Immune response, hematological traits, biochemical blood parameters, and histological status of laying hens influenced by dietary chitosan-oligosaccharides

Immune response, hematological traits, biochemical blood parameters, and histological status of laying hens influenced by dietary chitosan-oligosaccharides

Item Type Article

Authors Youssef, Islam M.;Khalil, Hassan A.;Shakoori, Afnan M.;Bagadood, Rehab M.;Alyahyawi, Areej Y.;Alhazzaa, Rasha A.;Fakiha, Khloud G.;Nasr, Samia;Abo-Samra, Maher A.;Hassan, Magdy S.;Halim, Haiam S.Abd El;El-Hack, Mohamed E. Abd;Jaremko, Mariusz;Al- Nemi, Ruba;Youssef, Khaled M.

Citation Youssef, I. M., Khalil, H. A., Shakoori, A. M., Bagadood, R. M., Alyahyawi, A. Y., Alhazzaa, R. A., Fakiha, K. G., Nasr, S., Abo- Samra, M. A., Hassan, M. S., Halim, H. S. A. E., El-Hack, M. E.

A., Jaremko, M., Al-Nemi, R., & Youssef, K. M. (2023). Immune response, hematological traits, biochemical blood parameters, and histological status of laying hens influenced by dietary chitosan-oligosaccharides. Poultry Science, 102(9), 102834.

https://doi.org/10.1016/j.psj.2023.102834 Eprint version Publisher's Version/PDF

DOI 10.1016/j.psj.2023.102834

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Immune response, hematological traits, biochemical blood parameters, and histological status of laying hens in fl uenced by dietary chitosan-

oligosaccharides

Islam M. Youssef,*Hassan A. Khalil,^yAfnan M. Shakoori ,^zRehab M. Bagadood ,^z Areej Y. Alyahyawi ,^x,kRasha A. Alhazzaa ,^k,{Khloud G. Fakiha ,^#Samia Nasr,**

Maher A. Abo-Samra,^yMagdy S. Hassan,*Haiam S. Abd El Halim,^yMohamed E. Abd El-Hack ,^yy,1 Mariusz Jaremko,^zzRuba Al-Nemi ,^zzand Khaled M. Youssef^xx

*Animal Production Research Institute, Agriculture Research Center, Dokki, Giza 12618, Egypt;^yAnimal Production Department, Faculty of Agriculture, Suez Canal University, Ismailia 41522, Egypt;^zLaboratory Medicine Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah, Saudi Arabia;^xDepartment of

Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia;^kKing Abdullah International Medical Research Center, Riyadh, Saudi Arabia;

{Basic Sciences Department, College of Science and Health Professions, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia;^#University of Jeddah, College of Science, Department of Biology, Jeddah,

Saudi Arabia;^**Chemistry Department, Faculty of Science, King Khalid University, Abha 61413, Saudi Arabia;

yyPoultry Department, Faculty of Agriculture, Zagazig University, Zagazig, 44511, Egypt;^zzSmart-Health Initiative (SHI) and Red Sea Research Center (RSRC), Division of Biological and Environmental Sciences and Engineering (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia; and

xxFood Technology Department, Faculty of Agriculture, Suez Canal University, Ismailia 41522, Egypt

ABSTRACT This experiment aimed to examine the effect of chitosan-oligosaccharides (COS) supplementa- tion in laying hens’diets affected their immune response, hematological characteristics, blood biochemical param- eters, and histological status. At the age of 34 wk, 200 laying hens and 20 cocks of the Mandarah chicken strain were allotted into four groups, each consisting of 50 hens andfive cocks. Thefirst group acted as a control group, fed on a basal diet. The second, third, and fourth experi- mental groups each received 0.1, 0.2, and 0.5 g/kg of COS in addition to a base diet. Birds received COS at various dosages had significantly (P ˂ 0.05) increased serum concentration of immunoglobulins, avian influ- enza, and Newcastle disease antibodies compared with the control birds. Moreover, adding COS at level 0.2 g/kg diet insignificantly enhanced immune response than the rest of the treatment groups. Also, treated birds with COS at different levels had insignificantly

improved hematological parameters such as red blood cells, white blood cells, hemoglobin and hematocrit com- pared to the control group. Birds fed COS at all levels had significantly decreased serum cholesterol, triglycer- ides, Ca⁺⁺and alanine aminotransferase concentrations compared with control birds. In addition, compared to the control group, chitosan-treated birds showed enhanced histological examination of the small intestine, isthmus, and testis, notably in birds given COS at 0.1 g/kg diet compared to other treated birds. Cocks fed COS at all levels improved testicular tissues and increased the number and diameter of seminiferous tubules compared with control birds Morphological examination of the ileum showed increased villi number, height, and crypt depth. It is possible to conclude that laying hens’ physiological performance and general health can be effectively improved by using chitosan at 0.1 or 2 g/kg diet levels enhanced immune response.

Key words:chitosan-oligosaccharide, immune response, physiological trait, laying hen

2023 Poultry Science 102:102834 https://doi.org/10.1016/j.psj.2023.102834

INTRODUCTION

Various feed additives have recently been developed to keep farm animals in good metabolic and physical condition while enhancing their reproductive and pro- ductive abilities. It has been recommended that natural

Ó2023 The Authors. Published by Elsevier Inc. on behalf of Poultry Science Association Inc. This is an open access article under the CC BY- NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Abbreviations:AI, avian influenza; ALT, alanine aminotransferase;

AST, aspartate aminotransferase; COS, chitosan-oligosaccharides;

HDL, high-density lipoprotein; IgG, immunoglobulin G; IgM, immuno- globulin M; LDL, low-density lipoprotein; ND, Newcastle disease;

RBCs, red blood cells; WBCs, white blood cells Received March 30, 2023.

Accepted May 31, 2023.

1Corresponding author:dr.mohamed.e.abdalhaq@gmail.com

1

feed additives such as pro, prebiotics, organic acids, phy- tochemicals, and enzymes are safer antibiotic substitutes (Swiatkiewicz et al., 2015;Abd El-Hack et al., 2020a,b, 2021; Kamal et al., 2023a). According to Alagawany et al. (2017,2021), adding probiotics, enzymes, synbiot- ics (pro+prebiotcs) and organic acids to the diet could help the immune system develop and improve. Chito- san-oligosaccharides (COS) are one of the newer and less popular feed additives.

Chitosan-oligosaccharides are the N-deacetylated chi- tin comprising N acetyl glucosamine and D glucosamine monomers. It is a deactivated form of variable degrees of chitin, prevalent from the exoskeleton components of shrimp, crabs, insects and other marine creatures in the crustacean family and is a good supply of COS (Singla and Chawla, 2001; Abd El-Aziz et al., 2022; Kamal et al., 2022,2023a).

The COS is one of the most widespread polymers and is a naturally positive-charged alkaline polysaccharide (Xu and Wang, 2005). COS has antimicrobial, anti- inflammatory, antioxidant, antitumor, immune-stimu- lating, and hypocholesterolemic properties because it has reactive functional collections such as amino acids and hydroxyl groups (Xia et al., 2011). The COS exhib- its growth-promoting activities based on the previously verified positive effects, and it can be employed as a powerful substitute for antibiotic growth promoters.

Moreover, oligosaccharides have been used as prebiotics to improve health status, minimize pathogen formation in the small intestine (Yaqoob et al., 2021), develop immunological role growth-promoting in broilers (Wang et al., 2003), and increase the productivity of laying hens (Sheoran et al., 2018).

Chitosan-oligosaccharides can stimulate humoral immunity and the complement system (Sarwar et al., 2021). Moreover, some researchers have suggested that COS possesses immune-enhancing properties and can be an animal immunological stimulant (Yoon et al., 2008;

Kong et al., 2014). Studies conducted in vitro and in vivo indicate that dietary COS is a potent and promising immunostimulatory. The immunostimulatory properties of chitosan depend on Toll-like receptor four and are mediated across interactions with membrane receptors on the surface of macrophages (Zhang et al., 2014).

Moreover, COS may reduce intestinal inflammation and oxidative stress in laying hens (Gu et al., 2022).

One of COS’s most notable impacts on blood metabo- lites is decreasing cholesterol levels, especially low-den- sity lipoprotein (LDL). This impact is most notable when animals are fed high-energy, high-fat diets, such as those that contain a lot of seed oil and/or fat comple- ments (Keser et al., 2012). According to several studies, dietary COS supplementation enhanced the reproduc- tive system and alimentary canal’s morphological and histological structure in farm animals. According to Nuengjamnong and Angkanaporn (2018), adding COS to broiler feed at the 2 g/kg diet level resulted in a signif- icantly higher ratio of villus tallness to crypt depth while a lower crypt depth. The present study hypothesized that enriching layer diets with COS may have positive

effects on health and performance. Therefore, this exper- iment was conducted to study the influence of COS administration in the diet on immune response, hemato- logical traits, blood biochemical parameters and histo- logical statues of Mandarah laying chickens.

MATERIALS AND METHODS

All procedures in the current study were approved by the Local Experimental Animal Care Committee and the Institutional Ethics Committee of the Animal Pro- duction Department, Faculty of Agriculture, Suez Canal University, Ismailia, Egypt.

Experimental Design

Individually weighed birds were allocated into four equal experimental groups at random (30 hens and three cocks each). Each group represented one treatment;

each was randomly divided intofive replicate pens. Each pen represented an experimental unit and contained ten hens and one cock. The first group acted as a control group, fed on a basal diet. The second, third, and fourth experimental groups were fed the basal diet plus 0.1, 0.2, and 0.5 g/kg of COS, respectively. Chitosan Egypt Agri- culture Corporation, located in Dokki, Giza, provided the COS.

Birds and Management

At 34 wk, 200 laying hens and 20 cocks of the Egyp- tian chickens’ Mandarah strain (a locally generated Egyptian chicken strain) were selected and, at random, allotted into four groups of three replicates (10 hens and one cock each). The daily egg production and average body weight of the hens in each group were comparable (1,428.56 3.27 g and 46.45 2.54%, respectively). The same administrative and sanitary practices were used to raise the birds, and they were fed a basal laying diet cre- ated in accordance with NRC (1994). Table 1 lists the basal laying ration’s feed components and chemical anal- ysis. The photoperiod was maintained throughout the experiment at 16 h of light and 8 h of darkness daily.

The experiment’s average inside ambient temperature and relative humidity were 23.51°C and 58.81°C, respec- tively. With a density of 5 birds per m², all birds were kept in a semi-closed housing system on thefloor laying pens (2 £ 1.5 m). The experimental duration extends from 34 to 50 wk of age (16 wk of egg production).

Measurements

Immunoglobulin Concentrations, Blood Biochemi- cal, and Hematological Parameters At the comple- tion of the experiment, six hens and three cocks from each experimental group were slaughtered (50 wk of age) by having their jugular veins severed. Each bird had two different tubes filled with blood samples that were taken. Thefirst sample was taken in a non-heparin

tube, and blood serum was centrifuged to separate it at 3,000 rpm for 15 min. The serum was kept at 20°C measuring biochemical characteristics and immunoglob- ulin concentrations. Commercial Eliza kits from Thermo Scientific Companies Serving, Egypt, were applied to measure the concentrations of serum immunoglobulin G (IgG) and immunoglobulin M (IgM). Also, humoral immune responses were investigated by detecting serum antibody levels against Newcastle disease and avian influenza by hemagglutination inhibition test as described byAlexander (2000).

Additionally, commercial kits (Reactivos GPL Chem- elex, S.A. Pol. Ind. Can Castells. C/Industria 113, Nau J 08420 Canovelles-Barcelona), the total protein, total cholesterol, LDL, HDL, triglycerides, and the blood serum’s Alanine Aminotransferase (ALT) and Aspar- tate aminotransferase (AST) liver enzyme activity was measured calorimetrically (Young and Friedman, 2001).

Also, serum Ca⁺⁺ and phosphorus levels were deter- mined by spectrophotometry using commercially avail- able kits from an Egyptian biotechnology company (S.

A.E.). The second sample was obtained in a heparinized tube and kept at +4°C till hematological parameter examination. Using the improved Neuober hemocytome- ter and the Natt and Herrick solution as the diluent, the counts of red blood cells (RBCs) and white blood cells (WBCs) were done (Natt and Herrick, 1952). Hemoglo- bin determination followed Lamberg and Rothstein’s (1977)instructions. During differential leukocyte count, platelets were counted using a compound microscope (Mayengbam et al., 2020). According toGaertner and Pazdro (1969), the microhematocrit technique was used to calculate hematocrit values.

Histological Status After slaughtering, the abdominal cavity of each bird was opened, and histological samples

were taken from the isthmus and small intestine (ileum) from hens and the right testis from cocks. For histologi- cal examination, each bird in the control and treatment groups had samples made of them. The sample of each organ (0.5 cm) was fixed in 10% neutral formalin, seri- ally dehydrated in increasing alcohol concentrations, and then embedded in paraffin wax. Hematoxylin and Eosin stains were used to segment the samples and stain them (Bancroft et al., 1990). Sections were clarified and inspected under a microscope.

Statistical Analyses

The general linear model technique was used to exam- ine the data procedure of SPSS (SPSS, 2019). The Dun- can’s multiple-range test was utilized to estimate mean differences (Duncan, 1955). Using the following model, a one-way ANOVA test was performed:

Yij¼mþTiþeij

Where:

Yij= the observation on the jth individual from the ith treatments.

m= the overall mean.

Ti= thefixed effect of thei^thtreatments (control and COS treatments).

e_ij= the random error associated with the individual ij.

RESULTS

Immune Response and Hematological Parameters

Table 2 displays the effects of dietary COS supple- mentation on serum immunoglobulin antibody concen- trations of IgG and IgM, as well as blood serum antibody titers against avian influenza and Newcastle disease. IgG, IgM, AI, and ND antibodies were signifi- cantly different (P ˂ 0.05) across treatment groups, according to variance analysis. Birds fed COS at all lev- els had significantly increased serum concentrations of IgG, IgM, ND, and AI antibodies compared with these recorded in control birds.

As well, the percent changes in IgG, IgM, AI, and ND antibodies are illustrated in Figure 1. However, com- pared to the control group using COS enhanced the per- cent change of IgG by 40.90, 59.53, and 55.23%, IgM by 29.66, 45.96, and 48.68%, AI by 75.00, 138.89, and 94.44%, and ND by 77.78, 100.00, and 77.78% at levels 0.1, 0.2, and 0.5 g/kg diet, respectively.

Moreover, the lowest percentage changes of IgG and IgM antibodies were detected in the serum of birds fed COS at the level of 0.1. Still, the highest values were esti- mated in the serum of birds fed COS at 0.2 and 0.5 g/

kg/diet, respectively. Also, the highest percent changes of both antibodies to ND and AI were obtained in the serum of birds fed COS at level 0.2, but the lowest values Table 1.Ingredients and the basal diet’s determined chemical

composition.

Ingredients %

Yellow corn (8.5%) 63.14

Soybean meal (44%) 27.10

Limestone (CaCo3) 7.60

Di-Ca⁺⁺phosphate 1.50

Dl-Methionine 99% 0.06

(NaCl) 0.3

Vit + Min. premix* 0.3

Overall 100.00

Calculated analysis**

Crude protein, % 17.33

ME, Kcal/kg 2722.00

Ca⁺⁺, % 3.35

Available phosphorus, % 0.40

Lysine, %. 0.88

Methionine, % 0.34

Methionine plus cystine, % 0.64

Zinc (mg/kg) 22.21

*Vitamins and minerals premix: Given per Kg of diet: Vit. A, 12000 IU;

Vit. E, 10 mg; Vit. D3, 2200 ICU; Vit K3, 2 mg; Vit. B1, 1 mg; Vit. B2 5 mg; B6 1.5 mg; B12 10 mcg; Pantothenic acid 10 mg; Nicotinic acid 30 mg; Folic acid 1 mg, Biotein 1.5 mcg; Choline 250 mg; Copper 10 mg; Sele- nium 0.1 mg; Cobalt 0.1 mg Manganese 60 mg; Iron 30 mg; Zinc 50 mg;

Iodine 1 mg.

**Calculated according toNRC (1994).

IMPACTS OF CHITOSAN-OLIGOSACCHARIDES ON LAYER 3

were recorded in the serum of birds fed COS at 0.1 g/kg/

diet.

Also,Table 2 presents the influences of dietary COS supplements on blood parameters in laying hens. In all hematological variables, variance analysis revealed no

statistically significant differences between treatments.

However, at various levels, hematological indicators such as RBCs, WBCs, hemoglobin, and hematocrit were only slightly higher in treated birds with COS than in the control group.

Table 2.Effects of dietary chitosan-oligosaccharides supplementation on immune response and hematological parameters of Mandarah laying hens (means§SE).

Traits

Treatments (g chitosan/kg diet)

P-Value

Control COS 0.1 COS 0.2 COS 0.5

Immune response

IgG (mg/mL) 82.00^b§3.78 115.66^a§8.87 130.33^a§1.76 127.00^a§4.61 0.001

IgM (mg/mL) 174.66^c§2.18 226.33^b§5.89 254.66^a§8.81 259.66^a§3.52 0.000

AI¹(log2) 3.33^b§0.33 5.66^a§0.33 7.66^a§0.88 6.33^a§0.88 0.030

ND²(log2) 3.00^b§0.00 5.33^a§0.33 6.00^a§0.57 5.33^a§0.66 0.024

Hematological traits

RBCs (10⁶/mm³) 2.56§0.12 2.82§0.34 2.60§0.34 3.26§0.09 0.260

Hemoglobin (g/100 ml) 12.60§0.70 14.80§2.40 13.36§2.38 15.06§2.58 0.827

Hematocrit (%) 41.58§2.31 48.84§7.92 44.11§7.85 49.72§8.52 0.827

WBCs (10³/mm³) 6.57§0.65 7.76§0.10 7.30§0.70 7.43§0.80 0.613

Platelets (10³/mm³) 141.33§10.41 137.33§16.34 140.01§11.54 137.00§8.50 0.992

Abbreviations: COS, chitosan-oligosaccharides; IgG immunoglobulin G; IgM, immunoglobulin M; RBCs, red blood cells; SE, standard error; WBCs, white blood cells.

a, b, c

Means within the same row with different superscripts are significantly different (P≤0. 05).

1AI: avian influenza.

2ND: Newcastle disease.

Figure 1. (A) IgG antibodies concentration, (B) IgM antibodies concentration, (C) antibody titers to AI, and (D) antibody titers to ND of Man- darah laying hens between control and COS treatment groups. Abbreviations: AI, avian influenza; COS, chitosan-oligosaccharides; ND, Newcastle disease.

Blood Biochemical Parameters

The results presented inTable 3illustrate the influen- ces of dietary COS supplementation on biochemical blood variables such as total protein, albumin, globulin, cholesterol, triglycerides, HDL, LDL, glucose, ALT, AST, Ca⁺⁺, and phosphorus concentrations of the Man- darah chicken strain. Variance analysis revealed signifi- cant variations between groups for serum cholesterol, triglycerides, AST, ALT, and Ca⁺⁺levels. At the same time, no statistically significant differences between treatments were identified for the remaining parame- ters.

The results revealed that adding COS at all levels had significantly reduced serum cholesterol, triglycerides, Ca⁺⁺and ALT levels compared with control birds. The birds receiving COS at 0.1g/kg diet had the lowest value, while those receiving COS at 0.5 g/kg diet had the highest blood AST concentration. Moreover, com- pared to control birds, birds given COS at various doses showed insignificantly increased serum concentrations of glucose, phosphorus, albumin, globulin and total pro- tein.

Figure 2 also shows the percentage changes in blood biochemical parameters affected by the treatments.

When compared to the control group, using COS decreased serum cholesterol by 23.88, 27.70, and 29.52%, triglycerides by 16.33, 18.05, and 27.20%, ALT by 17.73, 28.16, and 7.72%, AST by 12.89, 10.68, and 13.59%, and Ca++ by 18.51, 13.31, and 24.78%, respectively.

Histological Status

Testicular Development The histological evolution of testicular tissue and seminiferous tubules after 16 wk from treatments are illustrated inFigures 3 and 4. Cocks fed COS at all levels improved testicular tissues and increased the number and diameter of seminiferous tubules compared with control birds. Also, compared to the control group, all the seminiferous tubules in all COS groups displayed full spermatogenesis. Also, when

compared to other treatment groups, cocks that received COS at a dosage of 0.1 g/kg diet showed better testicu- lar development.

Small Intestine (Ileum) Histological evolutions of the ileum as affected by treatments are demonstrated in Figure 5. When comparing treated birds with COS to the control group. Morphological examination of the ileum showed increased villi number, height, and crypt depth. Moreover, higher villi numbers, heights and crypt depth were seen in birds fed COS at diet levels of 0.1 and 0.2 g/kg than in birds received COS at level 0.5 g/kg diet. Also, the intestine of birds fed COS at levels 0.1 and 0.2 showed less disturbed architecture, with mild flattening of villi and preserved crypts. Also, there was moderate chronic inflammation in lamina propria and intestinal glands were uniform compared with the con- trol group.

Oviduct (Isthmus) Histological evolutions of the ovi- duct (isthmus) as affected by treatments are illustrated in Figure 6. Morphological observation of the isthmus appeared an increase in the number of glands and the height and width of the folds in COS-treated birds com- pared to the control group. Moreover, birds with COS at level 0.1 g/kg diet increased in glands number, height and width, and regular luminal columnar epithelial cells and uniform glands compared with other treated groups.

DISCUSSION

Results of the present study confirmed that using COS at different levels enhanced the immune response of Mandarah laying hens, as shown by rising serum IgG, IgM, and AI and ND antibodies compared with the con- trol group. These findings are consistent with previous investigators (Li et al., 2015;Miao et al., 2020;Xu et al., 2020).

In this respect, Huang et al. (2007) and Deng et al.

(2008) reported that adding COS to the diet improved chicken serum IgA, IgG, and IgM concentrations and developed immune systems organs. Moreover,Jan et al.

Table 3. Effects of dietary chitosan-oligosaccharides supplementation on blood biochemical parameters of Mandarah laying hens at 50 wk of age (means§SE).

Traits

Treatments (g chitosan/kg diet)

P-Value

Control COS 0.1 COS 0.2 COS 0.5

Total protein (g/dL) 4.65§0.72 4.60§0.52 5.00§0.86 4.99§0.43 0.953

Albumin g/dL 1.36§0.18 1.47§0.16 1.44§0.13 1.40§0.16 0.968

Globulin (g/dL) 3.29§0.55 3.12§0.40 3.56§0.74 3.59§0.33 0.910

Glucose (mg/dL) 210.33§9.93 232.01§8.08 253.66§21.79 233.66§11.89 0.262

Cholesterol (mg/dL) 170.00^a§16.07 127.00^b§1.73 120.66^b§0.88 116.33^b§8.21 0.010

Triglyceride (mg/dL) 212.33^a§9.02 177.66^b§8.26 172.33^b§13.22 154.00^b§6.55 0.031

HDL (mg/dL) 40.00§8.66 29.00§4.16 25.00§5.13 25.33§1.20 0.255

LDL (mg/dL) 88.66§4.63 70.2§16.19 75.66§4.25 71.33§1.45 0.463

AST (IU/L) 50.66^a§0.88 41.66^ab§3.71 36.33^b§2.90 54.33^a§6.06 0.012

ALT (IU/L) 97.00^a§2.08 84.33^b§2.02 86.66^b§3.17 83.66^b§1.76 0.038

Ca⁺⁺(mg/dL) 10.70^a§0.27 8.69^b§0.58 9.27^b§0.21 8.02^b§0.83 0.035

P (mg/dL) 6.75§0.30 7.03§0.25 7.00§0.13 7.13§0.37 0.807

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; COS, chitosan-oligosaccharides ; HDL, high-density lipoprotein;

LDL, low-density lipoprotein; SE, standard error.

a, bMeans within the same row with different superscripts are significantly different (P≤0. 05).

IMPACTS OF CHITOSAN-OLIGOSACCHARIDES ON LAYER 5

(2012)demonstrated that dietary COS raised antibodies against ND and serum IgG antibody concentrations in broilers. According toXu et al. (2020), COS supplemen- tation raised the concentration of IgG and IgM antibod- ies in laying hens. The presence of the amine group during COS production may be to blame for the improvement in immunological response since it stimu- lates the immune system to make antibodies (Li et al.,

2015). On the other hand, COS enhanced the immune status in the developing Huoyan geese by increasing the weightiness of immunological organs and blood levels of immunoglobulins, hormones, complements, and cyto- kines (Miao et al., 2020).

Moreover, immunohistological investigations have demonstrated that COS can open the tight junctions that connect cells by influencing F-actin filaments.

Figure 2. (A) Serum cholesterol, (B) triglycerides, (C) ALT levels, (D) AST levels, and (E) Ca⁺⁺levels of Mandarah laying hens between con- trol and COS treatment groups. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; COS, chitosan-oligosaccharides.

Consequently, the contact of the antigen with the sube- pithelial lymph nodes of the nasal cavity and better immune responses are most likely made possible by com- bining COS bioadhesion and paracellular transport effects. Combining COS with a strong adjuvant may help improve vaccine effectiveness further because it can potentially lower both the antigen and adjuvant doses, minimizing any irritation or systemic toxicity (Illum et al., 2001).

According to the results, adding COS to birds’ diets had no significant influence on any hematological parameters. These results indicated that COS treat- ments had no detrimental or adverse impact on the blood hematological of Mandarah laying hens. These findings are consistent with those that Doriane et al.

(2017) stated that COS treatments and the control group had no statistically significant influence on the

hematological parameters in the broiler. However, our results recorded that treated birds with COS at different levels had insignificantly improved hematological parameters such as RBCs, WBCs, hemoglobin and hematocrit compared to the control group. In contrast, Meng et al. (2010)reported that laying hens nourished COS at a level of 0.4% had an increased number of WBCs than the control group. Moreover, the broiler given COS at a dose of 1 and 2 g/kg diet increased WBC number compared to the control group, according to Nuengjamnong and Angkanaporn (2018).

These findings showed that, in comparison with the control group, using COS had a hypocholesterolemic impact on laying hens. Thesefindings concur with those published byOsman et al. (2010)andTufan and Arslan (2020), who indicated that COS lowered total choles- terol concentrations in diets for broilers. Moreover, Xu

Figure 3. Testes’histological development 16 wk after therapy (at 50 wk of age).

IMPACTS OF CHITOSAN-OLIGOSACCHARIDES ON LAYER 7

et al. (2020)reported that chitooligosaccharide adminis- tration might enhance the immune system, lower serum lipids, and boost antioxidant activity in chickens through the late-laying season. The causes of hypolipide- mia might be connected to higher intestinal viscosity and a coincidental reduction in lipid assimilation (Raz- dan and Pettersson, 1994; Razdan et al., 1997). More- over, results showed reduced lipase action in the small intestine (Kobayashi et al., 2002) and rising fecal choles- terol due to bile acids associated with COS (Razdan and Pettersson, 1996;Zheng and Zhu, 2003).

Furthermore,Chang et al. (2020) demonstrated that COS reduced broilers’AST and ALT serum levels. Simi- larly,Qiao et al. (2011)found that COS reduced blood AST and ALT levels, defending against the liver’s nega- tive effects of lipopolysaccharide. Moreover,Kong et al.

(2018) found that COS prevented the D-galactose

caused by the upregulation of the serum AST and ALT levels.

Also, the results showed that COS at all levels decreased serum Ca++ concentrations in laying hens significantly compared to the control group. This is probably because Ca⁺⁺ is withdrawn from the blood into the uterus gland to form eggshells, especially in birds with high egg production rates. Because Ca⁺⁺

metabolism has an important impact on the formation of the eggshell, and the amine groups on COS serve as a chelation site for metal ions (Gotoh et al., 2004).

On the other hand, results recorded that birds fed COS at all levels had improved oviduct, testis and intes- tinal morphology and maintained tissue integrity com- pared to the control group. Our findings concur with those of Nuengjamnong and Angkanaporn (2018), who observed that including COS in broiler diets

Figure 4. Testes’histological development 16 wk after therapy (at 50 wk of age).

significantly enhanced the ratio of villus height to crypt depth while decreasing crypt depth. Moreover, Tufan et al. (2015)reported that Japanese quail-fed COS had considerably longer villus and crypt lengths than the control group. Moreover,Kamal et al. (2023b)obtained that growing rabbits fed COS had more villi and deeper crypts than control animals. Also, data showed that compared to control and other treatment groups, cocks given COS at a dose of 0.2 g/kg diet had improved tes- ticular structure and development, including an increase in the number and diameter of seminiferous tubules.

These results closely match those ofZalok et al. (2019) and Nuengjamnong and Angkanaporn (2018), who

observed that COS enhanced reproductive ability and increased gonadal development improved testicular tis- sue in experimental animals. Also, growing rabbits with COS had enhanced testicular shape and function (Kamal et al., 2023a).

This enhancement in the histological structure of the small intestine, testis, and oviduct may be attributable to COS’s function as an antioxidant that protects the tissue’s integrity from the damaging effects of free radi- cals. Since dietary COS supplementation improved glu- tathione peroxidase activity in the duodenum and jejunum, chitosan supplementation can probably decrease heat stress-induced oxidative damage in the

Figure 5. Histological evolution of small intestine (ileum) after 16 wk from treatments (at 50 wk of age).

IMPACTS OF CHITOSAN-OLIGOSACCHARIDES ON LAYER 9

gut (Li et al., 2019;Lan et al., 2020). According to chito- san’s antioxidant capabilities, supplementation improved the intestine muchitosana’s antioxidant capacity in heat-stressed broilers (Naveed et al., 2019).

According to Osho and Adeola (2020), dietary COS improved intestinal health in broiler hens by lessening the detrimental consequences of stress. Furthermore, Japanese quails’ antioxidative state was improved by adding COS and nano chitosan, which also increased catalase activity, a characteristic enzymatic antioxidant in poultry (Hassan et al., 2021). Moreover, COS can help improve nutrition transport (Chen et al., 2022), maintain the intestinal barrier afterward, and minimize cellular apoptosis (Na et al., 2022), converting nutrients into hormones used in steroidogenesis. Enhancing sexual hormones undoubtedly improved spermatogenesis, ulti- mately, sperm quality and testicular growth (Swelum et al., 2021).

CONCLUSION

Thefindings of this research clearly showed that the addition of COS to the diets of Mandara chickens had positive and significant effects on the immune response and physiological performance compared to control birds. These findings from the present study suggested that COS at levels of 0.1 or 0.2 g/kg diet may be a good substitute for antibiotics as a growth promoter to enhance the performance of laying hens.

ACKNOWLEDGMENTS

The authors extend their appreciation to the Ministry of Education in KSA for funding this research work though the project number KKU-IFP2-P-1.

Figure 6. Histological evolution of isthmus after 16 wk from treatments (at 50 wk of age).

Author Contributions: All authors equally contrib- uted to this work. All authors reviewed and approved thefinal version of the manuscript.

DISCLOSURES

The authors declare that they have no known compet- ing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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