Effects of mineral premix feed supplement on immunity of laying white leghorn

The aim of the study is to ascertain how White Leghorn laying eggs are affected by supplemental organic mineral premixes on their immune systems. 72 White Leghorn laying hens that were 3 weeks old and had not received the avian influenza (AI) vaccine were used in this study. Three treatment groups were created from the samples. Each treatment in the study consisted of eight hens that were grown for 42 days in three duplicates. A control group (PM-0), which received commercial feed without any added mineral premix, a PM-0.25 group, which received commercial feed with a 0.25% mineral premix, and a PM-0.50 group, which received commercial feed with a 0.50% mineral premix, were the three groups that received the experimental treatments. Immunity, with an emphasis on hematology and lymphoid organ weight, was measured in this study. The acquired data were analyzed using analysis of variance in a one-way design. The proportion of eosinophils in the blood hematology profile indicated a significant difference, with the maximum value recorded in the treatment comprising the addition of 0.5% mineral premix (3.4%) and the lowest value observed in the control treatment (0.5%). There were no statistically significant differences in lymphocyte, monocyte, or heterophil percentages across the three treatment groups. There was no discernible effect of the substances on thymus and spleen weights or percentage weights. As a result, the inclusion of mineral premix in animal feed at a maximum concentration of 0.50% did not trigger the immune response but effectively maintained the eosinophil level within the established acceptable range.


Introduction
Laying hens are chickens specifically raised to produce eggs commercially.Currently there are two groups of laying hens, namely the medium type and the light type.The medium types generally lay eggs with a brown shell color while the light types lay eggs with a white shell color [1].The first chicken to enter and start breeding in Indonesia was the White Leghorn layer chicken which was thin and generally used as broiler chickens after its productive period was over.There are three types of chickens, namely the light type from the White Leghorn nation, the medium type from the Rhode Island Reds, and Barred Plymouth rock and the heavy type from New Hampshire, White Plymouth rock, and Cornish [2].White Leghorn laying hens produce white eggs and are often used as a medium for virus propagation which is used as raw material for vaccines or used for field testing related to viruses in poultry.The chickens were not vaccinated in their maintenance.The productivity of laying hens can be disrupted by various factors, one of which is disease.Diseases in laying hens can reduce the quality and quantity of eggs, even causing death.Therefore, efforts are needed to increase the immunity or body resistance of laying hens in order to prevent and overcome disease.One way to increase the immunity of White Leghorn laying hens is by providing feed that contains sufficient and balanced minerals.Minerals are inorganic substances that play a role in various metabolic processes, including in the immune system.Some minerals that are important for the immunity of laying hens include zinc (Zn), copper (Cu), iron (Fe), selenium (Se), and cobalt (Co).These minerals can function as antioxidants, coenzymes, cofactors, and components of immune cells.For this reason, additional feed ingredients are needed, especially minerals needed for metabolic processes and to increase the resistance of chickens to the surrounding environment [3][4][5][6].One of the materials that can be used is organic minerals (premix).
The level of immunity can be seen from the blood variables in the form of complete leukocytes and leukocyte differentials.Leukocytes are cells that play a role in the body's defense system which is very responsive to infectious agents.Leukocytes function to protect the body against various diseases by phagocytes and produce antibodies [7].The ability of body resistance in poultry can be seen through the weight of lymphoid organs (bursa, thymus and spleen) and the ratio of heterophil lymphocytes (ratio H/L).Bursa functions as a forming antibody.The more often the bursa forms antibodies, the depletion and shrinkage of lymphoid follicles is followed by a decrease in the number of lymphocytes, so that the antibodies produced are low.A decrease in the number of lymphocytes results in an increase in the H/L ratio and vice versa [8].However, giving minerals to laying hens' feed must be done carefully, because a deficiency or excess of minerals can have a negative effect on the health and productivity of laying hens.Therefore, it is necessary to know the optimal dose and the right proportion of these minerals in the feed of laying hens.

Materials and Methods
The animal experiments in this study were carried out with the approval of the Ethical Clearance Committee of the Wates Veterinary Center (BBVet Wates), Ethical Clearance No: 001/KE/BBVet/VII/2022.

Materials
The research tools used included chicken rearing cages with a litter system in the form of husks, analytical scales, tools used for feed proximate testing, tools used for mineral testing, tools of deferential leucocyte, scales, tools for necropsy.Seventy-two white leghorn chickens consisting of 1 male and 7 females aged 43 weeks, mineral premix, production chicken feed, and cages in the Experimental Animal Cage Installation (IKHP) at BBVet Wates Yogyakarta.Mineral premix, chicken feed production, ingredients used for feed proximate test, materials used for mineral tests, materials used for hematology tests.

Methods
The composition of nutrients in the feed listed in Table 1 and Table 2 is the result of feed analysis from the Clinical Pathology Laboratory of the Wates Veterinary Center and the Center for Quality Testing and Feed Certification, Bekasi (2022).The method used in this study was an experiment using a one-way Completely Randomized Design (CRD) consisting of three treatments and three replications, each replication consisting of eight chickens.The treatments given were: Commercial feed without feed additives (control), commercial feed with 0.25% additional premix, commercial feed with 0.5% additional premix

Animal preparation and placement.
A total of 72 laying hens of the White Leghorn strain were reared for 49 days.Chickens were divided into three groups (24 individuals each) based on the treatment given.Furthermore, in each treatment group, the chickens were further divided into three sub-groups, each tail, according to the number of treatments.So, each treatment consisted of three groups as replicates and each replicate consisted of eight chickens.Each cage is equipped with a place to feed, lay eggs and drinking water.The rations were given ad libitum according to the treatment.

Blood hematology examination.
The fourth week before to surgery, the inner wing was cleaned with alcohol, and blood samples were obtained by injecting a syringe into the brachial vein.These samples were used to determine the heterophil-lymphocyte ratio.One milliliter (ml) of blood is drawn, placed in a tube that has been treated with EDTA, an anticoagulant, to prevent blood clots, then stored in a colling box and subjected to a manual deferential leucocyte analysis.

Observation of Lymphoid
Organs.Chickens are decapitated (slaughtered) and then dissected the carcass (necropsy) to remove lymph and thymus organs, and histopathology that leads to AI disease.Lymphoid organs including the spleen and thymus are obtained from chickens that have been decapitated (slaughtered) and then carefully and cleanly separated from meat and fat.Lymphoid organ weights were weighed using an analytical balance.

Parameters.
The variables measured in this study were immunity: Hematology and Lymphoid organ.

Statistical Analysis
The research data were analyzed using a one-way design and if there were differences between treatments, a further test was carried out in the form of Duncan's Multiple Range Test (DMRT).

Blood hematology based on the observations.
The data obtained for lymphocytes, monocytes, heterophiles and eosinophiles were presented in Table 3.Based on Table 3 it was known that the use of premix minerals in the rations did not significantly affect the values of lymphocytes, monocytes and heterophils, but significantly different for the values of eosinophiles (p <0.05).
The lymphocyte percentage obtained was within the range of 79.6-82.3%, and these outcomes were normal.According to Smith and Mangkoewidjojo, the proportion of lymphocytes in the body typically ranges from 24 to 84 percent [9].Lymphocytes are responsible for responding to antigens and stress by increasing the circulation of antibodies in the development of the immune system [10,11].Monocyte values in the study did not show significant differences between treatments.The percentage of monocytes obtained in this study ranged from 1.4-2.1% which was included in the low category.Eroschenko states that the normal limit for monocyte values in chicken blood is 3-10% [12].Monocytes are a distinct type of white blood cell that are part of the agranulocyte group.They develop in the bone marrow and mature once they are released into the bloodstream, turning into macrophages that can penetrate tissues.When inflammation and the immune response happen, according to Frankson et al., monocytes can phagocytose 100 harmful bacterial cells and turn into a regulatory system [13].The second line of defense against inflammation is made up of monocytes and heterophils, which are mobilized together.The study's low monocyte value suggested that heat stress had a negative impact on chickens' ability to create monocytes.The environment and stress were found to have the greatest effects on the number of lymphocytes and monocytes, according to Puvadolpirod and Thaxton.This is because heat stress reduced the weight of the thymus lymphoid organs and bursa of fabric, which in turn reduced the number of lymphocytes and monocytes [14].The percentage of heterophiles did not show significant differences between treatments.
The percentage of heterophils in the blood of chickens from the study was in the range of 15.2-15.8%,which is slightly below the standard.Hendro et al. stated that the normal percentage of heterophils in chicken blood is in the range of 20-40% [15].Heterophils are part of the leukocytes that belong to the granulocyte group and are on the front line (first line) which functions as the initial defense against diseases that can cause infection or inflammation [16].He et al. reported that heterophiles contain anti-microbial substances which are associated with disease resistance in the body and are influenced by the genetic control of these livestock [17] .Factors that determine the level of heterophile include environmental conditions, stress levels in livestock, genetics and nutritional adequacy of feed [14].
The percentage of eosinophiles was significantly different between treatments (p<0.05).The best eosinophil value was in the 0.5% treatment using mineral premix in the ration, namely at 3.4%.This value is still considered standard.Jain and Carrol stated that in the normal range the number of eosinophils is 2-8% of the total white blood cells and can survive 3-5 days.Eosinophils are part of the differential leukocytes formed in the spinal cord which function as parasitic, inflammatory and allergic responses [24].Lokapirnasari and Yulianto stated that eosinophils have two main functions, namely being able to attack and destroy pathogenic bacteria and being able to produce enzymes that can neutralize inflammatory factors [18].In preventing the entry of infection in the body, eosinophils work with chemical functions enzymatically.This is in accordance with the opinion of Moyes & Schute and Isroli et al. which states that eosinophils perform an immune function against microorganisms in the same way as a chemical function, namely enzymatically [19,20].

Lymphoid organ weight
Based on the results of the study, data on body weight, thymus and spleen were obtained as presented in Table 8.The results showed that the treatment given did not affect the weight of the thymus and spleen.Likewise, the results of the percentage weight of thymus and spleen were not affected by the treatment given.The weight percentage of these immune organs is still below the normal weight range of the spleen, which is 0.18-0.23%and the thymus is 0.1% and the thymus is below 0.18% -0.23% [21,22].The weight and percentage of organ weights that were still below the normal range indicated that the study chickens were reared in an environment that was still experiencing heat stress which caused the release of the hormone corticosterone into the blood vessels to help the chicken's metabolism.The hormone corticosterone causes failure of mediation cells and humoral immunity due to changes in lymphoid concentration which can reduce the weight of the immune system, spleen, and bursa of Fabricius [23].Chickens' immune systems can be either non-specific (natural) or specific (adaptive).The non-specific immune system is the body's naturally acquired immune system, and it offers only moderate protection.The immune system will eliminate any disease agent that enters the body, making the protection offered universal.The specific immune system consists of a cell-mediated system and an antibody-mediated immune system.Immunity organs consist of primary immunity organs (bursa fabricius and thymus) and secondary immunity organs (lymph).Indicators of body resistance as a form of chicken response to factors that cause stress can be determined from blood components such as the heterophil lymphocyte ratio (H/L).Blood is a circulatory system in the body which has the function of transporting nutrients and defending the body against foreign objects [25].The H/L ratio is the easiest stress indicator to detect early.The higher the ratio number, the higher the level of stress as a form of stress in poultry [26].

Ratio H/L
The research results show that the H/L ratio value is 0.20.This means that the chickens in the study were not under stress.According to Emadi and Kermanshahi [27] the level of body resistance in poultry can be determined by the H/L ratio value, around 0.2 (low), 0.5 (normal) and 0.8 (high) to environmental adaptation.Based on the research results, it shows that there is no real influence between treatments on the H/L ratio value, this shows that the use of 0.5% mineral premix in feed does not cause stress in chickens.

Conclusion
The addition of mineral premix in feed up to 0.50% has not been able to increase the immunity but keep eosinophils within standard range.

Table 1 .
Mineral Composition in Premix

Table 2 .
Feed Nutrient Composition

Table 4 .
Body Weight, Thymus and Lymph Organs