Time, site and Broodfish preparation
The study was conducted from February 2017 to January 2018, at the hatchery of Research Institute for Ornamental Fish Culture, Depok, West Java Province, Indonesia. Forty mature males of botia with total body weight of 40 - 80 g were acclimated to laboratory conditions and fed on a commercial diet in 45 × 30 × 30 cm plastic containers for 15 days. Then the fish were grouped into four groups with average initial weights of 60.47 ± 10.34 g, and stocked in six plastic containers (45 x 30 x 30 cm). Each box was equipped with continuous aeration and a black plastic mesh lid to minimize disturbance and prevent fish from jumping out. Six experimental groups were assigned to four replicates in a completely randomised design. Fish were fed once daily to satiation at 09:00 AM.
Health Research Ethics Committee Faculty of Medicine Univerity of Indonesia Cipto Mangunkusumo Hospital approved the study. Ethical approval number: KET-919/UN2.F1/ETIK/PPM.00.02/2019
Preparation extender and cryoprotectant
The Ringer’s solution and honey were used as extender and cryoprotectant, respectively. A stock of Ringers solution was prepared by dissolving 3.25 g NaCl; 0.125 g KCl; 0.175 g CaCl2.2H2O; and 0.1 g NaHCO3 in distilled water up to 500 ml, and the solution was kept at 4 °C . The pure honey was purchased from local market. Six concentrations of honey were tested, namely 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%, for these purposes, the respective volume of honey of 0, 0.1, 0.3, 0.5, 0.7, and 0.9 ml were added into Fish Ringer’s solution up to 100 ml .
Preparation of activator and eosin-Y solutions
The activator solution was prepared by diluting 0.263 g NaCl; 0.037 g KCl and 0.363 g Tris-HCl with aquabidest up to 100 ml. The solution was kept at 4 ℃ prior to use in the experiment . The 0.5% of eosin-Y solution was prepared by diluting 0.5 g of the eosin-Y with distilled aquabidest up to 100 ml.
Four males weighing 60.47 ± 10.34 g were treated intramuscularly with Ovaprim (Syndel Laboratories Ltd. Nanaimo, Canada) at dosage of 0.2 ml kg-1 body weight. After 18 h, sperms were collected from individual male donors by a gentle abdominal stripping method  and placed in 2 mL vials (Cryogenic storage vial, Nalgene Nunc International).
Fresh sperm was suspended in the diluent mixtures containing Ringers solution, 10% methanol, and the respective honey solution where applicable (Table 1). The composition of the solution was modified from . The dilution ratio of the fresh sperm and diluent solution was 1:9 based on Sunarma et al. . The compositions of each component of the diluent solution and the ejaculated sperm are presented in Table 1.
Equilibration, freezing and thawing
The diluted sperm was equilibrated at 4 − 5 °C in an ice box for 15 min then frozen at −80 ℃ in freezer for 48 h. Thereafter, the frozen sperm was thawed at 40 °C for 10 min in a water batch .
Sperm quality evaluation.
The fresh sperm was evaluated for colour and pH. The preserved sperm was analyzed for motility, viability, and abnormality rates using a Boeco Trinocular Microscope (Boeco, Germany) equipped with a digital eyepiece camera (MDCE-5a). The microscope was connected to a computer equipped with an image driving software (Scopephoto 2.0.4).
Egg Collection fertilization
The eggs were collected from the mature female by gentle abdominal pressure then put in the plastic basin and kept at 5 ℃ prior to use for fertilization. A total of 2 ml of eggs were mixed with 0.6 ml of thawed sperm (1:3 v/v) and two drops of tap water was added then mixed with a soft feather and left in contact for 5 min. A total of 100 eggs were taken randomly and incubated in a plastic basin. The fertilization rate was observed two hours after incubation. The fertilized egg was transparent, while the unfertilized egg was opaque. The fertilization rate was calculated using the following formula: Fertilization rate (%) = Fertilized eggs/ Total number of incubated eggs x 100 .
The percentage data were arcsine transformed prior to analysis . The data of sperm motility, viability and fertilization were analyzed using one-way ANOVA then followed by the Duncan's multiple range test to determine the best treatment. The analysis was conducted using SPSS 14. (SPSS, Chicago, IL, USA). The qualitative data such as semen color, volume, pH, and spermatozoa abnormality were analyzed descriptively.