Research Article

Protective Response of Zinc Oxide Nanoparticle with Chitosan Oligosaccharide on Intestinal Integrity, Goblet Cell Count, and Meat Quality of Broiler Chicken under Heat Stress

Syed Abdul Hadi1, Jameel Ahmed Gandahi1*, Muhammad Ghiasuddin Shah1, Saima Masood2, Noor Samad Gandahi1

1Department of Anatomy and Histology, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam, Pakistan; 2Department of Anatomy and Histology, University of Veterinary and Animal Sciences Lahore, Pakistan.

Received | April 16, 2024; Accepted | August 05, 2024; Published | December 05, 2024

*Correspondence | Jameel Ahmed Gandahi, Department of Anatomy and Histology, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam, Pakistan; Email: [email protected]

Citation | Hadi SA, Gandahi JA, Shah MG, Masood S, Gandahi NS (2024). Protective response of zinc oxide nanoparticle with chitosan oligosaccharide on intestinal integrity, goblet cell count, and meat quality of broiler chicken under heat stress. J. Anim. Health Prod. 12(4): 621-629.

DOI | http://dx.doi.org/10.17582/journal.jahp/2024/12.4.621.629

ISSN (Online) | 2308-2801

 

 

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction 

In recent years, the poultry industry has observed outstanding growth to meet the soaring global demand for high-quality protein sources. However, the intensification of poultry production has brought forth new challenges, particularly considering escalating environmental temperatures attributed to shifts in the global climate (Mendes et al., 2022). This surge in temperatures has led to an increased incidence of cyclic heat stress, presenting a significant threat to the well-being and performance of broiler chickens (Chang et al., 2022). Adding zinc to the diet of broilers under heat stress improved their production performance, show varying degrees of antibacterial effectiveness, and possess good thermal stability. They also exhibit enhanced hydrophobicity, reduced solubility, and, in certain instances, better barrier properties compared to current commercial packaging. This highlights the potential importance of zinc supplementation in mitigating the adverse effects of high temperatures on broiler production (Mahmood et al., 2023). Over the last six decades, antibiotics have been extensively employed in feeding to promote gut well-being. Nevertheless, with the prohibition of antibiotic addition in feed and the growing consumer demand for antibiotic-free production, there is a progressive reduction in the inclusion of antibiotics in poultry feed (Abuajamieh et al., 2020). The alteration in gut microbiota composition may lead to diverse implications for intestinal well-being, potentially resulting in disruptions to the intestinal barrier and elevated stress levels (Fatima et al., 2023). Consequently, there is a heightened emphasis on the management of gut health in poultry. However, a singular, highly effective alternative has remained elusive thus far (Salem et al., 2022). In response to this concern, the integration of nanotechnology into animal nutrition has emerged as a promising area of investigation.

Zinc oxide nanoparticles (ZnO-NP), among various nanoparticles, have received significant attention due to their multifaceted benefits for health (Dumlu, 2024). These properties encompass an augmented antimicrobial efficacy, potentially aiding in the prevention of microbial challenges, as well as immunomodulatory effects (Ramiah et al., 2020). Given its pivotal role in nutrient absorption and immune defense, ensuring the health and functionality of the intestinal tract becomes paramount under such circumstances (Dosoky et al., 2022). This exploration into the integration of nanotechnology within animal nutrition has revealed promising and potential approach (Abdel-Wareth et al., 2022).

Chitosan, a natural biopolymer derived from chitin, exhibits notable biocompatibility, and holds substantial therapeutic promise (Lochi et al., 2023). Chitosan is a non-toxic prebiotic that originated from chitin. The functional chitosan oligosaccharides (COS) are a natural alkaline polymer of glucosamine obtained by chemical and enzymatic hydrolysis of chitosan (Khambualai et al., 2009; Shi et al., 2005). In the face of these challenges, preserving the health and functionality of the intestinal tract emerges as a critical focal point. The gastrointestinal tract, comprising a complex network of tissues and cells, plays a pivotal role in the digestive process, nutrient absorption, and immune defense mechanisms (Lan et al., 2019). Any disruption in the integrity of this vital organ system can lead to a cascade of detrimental effects on the overall health and productivity of the birds (Ali et al., 2023). The positively charged surface of COS has an affinity for the negatively charged cell membrane surface. This mucoadhesive property of COS serves to enhance the absorption (or diffusion) of metal oxide nanoparticles, such as zinc oxide, by extending their residence time in the gut epithelial environment (Alalaiwe et al., 2019). In various instances, COS has been utilized to regulate the delivery of substances. For example, they were employed for controlled delivery of vitamin C in rainbow trout (Oncorhynchus mykiss) (Alishahi et al., 2011).

Chitosan oligosaccharide exhibits promise as a therapeutic agent, known for its ability to mitigate inflammation and support tissue regeneration. When employed in conjunction with ZnO-NP, this combination may hold the key to a synergistic protective response against the challenges posed by cyclic heat stress (Mohapatra & Limayem, 2020). The rationale underlying this inquiry is grounded in the belief that this combined intervention possesses the capacity to offer a multi-dimensional approach in alleviating the adverse effects of heat stress on avian physiology. A meticulous assessment of these interconnected variables gives insights into the underlying mechanisms that drive this protective response (Abdel-Wareth et al., 2022). Current research is aimed to examine the combined influence of ZnO-NP and COS on the protective response against heat stress in broiler chicken, with a primary emphasis on jejunum histomorphology, goblet cell count, and meat quality. This incorporation might present a multifaceted approach to alleviate the detrimental effects of heat stress. Unraveling the mechanistic foundations of this protective response carries considerable implications for the development of sustainable and healthy strategies in poultry production.

Materials and Methods

Animal welfare statement

This study’s ethical clearance was approval by the Directorate of Advanced Study and Research and Animal Welfare Committee (DAS) under the reference (No. DAS/ 353 / of/ 2021 on Dated 12/02/2021) from Sindh Agriculture University, Tando Jam, Pakistan.

Birds diet, heat stress management

The present study involved Ross day-old broiler chicks (n=336) obtained from a commercial hatchery. Upon their arrival, the birds were weighed and subsequently allocated randomly into seven groups, number of birds per group was 48, with six replicates and each having eight birds per replicate. Negative control (NC) group received basal diet (BD) only; positive control (PC) group received BD + HS; heat-stress-zinc (HZ) group received HS + ZnO (60 mg/kg); heat-stress-zinc-nanoparticles (HZN) group received HS + ZnO-NP (60 mg/kg); heat-stress-chitosan (HC) group received BD + HS + COS (200 mg/kg); heat-stress-zinc-chitosan (HZC) group received HS + ZnO (60 mg/kg) + COS (200 mg/kg); and, heat-stress-zinc-nanoparticles-chitosan (HZNC) group received HS + ZnO-NP (60 mg/kg) + COS (200 mg/kg). Wood shaving was used as litter for rearing the chicks. Feed and water were provided without restriction for 24 h. The diet formulation followed the recommendations of the National Research Council (Council and Nutrition, 1994).

Vaccination

The vaccination was done against Infectious Bursal Disease Virus and Newcastle Disease Virus. On day 4 live attenuated Vaccine was administered and was repeated on day 20th day in drinking water. Birds were vaccinated intraocular for IBDV on 8th day and booster dose was given on 24 in drinking water.

Heat challenge

On the day first the chicks arrived; the ambient temperature was maintained at 35 °C with a relative humidity (RH) of around 65%. However, a weekly reduction of 2.8 °C in temperature was implemented to reach a thermoneutral zone of 26 °C with a similar RH of 65% up to 21st day. After this period, one group was kept at the established thermoneutral zone as a control group. Other groups were subjected to a cyclic temperature of 35°C for 8 hours daily, while the temperature for the remaining hours was kept the same as the control group. The birds were vaccinated against Newcastle disease virus and infectious bronchitis disease at specific intervals.

Preparation of zinc oxide nanoparticles

The process of preparing nano-zinc oxide particles was conducted by the National Center of Excellence in Analytical Chemistry at the University of Sindh Jamshoro following the published protocol (Ramiah et al., 2019).

Sampling and processing

At the end of 42-day trial period, two birds were randomly collected from each replicate, resulting in a total of 16 birds per group. The collected birds were sacrificed through cervical dislocation, and specimens were collected.

Muscle tissue processing

Specimens of Pectoralis major were prepared for light microscopy following formalin-fixed paraffin-embedding technique. For muscle fascicle diameter (MFD), H&E images of Pectoralis major were captured at 4X with the help of Progress capture Pro 2.7.7 (Labomed, USA) software.

Measurement of pH

Muscle pH of Pectoralis major samples was measured with digital portable meat pH meter HI 99163 (Hanna, Italy) according to the method described by (Bian et al., 2024).

Measurement of Water Holding Capacity (WHC)

Honikel’s gravimetric drip loss method was used as described by (Kaić et al., 2020). The formula used for WHC calculation.

WHC = (W1-W2/W1) *100

Histomorphometry of jejunum

For histomorphometry analysis, slides were observed under a microscope, and a specific software program (Prog Res®2.1.1 Capture Prog Camera Control Software) was used to measure various parameters in the small intestine following the procedure of (Ali et al., 2017).

Goblet cell count

For counting the goblet cells, the slides were stained by combined Alcian blue-PAS stain. Sections were observed at 10X under bright field microscope (Labomed, USA), according to a published procedure (Bancroft, 2013).

Weight of visceral and immune organs

The weight of various organs such as heart, liver, spleen, and kidney were collected and weighted through digital weight balance.

Statistical analysis

Statistical analysis was performed using one-way ANOVA with SPSS software, and the data were presented as mean ± standard error. Duncan’s multiple range tests were used to identify significant differences among treatment groups (P≤0.05).

Results

Histomorphometry of jejunum

The results of current study show that dietary supplementation of zinc oxide or nano-zinc, alone or in combination with COS affecting to the small intestine, led to significant improvements in various parameters of jejunum, like villus height (VH), villus width (VW), villus surface area (VSA), and crypt depth (CD) were significantly higher (P≤0.05) in all supplemented groups compared to PC. Similarly, the effect of zinc oxide and COS significantly enhanced (P≤0.05) the thickness of lamina propria (LP), muscularis mucosa thickness (MM) and muscularis externa (ME) in all supplemented group compared to PC. The villus-height: crypt-depth ration (VH:CD) was higher (P≤0.05) in PC compared to supplemented groups. Among supplemented groups the best results were found in HZNC compared to PC (Table 1) and (Figure 1).

 

 

Table 1: Single and combine effect of zinc oxide nanoparticle and chitosan supplementation on jejunum histo-morphometry of heat-stressed broiler chicken (Mean ± SEM)

Group	NC	PC	HZ	HZN	HC	HZC	HZNC	P-Valve
VH (μm)	980±3.4b	820±3.6d	930±6.4c	1010±8.68b	920±5.3c	1160±4.46a	1195±7.5a	0.001
VW (μm)	170.7±3.6b	125.3±4.6d	140.4±5.4cd	160.7±2.3c	155.6±4.3c	175.77±4.3b	185.7±2.1a	0.002
VSA (mm²)	0.13±0.03cd	0.10±.02e	0.12±0.04cd	0.14±0.03c	0.11±0.01e	0.15±0.03b	0.17±0.04a	0.001
CD (μm)	230.08±1.6b	105.8±1.3d	180.2±5.4c	205.4±2.3c	195.8±1.4c	240.6±5.3b	270.4±4.1a	0.021
LPT (μm)	155.9±1.6b	105.2±1.3d	130.4±5.4c	146.3±2.3c	140.6±1.3c	160.51±4.3b	190.2±1.1a	0.005
MM (μm)	30.51±4.6c	22.40±1.7e	25.41±1.8d	29.6±2.9c	26.6±3.1d	32.8±1.6b	36.9±.90a	0.003
ME (μm)	260±8.5b	166.3±5.36d	260.2±5.4bc	270.4±6.4bc	250.7±4.3bc	280.3±5.1b	310.5±.4.1a	0.018
VH:CD	4.26±.12c	7.80±.07a	5.16±.27b	4.92±.26c	4.71±.08c	4.83±.19cc	4.42.25±.0c	0.001

a–e Means within the same row displaying distinct superscripts exhibit significant differences (P<0.05). Values represent the Mean ± SEM of five replicates.

VH: villus height, VW: villus width, VSA: villus surface area, CD: crypt depth, LPT: lamina propria thickness, MM: Muscularis mucosa, ME: Muscularis externa. VH:CD: ratio of villus height to crypt depth. G: Groups.

Negative control (NC) group received basal diet (BD) only; positive control (PC) group received BD + heat stress (HS); heat-stress-zinc (HZ) group received HS + ZnO (60 mg/kg); heat-stress-zinc-nanoparticles (HZN) group received HS + ZnO-NP (60 mg/kg); heat-stress-chitosan (HC) group received HS + COS (200 mg/kg); heat-stress-zinc-chitosan (HZC) group received HS + ZnO (60 mg/kg) + COS (200 mg/kg); and heat-stress-zinc-nanoparticles-chitosan (HZNC) group received HS + ZnO-NP (60 mg/kg) + COS (200 mg/kg).

 

Table 2: Single and combine effect of zinc oxide nanoparticle and chitosan supplementation on goblet cells and cecal tonsils histochemistry of heat-stressed broiler chicken (Mean ± SEM)

Group	NC	PC	HZ	HZN	HC	HZC	HZNC	P-Valve
AGC (Nos.)	78.70±3.04b	61.70±3.04d	75.40±3.04bc	83.80±3.04b	73.20±3.04bc	89.20±3.04a	92.90±2.08a	0.001
MGC (Nos.)	63.80±1.3c	52.40±1.3d	65.30±2.9c	72.30±1.6b	64.40±2.6c	75.50±2.3b	81.20±1.42a	0.003
TGC (Nos.)	142.5±3.04c	114.1±5.04d	140.7±4.84c	156.1±4.4b	137.60±5.20c	164.7±4.8b	174.1±3.6a	0.001
LLN (mm)	0.15±0.014c	0.12±0.023d	0.14.60±0.012bc	0.15±0.017c	0.14±0.01bc	0.17±0.015b	0.19±0.019a	0.004
WLN (mm)	0.09±0.012b	0.06±0.015d	0.08±0.018c	0.10±0.013b	0.09±0.018b	0.11±0.014b	0.13±0.011a	0.000
ALN (mm2)	0.09±0.02d	0.07±0.05e	0.015±0.05d	0.019±0.08c	0.016±0.07c	0.023±0.04b	0.028±0.09a	0.001
LNN (Nos.)	6.5±1.4c	4.3±1.2d	5.5±1.6cd	6.1±1.3c	5.4±1.6c	7.4±1.9b	8.6±1.2a	0.001

a–d Different superscripts in the same row indicate significant differences (P<0.05). Values presented are the Mean ± SEM of six replicates. Acidic Goblet Cell (AGC), Mixed Goblet Cell (MGC), Total Goblet Cell (TGC), Length of Lymphatic Nodule (LLN), Width of Lymphatic Nodule (WLN), Area of Lymphatic Nodule (ALN), lymphatic nodule number (LNN). For grouping see footnote of Table 1.

 

Table 3: Single and combine effect of zinc oxide nanoparticle and chitosan supplementation on visceral organ, and meat quality of heat-stressed broiler chicken (Mean ± SEM)

Group	NC	PC	HZ	HZN	HC	HZC	HZNC	P-Valve
pHi	6.50±.0b	6.25±.03c	6.45±.04b	6.57±.05b	6.42±.02b	6.56±.05b	6.66±.04a	0.001
pHu	6.12±.03a	5.43±.04b	5.73±.03b	5.85±.03b	5.71±.03b	5.82±.03b	6.15±.07a	0.005
WHC, % MFD, mm	4.40±.31b 0.99±0.34b	4.95±.62a 0.456±0.21d	4.25±.33b 0.652±0.32c	3.11±.39c 0.798±0.21c	3.56±43c 0.766±0.41c	3.52±.60c 0.989±.96b	2.97±.36d 1.05±.56a	0.000 0.000
Heart, %	0.70	0.68	0.68	0.69	0.68	0.66	0.69	0.23
Kidney, %	0.54	0.52	0.53	0.53	0.54	0.55	0.55	0.41
Liver, %	2.93a	2.21d	2.72c	2.93a	2.68c	2.88ab	2.95 a	0.03
Spleen, %	0.14	0.14	0.14	0.13	0.14	0.13	0.13	0.23

a–d Distinct superscripts within the same row indicate significant differences (P<0.05). The presented values represent the Mean ± SEM of five replicates. pHi: initial pH, pHu: ultimate pH, WHC: Water holding capacity, MFD: Muscle fascicle diameter. For grouping see footnote of Table 1.

Histomorphometry of cecal tonsils and goblet cell counts

The combination of nano-zinc oxide and COS showed particularly favorable outcomes. Goblet cells count of acidic, mixed, and total were significantly enhanced (P≤0.05) in all supplemented groups compared to PC and cecal tonsil morphometry of (LLN), WML, and ALN were significantly improved (P≤0.05) in all supplemented groups compared to PC. Amongst groups the best results were found in HZNC (Table 2) and (Figure 1).

 

Figure 1: Jejunum morphometric measurement of villus height, width, lamina propria, crypt depth, lamina muscularis and tunica muscularis.

A – positive control group, B &C – HZNC group showing improved villus structure (H&E), and goblet cells (Alcian blue-PAS) respectively. For grouping see footnote of Table 1.

Meat quality parameters

In terms of meat quality parameters from Pectoralis major muscle, dietary supplementation with zinc oxide or nano-zinc in combination with COS showed significant (P≤0.05) improvements in muscle pH levels, water holding capacity (WHC), and MFD (Figure 2). These improvements were more pronounced when ZnO-NP and COS were used together. Among supplemented groups the best results were found in HZNC as compared to PC group (Table 3).

 

Figure 2: Morphometric measurement of Pectoralis major muscle for fascicle diameter (MFD) at 4X.

A – positive control group, and B – HZNC group showing increased muscle fascicle diameter. For grouping see footnote of Table 1.

Weights of different visceral organs

The effect of dietary zinc oxide nanoparticle and COS on the weight of visceral organs (heart, kidney, and spleen) did not significantly affected (P>0.05) when compared to PC. While the weight of liver increased significantly (P≤0.05) in all supplemented groups compared to PC (Table 3).

Discussion

More attention has been given to trace minerals as an alternative to antibiotics. Their multiple properties plus high chemical stability and antimicrobial activity, while electrical optical and anti-bacterial properties are closely related to the size and morphology (Dogra et al., 2023). Zinc, an essential trace element for broiler chicken has a critical role in growth, intestinal morphology, meat quality, metabolism, immune organ morphometry and antioxidant enzymatic activity (Ogbuewu & Mbajiorgu, 2023). During heat stress jejunum mucosa plays an important role in absorption, while the height of the villus and its ratio with crypt depth are good indicators for better absorption of minerals (Ghulam et al., 2022). Current study showed that birds supplemented with 60 mg/kg single or combination with COS 200mg/kg improved histological parameter in the jejunum are line with Ali et al. (2017) who investigated the use of zinc oxide nanoparticle 40 to 80mg/kg increased villus surface area and villus height these both are associated with greater absorption on available nutrients, long villi are correlated with gut health, but due to oxidative damage jejunum integrity is affected by heat stress while in the supplemented group increased villus height may be due to bioavailability on ZnO-NP and COS which are correlated with the oxidative effect of Zn and antimicrobial effect of COS during heat stress. In the current study, the extended height of the villi in the intestinal jejunum is associated with improved gastrointestinal well-being. This phenomenon could potentially be attributed to the enhanced bioavailability of ZnO-NP, which contribute to the preservation of the integrity of the epithelial barrier. This effect subsequently leads to a decrease in the cellular turnover rate within the villi, resulting in heightened villus height and crypt depth (Hu et al., 2012). In present study, the increased presence of goblet cells in the jejunum offers an additional explanation for the observed elevation in villus height in the afore mentioned intestinal segment. The abundance of acidic mucin, which is less susceptible to bacterial degradation, may be accountable for this outcome, thereby potentially reducing cellular damage (Ali et al., 2017; Ashraf et al., 2013; Deplancke & Gaskins, 2001). During episodes of heat stress, the crypt depth plays a pivotal role in the ongoing renewal of villi. This is chiefly attributed to the existence of stem cells within it, perpetually dividing throughout the lifespan to facilitate the replenishment of cells in the epithelium of the small intestine villi, this phenomenon might be attributed to the antioxidative characteristics of zinc (Zn) and the antibacterial effects of COS (Ashraf et al., 2013; Hu et al., 2012). The present examination of jejunum structure revealed that the ratio of goblet cells to villus height was greater in cases of ZnO-NP supplementation alone or when combined with COS, in contrast to the positive control groups. This could potentially be linked to the fact that the ratio of villus height to crypt depth exhibits an indirect relationship with heightened epithelial cell turnover, triggered cell division, and the fortification of the protective barrier (Duritis & Mugurevics, 2016).

In the current study, increased villus height and crypt depth indicates enhanced antimicrobial activity in the intestinal lumen due to decreased particle size and increased concentration. These characteristics of ZnO nanoparticles (ZnO-NP) were analyzed with an electron microscope. The particle size primarily ranged from 10 to 40 nm, which is consistent with the sizes used in previous studies with mice (Wang et al., 2016; Wang et al., 2017). Intestinal health is vibrant for the well-being and production of broiler. Large and small surface areas are related to intestinal villi that make birds for minerals strong and poor absorption, respectively (Hu et al., 2012; Hung et al., 2012). Our findings are consistent with those of Hu et al. (2012), who reported that a higher ratio of intestinal crypt depth to villus height indicates increased tissue turnover, reflecting a higher demand for new tissue during stress. In the current study, the ratio of crypt depth to villus height increased in groups supplemented with Zn or nano-zinc, either alone or in combination with COS. This may be attributed to ZnO’s role as an essential component of structural and functional proteins, which are classified as regulatory and structural proteins. Similarly, Ahmadi et al. (2013) examined that supplementation of ZnO-NP single and combine with lysine had significantly increased the jejunum morphology of villus height, width, crypts depth, and the ratio between villus height and crypt depth in the broiler. In the current study increased villus length and ratio between villus height crypt depth in broiler fed with ZnO-NP 60 mg/kg single or combination with COS 200mg/kg may be due to their synergistic effect or may be due to cell multifaction synthesis in small intestine (Neto et al., 2011). Increasing of thickness of lamina propria, muscularis mucosa and muscularis externa had significantly in all supplemented groups as compared to NC and PC groups, this might be attributed to the increased zinc oxide absorption from small intestine which ultimately effect on intestinal morphology and intestinal health (Tsai et al., 2016). Increased villus height with LPT, MM and ME in COS group may be due to antimicrobial and antioxidant activity of COS (Lan et al., 2021).

The current study results reviled that heat stress decline meat pHi, pHu and water holding capacity in PC group are line with Shah et al. (2019) who showed that decreasing of pH in heat stress groups may be due to lactic acid production as result of glycolysis after postmortem. Lactic acid is produced by adrenaline triggered by heat stress and a faster glycolysis cycle. In the current study decline of pH and WHC may be due to protein degeneration and lactic acid production. Increased pH and WHC in all supplemented groups may be due to better absorption of Zn fed utilization that improved muscle development which ultimately effect on muscle pH and WHC in all supplemented groups (Shah et al., 2019; Sharma et al., 2012). The higher pH in related to the COS group may be due to better absorption of zinc oxide and feed utilization that improved muscle development. The development muscle has greater muscle cross-section area and muscle fascicle diameter, in our experiment results slow decline of pH and water holding capacity may be due to antioxidant activity of zinc and synergistic effect of prebiotic (Kim et al., 2018; Shah et al., 2019). Increased pH and WHC in combination form may be due to their synergetic effect because ZnO-NP enhance the efficacy of COS. The decreased MFD and WHC in heat stress and increased in the supplemented groups may be due to many reasons including decreased oxidative stress which is the property of Zn in heat stress (Prasad, 2008), antimicrobial activity of prebiotic and zinc (Ogidi & Oyebode, 2023) and potential role in digestive enzyme secretion (Naz et al., 2016). The higher MFD in the prebiotic supplemented group can be linked with COS in role as a strong muscle growth development (Khan et al., 2022) The muscle MFD decreased in heat stress but increased with dietary supplementation of ZnO-NP and COS may be due to an increase in muscle fiber diameter and area is composed of muscle fiber (Shah et al., 2019). In the current study serum, zinc concentration had significantly increased in all supplemented groups as compared to negative control and positive control groups. It may be due to longer villi in the present study, the increased surface area that allowed greater absorption of available nutrients in the distal intestinal segment, longer villi may promote growth, and tissue damage is induced by zinc oxide (Ramiah et al., 2019; Sharma et al., 2012).

Zinc (Zn) might have a significant function in inhibiting the generation of free radicals by virtue of its capability to displace iron (Fe) and copper (Cu) from binding sites. This action could result in decreased production of free radicals in broiler chickens experiencing heat stress (Ghazi et al., 2012). The elevation observed across all types of goblet cells in the jejunum suggests enhanced bactericidal activity and a metaplastic response, possibly due to the presence of ZnO-NP. Their superior immune-stimulating potential, attributed to their diminutive size, and the up-regulation of mucin genes could contribute to this effect. Moreover, the heightened count of acidic goblet cells in the jejunum implies an increased output of acidic mucin, which is known for its resilience against bacterial enzymatic breakdown and its role in reducing bacterial translocation (Ali et al., 2017; Deplancke & Gaskins, 2001). Furthermore, cecal tonsils furnish immunity against identical viral infections like Avian Influenza (AI) and Newcastle Disease (ND). In this present investigation, the addition of ZnO-NP either individually or combined with COS lead to improved width, surface area, and overall count of lymphatic nodules within of cecal tonsils. This might be attributed to the potential immunostimulatory impact of ZnO-NP and COS on the percentage of lymphocytes (Altan et al., 2003). The reduction in lower, width, and upper lymph node (LLN, WLN, and ALN) dimensions observed in this study could be attributed to the influence of heat stress, which tends to lower lymphocyte concentrations. Conversely, the escalation in the count of cecal tonsils could potentially result from a synergistic effect between zinc oxide and COS, contributing to an elevated concentration of lymphocytes (Ali et al., 2017; Shah et al., 2020).

Conclusion

In the present study, the research focused on examining the adverse effects of heat stress on jejunal histomorphometry, goblet cell count, meat quality, and the relative weight of visceral and immune organs in broiler chickens exposed to heat stress. The findings indicated that heat stress had a detrimental impact on these parameters, suggesting compromised intestinal health, an increased goblet cell count, and reduced meat quality. However, dietary supplementation with zinc oxide nanoparticles at a concentration of 60 mg/kg, combined with COS at 200 mg/kg, appeared to mitigate the negative effects of heat stress and improve jejunal histomorphometry, goblet cell counts, and meat quality in broiler chickens under cyclic heat stress.

Acknowledgements

The authors express their sincere gratitude to the Higher Education Commission for funding this project. Heartfelt thanks go to the members of the Center of Excellence in Analytical Biochemistry in Jamshoro for their involvement in the preparation of zinc oxide nanoparticles. The National Veterinary Laboratory (NVL) and the Sindh Institute of Animal Health are warmly acknowledged for their invaluable laboratory support.

Conflict of interest

The authors declare no conflict of interest.

novelty statement

Current research used novel methodology of supplementing zinc oxide nanoparticles (ZnO-NPs) with chitosan oligosaccharide (COS) to broiler under heat stress, with an aim to address the dual challenge of maintaining intestinal integrity and meat quality. This research fills a critical gap by focusing on the effects of this innovative combination on intestinal integrity, goblet cell count, and overall meat quality, which are crucial for welfare of broiler and economic outcomes in the poultry industry. The supplementation is particularly useful for the broilers grown in hot climates where it acts synergistically and helps mitigate the harmful effects of heat stress by reducing the oxidative stress, and improves hematology, intestinal integrity, bone health, growth performance and meat quality parameters in broiler chickens.

Authors Contribution

Every author played a role in formulating and shaping the concept and design of the study. The conception and research design were formulated by (S.A Hadi, J.A Gandahi, M.G Shah, S. Masood), and the experimentation was carried out by (S.A Hadi, and J.A Gandahi). Data collection and analysis were conducted by (M.G Shah and N.S Gandahi), while the initial draft of the manuscript was composed by (S.A Hadi and J.A Gandahi) and reviewed/revised by (S.A Hadi, J.A Gandahi and N.S Gandahi). All authors provided feedback on earlier manuscript versions. Ultimately, all authors reviewed and endorsed the final manuscript.

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