INTRODUCTION
MATERIALS AND METHODS
Preparation of B. subtilis TLRI 211-1
Experimental animals and treatment
Measurements
Body weight was measured at the beginning and end of the experiment to determine the weight change of hens.
Egg weight was measured two days a week and feed consumption was recorded every week. The number of eggs produced, and abnormal egg count were recorded every day. The hen-day egg production, egg mass and feed conversion ratio were calculated as follows:
-
Egg quality: Twelve eggs were collected per group every four weeks for measurement of eggshell quality, egg quality and Haugh unit using Digital Egg Tester, DET6500 (Nabel Co., Ltd. Kyoto, Japan).
Eggshell quality determination included eggshell strength, eggshell thickness, eggshell weight, and eggshell strength. Egg quality included yolk percentage, yolk color, yolk height, yolk diameter and albumen height.
Eggshell thickness determination: A piece of eggshell at the blunt end, tip and equatorial part of the egg were taken and measured using micro measuring instrument to a decimal point of 3 digits. The average of the three measurements was the thickness of eggshell [18].
After the eggs were weighed, the eggshell, yolk and albumen weight were taken separately, and the eggshell weight, yolk weight and albumen weight were calculated as the percentage of egg weight.
Blood characteristics: Eight hens from each group were randomly selected for blood test every four weeks. Five mL of blood samples were taken from the wing vein. Serum was separated by the Centrifugal Separator (1,700×g, 15 min) and stored in a −20°C freezer for analysis. Immunoglobulin (IgA, IgG, and IgM) were determined using chicken ELISA quantitation set (Bethyl Laboratories Inc., Montgomery, TX, USA). Blood glucose, bilirubin, creatinine, uric acid, urea nitrogen, creatine phosphatase, glutamate oxaloacetate transaminase (GOT), pyruvate transaminase (GPT), total protein, albumen, alkaline phosphatase, total cholesterol, triglycerides, high density lipoprotein (HDL), low density lipoprotein (LDL), inorganic phosphorus, and amylase activity were analyzed using automatic analyzer (Hitachi 7176A; Tokyo, Japan) [19].
Determination of egg quality during egg storage: At the end of the experiment, 48 eggs were selected from each treatment group and stored at room temperature (23°C to 30°C) for 4 weeks. Twelve eggs from each group after 2, 3, and 4 weeks of storage were selected for the determination of egg quality and Haugh unit as the above egg quality determination method.
Determination of the odor of laying hen excreta: At the end of the experiment, 2 replicates of fresh feces were taken from each group, 10 hens per replicate. Each excreta sample was mixed well and 5 g were placed in a 500 mL Erlenmeyer flask, and the bottle sealed with a parafilm for 5 min. One hundred mL of gas was suctioned with a gas sampler (Kitagawa AP-20) from 5 cm above the bottom of the bottle and poured into the ammonia detection tube (Kitagawa Gas Detector Tube Ammonia 105SC or 105SD) for determination of ammonia concentration. Each sample was conducted 3 replicate tests.
Approximate nutrient contents of experimental diets: Diet samples were sent and analyzed at the Feed Analysis Center, Animal Nutrition Division, TLRI using AOAC [20] methods.