Birds & Experimental Diet
Abstract
This experiment was conducted to evaluate the effects of dietary Salvia mirzayanii (SM) supplementation on immune responses, blood factors and meat quality in broilers. A total of 200, one-day-old broiler chicks were fed five experimental diets containing different levels of SM (0, 0.25, 0.50, 0.75 and 1 percent of diet) until 42 days of age. Antibody titer against sheep red blood cells in 0.50% and 0.75% treatments was greater than control (P < 0.05). Antibody titer against Newcastle disease virus in 0.50% and 0.75% treatments was greater than control (P < 0.05).
Feeding with SM at levels of 0.50% and higher increased skin thickness after challenging with dinitrochlorobenzene (P < 0.05). The greatest bursa of Fabricius relative weight was observed in 0.50% treatment (P < 0.05). Broilers fed SM leaf powder had higher serum triglyceride and cholesterol when compared with those birds in control, however, serum glucose decreased when birds fed SM greater than 0.50% in diet (P < 0.05). Using 0.25, 0.50 and 0.75 percent of SM in diet decreased meat malondialdehyde concentration compared with control (P < 0.05). Using 0.50% SM in diet of broilers could improve humoral and cellular immune responses, and enhance meat quality.Key words: Salvia mirzayanii, immune response, albumin, meat quality, broilers.
Introduction
Remaining of growth-promoting antibiotics in poultry products for human consumption has challenged the use of these compounds in the diet of broiler chickens. Considering that the prohibition of the use of growth-promoting antibiotics leads to a decline in the production and yield of food, many researches has been held to find suitable alternatives for them. Medicinal plants have played a major role in preserving the health and improving the quality of life of humans for thousands of years and are among the ingredients introduced as alternatives to growth-promoting antibiotics in poultry. The usefulness of many medicinal plants has been reported to improve blood metabolites and to enhance immune system.
Mint family has a great importance among the medicinal plants. Salvia mirzayanii (SM) is one of the plants of this family, whose habitat is warm and semiarid desert climate in southern Iran. The most important compounds in the essential oil of SM are cineole, linalool, alpha pinene, carvacrol, thymol, linalyl acetate, spathulenol, delta-cadinene, alpha-terpinyl acetate, alpha-cadinol, alpha-terpineol, beta-eudesmol, cubenol and eucalyptol. The properties of boosting immune system, improving blood factors and improving the quality of meat have been reported with the use of medicinal plants of the mint family. However, studies on the properties of SM medicinal plant and the way it works are very limited. Therefore, this study was carried out to evaluate the effect of using SM leaf powder in the diet on immune response, blood factors and meat oxidation of broiler chicken.
Birds and experimental diet
Salvia mirzayanii leaves were manually harvested in the Fasa region of Fars province, Iran in late May, before the flowering stage of the plant. The fresh plant leaves were dried for five days in a dark room with good ventilation, relative humidity of 40% and temperature of 28° C and then they were ground. The resulting leaf powder contained 10% moisture content, determined in four replicates using an oven of 105 °C (11).Two hundred one-day old Ross 308 broiler chicks were distributed in a completely randomized design into five treatments and four replicates with 10 chicks per replicate. The basal diet was adjusted for the Starter (1-10 d), grower (11-24 d) and finisher (25-42 d) periods based on recommended dietary requirements for broiler chickens (Table 1). Five SM diets (0, 0.25, 0.50, 0.75 and 1 percent of diet) were used in this experiment and different levels of dried SM leaves were added to the basal diet at the expense of corn starch.
Humoral and cellular immunity
At the age of 17 and 27 days, three birds from each replicate were injected through a wing vein with a volume of 0.2 mL of 1% sheep red blood cells’ suspension washed in a sterile phosphate buffer. After 15 days of the second injection, 2 mL of blood was taken from the bird’s wing vein and blood serum samples was separated by centrifuging for 10 minutes at a speed of 1500 rpm and kept frozen at a temperature of -20° C until the time of evaluating antibody titer. Antibody titers against sheep red blood cells were performed by using agglutination test using U-shaped 96 well microplates. Newcastle disease vaccine (B1 Strain) was given at the age of 7 days, Newcastle disease vaccine (LaSota Strain) was given at the age of 21 days, and infectious bronchitis vaccine H-120 for poultry was given at the age of 14 days via eye drop. At the age of 42 days, blood was taken from the wing vein of three birds from each experimental group. After the separation of serum from the blood clot, the generated antibody titer against Newcastle virus and bronchitis vaccines was measured by the HI method. On day 42, three birds with weight close to the average weight of the pen were selected from each replicate, weighed and killed.
After defeathering, the bursa of Fabricius was separated and its weight was measured by a digital scale with a precision of 0.001 g and reported as a percentage of live weight.At the age of 40 days, three birds from each experimental unit were labeled with different colors by skin-ditching with 0.1 mL of dinitrochlorobenzene 0.1% (containing 1 mg of dinitrochlorobenzene in 1 mL of Acetone and olive oil mixture by a ratio of 4:1). In this method, a region with relatively no feather with an approximate area of 10 cm2 was selected on the right side of the body for ditching with dinitrochlorobenzene. In order to evaluate the reaction rate, the skin thickness was measured with an electronic micrometer with a precision of 0.01 mm before ditching and 12, 24, and 48 hours after ditching. The average increase in skin thickness in each bird was obtained from the difference in thickness before and after each ditching. Each measured number, which was an average of 3 replicates of the considered area, was considered as the average of each bird within each experimental unit.
Blood factors
At the age of 42 days, three birds were randomly selected from each experimental unit, and 2 mL of blood was taken from their wing vein. After serum separation, the levels of triglyceride, cholesterol, total protein, albumin and glucose were determined using Pars Azmun (Tehran, Iran) biochemical kits and by spectrophotometric method (UNIKON 933, Kontron Co. Ltd., Milan, Italy).2.4. Meat oxidation1 g of chicken breast meat were homogenized in the presence of 5 mL of aqueous solution with 5% of Trichloroacetic Acid (Merck, Darmstadt, Germany) as well as 5 mL of BHT solution in hexane (Merck) at a concentration of 0.8 g per 100 mL.
The resulting mixture was then centrifuged at 3000 rpm for 10 minutes, and after dispensing the upper layer, 2.5 mL of the lower layer was mixed with 1.5 mL of aqueous solution of 2-Thiobarbituric Acid (Merck) at a concentration of 0.8 g per 100 mL, and the resulting mixture was incubated for 30 minutes in a warm-water bath of 70° C. The optical absorption of the resulting mixture after cooling was observed in a wavelength of 532 nm, and the amount of malondialdehyde was calculated using the standard material 1, 1, 3, and 3-Tetraethoxypropane (Merck, Hohenbrunn, Germany) in terms of μg/g of meat.
Statistical analysis
Data analysis was performed using the SAS software, and averages of characteristics were compared using Duncan test (DMRT). The analysis of the averages related to skin thickness increase in ditching with dinitrochlorobenzene was performed by the repeated measurement method. A significant difference level between treatments’ average was considered to be 5%.3.
Results. Immune response
Experimental treatments significantly influenced the antibody titer produced against sheep red blood cells, Newcastle disease and bronchitis viruses at age of 42 days (Table 2). Antibody titers against sheep red blood cells in birds fed with levels of 0.50% and 0.75% of SM were higher than control birds (P < 0.05) and 1% treatment was not different from 0.50% and 0.75% treatments. The highest antibody titer against Newcastle virus was also observed in birds fed with 0.50% and 0.75% treatments of SM, which was higher than antibody titers in control birds (P < 0.05). Antibody titers against bronchitis virus was greater in the SM groups than in the control group (P < 0.05). The use of SM leaf powder in the diet at a level of 0.50% and higher resulted in increased skin thickening due to the ditching with dinitrochlorobenzene (P < 0.05). The effect of experimental treatments on the relative weight of spleen was not significant (p = 0.856). The relative weight of the bursa of Fabricius was affected by experimental treatments, so that the highest relative weight of the bursa of Fabricius was observed in birds fed with 0.50% of SM treatment.
Blood Factors
The use of SM leaf powder in the diet increased triglyceride and blood serum cholesterol levels in birds compared to control birds (P < 0.05). Total protein in blood serum of chickens fed with a diet containing a level of 0.50% of SM was higher than that of the control group (P < 0.05). Consuming diets containing 0.25%, 0.50% and 0.75% of SM increased serum albumin in comparison with the control group (P < 0.05). Consuming SM leaf powder in the diet at a level of 0.50% and above reduced blood glucose levels significantly in comparison with the control group (P < 0.05).3.3. Meat oxidationThe experimental treatments had a significant effect on the level of malondialdehyde (MDA) in broiler chicken meat after 60 days of storage at -20° C (Table 5). Broiler chickens fed with levels of 0.25%, 0.50% and 0.75% of SM had a lower MDA level in their meat in comparison to control birds (P < 0.05).
Discussion
The results of this research show that the use of 0.50% of SM leaf powder in the diet of broiler chickens improves humoral and cellular immune response. Improvement of immune system response by medicinal plants is attributed to various mechanisms. One of these mechanisms is attributed to the antioxidant properties of medicinal plants that prevent oxidative damage to cells. It should be noted that in the present study, the lowest malondialdehyde concentration (oxidation index) and the highest immune system response were observed in the level of 0.50% of SM leaf powder. Another mechanism of boosting immune system due to the consumption of medicinal plants has been reported to changing the microbial ecosystem of the digestive system in favor of beneficial bacteria. In a study by Bagherzadeh-Kasmani et al., an increase in the population of lactic acid bacteria and a decrease in the population of whole-form bacteria was observed with the addition of 0.50% of SM leaf powder to the diet of broiler chickens. In agreement with these results, Awaad et al. and Mehri et al. reported that birds fed with mint had better immune responses than chickens of control groups.
The results of the present experiment show that the use of SM leaf powder increases triglyceride and serum cholesterol but decreases serum glucose level. Alpha-amylase is one of the key enzymes in the digestion of carbohydrates, which causes starch to break down into simple sugars. Inhibition of this enzyme reduces the digestion of carbohydrates and, consequently, reduces the absorption level of glucose from the digestive system. It has been shown that SM decreases the activity of this enzyme. A higher concentration of serum glucose is associated with the expression of SREBP-1c gen in skeletal muscles and the increase of lipogenesis. In fact, reducing serum glucose concentration due to the consumption of SM reduces the process of lipogenesis in skeletal muscles, which is associated with higher levels of these compounds in the serum due to the reduction in the consumption of triglyceride and serum cholesterol in this process.
The power of phenolic compounds of medicinal plants in improving the oxidative stability of meat has been proved in numerous studies. In a report by Farag et al., the high level of antioxidant activity of thymol was due to the presence of hydrogen supplying groups to the radicals generated during the first phase of oxidation of OH-phenolic lipids, resulting in a delay in the formation of hydrogen peroxide. Linalool and alpha terpineol in SM may also help prevent oxidative damage such as lipid peroxidation. The antiperoxidative activity of these flavonoids depends on the concentration and number of hydroxyl groups in their structure. According to the results of this experiment, the use of SM leaf powder at a level of 0.50% in broiler chicken diets can be recommended, because it improves the humoral and cellular immune response, increases the amount of total protein and serum albumin and reduces the amount of meat oxidation.
