Effect of enzyme activated guanidinoacetic acid supplementation with different levels of metabolizable energy containing diets on serum biochemical profile and immunity index in laying hens

Introduction

India ranks second in the world for egg production, fourth in meat production and it leads the world in milk production. The nation's total egg production is projected to reach 149.11 billion eggs in 2024-2025, representing a 3.18% increase over the previous year compared to predictions of 142.77 billion eggs in 2013-2014 (DOAH, 2025) [11] . Hybrid layers of today lays on an average of 310-320 eggs per year compared to 240-250 eggs 30 years back. Similarly, present day broilers attain 1.8 to 2.0 kg mean body weight with FCR of 1.6 to 1.8 in only 38 days (Rao, 2020) [25] . The per capita availability of eggs was increased from 62 nos/annum in 2014-15 to 106 nos/annum in 2024-25. The expansion of the poultry sector is significantly dependent on the availability of high-quality with required quantity of feed for both producers and consumers and at economic rate (Adejinmi et al., 2011) [1] . In developing countries, poultry production industries face the challenge of raising feed costs by 60-70 percent (Tesfaye et al., 2013) [28] . For economic reasons, this supply of nutrients should be at least cost and so we must supply only enough for requirements without there being any major excesses. In formulating diets, the necessary nutrients are energy, protein, fat, vitamins, minerals and water. Among these in poultry diets energy and some of the essential amino acid such as Methionine and Lysine are considered as limiting nutrients (Alagawany et al., 2021) [3] . Metabolizable Energy (ME) is the part of energy remaining after faecal and urinary energy loss and represents the energy available for growth or reproduction. It is considered as the standard measure used to describe energy requirements of the birds and dietary content of energy (NRC, 1994) [23] .

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The dietary ME level is one of the major factors, which play an important role in regulating feed intake and feed efficiency in chickens (Lopez and Leeson, 2008) [19] . Guanidinoacetic acid is a stable and highly available feed supplement that is mainly used to boost energy utilisation. It is a direct precursor of creatine which has recently attracted attention as a feed additive due to its high thermal stability (Vranes et al., 2017) [29] . The European Food Safety Authority (2009) [12] concluded that GAA, if administered at a dosage not higher than 600-800 mg GAA per kg feed could be considered as an effective and safe feed additive for chickens for improving performance characteristics. Hence, GAA has been officially registered as an animal feed additive by American and European regulatory agencies (Khajali et al., 2020) [18] . Now a days, researchers are gaining more attention to evaluate the potential of enzyme activated Guanidinoacetic acid (eGAA) instead ordinary exogenous GAA for their role in energy metabolism.

According to metabolic pathways of GAA in animals, the activity of transporter enzyme and methyl group has been enhanced to make activated GAA which can be quickly and efficiently convert into creatine in young animals or when animal suffer in stress condition. Therefore, this study was conducted to determine effects of supplementing enzyme activated GAA with different levels of ME on blood biochemical profile and immunity index in laying hens.

Materials and Methods

Experimental details

The experiment on layers was carried out at Biotech Poultry Farm, At. & Post, Vadod, Dist. Anand, Gujarat for 56 days (24 th week to 31 st week). Geographically, this experimental station is located at 22.30° N latitude, 73.19° E longitude and 39 meters above sea level with subtropical climatic conditions. A total one hundred and sixty (n = 160) layers of 24 weeks old of BV300 strain reared under cage system were used as experimental birds. They were weighed individually and randomly assigned into five homogenous groups on the basis of their live body weight. Each group (32 birds) comprised of 4 replicates (08 birds/replica). The 24 weeks old layers were weighed individually with standard electronic weighing balance and it was considered as initial body weight of layers. Dietary treatments include basal diet (T 1 ), 5% and 10% reduced energy diet with GAA supplementation at 0.06% (T 2 & T 3 ) feed and eGAA supplementation at 0.06% (T 4 & T 5 ) feed.

Birds were adapted to respective treatment diet for a week before the commencement of the actual data collection.

Standard vaccination schedule was strictly followed. The required quantity of feed used in present experiment was formulated by using available feed resources and prepared in a single batch by uniform mixing of ingredients. Feed and water were provided with feeder and drinker channel. The entire replica under each group were separated to each other by kept one blank cage for avoid mixing of feed. Light bulbs were placed for the lighting system to increase the lighting period to 16 h per day in order to increase feed intake and laying. They were fed standard diet formulated as per BIS (2007) [7] by using ingredients listed in Table 1.

Health status

Blood was collected randomly from eight birds of each group (two birds/replica) at the end of experiment for collection of serum and plasma separately. Biochemical parameters viz. glucose, total protein, albumin, globulin, total cholesterol, triglycerides, uric acid and creatinine were estimated from serum samples by using commercially available analytical kits (Q-Line Biotech Pvt. Ltd.) using spectrophotometer (Model No. 4001/1 Gensys 20, Thermo Fisher Scientific, USA). Hematological parameters viz. hemoglobin (Hb), packed cell volume (PCV), total leucocyte count (TLC) and total erythrocyte count (TEC) were analysed using auto-haematology cell counter (Exigo EOS H400 hematology analyzer, Spanga, Sweden). Antioxidant indices including superoxide dismutase (SOD), reduced glutathione (GSH-Px), lipid peroxidation (MDA), total antioxidant capacity (TAC) and Malondialdehyde (MDA) was carried out with the help of chicken ELISA kit (G Biosciences, Geno Technology, Inc.) and reading was undertaken in ELISA reader (Cyber ELISA-R01 microplate reader, Cyberlab corp., USA).

Statistical analysis

Data collected from the above experiments were analysed using SPSS (20.0) version. One-way analysis of variance and Tukey least significant difference test for post-hoc comparisons (Snedecor and Cochran, 1994) [27] .

Results and Discussion

Hematological Parameters

The effects of dietary supplementation of enzyme activated Guanidinoacetic acid (eGAA) with different levels of metabolizable energy (ME) on hematological parameters of laying hens are presented in [14] . In the support of present findings, chickens were fed low ME (2700 Kcal/kg) basal diets and the GAA at 0, 0.6, and 1.2 g/kg observed improved the health of broilers with no negative effects (Ayeni et al., 2023) [5] and hypothesized that GAA could functioned as an antioxidant in the blood to safeguard the structure of the erythrocyte membrane and increase the deformability and transportation capacity of erythrocytes. According to Oloruntola et al. (2019) [24] , the PCV value fall within the normal range for broiler chicken and suggested that GAA/eGAA supplementation with reduced dietary energy does not adversely affect erythropoiesis, oxygen-carrying capacity and immune cell profile. This indicates that GAA supplementation supports normal physiological and metabolic functions without inducing hematological stress (Mousavi et al., 2013) [22] . In contrary, DeGroot et al. (2018) [10] reported improved hematological profiles in broilers supplemented with GAA, attributing it to better metabolic efficiency and oxygen transport capacity.

Blood biochemical status

Blood biochemical status (Table 3) revealed that the albumin, uric acid and AST were significantly differed among the treatment in layer chicken while other blood biochemical were comparable. However, the overall biochemical profile remained within the normal physiological range (Kaneko et al., 2008) [17] , indicating that supplementation of 0.06% enzyme-activated GAA even with reduced energy diets did not negatively affect liver and kidney function. The absence of significant variation (p > 0.05) in biochemical indicator suggests that protein metabolism was not markedly influenced by dietary supplementation of GAA (Michiels et al., 2012) [21] . These findings agree with Fosoul et al. (2018) [16] and Majdeddin et al. (2020) [20] , who observed improved protein metabolism and serum protein profiles in broilers receiving GAA under energy-deficient conditions. In contrast, Emami et al. (2017) [13] , Cordova Noboa et al. (2018b) [08] and Al-Abdullatif et al. (2024) [02] did found significant difference (p > 0.05) in albumin concentration between treatment groups when GAA was supplemented at 0.6 g/kg to chicken which indicates the bioavailability of dietary protein and albumin were similar during the whole experimental period. The values of all serum antioxidant markers (Table 4) in laying hens were found at par (p > 0.05) among the treatment groups which indicates that dietary eGAA did not notably alter the oxidative balance in chickens. Similar comparable trends were observed by Salah et al. (2020) [26] where non-significant linear increase in the liver GSH level with the increase in the dietary supplements of the GAA. However, the GAA supplements resulted in a significant reduction in the liver MDA levels. The positive effects of GAA on the liver antioxidant activity may be attributed to the expected higher creatine level. Similarly, the study of Azizollahi et al. (2024) [6] provides evidence that the administration of GAA results in a decrease in MDA levels and a numerical increase in TAC value. Previous studies have also highlighted the potential antioxidant effects of supplemental GAA in low to moderate quantities (Amiri et al., 2019; Cui et al., 2022) [4,9] . In contrast to present findings, Ezzat et al. (2018) [15] observed that as increased the level of GAA there were parallel increased the superoxide dismutase (SOD) and glutathione peroxidase (GPX) activity on betaine (1600 mg/kg) and GAA (600, 800 and 1000 mg/kg) supplementation with basal diet in laying hens. Likewise, Wang et al. (2012) [30] demonstrated that dietary GAA improved antioxidant status by elevating total antioxidant capacity and the activities of several antioxidant enzymes. This could be due to GAA-related metabolites (creatine and arginine) might be able to quench free radicals after GAA ingestion, suggesting indirect antioxidant effect of GAA utilization.

Immunity Index

Conclusion

It was concluded that supplementation of GAA and eGAA with reduction level at 5 and 10% in ME level did not affect the hematological indicators of laying hens. Moreover, serum glucose, total protein, globulin, albumin: globulin, creatinine, total cholesterol and triglycerides remained statistically similar between treatment groups. While, albumin and uric acid were statistically higher in T 5 group containing eGAA with 10% reduced ME diets. Further, liver enzymes, ALT remained unchanged but AST became significantly differed (p < 0.05) in treatment group. In addition, the serum antioxidant including total antioxidant capacity (umol/L), Superoxide Dismutase (ng/ml), Glutathione Peroxidase (umol/L) and Malondialdehyde (umol/L) in laying hens were revealed comparable. Thus, eGAA supplementation might have potential to cover the gap of energy reduction in the feed at 5 and 10% in layer chicken without affecting the performance of laying hens.

Acknowledgement

AcknowledgmentThe author extends his gratitude to The Principal, Collage of Veterinary Science & Animal Husbandry, Kamdhenu University, Navsari for providing necessary research amenities to accomplish the present research study and declare that there are no conflicts of interest regarding the publication of this article.

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