Lipid regulation by early administration of irradiated chitosan and glutathione in heat-stressed broilers

This research was conducted to determine the effect of irradiated chitosan and glutathione on lipid regulation of broiler and its impact on free radical compounds. One hundred broiler starter phase as the objects of research. The study used a completely randomized design (CRD) with experimental protocols method, consisting of nine treatments and three replications. Each replication consist of five broilers, namely P0 = without giving chitosan and glutathione, PA1 = given 100 ppm chitosan from the age of 4-35 days, PA2 = 450 ppm chitosan from the age of 4-35 days, PA3 = 100 ppm chitosan + 150 ppm glutathione from the age of 4-35 days, PA4 = 100 ppm chitosan + 150 ppm glutathione from the age of 4-35 days, PB1 = 300 ppm chitosan from the age of 20-35 days, PB2 = 450 chitosan ppm since the age of 20-35 days, PB3 = 100 ppm chitosan + 150 ppm glutathione since the age of 20-35 days and PA4 = 100 ppm chitosan + 150 ppm glutathione since the age of 20-35 days. Parameters observed in this study, namely regulators and broiler lipid transport (Apo A1, Apo A2, Apo B, Apo C, HDL, LDL), plasma lipids (triglycerides, total cholesterol, NEFA), The results showed that the administration of irradiated chitosan and glutathione had a significant effect (P<0.05) on regulators and broiler lipid transport by fed 450 ppm of irradiated chitosan with 150 ppm of glutathione. Based on the research results, it can be concluded that the administration of irradiated chitosan accompanied by glutathione induction in broiler rations can affect lipid regulation and biochemical aspects and performance as a result of this regulation. Giving chitosan as much as 450 ppm with 150 ppm induced glutathione since the early age of the finisher phase (20 days) was able to regulate lipid biosynthesis optimally with the best final body weight.


Introduction
A rapid broiler growth is followed by an increase in abdominal fat, sub-cutaneous fat and intramuscular fat, as well as tissue lipids (fats and sterols).The percentage of sub-cutaneous fat that has been reported by many researchers consists of 0.5-1% of body weight, while abdominal fat can reach 6% of body weight at 6 weeks of age [1][2][3][4].Tissue fat and cholesterol profiles also increase with broiler age [5][6][7].Both types of lipids are needed for the development of cells in all tissues.On the other hand, fat and 2 cholesterol are unfavorable for producing a healthy product.An excessive proportion of abdominal fat is unfavorable to consumers.
Efforts to produce broilers with lower proportions of abdominal and tissue fat and tissue cholesterol are an interesting research concern, while not reducing growth.Giving natural ingredients (herbs) to broilers has been widely done, and shows very significant results in reducing broiler lipids, but accompanied by a decrease in body weight, for example by giving kayambang (Pistia stratiotes) leaves [8].The results of other studies show that giving natural ingredients that are mixed into traditional herbs to stimulate broiler growth appears to be effective in increasing body weight but also accompanied by an increase in the proportion of fat with carcass proportions that are not significantly increased [9][10][11][12].The results of these previous studies did not show a metabolic balance between tissue formation [13,14] and inhibition of lipogenesis [2,5,6,[15][16][17].These results have also not shown the effectiveness of using these herbs as lipogenesis inhibitors.
A feed additive that can be an inhibitor of lipogenesis is chitosan.Chitosan consists of glucosamine polymer, accompanied by minerals with amine and acetyl groups [10].Chitosan in this form is not effective as an inhibitor of fat formation and lipogenesis-related protein (gene and enzyme) activities [3,16].Irradiation technology has been used to degrade the polymer into lower molecular weight glucosamine monomers [3], which can be better absorbed in the gastrointestinal tract.
The effectiveness of irradiated chitosan as a lipogenesis inhibitor has been indicated by previous researchers.Inhibition of lipogenesis (lipid formation) or increased lipolysis is always accompanied by oxidation which causes an increase in radical compounds [18,19].Previous studies have shown that excessive lipolysis leads to reduced growth, as a direct lipid deprivation effect, as well as increased tissue inflammation by radical compounds from lipid oxidation.However, this condition can affect hematologic conditions, as well as alter the antioxidant activity profile in order to prevent excessive inflammation.
A strategy that can be applied to prevent the adverse effects of using irradiated chitosan is the addition of glutathione.Glutathione is an enzyme that contains selenium as its basic component so it is classified as a seleno-protein [4,20,21].The enzyme glutathione consists of 4 selenium atoms bound as selenocysteine.Glutathione peroxidase can form a defense against oxidants or free radicals in the body and prevent cell damage or inflammation [22][23][24][25] and can stimulate growth through cell repair [26][27][28].Indications of a positive impact with glutathione administration are improved hematologic conditions and increased antioxidant activity.Studies on the pattern of chitosan and glutathione administration need to be researched and studied comprehensively.This study shows the impact of early administration of irradiated chitosan and glutathione on hematologic condition and antioxidant activity of broilers.

Animal
The experiments related to animals in this investigation were conducted based on the Guideline of Animal Ethics and Welfare and approved by the National Nuclear Energy Agency of Indonesia (BATAN) with decree No. 34/AR/2022.One hundred day-old, mixed-sex broiler DOCs were used in this study.The average body weight of the study sample used was 40.93±1.09.Broiler samples were reared in flocks measuring 50 x 50 cm, an experimental unit containing 5 DOCs.Flock temperature was set to 40 0 C after brooding, using incandescent wire heater and 40 watt incandescent lamp in each experimental unit.The research was carried out from September to October 2022 at poultry coop of Faculty of Animal Science, University of Padjadjaran.Blood samples were tested at the Laboratory of Livestock Physiology and Biochemistry, Faculty of Animal Husbandry, University of Padjadjaran, Jatinangor, Sumedang, West Java.

Experimental and treatment
The experiment design used a CRD with experimental protocols method, consisting of nine treatments and three replications.Each replication consists of five broilers.Each bird in the experimental unit was given a wing tag.On days 1 to 3, broilers were fed commercial crumble rations, followed by mash rations after the third day until day 35 The experimental basal ration was fed indefinitely.The food composition and metabolic energy of the rations used can be seen in Table 1.

Results and discussion
Based on the results of this study, it showed that the administration of irradiated chitosan and glutathione had an effect on the levels of lipid homeostasis regulators for broilers.It was seen that the levels of chitosan and the age of administration also contributed to variations in the concentration of the PPAR© gene expression.The results of the present study seem to demonstrate the ability of irradiated chitosan to inhibit (prevent) the expression of the lipid regulatory gene (PPAR©).Previous research [15] found that chitosan inhibits lipid regulators in the body by decreasing the expression of the PPAR© gene.
Although used non-irradiated chitosan with a higher level [15].This fact also shows that the lower molecular weight of chitosan through the irradiation process appears to be more effective in preventing the expression of these regulatory genes.The effect of giving irradiated chitosan and glutathione levels in feed on broiler lipid regulators and transport based on the results of the study can be seen in Table 2.
Regarding the impact of glutathione which can stimulate PPARα expression in the broiler group with the combination of 100 ppm irradiated chitosan, these results indicate that glutathione is related to specific proteins, especially those that play a role in lipid regulation.These results can be strengthened by the results of a study reported by [29] that glutathione levels have a strong association with proteintyrosine phosphatase (PTP)-1B activity.PTP-1B is a redox-sensitive protein and has been identified as a potent inducer of SREBP-1 gene expression and as a novel regulator of lipogenesis.Increased expression of SREBP-1 also stimulated increased expression of PPAR©.
The concentration of lipid regulators decreased as a result of irradiated chitosan administration and the effect of glutathione administration on increasing PPAR© also seems to have an impact on the concentration profile of lipids circulating in the circulation system.This is indicated by differences in lipid transport changes in the circulation system, namely HDL and LDL and apolipoprotein (APO) which acts as an activator of HDL and LDL [3].Based on the results of this study (Table 2) it appears that overall the administration of the combination of chitosan and glutathione increased (P<0.05)levels of APO A1,2, APO C and HDL especially in the broiler group entering the finisher phase.In contrast, the combination of the two treatments reduced (P<0.05)APO B and LDL levels.The same changes in APO A, C and HDL profiles as well as APO B and LDL could be due to the relationship between these compounds.It is known that apolipoprotein (APO) is a protein component of lipoprotein, where apo A and apo B, both lipoprotein, are important in regulating cholesterol function in the body.APO AI and II and APO C activate enzymes that play a role in converting cholesterol in tissues to HDL and recognition of HDL on receptors in the liver [23,28].The two APO A subtypes, the levels of APO A-I are more than apo A-II.APO A-I and II, both are related to HDL levels, the higher the activity of APO A and C, followed by an increase in HDL activity [30].Likewise, apolipoprotein (APO) B also consists of two subtypes, apo B-100 and apo B-48, both of which form VLDL in the liver and then become LDL.APO B-100 is related to LDL levels.
Fluctuations in APO A, C profiles with HDL, as well as fluctuations in APO B with LDL, are closely related to changes in PPAR© concentrations as a regulator to maintain lipid homeostasis.The research results of [31] reported that PPAR© is the main regulator of intra and extracellular lipid metabolism.Low PPAR© levels affect the regulation of hepatic apolipoprotein C-III and APO B and increase lipoprotein lipase and LDL gene expression.In addition, the low activation of PPAR© actually stimulates an increase in plasma HDL through the induction of hepatic apolipoprotein A-I and the expression of apolipoprotein A-II and APO C. PPAR© also plays a role in intracellular lipid metabolism by upregulating the expression of enzymes involved in fatty acid conversion [10,23].
The effect of giving various irradiated chitosan and glutathione in feed on the lipid profile of broiler blood plasma in current experiment shows in Table 3. Chitosan contributes to lipogenesis regulation, as it has been reported by previous researchers that chitosan can reduce fat content in livestock.Other studies have shown that chitosan administration activates related lipogenesis in the liver, as well as in adipose tissue significantly.This current experiment, show that broiler fed the chitosan was significantly inhibited the endogenous expression of lipogenic genes, including Fatty Acid-Transport-1 (FA-TP1), 3-Hydroxil-3-methyl-glutaryl Co-A Reductase (HMGCRase), and FA-Binding Protein-4 (FA-BP-4), FA-Synthase (FASase) in liver and adipose tissues of experimental animals fed a high energy.The results of this study suggested that chitosan supplementation could reduce lipogenesis through activation of AMPK and inhibition of lipogenesis-related genes according to [31].An increase in the rate of lipolysis causes fat as an energy store to be catalyzed into NEFA and glycerol.NEFA is one of the precursors of triglyceride (TAG) formation in adipose tissue, liver and muscle through the esterification process [32].NEFA was not formed into triglycerides (TAG) in this study because irradiated chitosan plays a role in reducing the activity of lipoprotein lipase that hydrolyzes, oxidizes chylomicrons and free fatty acid or NEFA into TAG/triglycerides.[33] states that triglyceride synthesis in the exogenous pathway comes from food and takes place in the ileum (small intestine).Triglycerides (TAG) are formed as chylomicrons which are then transported in the blood through the thoracic duct.Free fatty acids absorb into muscle cells and/or adipose cells by crossing the endothelium system.These fatty acids are converted back into TAG for energy storage.
The level of blood cholesterol decreased as the level of IC (irradiated chitosan) concentration in the feed increased during the study.Based on research by [34] stated that the decrease in cholesterol transport activity through blood plasma by HDL and LDL is the impact of chitosan injection.The results of other studies also showed an increase in cholesterol uptake by liver cells with chitosan administration [32,35,36].
As the results of the current study, it can be seen that the total cholesterol profile of broiler chickens without chitosan irradiation has the highest mean value compared to the treatment given chitosan irradiation.Based on research [15] showed a decrease in the hypertoric activity of adipocyte cells and the accumulation of triglycerides, on the contrary, there was also an increase in the rate of lipolysis, which was accompanied by a decrease in the activity of lipoprotein lipase in adipose tissue in the epididymal layer.The results of previous studies, as a whole, reported a decrease in the activity of contrast enzymes in the liver related to specific proteins that are responsible for the biosynthesis of lipids and their precursors.The addition of chitosan in the diet was reported to significantly stimulate several genes related to energy homeostasis, such as AMPK, and other studies showed that high-fat diets induced the expression of lipogenic transcription factors PPAR-γ and SREBP1c in liver and adipose tissue.Cholesterol levels in liver cells contribute to the inhibition of the synthesis of the enzyme 3 hydroxylmethyl glutaryl Co A (HMG Co-A).This enzyme plays a role in cholesterol synthesis, so a decrease in its expression causes endogenous cholesterol synthesis and the amount of cholesterol circulated into the vascular system also decreases.

Conclusion
Based on the research results, it can be concluded that the administration of irradiated chitosan accompanied by glutathione induction in broiler rations can affect lipid regulation and biochemical aspects and performance as a result of this regulation.Giving chitosan as much as 450 ppm with 150 ppm induced glutathione since the early age of the finisher phase (20 days) was able to regulate lipid biosynthesis optimally with the best final body weight.

Table 1 .
Nutrient composition and metabolic energy (ME) of the research basal ration

Table 2 .
The response concentration regulators and broiler lipid transport Means in the same row with a different letter of superscripts are significantly different (p<0.05);PPAR©= Peroxisome proliferator-activated receptor gamma; APO = Apo-lipoprotein; HDL = high density lipoprotein; LDL= low density lipoprotein

Table 3 .
The response of broiler blood plasma lipid levels