Dried probiotics enhances immunity and liver function of final production laying hens maintained at upper temperatures zone

Dried probiotics (DP) are feed additives, capable of increasing body resistance and maintaining the balance of the microbial ecosystem in the digestive tract against pathogenic bacteria. Biological age and environmental temperature are important factors determining immunity and liver function. This investigation was performed to evaluate the effect of dry probiotics on immunity (lymphocytes, neutrophils and their balance) and liver function of layer-phase laying hens. This study used 40 layer-phase hens aged 70 weeks with the Completely Randomized Design (CRD) method. It was divided into 4 treatments and 5 replicates., each treatment was without dry probiotics in the diet, 2% dry probiotics in the diet, 3% dry probiotics in the diet, and 4% dry probiotics in the diet. Blood sampling was done every month during the three-month experimental period. The results showed that feeding 4% dry probiotics in the diet had a significant effect (P<0.05) on the levels of lymphocytes, neutrophils and transferase enzyme profile. The immunity and liver function of laying hens appeared to be optimized by feeding 4% dry probiotics in the ration.

Anticipatory measures need to be pursued through the provision of natural substrates to laying hens.One of these efforts is the provision of dried probiotics as feed additives [1][2][3].Dried probiotics have been developed into ration components to maximize their function for livestock.Dried probiotic is the result of yogurt development [10][11][12][13].
Previous studies have shown that probiotics can act as anti-stress agents, and some previous reports have also shown their effectiveness as antimicrobials and antibiotics, so they are expected to prevent molds and aflatoxins from entering the absorptive cells of the ileum through feed [14].Immune system stimulation triggered by probiotics can increase immunity-related plasma proteins with 2-4% in animal diet [15][16][17].This increase in plasma protein is indicated by high production of total protein with fed 4% probiotics [4], albumin and globulin as well as leukocytes and their differentiation [11].The improvement in immunity function also reduces tissue inflammation by reducing proinflammatory protein [18,19].This condition can be observed through liver function as indicated in the amine group transfer enzymes in the liver [3,6].
Therefore, researchers are interested in examining the effect of giving dried probiotic on immunity, through indicators of leukocyte levels and their differentiation (lymphocytes and neutrophils) and blood proteins (albumin, globulin and total protein) in the blood plasma of laying hens.

Animal samples
A total of 40 70-week-old laying hens were used in this experiment.The study was conducted from July to August 2022 at the Experimental Cage of Poultry Production Laboratory, Faculty of Animal Husbandry, Padjadjaran University.The chickens were kept in semi close house type cages with individual battery form, partitioned into 40 cages and each cage was filled with one chicken.The feed used was in accordance with the recommendations of the type of chicken being raised, with crude protein 22%, metabolism energy 2900 Kcal/kg diet.The average temperature and humidity recorded during the study ranged between 37-38.50C and 75-85%, respectively

Experimental design
This study has been conditioned with a completely randomized design (CRD).It consisted of 4 treatments, namely without and with the provision of dried probiotic, which was given from the beginning of the study.Each treatment consisted of 10 replications so that there were 40 flocks of experimental units.Each experimental unit consisted of 1 laying hen.The four treatments applied were P0: Basal ration without dried probiotics, and without heat stress; P1: Basal ration containing dried probiotics (DP) 2% of total ration, with cage temperature 38 0 C; P2: Basal ration containing DP 3% of total ration, with cage temperature 38 0 C; P3: Basal ration containing 4% DP of the total ration, with a cage temperature of 38 0 C.

Experiment protocols 2.3.1. Blood sample collected
Blood samples were taken at the end of rearing after 30 days of administration of dried probiotic, using a syringe inserted from the flank vein (pectoralis vein externa) as much as 3 ml.Blood samples enter the container with EDTA then shaken gently and then stored in a cooler before the blood was analyzed to avoid blood clotting.

Sample analysis
At the end of the research, chicken blood was retrieved from the external pectoralis vein using a venojete tube containing EDTA anti-coagulant, as much as 3 mL.The blood that had been collected was first analyzed for leukocyte, neutrophil, and lymphocyte levels using a haematology analyzer.The whole blood was centrifuged at 3500 rpm for 15 minutes, for plasma fluid.Plasma was separated and collected into a 3 mL appendof tube, using a micropipette.
Plasma was used for the analysis of total protein, albumin, globulin, SGOT and SGPT.Biochemical analysis of this virgin used spectrophotometric methods with analysis techniques based on the instructions on the Biolabo KIT, following the protocols previously research [4,10].

Statistical analysis
The ANOVA (one-way analysis of variance) technique was used in this experiment, based on the analysis protocol by the General Linear Models (GLM)of SAS Version 8.216 for a completely randomized design, and Duncan test to determine difference between treatment group (SAS Institute, 2001).

Results and discussion
Plasma protein levels, leukocytes and their differentiation with the administration of dried probiotics at the end of the finisher phase of laying hens experiencing heat stress, based on the results of the study can be seen in Table 1.The effect of DP level feeding in laying hens on total protein, albumin and globulin levels based on the this experiment, overall showed a significant effect on these blood biochemical parameters (Table 1).There is a similar trend that the higher the level of DP administration causes the levels of total protein and blood plasma albumin to also increase, although different from the globulin response.Total protein levels without DP administration, which were 4.51 mg/dL and albumin levels of 6.45 mg/dL, were both significantly lower (P<0.05)compared to the groups of chickens that received DP in the diet at 4% ppm (P3).Table 1 also shows that the globulin level without DP, which was 3.16 mg/dL, was not significantly different (P>0.05)compared to the groups of laying hens treated with DP level in the ration.However, the group of hens treated with 4% DP level showed higher blood plasma globulin levels of 4.63 mg/dL (P<0.05)compared to the groups of laying hens without DP and with 2-3% DP.
Overall, based on the results of this study (Table 1), it still shows the linearity of the effect of DP level on increasing the levels of total protein, albumin and globulin in the blood plasma of laying hens.This increase is related to the function of liver cells.It is known that liver cells are responsible for the production of albumin [15][16][17][18][19][20][21][22][23], so the higher the albumin, the higher the plasma total protein levels.
As for the role of DP administered through feed, several researchers have previously reported that the administration of DP in feed is able to improve the rhythm and rate of metabolism in livestock, and show good physiological responses [17,18,[25][26][27][28][29].Other researchers have shown that feeding probiotics to animals and livestock has an impact on their biochemical profiles.These studies demonstrated the ability of probiotics to affect the regulation of protein biosynthesis (hormones and enzymes), as well as specific proteins [30][31][32][33].
Based on the results of this study (Table 1) shows that the number of leukocytes is the highest (P <0.05) in the group of laying hens given 4% DP, which is 8,832 x 103 cells, compared to the group of chickens without DP, and with DP 2 and 3%, namely 8,224; 8,353; 8,531x 103 cells.The same phenomenon also occurred with the number of lymphocytes, where the number of lymphocytes in the provision of 4% DP in laying hens through the highest ration (P < 0.05) was 7.93 x 102 cells compared to the others.In contrast, the number of neutrophils decreased with increasing levels of DP.
The description of the research results above clearly shows that DP is able to function as an immunostimulator which is indicated by the increase in the number of leukocytes and lymphocytes, on the contrary, it decreases neutrophils.Based on these results, it can be explained that DP increases the growth of leukocyte cells.The increase in leukocytes stimulates the increase in lymphocytes, both B and T lymphocytes.The increase in both types of lymphocytes physiologically suppresses the growth of neutrophils.The results of [18,19,[20][21][22][23] showed that dried probiotics can protect against the activation of ROS (Reactive Oxigen Species) by turning on Stimulator of Interferon Genes (STING) and Cyclic Guanine Monophosphate-Adenyn Monophosphate Synthase (cGAS) in dendrite cells.It was further reported that activation of these two types of specific proteins (cGAS and STING) led to dendrite cell initiation.showed that dried probiotics can protect against the activation of ROS (Reactive Oxigen Species) by turning on Stimulator of Interferon Genes (STING) and Cyclic Guanine Monophosphate-Adenyn Monophosphate Synthase (cGAS) in dendrite cells.It was further reported that activation of these two types of specific proteins (cGAS and STING) led to dendrite cell initiation.
Related to the activation of dendrite cells, [22,24,25] suggested that the activation of dendrite cells causes a physiological impact on the stimulation of immune activity through the growth of leukocytes and lymphocytes.The growth of leukocytes and lymphocytes is related to the initiation of immunoglobulin production, which is stimulated by dendrite cells through the production of T helper cell proteins.
The concentration of biochemical biomarkers of cell damage in broiler liver with and without DP and glutathione induction can be seen in Table 2 below.Fed DP in ration at the current investigation showed a significant effect result on the levels of SGOT, SGPT and Gamma GT.The provision of higher levels of DP decreases the levels of inflammatory markers even in a state of heat stress.Based on Table 2, the average level of SGOT without giving DP is 119.63 IU/L, which is not significantly different from the average level of SGOT in the experimental chicken group given DP4%, even in heat stress, which is 121.19IU/L.
The same results were also seen in SGPT levels.The provision of higher DP caused a decrease in SGPT levels in the blood plasma of hens.Based on Table 2, the SGPT level without the provision of DP was 15.48 IU/L, this result was not significantly different from (P>0.05) that was given 4% DP, which was 16.12 IU/L.The same phenomenon also occurred with gamma GT levels.The results show the effectiveness of DP in reducing the risk of inflammation as a result of heat stress.
As results in this study, it can be confirmed that heat stress for laying hens can increase the levels of SGOT, SGPT and γ-glutamyl transpeptidase, indicating that liver damage increases with heat stress.However, DP with a concentration of 4% was able to reduce the levels of SGOT, SGPT and γ-glutamyl transpeptidase in the blood which initially increased with heat stress treatment.Increased levels of SGOT, SGPT and γ-glutamyl transpeptidase are caused by cell damage [21].As according [34,35] states that SGPT and SGOT levels increase in proportion to cell damage.
According to [36,37] SGOT levels in laying hens increased SGOT levels can be caused by fatigue in certain organs, causing free radicals.Fatigue, inflammatory conditions of lipid peroxidation, infection can cause increased free radicals.According to [38][39][40] free radicals can be prevented by probiotics, because some of the peptide content in probiotics, are antioxidants that can donate hydrogen atoms [40,41] to hydroxyl to neutralize so as to prevent the formation of free radicals [42].The use of DP as an antioxidant provides benefits but there are challenges on how to use antioxidants effectively.

Conclusion
As a result of the current study, it can be affirmed that DP with a level of 4% in the ration of laying hens is able to stimulate immunity and reduce inflammation, even though laying hens are under heat stress.

Table 1 .
Effect of dried probiotic on protein and leukocyte levels and their differentiation at the end of the finisher phase of heat stressed laying hens

Table 2 .
Biochemical Markers of Heart Inflammation with and without DP