The effect of using effective micro-organisms on the histopathological, immunohistochemical expression of kidneys in quails under thermal stress

Introduction

One of the environmental factors that affects the poultry industry all over the world is heat stress (1). Poultry is very sensitive to thermal stress and their capacity to dissipate the heat of the body is low (2). Thermal stress decreases weight of egg and quality of shell in laying hens (3). Furthermore, laying hens exposure to high temperature causes a reduction in level of phosphorus and plasma calcium (4-6) which are essential minerals for hens that affect production of egg and quality of shell (7-9). The renal tissue plays an important role in preserve phosphorus and calcium homeostasis which is equiponderant by renal excretion and absorption of gastrointestinal tract. Effective microorganism (EM) is a mixed cutler cell which is consist of yeast, fungi, photosynthetic bacteria and actinomycetes (10,11). EM is considered a new technology were contrived in Japan. It’s a combination of 65-75 types of perfect microorganism contributing to a wide ambit of applications. EM microorganism is not engineered genetically, but they are usually found in food and healthy soils forest. EM mutually live together to form useful relationships with each other in a runny medium (12). EM microorganism helps one other for survival in a chain of food and whereby form synergy that struggle the rotting and pathogens. Effective microorganisms are sterilizing (PH between 3. 3-3. 6), thus pathogenesis cannot survive inside its. Chronic thermal stress accountable for poor growth of body weight and gain rate and week animal body immune system (13,14) Moreover in different stress state, pro-inflammatory and anti-inflammatory cytokine excrete from various immune cell and play important role to limited the immune condition of an organism (15,16) mostly, pro-inflammatory cytokines intercede inflammatory damage, while anti-inflammatory cytokines improve inflammation and spur healing process (17). during inflammatory response, several interleukin and cytokine such as IL-1B, IL-1, 2, 6 are involved on increasing immune response exacerbation. IL-1B and IL-6 is a main inducer of acute inflammatory response.

The aim of this study was to evaluate the effects of heat stress and effective microorganisms on kidney weight, renal tissue pictures and interleukin expression.

Materials and methods

Ethical approval

Empirical design

Forty one-day old quails were used, after adaption period they partitioned as follow, M1 (control groups), M2 exposed to heat stress (36℃for sixty hour), M3 were giving both (EM and exposed to stress) and M4 remedy with Effective Microorganisms with 1000ppm in drinking water (1gm/L) according to (18), and throughout the experiment period all birds received the same food ration, Birds were sacrificed at 20 and 40 days of the experiment respectively.

Relative weight of the kidney

Kidney tissue was collected from quails after performing an microscopical description of it, and the relative weight of it was calculated based on the following equation: Relative weight of the organ = (weight of the organ/weight of the living body)*100

Histopathology findings

Immunohistochemistry

The kidney samples were pre-fixed overnight and processed using an automated tissue processor. The embedding station was used to embed the paraffin-treated kidney samples. Using a rotary microtome, kidney slices were cut to 3-4 μm thick and the sections were fixed on hard-frozen slides. To remove paraffin, distilled water was used with ethanol and xylazine in graded concentrations. section was heated using citrate buffer to restore antigen efficiency using a microwave for five minutes. The detection kit of the HRP/DAB type (Abcam) was used, and the pro-inflammatory cytokines (IL-1B) was used at 1:500dilution, with primary antibody the segment were incubated, follow by horseradish peroxidase conjugate were incubated and substrate with DAB eventually the segment were kowtow to counterstaining with Hematoxylin for examination under the light microscope and photographed by camera, the intensity color of immunohistochemistry was graded from 1-4 scale in which 1 no staining color and 4 high intensity color (19).

Statistical analysis

A student’s t- test the data analyzed, at mean ±SE. the significant level was at probability level P<0.05 (20).

Results

Relative weight of the kidney

The kidney relative weight statistical analysis result showed a significant increase at the probability level P<0.05 in the group treated with EM 3.00%, and a significant decrease was observed in the group exposed to heat stress 1.52% compared to the first and the fourth groups at 20 days of the experiment. Additionally, the third group recorded a significant increase in the relative weight of the kidney at the 40 days of the experiment 1.62% than other. A significant decrease in the relative weight was observed in the second group 1.33% at the 40 days of the experiment (Figure 1).          

Figure 1: Kidney relative weight between 20 and 40 days of the experiment.

Histopathology finding

The result showed that, the general histological characteristics of the kidney in the M1 was normal, in second group the renal tissue section showed bleeding, atrophy, glomerular tissue necrosis in addition to sever destruction in the tubular epithelium at middle and the end of the experiment (Figures 2 and 3). Area of bleeding with infiltration of inflammatory cells, coagulative necrosis and atrophy of glomerular tubules are showed in M3 groups (Figures 4 and 5), as well as the effective microorganism treated group showed Pyknosis and hyper cellularity (Figures 6 and 7). Table 1 showed renal tissue lesions, categories, grade of lesions and figure number

Figure 2: Kidney micrograph section showed Presence of glomerular tuft atrophy (red arrow) and glomerular tissue bleeding (black arrow), tubular cells necrosis (green arrow), 20 days of the experiment, M2 groups, X100.

Figure 3: Kidney micrograph section showed sever destruction of tubular epithelium (black arrow) and bleeding (red arrow), 40 days of the experiment, M2 groups, X100.

Figure 4: Kidney micrograph section showed Presence of bleeding (black arrow) coagulate necrosis (green arrow) and atrophy of glomerular tubules (red arrow), 40 days of the experiment, M3 groups, X100.

Figure 5: Kidney micrograph section showed Presence of bleeding (blue arrow), infiltration of inflammatory cell (black arrow), 40 days of the experiment, M3 groups, X100.

Figure 6: Kidney micrograph section showed Presence of pyknosis (green arrow) and hyper cellularity (black arrow), 20 days of the experiment, M4 groups, X400.

Figure 7: Kidney micrograph section showed Presence of bleeding) black arrow), infiltration of inflammatory cell (blue arrow), 40 days of the experiment, M4 groups, X400.

Table 1: Kidney microscopic lesions description

Immunohistochemistry finding

The immunohistochemistry result showed that IL-1B expression was positively stained with moderates and sever grade inside glomerular and cytoplasm of the tubular lining cells in the all treated groups (Figures 8-13).

Figure 8: Tubular and glomerular expression (moderates) of IL-1B in quail’s kidney, M2, 20 days of the experiment, 400X.

Figure 9: Tubular and glomerular expression (sever) of IL-1B in quail’s kidney, M2, 40 days of the experiment, 400X.

Figure 10: Tubular and glomerular expression (moderates) of IL-1B in quail’s kidney, M3, 20 days of the experiment, 400X.

Figure 11: Tubular and glomerular sever expression of IL-1B in quail’s kidney, M3, 40 days of the experiment, 400X.

Figure 12: Tubular and glomerular (moderates) expression of IL-1B in quail, s kidney, M4, 20 days of the experiment, 400X.

Figure 13: Tubular and glomerular expression (sever) of IL-1B in quail’s kidney, M4, 40 days of the experiment, 400X.

Discussion

The current study aimed to discuss the incidental renal tissue Histopathological pictures and the effects of EM in quails exposed to thermal stress. A significant increase in the renal tissue weight in EM groups, A non-significant increase in the renal tissue weight was observed in the thermal stress and mixing groups, these ascribes to kidney role in participates in many vital activities of the body and exposure to stress factors (such as heat, transportation, overcrowding) affects the metabolic processes of the kidney (21,22) and decreased blood flow and decreased levels of calcium and phosphorus in the plasma, as the kidney requires a large amount of energy in order to perform its functions (23). Effective microorganism reduces kidney damage and weight loss as these organisms increase blood flow to the kidney and its production of protein-forming enzymes in the digestive system and the absorption of these enzymes attributed to increase in protein and amino acids decomposition by liver during the urea cycle, which leads to an increase in blood urea levels resulting within normal limits. The increase in protein is followed by an increase in Creatinine levels due to the digestion of protein and the production of many essential amino acids (24). The increase in urea and Creatinine within the normal range shows a positive effect because it indicates an increase in the digestion process and protein formation to support the growth process and increase kidney weight (25). Heat stress causes pathological changes in the kidney tissue, represented by degeneration, necrosis, due to oxidative stress resulting from the lack of oxygen and nutrient supplies return to lack of blood supply to the kidney (26,27), which causes damage to the mitochondria and affects oxidative phosphorylation and leads to rapid depletion of adenosine triphosphate (ATP) and failure of enzymes and energy-dependent ions, especially Na⁺, K⁺, ATPase, with which leads to an imbalance in the concentration of Na⁺, K⁺, and electrolytes ions inside and outside the cell, and it is no longer possible to provide better electronics to maintain kidney functions. infiltration of inflammatory cells, and congestion were also detected in the renal tissue due to hypoxia (28,29). The result showed that EM caused histological changes in the renal tissue represented by necrosis, congestion of blood vessels with infiltration of inflammatory cells. This is attributed to the fact that effective organisms contain bacteria and yeasts, and giving of these organisms for a long period led to fermentation and decomposition of all easily fermentable starches and carbohydrates, causing an increasing breakdown of protein into amino acids (30,31) and an increase in the formation of glutathione, which is the main metabolic source for the formation of ammonia

 IL-1B is a group of cytokines, proteins and gene. There are two types of them IL-1 Β and α. It produced by mononuclear cells, macrophages, and lymphocytes (32,33). Heat stress causes an imbalance between oxidation and antioxidants due to the production of many reactive oxygen species, production of free radicals, the formation of lipid peroxide, oxidative damage to membranes and activation of cellular immunity. Histological examination of kidney tissue in quails showed positive expression (moderate and severe degree) during the first and second killings, and it was highest in the second killings. It’s correlated with the impact of heat as a stress factor and its effect on the blood supply of the digestive system, the construction of blood vessels, occurrence of hypoxia, decrease of nutrients and increase oxidative stress by free radicals (RNS and ROS) that leads to the weakness of the intestinal barrier resistance to stress factors, parasitic and bacterial infections and the occurrence of inflammation represented by many responses to pro-inflammatory cytokines. Alternative antibiotic like probiotics, antioxidants and effective microorganisms have beneficial effect on poultry production through improving intestinal balance and stimulating the immune response (34). IL-1B intensity expression moderate to severe, this is attributed to the expression balance between pro- and anti-inflammatory cytokines, regulating the physiological response for the body's immune function (35), and maintaining a stable balance between the effectiveness of additives, alternative antibiotics and the ability to suppress and invade pathogens (36-38).

Conclusion

Quail Renal Tissue showed a series of Histopathological lesions that characterized by 7 types of lesions, a majority of specimen having bleeding, infiltration of inflammatory cells and necrosis, although quails are resistant to environmental conditions, heat stress had a negative effect on kidney tissue and IHC found moderate to severe immune reactivity of IL-1B specially after 40 days of the experiment. We propose to investigation the molecular pathways of such material and find the notch genes responsible of such improvements and repair of tissue damage resulting from any type of microbiological infections

Acknowledgment

The researchers thank the College of Veterinary Medicine, University of Mosul for providing all the easiness to complete the research paper.

Conflict of interest

There is no conflict of interest