Air and rectal temperatures
Although several factors including air temperature, relative humidity, wind velocity and previous acclimatization (48, 49) influences bird’s responses to temperature fluctuations, indoor temperatures above 300C have been associated with heat stress conditions in laying hens (49). For our study, we focused on the influence of ambient temperature changes encountered during summer season on laying hens performance.
A relatively wide temperature range between 100C to 270C can allow for optimal growth whereas highest feed efficiency is achieved at 270C (50, 51). Similarly, Abbas et al. (52) reported that layers housed within 14-280C had improved feed conversion, body weight and body weight gains. Compared with our results, this indicate that during the summer experimental period, indoor pen temperatures of laying hens were beyond thermal comforts for maintenance and production which may accrue to heat stress effects. For sustained productivity, Charles (50) recommended maintaining optimum indoor temperature of 19-220C for laying hens, whereas, we recorded average minimum and maximum indoor temperatures of 25.020C and 31.01 0C respectively during the test period, suggesting birds were exposed to summer heat.
Throughout the study period, we recorded no significant changes of dietary L-Cit supplementation on rectal temperatures of laying hens. This findings differs from (44) who found that oral administration of L-Cit could reduce rectal temperature in chicks. Also, similar report by Kvidera et al. (53) showed that citrulline supplementation had tendency (P = 0.07) to decrease rectal temperature in pigs during heat stress. This differences may be attributed to the route of administration and severity of heat stress conditions.
Laying performance and Egg quality
Our findings indicate that L-Cit supplementation had no significant effects on the growth and production performances of laying hens. No significant differences in average daily intake, body weight, egg weights, egg mass, laying rates, and feed conversion ratios were observed among groups. Likewise, supplemental citrulline did not affect production variables of pigs under thermal stress (53). 1% L-Cit fed to farrowing sows during summer increased urinary nitrite/ nitrate concentrations, reduced respiratory rates and pre-weaning mortality but had no effects on feed intake, body weights, or rectal temperature (54). Although our studies showed no changes on food intake with L-Cit treatment, studies in rats have shown that L-Cit can suppress appetite by activating hypothalamic proopiomelanocortin levels (55), resulting in significant declines of food intake.
The daily fluctuations in diurnal temperatures along with time span of these heated periods during summer stress could result for the absence or insignificant impacts recorded on laying hens performances. Constant temperatures result in higher reductions in egg laying performance compared to diurnal fluctuating temperatures (56). With upper temperature limits of 280C, Tumova and Gous (57) reported no changes in egg production. With these findings, we may suggest that during the study period, the severity of heat exposure was not sufficient to initiate severe adverse effects on laying hens performance.
The external and internal characteristics of eggs provide important criterion for evaluating egg quality. Increased temperature and humidity index, a common measure of environmental stress, can result in prolonged alterations in egg production up to 25% possibly due to reduced feed intakes and lowered nutrient availability for egg production (49). The effects of temperature changes on laying performance has often been examined, however, Al-Saffar and Rose (56) reported that hens were better able to tolerate rise in diurnal temperatures without marked effects on their productive performances. The egg weights, shell thickness, egg shell strength, yolk color, yolk grades, yolk weigh, albumen weights and shell weights were unchanged with treatment groups in this study. Our findings corroborate with Kilic and Simsek (49), who showed that during summer stress, there were no significant variations in egg quality characteristics of laying hens
Egg shape index depicts the proportion of egg breadth to egg lengths, as such, a vital tool in egg grading (58). It is an important determinant of egg quality since it significantly affects the egg weight, eggshell surface area and egg volume (59). Egg internal egg quality is dependent on traits such as albumen height and Haugh unit which both exist in a highly positive relationship (60) and are genetically correlated (61), inferring that improvements of albumen height would affect Haugh unit. Likewise, positive correlation exist between shape index and albumen height of eggs (62), and between shape index and Haugh units (58) such that, Haugh units improved with increase in egg shape index. With different laying hen genotypes, Sarica et al. (63) reported highly significant and positive correlations between egg shape index and albumen height (r2 = 0.089), as well as between shape index and Haugh units (r2 = 0.086). In contrast, Olawumi and Ogunlade (60) had reported shape index to exist in a negative and non-significant correlation with egg internal quality.
Our results showed that dietary L-Cit increased egg shape index, and tended to improve albumen heights and Haugh units, suggesting that L-Cit increment of egg shape index may improve the albumen heights which directly influenced the Haugh unit, thus, promoting internal egg quality. The relationship between these measures of egg quality demonstrates that L-Cit increment of egg external shape index may correspond to improved egg internal quality. Similar to our findings, dietary supplementation with natural antioxidants (thyme, oregano, curcuma longa and rosemary) increased egg shape index of laying hens (64).
Liu et al. (65) reported that increased circulating free calcium (Ca2+) in the blood in combination with other plasma proteins which are involved in egg shells formation resulted in improved egg shell quality. Likewise, there exist a quantitative relationship between calcium concentrations and citrulline synthesis in the mitochondrial matrix (66). L-Cit stimulated cytosolic Ca2+ concentrations in a Nitric-oxide dependent manner in pancreatic β-cells (67). This findings imply that L-Cit is metabolized by NO synthases to produce NO, which in turn may act to increase intracellular Ca2+ ions responsible for improving egg shape index. This coincides with Sahin et al. (68) who demonstrated that enhanced calcium metabolism results in improved eggshell quality.
Plasma metabolites and circulating NO levels
In this present study, the addition of L-Cit to the diet of laying hens did not alter the blood biochemical parameters of the birds. No significant changes in plasma triglyceride, total cholesterol, total protein, urate and aspartate aminotransferase observed in the current study coincides with Ismail et al. (69) who reported that ascorbic acid and zinc bacitracin (anti-oxidant supplements) did not affect the serum metabolites of heat stressed broilers. Similarly, Chowdhury et al. (45) observed that orally administered L-Cit did not affect plasma total cholesterol and triacylglycerol levels in chicks. From this study, the insignificant differences in plasma aspartate aminotransferase levels, an indicator of liver and kidney functions, indicates that L-Cit feeding did not alter hepatic and renal functioning of laying hens.
Several studies in porcine and human models have reported L-Cit as a potent precursor for NO synthesis (70, 35, 71, 72), however, this is yet to be ascertained in poultry. Our study hypothesized that L-Cit, as an endogenous precursor of arginine, would modulate NO synthesis when supplemented in diets. Since Arginine biosynthesis in poultry is limited due to lack of carbamoyl phosphate synthase (73), and the several precursory roles of arginine including protein synthesis, urea cycle, production of polyamines, creatine and nitric oxide, may affect its priorities of use and bioavailability (73), the ability of supplemental L-Cit to facilitate the Arg- Cit- Arg recycling for NO synthesis will be of significant impacts for both arginine and NO-mediated functions (74, 75). Earlier investigation on citrulline utilization in poultry reported by Klose and Almquist (76) demonstrated that citrulline supplementation to an arginine deficient diet effectively improved chick growths with a similar response as arginine addition.
Our study revealed that dietary L-Cit was able to increase systemic nitric oxide concentrations, as well as the activities of NO synthases in a dose specific manner with highest L-Cit levels (1%) producing largest NOx concentrations and NOS activities. NOx concentrations are positively associated with citrulline levels (77). This association was demonstrated in our study by the increment observed between L-Cit levels and NO production since the quantitative assessment of plasma NO concentrations (NO2− +NO3−) depicts the systemic NO synthesis (78).
Our results indicated that peak in plasma NOx can be attributed to generation of NO by NOS since both total NOS and inducible NOS activity were upregulated at end of experiment (week 8). Several factors are involved in the regulation of iNOS expression including stress signals and inflammatory cytokines, however, iNOS expression has been implicated in both stimulatory and inhibitory actions (79). Thus, asides its role in NO synthesis, further research is necessary to ascertain the implications of L-Cit induced iNOS expressions. Similar to our findings, Ham et al. (35) demonstrated that L-Cit induced iNOS mRNA expression were associated with increased mRNA expressions of endogenous antioxidants such as SOD1, SOD3 and catalase. Similar report by Kim et al. (80) showed that citrulline ingestion improved NO synthesis rate. Also, NG-nitro-L-arginine methyl ester (L-NAME), a non-selective NOS inhibitor, when administered to chicks was able to inhibit heat stress induced hyperthermia, however, the plasma levels of Nitrite/ Nitrate levels were unaffected (22). This findings differ from Chowdhury et al. (45) who reported that oral injections of L-Cit did not significantly increase plasma NOx concentrations both under ambient temperatures or heat stressed conditions.
Antioxidant enzymes (CAT, GSH-Px, and SOD) activities are typically upregulated after heat stress to act as cyto-protective measures against excess superoxide formation (15, 81). An increase in oxidative stress may be expressed by the elevation in plasma levels of lipid peroxidation products (including malondiahaldehyde (82, 83). Increased activity of antioxidant enzymes has been associated with decreased MDA levels (84). Likewise, curcumin was shown to improve antioxidant status in heat stressed hens by lowering serum MDA levels while promoting enzymatic activities of SOD and GSH-Px (65).
Our study revealed that MDA levels were reduced, whereas anti-oxidant enzymes activities of CAT, SOD, and T-AOC were elevated in laying hens fed L-Cit. This implies that dietary L-Cit initiated antioxidant defenses to combat the excessive production of ROS. Previous research have reported that L-Cit can inhibit ROS formation through direct scavenging of hydroxyl radicals via H+ ion donation to form water and/or chelation of copper to inhibit hydroxyl radicals production via the Fenton reaction (85).
Our results showed that increasing L-Cit levels inhibited GSH-Px actions in a dose specific manner. This findings could be from L-Cit elevation of NO concentrations, as this would result in improved reactivity between NO and oxygen to yield oxidant metabolites of NO which can suppress GSH-Px activity (86). The GSH-Px enzyme belongs to the family of selenoproteins, as such carries a selenocysteine at its active site (87). Research has shown that S-nitroso-N-acetyl-D, L-penicillamine (SNAP), an NO donor can irreversibly inactivate GSH-Px enzymes, since the NO released acts to modify the cysteine-like active site on GSH-Px (88). Similarly, administration of peroxynitrite precursor, 3-morpholinosydnonimine N-ethylcarbamide, as well as synthetic peroxynitrite inactivated bovine GSH-Px activity by modifying its catalytic center (89). These infers that Nitric oxide and its derivatives yield Nitrosonium ions (NO+) which actively oxidizes the GSH-Px active site of selenocysteine residue to yield selenenyl sulfide thus inactivating the enzyme (86).
These findings suggest that increasing nitric oxide availability would directly attenuate GSH-Px activity which may result to increased intracellular peroxides. However, since L-Citrulline initiates the activity of other anti-oxidant enzymes such as SOD and CAT which also actively catalyze peroxides, we speculate that this would achieve a significant balance between oxidants and antioxidants for cellular homeostasis. Anti-oxidant enzymes interact in concert with each other, such that SOD catalytic product of hydrogen peroxide from superoxide ions is eliminated by CAT or GSH-Px activity (81).
Researches have also linked L-Cit induction of Nitric oxide production alongside with its anti-oxidant ability. L-Cit directly attenuated ROS production by downregulating the protein expression levels of p67phox, a critical component for NADH/NADPH superoxide generation (90). In vitro studies using asymmetric dimethylarginine (ADMA), a non-selective NOS inhibitor, demonstrated L-Cit effectiveness to restore NO production and attenuate nitrosative stress (91). Also, in atherosclerotic rabbits fed orally with L-arginine + L-Citrulline alone or with antioxidants, there was a reduction in superoxide production, downregulation of oxidation-sensitive (Elk-1 and p-CREB) genes with associated increments in NO synthase (eNOS) expressions and NOx plasma concentrations (71).