CC BY
02019, Scienceline Publication
World's Veterinary Journal
World Vet J, 9(3): 211-217, Sept 25, 2019
https://dx.doi.org/10.36380/scil.2019.wyj27
Histopathological Alterations of Ceca in Broiler Chickens (Gallus gallus) Exposed to Chronic Heat Stress
Antonia Vania Adji1, Hani Plumeriastuti2, Anwar Ma'ruf3* and Djoko Legowo2
'Faculty of Veterinary Medicine, Airlangga University, Surabaya 60115, Indonesia
2Department of Veterinary Pathology, Faculty of Veterinary Medicine, Airlangga University, Surabaya 60115, Indonesia 3Department of Basic Veterinary Medicine Science, Faculty of Veterinary Medicine, Airlangga University, Surabaya 60115, Indonesia ♦Corresponding author's E-mail: anwarunair@gmail.com; ORCID: 0000-0001-7158-9156
ABSTRACT
Heat stress has been found to cause adverse effects on small intestinal microstructure, but little is known about its impact on chicken's cecum. In this research, the histopathological alterations of broiler chicken's cecum following chronic heat stress were evaluated. 20 broiler chickens were randomly divided into control group and treatment group containing 10 replicates, respectively. Both groups were reared under standard conditions until 21 days of age. From day 22 to day 42, the control group was kept at 24-280C as well as relative humidity of 40-55%, while the treatment group was exposed to high temperature of 36-400C and relative humidity of 45-65% for eight hours per day. At the end of the period, proximal part of each chicken's cecum was collected and made into histopatholo gic al slides with Hematoxylin and Eosin staining. Villus height, villus width, crypt depth, villus surface area, and villus height to crypt depth ratio were examined from 10 villi per replicate. Results analysis revealed that chronic heat stress profoundly (P<0.05) reduced the crypt depth. Insignificant (P>0.05) changes of the villus despite the long-term heat exposure might imply that the damage is at its early phase. In conclusion, chronic heat stress can produce morphological alterations in the ceca of broiler chickens, though requiring longer duration due to cecum's durability. Key words: Broiler chicken, Cecum, Heat stress, Intestinal morphology
INTRODUCTION
Heat Stress (HS) is defined as biological response to high ambient temperature which disrupts the heat exchange equilibrium (Lara and Rostagno, 2013). Over the past decades, the Earth's surface temperature has gone beyond the former baseline, going as far as nominating 2016 as the warmest year since the recording was initiated in 1880. Consequently, the risk of HS' emergence in livestock projects to a higher level (Thornton et al., 2009). Broiler chickens are particularly more prone to contract heat stress because their cardiovascular and respiratory systems, which are vital for heat loss mechanisms, are unable to cope with their heavier body weight and high metabolism rate resulting from genetic selection (Sozcu, 2019).
HS has generally been found to be detrimental to small intestine's morphology in broiler chickens (Al-Fataftah and Abdelqader, 2014; Yi et al., 2016). The underlying mechanisms involve hypothalamic-pituitary-adrenal (HPA) axis activation, ischemia, oxidative stress, or perturbation of intestinal microbiota (Bolek, 2013; Song et al., 2014; Yi et al., 2016). The activated HPA axis subsequently elevates corticosterone level which in turn undermines immunity (Mishra and Jha, 2019). It has also been discovered that HS can suppress the number of goblet cells and the mRNA expression of tight junctions and adherence junctions in broiler chickens subjected to 330C, 10 hours/day for 21 consecutive days (Zhang et al., 2017). These occurrences along with the impaired immune system facilitate the penetration of both toxins and pathogenic bacteria into the circulation afflicting various organs. Meanwhile, cecum is a part of chicken's intestine with the most abundant and diverse microorganisms colonizing its lumen (Ijaz et al., 2018). Furthermore, cecum regularly receives the backflow of urine rendering potentially more severe damage by the pathogens trespassing (Kum et al., 2015).
The available research on the influence of HS on cecal morphology produced conflicting results. Reduction in villus length and crypt size in the ceca of Japanese quails (Coturnix coturnix japonica) exposed to chronic HS was demonstrated by El-Daly et al. (2014). In contrast, negative results were obtained in Jaafar (2013)'s research. Other experiments, for the most part, are concerned with the microbiome in cecum, for instance HS was proven to be able to restrain the growth of normal flora colonies, like Lactobacillus sp., while enhancing the colonies of Escherichia sp., Salmonella sp., and aerobic bacteria in the cecum of broiler chickens (Park et al., 2013).
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It was proposed that HS influenced the intestine variably in compliance with susceptibility level of each segment and the duration of HS occurrence (acute or chronic) (Loyau et al., 2015; Varasteh et al., 2015). This was borne out by analogous findings in Cherry-Valley ducks exhibiting no substantial crypt depth discrepancy in the cecum despite pronounced changes in the jejunum and ileum (He et al., 2019). Taking into account the lack of consistent and adequate number of results, the current research had an objective to evaluate the impact of chronic HS for 21 days on the microstructure of broiler chicken's cecum.
MATERIALS AND METHODS
Ethical approval
This experiment was performed on the basis of approval by the laboratory animals use research ethics committee of faculty of veterinary medicine, Airlangga University, Indonesia.
Experimental animals
In this research, 20 one day old Cobb broiler chicks were housed at the cage of laboratory animals, Faculty of Veterinary Medicine, Airlangga University, Surabaya, Indonesia. The chicks marketed as Wonchick originated from Wonokoyo Jaya Corporindo Ltd. (Limited Company, Surabaya, Indonesia). The rearing temperature followed the guidelines provided by the referred company, namely the leaflet containing Wonchick broiler chicken rearing guide. The chicks were allowed ad libitum access to feed and water. This went on up to 21 days.
Experimental design
The chickens were randomly allocated to two groups (n= 10, respectively), the control (C) group and the treatment (T) group, from day 22 onwards. This starting point was chosen because broilers in the later stage of life have greater sensitivity to high surrounding temperature than the younger birds (He et al., 2018). In that, true experimental design, specifically the post-test-only control group design, was applied. The C group was maintained on thermoneutral conditions (24-280C and relative humidity (RH) 40-55%), while the T group was exposed to high ambient temperature and humidity (36-400C and RH 45-65%) for eight hours/d then returned to 24-280C and RH 40-55% for the rest of the day. On day 42, all the chickens were humanely slaughtered as affirmed by the research ethics committee and organ collection was carried out.
Cecal histopathology
The proximal part of cecum was dissected from 23.59% - 23.65% proximal of its length for all chickens. About one cm of the segment was fixed in neutral buffered formalin 10% and then routinely processed into slides with Hematoxylin and Eosin (H&E) stain. The sections were analyzed under light microscope with computer-assisted digital image analyzer (NIS Elements) for Villus Height (VH), Villus Width (VW), and Crypt Depth (CD) (Figure 1). Measurements of VH, VW, and CD were based on the methods used by Abdelqader and Al-Fataftah (2016); Godwin et al. (2016) and Shokryazdan et al. (2017). The Villus Surface Area (VSA) was calculated from the formula = n XVW x VH. The Villus Height to Crypt Depth (VH:CD) ratio is a comparison of VH to CD (Santos et al., 2015). Collection of all the variables was done on 10 intact and the longest villi with their associated crypts in one cecal cross section per chicken. These values were averaged to obtain mean value of each variable for each chicken.
Figure 1. Methodology for morphometric assessment of villus-crypt unit of cecum in broiler chickens, A: in straight unit and B: in irregularly-oriented unit (H&E, 200*), Villus height (yellow line) was measured from the tip of the villi to the villus-crypt junction (green line), Villus width (red line) was measured from side to side of the villi at its half height, Crypt depth (blue line) measurement was based on the depth of invagination between adjacent villi.
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Statistical analysis
Data analysis used the two-tailed T Test for independent samples, and p< 0.05 was the accepted significant value. It was conducted with SPSS (Version, 24) for Windows OS (SPSS, Chicago, IL, USA). The results are presented in mean ± standard error.
RESULTS AND DISCUSSION
From the five dependent variables examined, profound decline of CD (p<0.05) was observed in the chickens of T group compared to C group (Figure 2 and figure 3). The remaining variables did not express significant difference between the two groups (p>0.05) (Table 1).
Figure 2. Cecal morphology measurements of broiler chickens of control group from the study in Surabaya, Indonesia between August and September 2017. The yellow lines, red lines, and blue lines represent villus height, villus width, and crypt depth respectively (H&E, x100). VH: villus height; VW: villus width; CD: crypt depth
Figure 3. Cecal morphology measurements of broiler chickens from treatment group which were exposed to high ambient temperature and humidity from 22 to 42 days of age during the study in Surabaya, Indonesia between August and September 2017. The yellow lines, red lines, and blue lines represent villus height, villus width, and crypt depth respectively (H&E, x100). VH: villus height; VW: villus width; CD: crypt depth.
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Table 1. The effect of heat stress on histopathology of ceca in broiler chickens in Surabaya, Indonesia between August and September 2017
Variable Group C Group T
VH (^m) 518.66 ± 31.44 493.22 ± 45.01
VW (^m) 115.23 ± 2.86 122.92 ± 5.25
CD (^m) 116.67 ± 4.65a 98.84 ± 5.83b
VSA (^m2) 189,980.84 ± 14,538.90 195,195.07 ± 22,117.80
VH:CD 4.75 ± 0.28 5.38 ± 0.56
C: control; T: chickens were exposed to high ambient temperature and humidity from 22 to 42 days of age; VH: villus height; VW: villus width; CD: crypt depth; VSA: villus surface area; Values are provided with mean ± standard error; a,b Different superscripts within the same row mark significantly different means (p<0.05).
HS is able to delay epithelial turnover by means of decreasing feed intake as a result of HPA axis activation (Hu and Guo, 2008). Elevated plasma corticosterone in broiler chickens is indirectly held responsible for the decrease in VH and CD in duodenum and jejunum, but this only occurs in the acute phase instead of chronic (Aggarwal and Upadhyay, 2013). Another proposed mechanism is ischemia in the event of blood diversion to heat-releasing organs, such as the turbinates and the respiratory muscles (Song et al., 2014). Ischemia will lead to vacuolation, rapid sloughing, and loss of microvilli in the intestinal epithelium (Al-Fataftah and Abdelqader, 2014). Moreover, ischemia is also reported to cause oxidative and nitrosative stress which implicated damaged cell membrane, disrupted cellular ion homeostasis, opened tight junctions, and reduced integrity of intestinal mucosa (Al-Fataftah and Abdelqader, 2014). This may proceed to shorter villi but deeper crypts to compensate (Song et al., 2014). The injured mucosa also disables the attachment of commensal bacteria. In addition, the expression of Heat Shock Protein (HSP) may act as receptor for pathogens, while lack of goblet cells which equals falling mucin production promotes the adhesion of pathogens to mucosa (Burkholder et al., 2008; Shah et al., 2019).
The VH, VW, VSA, and VH:CD ratio in present research were barely affected by the heat exposure (36-400C and RH 45-65%) for eight hours/day along 21 days. Similar findings were attributed by Quinteiro-Filho et al. (2010) to the rapid periodical re-epithelization of intestinal mucosa in the absence of structural adaptation. This explanation is however inappropriate for this case given that it normally occurs in acute or shorter period of cyclic chronic HS, i.e. (seven days). Chronic HS for over one week has been recorded to consistently diminish VH in the absence of adaptation or compensation, although temperature exceeding 36±10C could accelerate this process into less than a week (Santos et al., 2015; Abdelqader and Al-Fataftah, 2016; Yi et al., 2016). Meanwhile, CD showed variable responses to HS (decreased, increased, or no difference) regardless of the HS duration. This denoted that there are different sequences of alterations, and their progression is dependent on the level of temperature and internal variation of the organisms subjected to HS. Within the earlier period of HS when the CD of cecum and small intestine have recovered, their VH often did not reach to a similar state, in other words changes in villi are initiated by the crypts (Deng et al., 2012; Abdelqader and Al-Fataftah, 2016).
Crypt of Lieberkuhn, being the location of stem cells, is the center for proliferation of intestinal epithelium. The newly generated enterocytes then migrate up and are ejected at the villus tip. Because crypts merely grow in length, the activity level of crypts is proportional to CD (Cummins et al., 2011). Significantly (p< 0.05) shallower CD in the T group thus indicated less proliferation rate. The crypts due to proliferative property, were the first part of intestine observed to suffer from stress as remarked by Burkholder et al. (2008). By combining this fact with current results, it can be interpreted that the cecum of chickens in T group is on early phase of damage despite the prolonged HS. The possibility that injury on the cecum of T group was a start can also explain the insignificant (p>0.05) alterations of other variables besides CD. This idea is reinforced by the inference that colon (including cecum) is one of the segments more resistant to HS characterized by lower expression of HSP70 and HSP90 mRNA (Varasteh et al., 2015). If the order of segment susceptibility is to be associated with epithelial migration time, they may lay the basics for cecum's resistance against HS. The proximal cecum of Leghorns had demonstrated longer turnover time than duodenum (four days compared with 72 hours) in the research performed by Takeuchi et al. (1998). Similarly, epithelial migration time in the ceca of Japanese quails was much higher than in their small intestine, and it was second only to rectum, as noted by Starck (1998).
The development of adaptations by the chickens subjected to present study can be considered as another reason. It takes longer period to impair the intestinal compensatory mechanisms in adaptive birds according to Santos et al. (2015). This was reflected by the zero mortality of the Cobb broiler chickens taking part in comparison to 12.5% death of Hubbard chickens following a shorter period of HS with slightly lower temperature applied in Al-Fataftah and Abdelqader (2014)'s research. The adaptive trait might have originated from the Cobb strain itself or from inherited
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adaptation subsequent to breeding attempts in the warm climate of Indonesia. Warm ambience could unknowingly perform an act termed as thermal manipulation on chickens prior to hatching which is capable of assisting thermotolerance acquisition (Al-Zghoul et al., 2019). Likewise, it is known that poultry are very likely to develop acclimatization when kept under tropical and subtropical climates with over two months of high temperature (He et al., 2018). Therefore, it can be deduced that the chickens in this study did not need deep crypts in their ceca to preserve their villi from potential casualties because they had created such an effective mechanism enabling them to withstand the lengthy HS (Biasato et al., 2018).
These results contradict those obtained by Deng et al. (2012) which found that the CD of layer hens' ceca was initially reduced by chronic HS on day six but returned to normal on day 12 (last day) while VH remained low throughout the experiment. This difference may be linked to breed, implying that broilers are more resistant than layers, or age of the chicken at the time of HS.
The retarded crypt activity might bring about decreased VH later as the HS progresses (Al-Fataftah and Abdelqader, 2014). CD, VW, and VSA can be enhanced in order to compensate the increased destruction, but still providing sufficient absorption area for nutrients (Santos et al., 2015). The VH:CD ratio inversely portrays the rate of epithelial cell turnover in the intestine, its value is smaller in heat stressed chickens due to induced inflammatory reaction (Laudadio et al., 2012; Shah et al., 2019). When the value is constant, the number of villi per unit area is required to be taken into account. It is recommended to eliminate the possibility that HS has reduced the number of villi instead of the villus morphometry (Marchini et al., 2016).
CONCLUSION
The resilience of broiler chickens' ceca to chronic HS was confirmed in this research. This feature can be credited to intestinal segment-specific resistance which may be explained by the slower epithelial turnover rate in cecum than in small intestine. Additionally, the current findings also suggest that broiler chickens in Indonesia have undergone an adaptation process to the high daily temperature. Nevertheless, it did not remove the possibility of damage progression if the heat exposure is extended.
DECLARATIONS
Acknowledgements
The authors are most grateful to the lecturers and staff of Veterinary Pathology Department, Faculty of Veterinary Medicine, Airlangga University for the technical assistance and facilities that were prepared. Also, the authors appreciate all participants who contributed to the completion of this work.
Competing interests
The authors have declared that no competing interests exist. Consent to publish
All the authors approved and agreed to publish the manuscript and declared that this work has not been previously published elsewhere.
Authors' contributions
Hani Plumeriastuti drafted and revised the manuscript while monitoring the course of the study. Anwar Ma'ruf was involved in revising the manuscript, also data analysis, presentation, and interpretation. Djoko Legowo devised the study and participated in its design and coordination, as well as guided the examination and analysis of the histopathological slides. Antonia Vania Adji participated in the execution of the study, data analysis, composition and revision of the manuscript. All authors read and approved the final manuscript.
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