recovered and enhanced EP, EW and EM when compared to the NC group, while no differences were recorded compared with the PC group. These findings are consistent with those of McDaniel et al. (1995), who found that egg production was 55.8% in heat stressed hens and 82.9% in control hens, indicating a 32.7% fall in laying rate. Heat stress also impacts hen welfare and negatively affects hen performance and egg production, according to Mack et al. (2013) and Mignon-Grasteai et al. (2015). Furthermore, Aswathi et al. (2019) showed that the heat-exposed group’s hen day egg production decreased compared to the control group. Similarly, Ani and Okpara (2019) and Aml and Abd-Elaal (2020), found that hen day egg production was inversely related to high temperature. Our findings are consistent with those of several other groups who found that adding Arg to the basal diet increased EP considerably. Silva et al. (2012) found that feeding 1.26% digestible Arg resulted in an increased egg production percentage, but the differences were not significantly varied from other Arg groups. Layer diets containing 1.36% of digestible arginine had the highest laying rate, according to Duan et al. (2015). According to Youssef et al. (2015), L-Arg supplementation at 2 and 4% over the control diet containing 0.700% Arg resulted in a considerable increase in egg production % compared to the control diet. Laying hens fed the basal diet supplemented with 500 or 1000 mg arginine-silicate-inositol complex for 90 days produced more eggs than the control group, according to Sahin et al. (2018). During the entire experimental period (33–44 wks of age), chickens exposed to CHS (NC) lay eggs with low EW and EM (Table 4) compared to those in the PC group by 7.36% and 20.2%, respectively. These findings are consistent with all prior findings published by (Attia et al. 2016; Aswathi et al. 2019, Aml and Abd-Elaal 2020; Barrett et al. 2019). Sabry et al. (2016) reported that at 40 weeks of age there were no significant effects of Arg supplementation on EW in Silver Montazah chickens. In addition, feeding varying doses of Arg from 0 to 2.5% did not significantly influence on average EW in local chickens (Basiouni et al. 2006; Youssef et al. 2015).

Experimental results showed that FI was not statistically different between PC and NC groups (120 g/d/hen), while it was dramatically reduced for the CHS groups fed on 1.37 and 1.50 Arg/Lys (Table 4). The reduction in feed intake represents an adaptation of laying hens’ abilities to high environmental temperatures (Felver-Gan et al. 2014) and, in or trial, is accompanied by an impaired FCR in the NC group compared to the PC group by 25.83% (Table 4). However, The FCR was significantly improved in the CHS groups when the Arg/Lys ratio increased (Table 4).

Barrett et al. (2019) found that FCR was reduced, and FI falling after two weeks of CHS exposure and stayed low for four weeks, compared to the two weeks prior to the start of CHS exposure. According to Attia et al. (2016), hens reared under optimal thermal conditions had a higher feed intake in comparison to hens kept under CHS. As described by Sahin et al. (2005), CHS may lower the digestibility of several diet components, resulting in an impairment in FCR. When the ambient temperature rises above the thermoneutral zone, nutritional digestibility declines, according to Sahin et al. (2005). In addition, Hai et al. (2000) found that mice housed at 32 °C had considerably lower trypsin, chymotrypsin, and amylase activity. These findings are consistent with the current findings, which show that the digestibility percentage of crude protein and dry mater of NC group was reduced by 2.84 and 2.35%, respectively, compared to the PC group. However, the protein digestibility of the CHS groups fed diets with varying levels of Arg/Lys was improved and not statistically different from that of the PC group (Table 5). Similarly, Attia et al. (2016) revealed that the nutrient digestibility of DM, EE, CF, and NEF remained comparable in hens grown under optimal thermal conditions or under CHS; however, the digestibility of CP was most negatively affected by CHS and fed a diet with no supplementation. According to Attia and Hassan (2017), CHS can induce alterations in hypothalamic peptides involved in appetite regulation, decreasing feed residue passing rate, trypsin, chymotrypsin, and amylase activity, changing intestinal morphology, and nutrient absorption. Youssef et al. (2015) found that providing poultry diets with two levels of L-arginine (2 and 4%) over the NRC (1994) recommended values significantly enhanced FCR. Fascinal et al. (2017) found that FI was reduced and FCR was improved when diets were supplemented with 1.056 mg of arginine per kg of feed. On contrast, diets containing higher Arg, did not impact feed consumption, according to Basiouni et al. (2006) and Wu et al. (2011).

Table 5. Effect of dietary arginine/lysine ratio on nutrient digestibility traits of Mandara breeding hens under cyclic heat stress condition.

Arg /lys ratio		Organic Matter %	Dry matter%	protein%	Ether extract%	Fiber%
1.25 (Control+)		58.11	82.56^b	81.84^a	77.28	27.67
Cyclic heat stress	1.25	57.87	80.62^c	79.11^b	77.97	26.52
	1.37	59.38	83.62^a	81.35^a	78.00	26.21
	1.50	54.68	84.42^a	81.69^a	78.34	26.25
SEM		0.363	0.077	0.235	0.695	0.753
p Value		0.5525	0.0001	0.0223	0.6757	0.9875

^a,b,cmeans having different superscripts in the same column are significantly different (p < 0.05).

SEM: standard error of means; p value: probability level; Arg/Lys: arginine/lysine ratio.

Results demonstrated that egg shape index, eggshell thickness, SWUSA, yolk colour and Haugh unit for all experimental groups were not statistically different. However, the shell weight % for the NC group significantly decreased by 8.8% compared with that recorded for PC group (Table 6). This may be due to a decrease in calcium and bicarbonate availability for eggshell formation (Attia et al. 2020). Eggshell weight and thickness were significantly reduced under high ambient temperature, according to Hamad (2010), Dhaliwal and Dhillon (2019), and Ani and Okpara (2019). Sartsoongnoen et al. (2018) found that eggshell weight from chickens kept at 35 °C was considerably lower than that of birds kept at 27 °C, and that shell thickness (mm) was not statistically different across treatment groups.

Table 6. Effect of dietary arginine/lysine ratio (Arg/lys) on some external and internal egg quality traits of Mandara breeding hens under cyclic heat stress condition.

(Arg /lys ratio)		Egg shape index, %	Shell weight, %	Shell^1 Thic, mm	SWUSA, mg/cm^2	Yolk index	Yolk color	Haugh unit score
1.25 (Control+)		75.23	10.20^b	34.09	103.45	47.3^a	6.64	84.92
Cyclic heat stress	1.25	74.51	9.30^c	32.62	110.72	44.0^b	6.23	83.98
	1.37	75.61	10.23^a	33.21	117.97	43.0^b	6.76	84.84
	1.50	73.71	10.22^a	33.98	112.13	43.2^b	6.65	84.45
SEM		1.17	0.693	1.13	6.68	1.62	0.30	2.82
P Value		0.8467	0.0001	0.7447	0.6435	0.0001	0.5513	0.5064

^a,b,cmeans having different superscripts in the same columns are significantly different (p < 0.05).

SEM: standard error of means; P value: probability level; Arg/Lys: arginine/lysine ratio; 1-ShelThic: Shell Thickness; 2-SWUSA: Shell weight per unit of surface area.

The decrease in eggshell weight and yolk index values caused by CHS is consistent with previous research regrading heat stress, this can result in an immediate drop in eggshell quality (Samara et al. 1996). Yolk index was significantly decreased by 6.98% compared with that recorded for PC group and manipulated diet with the other levels of Arg had no significant effect on yolk index. Specific gravity, shell weights, shell thickness, SWUSA, yolk weights, yolk/albumen ratio, yolk index, and Haugh unit were all impaired in CHS hens fed a diet without supplementation, according to Attia et al. (2018). Similar results were reported by Aml and Abd-Elaal (2020). According to Aswathi et al. (2019), the thickness of broiler breeder hens exposed to 37 ± 1 °C and 70% RH did not differ, but the egg shape index was drastically decreased. On different phases, they showed that in the heat stressed groups, albumen index, HU score, and yolk index were considerably lower than the control group. Similarly, when the body’s temperature rises, blood flow changes, and more blood flows to peripheral tissues to transmit heat from the core to the surface, potentially affecting blood flow to the oviduct (Etches et al. 1995). Furthermore, when birds are under heat stress, they paint in a variety of ways to release heat as water vapour (Kassim and Sykes 1982). Increased respiration rate causes a decrease in CO₂ and HCO₃ partial blood pressure, as well as an increase in blood pH, leading in respiratory alkalosis (Mahmoud et al. 1996). The amount of ionised Ca₂+ in the blood is reduced by a higher blood pH (Odom et al. 1986); Ca₂ + in used by the eggshell gland. The weight of eggshells was significantly reduced because of the high ambient temperature (Sahin et al. 2006). The decrease in eggshell weight could be related to a drop in plasma protein and calcium levels, both of which are essential for eggshell development (Mahmoud et al. 1996). In terms of the influence of Arg on egg quality, the results of various studies on arginine addition to laying hen diets were inconsistent. Dietary Arg and Arg/Lys ratio supplementation had no effect on egg quality indices in laying hens, according to Merzza (2012) and Youssef et al. (2015). Yang et al. (2016) found that adding 0, 8.5, or 17 mg of L-arginine/kg to the diet of 25-weeks-old brown Leghorn laying hens for 42 days had no effect on egg quality parameters. Silva et al. (2012) found that increasing Arg levels (0.943, 1.093, 1.243, 1.393, 1.543% digestible Arg) to broiler breeder hens improved egg quality.

Chickens exposed to CHS (NC) had considerably lower fertility and hatchability of total eggs and fertile eggs, with percentages falling from 91.3, 89.2, and 97.6% to 89.4, 76.1, and 85.0%, respectively, when compared to the PC group (Table 7). However, hens fed diets with 1.37 Arg/Lys had a higher fertility rate by 9.72% than the NC group and not statistically different to that of PC. In addition, the hatchability of total eggs and fertile eggs was lower in the NC group by 14.7 and 12.9%, respectively, compared to the PC group. These findings agree with Attia et al. (2006, 2009, and 2011) and Yoshida et al. (2011). Breeders exposed to CHS (NC) had considerably reduced fertility and hatchability of total eggs than those in the PC group, according to Attia et al. (2018). According to Daghir (2008), the fall in fertility and hatchability could be related to a deterioration of semen quality during the summer months. Sharideh et al. (2016) indicated that including Arg in the broiler breeder diet increased sperm penetration (SP) by 10%, and that higher fertility is positively connected with the SP and rate in the inner perivitelline layer overlaying the germinal disc. Youssef et al. (2015) found that supplementing the poultry diet (which contains 0.7% L-Arg) with 2 and 4% L-Arg to achieve a concentration of 0.714 and 0.728%, respectively, increased fertility and hatchability, but decreased female percent and early death percent compared to the unsupplemented control. According to Duan et al. (2015), an arginine-supplemented layer diet may protect offspring tissues from the negative effects of lipid oxidation products while also prolonging shelf life. The growth of embryos and Arg supplementation (from 0.943% to 1.543% digestible Arg) had no effect on fertility or hatchability. They also showed a numerical trend towards a higher rate of late mortality (18 to 21 days of incubation).

Table 7. Effect of dietary arginine/lysine ratio (Arg/lys) on some hematological parameters of Mandara breeding hens under cyclic eat stress condition.

Arg /lys ratio		RBCs	Hgb mg	WBCs x10^3/mm^3	Lym %	Het %	H/L ratio	pH
1.25 (Control+)		2.93^a	13.85^a	63.95^a	78.80^b	14.8^b	19.0^b	7.71^b
Cyclic heat stress	1.25	2.40^b	11.23^b	53.27^c	64.33^c	28.8^a	45.1^a	8.26^a
	1.37	2.65^ab	12.70^a	61.33^b	85.67^a	9.00^c	10.7^b	7.74^b
	1.50	2.79^a	12.75^a	62.85^ab	71.50^ab	10.6^c	14.8^b	7.63^b
SEM		0.09	1.34	1.27	3.40	1.22	1.564	0.46
P Value		0.0690	0.0026	0.0072	0.0092	0.0356	0.0001	0.0155

^a,b,cmeans having different superscripts in the same row are significantly different (p < 0.05).

SEM: standard error of means; p value: probability level; RBC: Red blood cells; Hgb: haemoglobin; WBCs: White blood cell; Lym: Lymphocyte; Het: Heterophi; H/L ratio: Heterophil/lympho; Arg/Lys: arginine/lysine ratio.

Layers exposed to CHS (NC) had a substantial drop in RBCs count, Hgb, and pH by 18.1, 18.91, and 7.1%, respectively, as compared to the PC group (Table 8). Furthermore, when compared to the NC group, layers fed various levels of Arg/Lys showed significant improvements in RBC count and Hgb values. The increase of pH value under CHS conditions needs further explanation; however, it can partly explained be due to the increases in carbon dioxide loss from the lungs, which cause a fall in carbon dioxide partial pressure (Wang et al. 1989), and hence blood bicarbonate. When a bird tries to eliminate the excess of heat through evaporative cooling, the blood ratio of bicarbonate to carbon dioxide rises, resulting in a rise in blood pH (respiratory alkalosis) (Franco-Jimenez and Beck 2007). The concentration of ionised calcium in the blood can be reduced as the pH of the blood rises. Ionised calcium is necessary to the hen for eggshell deposition (Odom et al. 1986). When the hen is subjected to extreme ambient temperatures, it is possible to have a decline in eggshell thickness and quality (Samara et al. 1996). In addition, CHS had a substantial negative impact on WBC count and lymphocyte percentages, whereas heterophil and heterophil/lymphocyte ratio (H/L ratio) were significantly higher in the CHS groups compared to the PC one (Table 7). At the same time, the H/L observed for the groups fed 1.37 and 1.50 levels of Arg/Lys was not statistically different than the PC group. However, layers fed different levels of Arg/Lys resulted in diminishing the negative effect of CHS on the pervious treats compared with the NC group. At the same time, the PC group was not statistically different than the H/L recorded for the layer’s groups fed 1.37 and 1.50 levels of Arg/Lys. This could elevate the adverse effect of CHS, which appears from the equalisation of the H/L ratio among the different experimental groups compared with the NC. The H/L ratio in a chicken’s blood is a good indicator of stress. These findings corroborated those of Mashaly et al. (2004), who found that hens subjected to cyclic heat stress show a significantly higher H/L ratio than those under thermoneutral conditions. Heat stress causes circulating WBC to be suppressed (Heller et al. 1979) and an increase in the H/L ratio, which is a stress signal (Gross and Siegel 1983). Heat stress reduced antibody formation, according to Zulkifli et al. (2000). This decrease may be related to an increase in inflammatory cytokines during stressful situations (Ogle et al. 1997), which promotes hypothalamic production of corticotrophin-releasing factor (Sapolsky et al. 1987). After 4 weeks, the total WBC of birds exposed to chronic heat stress was lower than the control group and significantly lower than the cyclic heat stress-group, according to Mashaly et al. (2004). Also, CHS considerably raised Hgb, according to Attia and Hassan (2017). Similar findings were also found by Jaiswal et al. (2017), Attia and Hassan (2017), and Aswathi et al. (2019). The results of RBC correspond with those of Al-Hassani (2011), who found no significant differences (p > 0.05) in RBC and Hgb, PCV, MCV, MCH, and MCHC in layers fed 0.0, 0.4, 0.7, and 0.9% arginine. Furthermore, Youssef et al. (2015) found that adding 0.728% L-Arg to laying hen diets, compared to the amounts suggested by the NRC (1994), had no adverse effects on the physiological performance of the birds, including RBCs, PCV, Hb, MCV, MCH, and MCHC.

Table 8. Effect of dietary arginine/lysine ratio (Arg/lys) on some biochemical parameters of Mandara breeding hens under cyclic heat stress condition.

(Arg /lys ratio)		Glucose (mg/dL)	Tpr (g/dL)	Alb (g/dL)	AST, U/L	ALT, U/L	ALP, U/L	Urea (mg/dL)	Creat (mg/dL)
1.25 (Control+)		176^a	6.26	3.32	51.8^b	60.4^b	102^b	2.57	1.22
Cyclic heat stress	1.25	171^b	6.46	3.40	55.4^a	64.2^a	119^a	2.50	1.16
	1.37	175^a	6.22	3.2	57.0^a	65.4^a	112^a	2.33	1.16
	1.50	177^a	6.22	3.2	57.1^a	65.2^a	114^a	2.40	1.19
SEM		0.325	0.284	0.34	0.3421	0.354	1.658	0.165	0.028
P Value		0.0001	0.0752	0.421	0.0005	0.0006	0.0024	0.7251	0.2172

^a,b,cmeans having different superscripts in the same row are significantly different (p < 0.05).

SEM: standard error of means; p value: probability level; Arg/Lys: arginine/lysine ratio; Tpr: Total protein; Alb: Albumin; ALT: Alanine amino transferase; AST: Aspartate amino transferase; Creat: creatinine; ALP: alkaline phosphatase.

The present results showed that NC group had significantly decreased glucose compared to PC group and the other experimental groups (Table 8). The decrease may indicate the use of glucose as energy source for paining. These findings are consistent with those of Hamad (2010), Attia and Hassan (2017) and Attia et al. (2016). On the other hand, Jaiswal et al. (2017), indicated an increase in serum glucose concentration in response to acute heat stress exposure and heat stress duration. In addition, Aswathi et al. (2019) found that blood glucose levels in broiler breeder hens exposed to heat stress (37 °C, and 70% relative humidity for 6 h daily) were higher (2.0-3.8%) than in controls on the 3rd and 10th days. Results in Table 8 showed that blood glucose concentrations in layers exposed to CHS and fed 1.37 and 1.50 Arg/Lys increased considerably in comparison to the NC group. This increase can indicate an improved glucose metabolism with increasing Arg/Lys ratio. However, Ebrahimi et al. (2014) did not find any effects of higher dietary arginine levels on glucose serum levels.

The plasma levels of total proteins and albumin did not differ among all experimental groups (Table 8). These findings were consistent with those of Yang et al. (2016), who found that dietary arginine supplementation had no influence on serum total protein and albumin levels in brown Leghorn laying hens fed diets supplemented with 0, 8.5, or 17 mg of L-arginine/kg for 42 days. However, all experimental groups exposed to CHS had significantly higher serum AST, ALT, and ALP activity as compared to the PC group. The concentrations of urea and creatinine were not statistically different across all experimental groups. The same findings were found by Attia and Hassan (2017) and Attia et al. (2018) who found that chronic heat stress enhanced AST activity, but liver and renal functions were unaffected. On the other hand, Yang et al. (2016) indicates that there were no effects of dietary arginine supplementation for brown Leghorn laying hens fed diets supplemented with 0, 8.5, or 17 mg of L-arginine/kg for 42 days. During the hot weather, Bozakova et al. (2015) found that feeding DeKalb Brown laying hens a basal diet supplemented with either 10 mg/kg L-arginine or 10 mg/kg L-arginine plus 250 mg/kg vitamin C significantly increased creatinine levels compared to the thermoneutral zone.

Laying hens exposed to CHS (NC group) showed significantly increases in total lipid, total cholesterol (TC), low-density lipoprotein (LDL), triglyceride constituents (TG), and malondialdehyde (MDA) activity by 11.2, 3.3, 2.7, 2.7 and 33.3%, respectively compared with PC group. The increase of Arg/Lys in the diets to 1.37 and 1.50 completely recovered of the previous treats as to PC group. However, HDL concentration was not significantly different among all experimental groups (Table 9). These findings corroborate those of Fouad et al. (2013) who found that chickens treated with L-Arg (0.25, 0.50 or 1.00% L-Arg for 3 weeks) had lower plasma TG and TC levels and they suggest that the reduction maybe due to decreasing hepatic 3-hydroxy l-3-methyl glutaryl-Co A reductase mRNA expression and enhancing CPT1, and 3HADH (genes related to fatty acid β-oxidation) mRNA expression in the hearts of broiler chickens. Yang et al. (2016) found that hens supplemented with 8.5 mg/kg L-arginine had lower total blood cholesterol and triglyceride concentrations than hens given 0 mg L-arginine/kg. Uunganbayar et al. (2005) and Addeo et al. (2021) reported that MDA, a soluble degradation product of lipids, could be used to monitor the extent of lipid peroxidation. Furthermore, Jiang et al. (2013) demonstrated that dietary supplementation of arginine decreased MDA levels in the layer’s serum and liver. The activity of triiodothyronine (T₃) was not different among the experimental groups, while T₄ activity for layer exposed to CHS (NC group) recorded the significantly highest activity compared with PC group (Table 10). Our results are in line with that obtained by Aml and Abd-Elaal (2020) who found that T₃, and T₄ concentrations of 40 wks old Hy-line layer hens were significantly lowered in heat stressed-hens (40 ± 1 °C for 5 days, 4 h daily) as compared with the control ones (25 ± 1 °C). However, Bowen and Washburn (1985) indicated that high ambient temperature causes a reduction in thyroid activity of poultry. Also, a reduction in the circulating concentrations of T₃ and T₄ is noticed at high temperatures (Hillman et al. 1985). However, Attia et al. (2016) found that heat stressed hens fed a diet with no supplementation of amino acids had the lowest T₃ and T₄. These results are in agreement with (Atakisi et al. 2009), who indicated that L-Arginine and zinc supplementation improved TAC and reduced MDA concentrations compared to the control. This may be due to the blood nitric oxide which concentrations were increased due to L-Arginine treatment. However, L-Arginine supplementation could be beneficial and useful for decreasing oxidative stress, boosting antioxidant capacity when the basal diet is supplemented with L-Arginine at 5 mg/kg, and zinc at 60 mg/kg. Duan et al. (2015) also observed that digestible dietary arginine had a significant effect on TAC levels and methane dicarboxylic aldehyde concentration in broiler breeder serum that may protect tissues against attack by lipid oxidation products. The values of IgG and IgM are statistically equal and did not have any differences among all experimental groups while the value of IgA was significantly decreased for the NC group compared with PC. However, the NC group showed significantly decreased IgA compared with PC and the other experimental groups (Table 10).

Table 9. Effect of dietary arginine/lysine ratio (Arg/lys) on lipid profile of Mandara breeding hens under cyclic heat stress condition.

(Arg /lys ratio)		T lipid g/dL	Cholest mg/dL	HDL mg/dl	LDL mg/dL	Trig mg/dL	CAT mg/dL	MDA mg/dL
1.25 (Control+)		4.66^b	210^b	44.2	94.8^ab	183^b	39.5^a	0.168^b
Cyclic heat stress	1.25	5.18^a	217^a	42.6	97.3^a	187^a	32.3^c	0.224^a
	1.37	4.38^b	212^b	46.6	91.3^b	181^b	37.8^b	0.167^b
	1.50	4.30^b	211^b	45.3	93.3^b	183^b	38.2^b	0.181^b
SEM		0.055	0.512	1.321	0.325	0.884	0.124	0.213
P Value		0.0001	0.0001	0.0954	0.0001	0.0001	0.0001	0.0001

^a,b,cmeans having different superscripts in the same columns are significantly different (p < 0.05).

SEM: standard error of means; P value: probability level; Arg/Lys: arginine/lysine ratio; Cholest: Cholesterol; HDL: high density lipoprotein; LDL: low density lipoprotein; Trig: Triglyceride; CAT: Catalase; MDA: Malondialdehyde.

Table 10. Effect of dietary arginine/lysine ratio (Arg/lys) on some hormone’s activity and immune parameters of Mandara breeding hens under cyclic heat stress condition.

(Arg /lys ratio) + EDB		T3 µg/dL	T4 µg/dL	TAC mg/dL	IgG, mg/L	IgM, mg/mL	IgA, mg/L	Ca mg/dL	P mg/dL
1.25 (Control+)		0.229	11.7^c	411	9.78	2.33	0.833^a	30.75^a	4.97^ab
Cyclic heat stress	1.25	0.224	17.0^a	405	9.80	2.36	0.663^c	24.5.0^c	4.24^c
	1.37	0.225	15.0^b	412	9.71	2.38	0.837^a	28.15^b	4.67^b
	1.50	0.224	15.1^bc	410	9.79	2.39	0.830^a	28.75^b	4.78^b
SEM		0.002	0.086	13.62	0.854	0.532	0.031	0.245	0.042
P Value		0.3215	0.0001	0.542	0.075	0.2541	0.0217	0.0001	0.0001

^a,b,cmeans having different superscripts in the same row are significantly different (p < 0.05).

SEM: standard error of means; P value: probability level; TAC: Total antioxidant capacity; IgG: Gamma immunoglobulin; IgM: Mu immunoglobulin; IgA: Alpha immunoglobulin; T3: Triiodothyronine; T₄: Thyroxine; Arg/Lys: arginine/lysine ratio; Ca: Calcium; P: Phosphores.

The NC group exhibited considerably lower plasma calcium (Ca) and phosphorus (P) concentration compared with the PC and other experimental groups (Table 10). Minerals are required for tissue protein synthesis, cellular acid-base balance, cellular homeostasis expression, gene regulation, detoxification systems, and structurally in bone metabolism, according to Borges et al. (2003) and Shahsavari et al. (2012). Our findings support those of Aswathi et al. (2019), who found that serum Ca and P levels in heat-stressed birds are considerably lower than in control birds. In addition, Attia et al. (2018) found that plasma Ca concentrations fell in hens grown under heat stress and fed a diet with no supplementation, whereas plasma P concentrations and Ca/P ratios remained similar. However, Aml and Abd-Elaal (2020) reported that serum Ca and P concentrations were lower on the first and third days of CHS exposure, before returning to normal on the fifth day. Exposure to high external temperature reduces calcium uptake by duodenal epithelial cells, according to Odom et al. (1986). Furthermore, due to poor blood flow, the high temperature may prevent the reproductive tract of laying hens from receiving proper nutrient supply, limiting nutrients reaching the reproductive tract for optimal egg formation (Alagawany et al. 2021). Furthermore, Belay and Teeter (1996) found that broilers fed at high-cycling ambient temperatures retained reduced levels of P, K, Na, Mg, S, Mn, Cu, and Zn. Balnave and Brake (2001) found that increasing the Arg/Lys ratio to 1.36 enhanced plasma Arg:Lys.

Conclusions

Our findings indicated that diets with an Arg/Lys ratio of 1.37 administered to laying hens farmed under cyclic heat stress conditions, are able to improve the laying performance, egg quality, fertility, hatchability, blood hematological and biochemical constituents, antioxidants, and immunity indices in comparison to the other groups, including the PC one. Thus, diets with an Arg/Lys ratio of 1.37 can be used to recover the adverse effect of CHS.

Ethical approval

The protocol for the present study was carried out at the meeting of the Animal Production Research Institute Scientific and Ethics Committee (Protocol No. 01-10-003-37) and record No.1563.

Authors’ contributions

Conceptualisation, YAA, and AAA; methodology, YAA, AAA, AEA, AShE, and RMZ; software, YAA, FB and AAA; data collection, AAA and RMZ, investigation, YAA, AAA, NFA, FB; AEA, AShE, and RMZ; resources, AAA, RMZ, RAA and FB; writing—original draft preparation, YAA, AAA, and RMZ; writing—review and editing, YAA, AAA, NFA, FB; AEA, AShE, RAA and RMZ; project administration and supervising, YAA; funding acquisition, RAA, and YAA. All the authors have read and agreed to the published version of the manuscript.

Institutional review board statement

The Institutional approval code Protocol no. (01-10-003-37) and record no.1563.

Informed consent statement

Not applicable.

Disvclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data are available upon official request from the principal investigator and with the permission of the funding agent.

Additional information

Funding

This research work was funded by Researchers Supporting Project number RSPD2024R581, King Saud University, Riyadh, Saudi Arabia.