The villi ileum growth of native quail fed by irradiated chitosan with glutathione from early age in high temperature

A total of 105 2-day-old quails (DOQ), reared at high temperature (390C) until 35 days of age, to investigate the effect of chitosan irradiated with early glutathione induction on ileal villus growth in quails. The research used a Completely Randomized Design (CRD) with experimental method, consist of 5 treatments with 3 replications. Each replication consist of 7 DOQs. Treatment was given from day 4 to 35, namely P0 = basal diet without irradiated chitosan and glutathione, P1 = basal diet with 100 ppm irradiated chitosan, P2 = basal diet with 450 ppm irradiated chitosan, P3 = basal diet with 100 ppm irradiated chitosan + 150 ppm glutathione, P4 = basal diet with 450 ppm irradiated chitosan + 150 ppm glutathione. Sample was collected by choosing randomly one quail in each experimental unit so there are 15 samples. Parameters observed in this research were ileal morphometrics (ileal length, number, height, and width of ileal villi) and ileal villi cell profiles (number of goblet cells, normal cells, apoptotic cells, and necrotic cells). The results showed that irradiated chitosan with glutathione induction had a significant effect (P<0,05) on morphometric and cell profile of ileal villi, except ileal length. The growth of villi ileum in quail optimal through 100 ppm irradiated chitosan with 150 ppm glutathione.


Introduction
Quail is a type of a bird that has very fast growth in a relatively short time.Factors that greatly influence the early growth of quail include micromolecular lipids, fatty acids, and sterols.High ambient temperature is one of the environmental factors that inhibit growth rate.Excessive lipid anabolism in quail has a negative impact on performance, which causes an increase in fat deposits, reducing the proportion of carcass to body weight [1,2].Efforts to overcome this as well as to spur quail growth, namely by adding feed additives to the ration.
One of the feed additives that has been developed is chitosan.Chitosan is composed of β-1-4-linked D-glucosamine and N-acetyl-D glucosamine, produced through the deacetylation process of chitin compounds which is the main component in crustacean shells [3].However, the administration of chitosan as a whole has a high molecular weight, making it difficult to be optimally absorbed by the digestive tract.Reducing molecular weight by applying radiation techniques can break the glycosidic chains between glucosamines contained in chitosan.The ability of chitosan to affect protein regulation, especially genes related to lipid synthesis [4][5][6] has an impact on livestock performance [5,7].Reduced performance with early administration of irradiated chitosan is due to low lipid availability and increased tissue inflammation as a result of chitosan's inhibition of lipogenesis.Inhibition of lipogenesis causes adverse effects on tissue growth, especially intestinal tissues that are involved in nutrient absorption, namely the ileum.Cell growth in the ileum requires lipids as precursors [8].In addition, inhibition of lipogenesis leads to increased free radical generation activity through nitrogen oxide-5 (Nox-5) and β-oxidation.These factors are detrimental to tissue formation as they stimulate protein translational failure and cell death.Overall, the administration of chitosan to avoid fatty liver in the abdominal cavity of quail has an impact on the morphometrics of the small intestine, especially the ileum [9].
Efforts to prevent these inhibitory effects can be made by adding antioxidants in quail rations, one of which is glutathione [10,11].Glutathione (γ-Glutamyl-L-cysteinyl-glycine) is a tripeptide consisting of the amino acids glycine, glutamate, and cysteine [11,12].Through the induction of glutathione, it is expected to prevent a decrease in lipogenesis and the formation of free radicals due to irradiated chitosan.Glutathione can prevent cell damage or inflammation and stimulate growth through cell repair [9].Thus, the role of cholesterol as a growth precursor continues so that the growth of ileal villi cells in the quail digestive tract remains optimal [10,12].Based on the above description, a study was conducted on the administration of irradiated chitosan with early glutathione induction to the growth of quail ileal villi cells.

Animal
The experimental animals used for this study were 105 quails that reared from DOQ until 35 days of age.The research was conducted from September to October 2022 in the Experimental Cage of the Poultry Production Laboratory, Faculty of Animal Husbandry, Padjadjaran University.On day 1 to day 3, quail were feeding the research basal ration for habituation.Irradiated chitosan with glutathione induction was started on day 4 until day 35.Feed was limited provided according to standard needs and drinking water ad-libitum.Quail were kept in semi close house type cages and randomly selected into each experimental unit.

Experimental design
This study was conducted using the completely randomized design (CRD) method.It consisted of 5 treatments, namely without and with irradiated chitosan and glutathione given from 4-35 days old.Each treatment consisted of 3 replicates, resulting in 15 flocks of experimental units.Each experimental unit consisted of 7 quails.The five treatments were P0: Basal ration without irradiated chitosan, without glutathione, and without heat stress; P1: Basal ration containing 100 ppm irradiated chitosan, with temperature 39 0 C; P2: Basal ration containing 450 ppm irradiated chitosan, with temperature 39 0 C; P3: Basal ration containing 100 ppm irradiated chitosan + 150 ppm glutathione, with a temperature of 39 0 C; and P4: Basal ration containing 450 ppm irradiated chitosan + 150 ppm glutathione, with a temperature of 39 0 C.

Experiment protocols 2.3.1. Irradiated chitosan and glutathione formulation
The chitosan solution to be administered in the study was prepared by mixing the parent chitosan into 125 mL of water, 0.25 mL and 1.125 mL respectively to obtain chitosan concentrations of 100 ppm and 450 ppm according to the treatment.Glutathione used in the study as much as 150 ppm was obtained by mixing 0.375 mL of parent glutathione solution into the chitosan solution that had been made.The chitosan solution that had been mixed with glutathione was sprayed little by little into each 1 kg of basal ration while stirring until smooth.

Sample collected
Samples were collected at the end of rearing, at 35 days of age, by randomly selecting 1 quail in each experimental unit, resulting in 15 samples.Ration feeding was stopped for 10 hours before cutting the quail samples.Quail were slaughtered and the ileum was immediately separated and washed with physiological NaCl solution.The ileum was cut into 3 cm lengths, and immediately inserted into a sample tube containing fixative solution.Furthermore, the ileum was fixed using a fixative solution for two days which aims to maintain the entire cell structure.

Sample analysis
Sample analysis was performed using procedures based on the Mallory-Azan staining protocol.Mallory-Azan is one of the methods of making Mallory antichrome preserved preparations.Determination of ileum morphometrics and the condition of villi cells in ileum preparations were performed using a microscope and its monitor.

Statistical procedures
The data were calculated with MS Exel and one way analysis of variance (ANOVA) technique using the General Linear Models (GLM) procedure of SPSS IBM 21 Version 5.83 for a Completed Romdomly Design [6].

Results and discussion
The effect of giving various doses of irradiated chitosan with glutathione induction in feed on quail ileum morphometrics including ileum length and the number, height, and width of ileum villi in the current results can be seen in Table 1.Based on Table 1, the ileal length of quail at P0 (42.22 cm) was not significantly different from the ileal length at P1 to P4 treated with irradiated chitosan and glutathione, which were 44.79 cm; 38.84 cm; 44.28 cm; and 42.83 cm, respectively.The results of the analysis of variance showed that the administration of irradiated chitosan and glutathione had no significant effect (P>0.05) on the length of the quail ileum.Thus, Duncan's multiple range test was not conducted.However, Table 1 and Illustration 1 show the growth of ileal villi in general.The shortest ileum was P2 (38.84 cm) treated with 450 ppm irradiated chitosan, while the longest was P1 (44.79 cm) treated with 100 ppm irradiated chitosan.The results showed a change due to the administration of chitosan with different doses.This supports the statement of [13][14][15] that the administration of chitosan with lower levels shows good anti-inflammatory.[16] reported the effect of giving 150 ppm irradiated chitosan in the diet on the morphometric improvement of the ileum of cihateup ducks.The optimal length of quail ileum remained in P3 which was given 100 ppm irradiated chitosan + 150 ppm glutathione.
The results of the analysis of variance showed that the administration of irradiated chitosan and glutathione had a significant effect (P<0.05) on the number, height, and width of quail ileal villi.Furthermore, to determine the average difference in the number of villi between treatments, Duncan's multiple range test was conducted.The number of villi in one field of view with 4x object magnification at P0 (27.33) was significantly (P<0.05)higher than P1 to P4 treated with irradiated chitosan and glutathione, which were 23.66; 14.26; 25.73; and 21.82, respectively.The lowest number of villi was P2 (14.26) treated with 450 ppm irradiated chitosan.Similar results were also obtained in the parameters of villi height and width, which showed that the higher dose of chitosan without glutathione induction resulted in lower villi morphometrics.The number, height, and width of villi remained optimal in P3 treated with 100 ppm irradiated chitosan + 150 ppm glutathione.
Treatment with irradiated chitosan tends to give lower results than the control.[17,18] states that the performance of small intestinal villi is influenced by several factors including the type of feed substance and feed chemicals.Chitosan supplementation decreased lipase activity and fat absorption in the small intestine, as well as reduced lipid absorption in the digestive tract due to binding of bile acids [19,20].Inconsistent results on intestinal morphology may be due to differences in species, age, and dose of chitosan administration.Quail growth performance results from several studies may differ due to various characteristics of chitosan, such as concentration, degree of acetylation, and molecular weight [21][22][23].
The administration of 450 ppm irradiated chitosan resulted in the lowest average growth of ileal villi (P<0.05)among all treatments.This shows that the intestine is less active so that villi atrophy occurs.The use of chitosan with higher doses can inhibit lipid synthesis, which is a precursor to the growth of the ileum and its villi.The polar and bipolar nature of chitosan (binds water and fat) will increase the viscosity of digesta, so that the feed digesta rate is faster and will reduce nutrient contact with the small intestine during absorption, thereby reducing villi development [24][25][26][27].Another factor is that early administration of chitosan inhibits the expression of genes or proteins that regulate lipid metabolism [27][28][29][30].As a result, villi growth and development are reduced, as well as suppressing the activity of the ileum in carrying out its function as the absorption intestine.
The role of glutathione as an antioxidant is crucial in inducing tissue growth in quail.Glutathione is able to offset the inflammatory effects caused by the inhibition of lipogenesis [10,31] and the increase in free radicals caused by chitosan [32].Glutathione induction in this study means adding concentrations of amino acids, especially glutamine, cysteine and glycine.The three amino acids are related to growth, as reported by [18] that amino acids, especially glutamine, play an important role for growth-related nutrient pathways and regulation of cell proliferation, preventing apoptosis [27], and even stimulating the synthesis of specific proteins related to tissue growth [28].
The effect of giving various doses of irradiated chitosan with glutathione induction in feed on the cell profile of quail ileal villi including the number of goblet cells, normal cells, apoptotic cells, and necrosis cells based on the results of the study can be seen in Table 2.As results of the analysis of variance showed that fed of irradiated chitosan and glutathione had a significant effect (P < 0.05) on the number of goblet cells, normal cells, apoptotic cells, and necrosis cells of quail ileal villi.Furthermore Duncan's multiple range test was conducted to determine the average difference between treatments.Based on Table 2, the number of goblet cells of quail ileal villi in one field of view with 100x object magnification in P0 (15.21 cells) was significantly different (P<0.05)higher than P1 to P4 treated with irradiated chitosan and glutathione, which were 13.31; 12.41; 13.29; and 11.18 cells, respectively.Similarly, the number of normal cells of quail ileal villi observed in 1,000 cells in P0 (535.67 cells) was significantly different (P<0.05)higher than P1 to P4 treated with irradiated chitosan and glutathione, which were 512.33 Based on these results, it can be seen that the administration of 450 ppm irradiated chitosan (P2) gave the lowest average number of goblet cells and normal cells among the treatments.
The number of apoptotic cells of quail ileal villi observed in 1,000 cells in P0 (13.67 cells) was significantly (P<0.05)lower than P1 to P4 treated with irradiated chitosan and glutathione, which were 59.00; 73.00; 46.67; and 51.00 cells, respectively.Then the number of necrosis cells of quail ileal villi in P0 (73.53 cells) was significantly different (P<0.05)lower than P2 (101.72 cells), but not significantly different (P>0.05) with other treatments.Based on these results, it can be seen that the administration of 450 ppm irradiated chitosan (P2) caused the most apoptotic and necrotizing cells among treatments, and decreased the number of goblet cells.
One of the factors that influence goblet cell growth is the availability of cell growth precursors, especially lipids.As explained in the previous discussion, irradiated chitosan is able to inhibit the synthesis of specific proteins, namely proteins that play a role in lipid regulation.Previous research has reported [29] that chitosan is able to inhibit the expression of genes that play a role and regulation of lipid metabolism, namely the genes PPAR and SREBP-1α.Inhibition of this gene causes the activity of proteins that play a role in lipogenesis to decrease so that the availability of fatty acids and endogenous cholesterol as a precursor to cell growth decreases.[30][31][32][33] and [34,34] reported the ability of chitosan to affect protein regulation, where inhibition of protein synthesis rate can trigger apoptotic cell death and decreased cell growth in ileal villi and goblet cells.
The cell profile of ileal villi remained optimal by administering 100 ppm irradiated chitosan with 150 ppm glutathione induction.Glutathione contains three amino acids related to growth as reported by [30,[35][36][37] that amino acids, especially glutamine, play an important role for growth-related nutritional pathways and regulation of cell proliferation [38], preventing apoptosis [37,39], and even stimulating the synthesis of specific proteins in the liver [33,36,40].Based on the ability of glutathione, the growth of goblet cells was better in the quail group that received a combination of glutathione administration compared to chitosan independently, as well as a decrease in the number of cells that experienced apoptosis and necrosis.

Conclusion
As the results of the study, it can be affirmed that the addition of irradiated chitosan with early glutathione induction has an effect on the growth of quail ileal villi.The growth of quail ileal villi remains optimal with the lowest adverse histology impact, namely the administration of 100 ppm irradiated chitosan induced by 150 ppm glutathione.

Table 1 .
Morphometrics of quail ileum without and with early administration of irradiated chitosan and glutathione *One field of view with 4x magnification; a,b,c,d Different letter annotations in the same column indicate significant differences (P<0.05)