Effect of lysophospholipid and sophorolipid supplementation on blood biochemical and antioxident status in broiler chicken

Introduction

The cost-effective production of the broiler business depends on increasing dietary fat consumption. By providing vital fatty acids and vitamins, may increase energy availability, feed efficiency, health status and carcass quality (Ravindran et al., 2016) [31] . For effective utilization of fats from diet, micelle production and process of emulsification are very crucial. Therefore, externally sourced emulsifier supplementation is alternative to accomplish this (Iqbal and Hussain, 2009) [16] . A class of chemicals known as emulsifying agents makes it possible for water in oil or oil in water to disperse uniformly as an emulsion. Emulsifiers are crucial because they break down dietary lipids into smaller droplets, which encourages the formation of micelle-like structures makes it easier for the body to absorb (Melegy et al., 2010) [23] . Numerous emulsifiers have developed to enhance broiler chicken development and nutrient digestion; however, public health concerns it is necessary to understand emulsifiers' toxicity and biodegradability (Upadhaya et al., 2018) [38] . Lysophospholipid (LPL) is a class of membrane lipids exhibits greater stability in the aqueous environment of the gastrointestinal tract and have efficient bio stimulant emulsifying qualities (Joshi et al., 2006) [18] . Furthermore, SPL might have a number of special effects, such as collagen neogenesis, fibroblast activation and immune regulation (Hardin et al., 2007) [14] . Therefore, the study was carried out to evaluate potential of Lysophospholipid (LPL) and Sophorolipid (SPL) as an exogenous surfactant on blood biochemical status of broilers. the Department of Animal Nutrition. Geographically, the experimental location is 9 meters above sea level with subtropical climatic conditions.

Treatment groups and feeding standards

A total of one hundred and sixty (160) a seven day -old broiler chicks of Vencobb-400 strain were used as experimental birds. Experimental chicks were weighed individually and randomly assigned into four homogenous groups on the basis of their live body weight. Each group (40 chicks) comprised of 4 replicates (10 chicks/ replica). All the birds were fed on standard diet formulated as per BIS (2007). The control group was fed basal diet (T1), where Lysophospholipid and Sophorolipid at 7.5, 12 mg/kg, 12.5, 20 mg/kg and 17.5, 28 mg/kg of feed were supplemented respectively in T2, T3 and T4.

Measures of Traits

• Feed intake: Weighed quantity of feed offer twice a day and leftover was collected in next early morning and weighed. The difference was considered as amount of feed ingested. • Blood sampling: Blood was collected early in the morning from the wing vein using clean, sterilized needles into two separate clean and dry test tubes for hematological and serum biochemical analysis. The first tube containing EDTA was used for estimation of hematological assay. In the second tube, blood was collected without anticoagulant and was allowed to clot for 1 h at room temperature (25°C) and then centrifuged at 2000 rpm for 15 minutes to harvest serum. The clear, non-hemolysed serum was then taken into a set of clean, dry, eppendorf tubes and stored at -40°C for further analysis. The voluntary feed intake was recorded as total feed intake (g) and average daily feed intake (g) are presented in Table 3 [26,20] . The results indicates that palatability of feed was unaffected on emulsifier supplementation; however, the level of energy, requirements, growth rate, type and composition of emulsifier, its dose rate, level and types of other incorporated nutrients/ additives, methods of formulation and forms of feed are factors can be associated with voluntary feed intake by chickens.

Results and Discussion

Feed Intake

Haematological parameters

Blood is a biological connective tissue primarily comprising of fluid portion and plasma in which major blood components (erythrocytes, leukocytes and thrombocytes) remain suspended. Blood represents a means of assessing clinical and nutritional health position of animals in feeding trials and the haematological variables most commonly in nutritional studies include PCV, RBC, Hb, mean corpuscular haemoglobin concentration (MCHC), Mean corpuscular volume (MCV) and clotting time (Osho et al., 2014) [29] . In this experiment, blood was collected at end of the experiment (42 nd days) for the analysis of hematological parameters. The findings of different hematological indices of treatment groups are presented in Table 3.2. The concentration of Hb, MCV, MCH, MCHC and monocytes did not display any significant changes among the different groups. All the haematological parameters found in present study were in normal range and reflected that inclusion of Lysophospholipid and Sophorolipid does not affect health status of birds. Similar to present results, Ani et al. (2024) [2] reported that dietary incorporation of lysolecithin induced no linear or quadratic effects (p>0.05) on all haemato-biochemistry attributes in broiler chickens. Our findings were supported by Cho et al. (2012) [6] and Mohammadigheisar et al. (2018) [24] , who observed that broilers fed diets supplemented with emulsifiers and lysolecithin in varying calorie diets did not exhibit any appreciable changes in their haematology attributes. There were no significant differences observed in blood total protein, total albumin, triglyceride, cholesterol, HDL and LDL levels (p>0.05) of broiler chickens fed an emulsifier-supplemented with or without low energy feed (Gholami et al., 2024) [13] . Our findings are further supported by Singh et al. 34 , who documented no dietary influence on the blood biochemistry and haematology of the broilers fed emulsifier at different energy levels. Kothare et al. (2021) [21] noticed no significant (p>0.05) effect of dietary energy levels and multi-enzymes between treatments on any of the haematological measures in broiler chickens. Consequently, the findings of Cowieson and Adeola (2005) [7] indicate the absence of any significant interaction between multi-enzyme supplementation and dietary energy levels. The current experimental findings are in agreement with the findings of Saleh et al. (2020) [32] who reported no significant (p>0.05) change for the total protein and albumin levels among treatment groups supplemented with multi-enzyme with different energy level (ME: 3151 and 3102 Kcal/Kg) diets. Alike to these, Attia et al. (2018) [3] noticed no significant (p>0.05) effect in broiler birds fed various levels of multi enzymes on the level of total protein, albumin and globulin in standard and low-density diets. However, contrary to the present findings, Abudabos (2012) [1] demonstrated significant increase in concentration of total protein in broiler birds fed different levels energy and multi-enzyme. Similarly, Sanhoury and Ahmed (2017) [33] also observed significantly (p<0.01) increased levels of total protein and globulin with multi-enzyme supplementation in broiler birds. [30] inferred that inclusion of lysolecithin did not affect serum total cholesterol, triglyceride, or free fatty acid concentrations. Similarly, serum total cholesterol, triglyceride, and HDL cholesterol levels of broiler chicken fed diets containing vegetable oil or animal fat were not affected by emulsifier supplementation (Lysophospholipid and Lipase enzyme supplementation to low metabolizable energy diets) as observed by Movagharnejad et al. (2020) [26] . Alike, Cai et al. (2024) [5] concluded that the differences in calorie content or the inclusion of LPC in the meals had no effect on the serum concentrations of TG, TC, and AST on day 21 (p>0.05). In the same way, Eid et al. (2025) [10] reported that discrete dietary supplementation of LPL at the level of 0.5 or 1g did not change the levels of AST, ALT, creatinine, LDL-cholesterol, and triglycerides parameters compared to the control group (-DEX).

On the other hand, Cho et al. (2012) [6] reported that broiler chickens fed with diet containing 0.05% emulsifier (sodium stearoyl-2-lactylate) had lower serum triglycerides than those fed with high-energy diets without emulsifier. They assume that the effect on lipid metabolites may depend on fat sources and emulsifier types. Similarly, Zhao and Kim (2017) [41] showed that LPC supplementation on day 14 decreased the TG levels. Because dietary fat is absorbed and metabolized more quickly in broilers given LPC, their serum levels of TG and NEFA may be lower. Furthermore, Eid et al. (2025) [10] reported that discrete LPL dietary supplementation at 0.5 or 1g did not change the levels of AST, ALT, creatinine, LDL-cholesterol, and triglycerides parameters compared to the control group (-DEX). Additionally, Kamran (2020) [19] have indicated that broilers fed diets containing emulsifiers or emulsifiers with lipase did not alter in the levels of cholesterol, glucose, and triglycerides.

In partial agreement, Mohiti-Asli et al. (2025) [25] reported that dietary supplementation with LP in broilers significantly reduced serum triglyceride (TG), total cholesterol, and low-density lipoprotein cholesterol (LDL-C) concentrations, while significantly increasing highdensity lipoprotein cholesterol (HDL-C) levels by day 42, compared with birds fed non-supplemented diets (p<0.05). This suggests that chylomicrons were either released into the serum more slowly or removed from the blood more quickly (Jones et al., 1992) [17] , which is indicative of better hepatic lipid metabolism (Tompkins and Parkin, 1980) [37] . Moreover, they specified that emulsifiers break down triglycerides for enhanced energy utilization. Based on these findings, it can be concluded that emulsifier supplementation effectively reduced blood triglyceride levels as birds aged, likely by enhancing energy utilization. Emulsifiers could also reduce cholesterol and triglyceride levels by stabilizing the phospholipid coating of portomicrons as suggested by Jones et al. (1992) [17] . This could in turn decrease the release rate of cholesterol and triglycerides (sequestered in the portomicron interior) into the blood; hence, low levels can be observed. Consistent with the observed increase in glucose concentration in the T3 group, Eiler (2004) [11] suggested that improvements in blood glucose levels reflect enhanced glucose uptake and utilization, thereby promoting metabolic activity in hepatocytes. In contrast, Cai et al. (2024) [5] reported that dietary lysophospholipid inclusion had no significant effect on serum glucose levels. Nasr et al. (2017) [27] suggested that serum biochemical parameters serve as fundamental indicators of oxidative stress, as reflected by alterations in blood biochemistry. The present findings may be attributed to the complex metabolic effects of emulsifiers, which vary according to dosage, mode of supplementation, and individual physiological differences. Some studies are partially consistent with the current results, as Oketch et al. (2022) [28] reported no significant dietary effects of emulsifier supplementation on total cholesterol and triglyceride levels at day 21. However, by day 35, marginal reductions in total blood cholesterol and triglyceride concentrations (p = 0.076 and p = 0.095, respectively) were observed in broilers fed emulsifiersupplemented NCL and PCL diets. These findings suggest that serum lipid fractions may be more susceptible to the effects of emulsifier supplementation over time. [5] reported a significant reduction in malondialdehyde (MDA) concentration in broilers fed lysophosphatidylcholine (LPC)-supplemented diets (p<0.01). However, LPC inclusion had a nonsignificant effect on hepatic superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities on day 42 (p > 0.05). Notably, there was a significant interaction between dietary energy content and LPC with respect to liver MDA levels (p<0.05), indicating that broilers fed low-metabolizable-energy diets were more responsive to LPC's effect on MDA. MDA, a product of [22,9,4] .

Previous studies support the antioxidant-enhancing effects of lecithin and LPL supplementation. Zangeneh et al. (2018) [40] observed improved oxidative balance in broilers, evidenced by increased TAC, GPx, and SOD activities alongside decreased MDA levels. [36] reported that including 0.5 g/kg LPL in breeder diets produced offspring with lower hepatic SOD (p<0.01), suggesting improved antioxidant status in the hens. Furthermore, El-Katcha et al. (2021) [12] demonstrated that supplementing 0.05% lysolecithin to 60day-old ducks increased GPx activity, aligning with the current study's results. Ani et al. (2024) [2] reported that dietary lysolecithin at 300-400 g/100 kg significantly decreased malondialdehyde (MDA; linear p = 0.003) while increasing superoxide dismutase (SOD; linear p<0.001), glutathione peroxidase (GPx; quadratic p<0.001), and glutathione (linear p<0.001) in broilers, suggesting a protective role against oxidative stress. The antioxidant effect of lysolecithin is attributed to its composition, which includes amino-alcohol phospholipids and tocopherols (γ, α, and δ). Sun et al. (2018) [35] suggested that the polar head groups of phospholipids, such as amino and choline groups, facilitate their use as lipophilic antioxidants, enhancing the capacity to counteract oxidative stress. Consistently, Das and Vasudevan (2006) [8] reported that lysolecithin exhibits antioxidant and neuroprotective properties, helping reduce liver damage, improve oxidative resistance, and increase the stability of oils and fats due to its phospholipid content. The efficacy of emulsifiers as antioxidants may depend on the type of oil, its tocopherol content, and fatty acid composition (Ilyas et al., 2016) [15] .

Conclusion

The present study demonstrated that dietary supplementation with lysophospholipid (LPL) and sophorolipid (SPL) had no adverse effects on hematological parameters in broiler chickens. Serum biochemical analysis revealed that glucose and albumin concentrations were significantly increased with emulsifier supplementation, while other parameters remained unaffected. Furthermore, the antioxidant status of the birds was significantly improved, as evidenced by reduced malondialdehyde (MDA) levels and increased activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in LPL-and SPL-supplemented diets. These findings indicate that LPL and SPL supplementation can improve antioxidant defense and overall physiological status in broilers.

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