All procedures were approved by the Department of Surgery and Obstetrics (DSO), Bangladesh Agricultural University’s (BAU) Animal Experimental Ethics Committee (AEEC/DSO-BAU/02/2015). The study was carried out from July 2016 to June 2017 in the Laboratory of Theriogenology, DSO, BAU, Mymensingh- 2202. All chemicals used in this study were purchased from Sigma (St. Louis, MO, USA) and Merck (Darmstadt, Germany). Ten Bangladeshi rams (Wera) aged 2.5-3 years old, bodyweight 26-32 kg, scrotal circumference 20-24 cm belonged to Sheep Research Farm, DSO, BAU were used as semen donors (Jha et al, 2018). Health management like; deworming (internal and external parasites), vaccinated (Tetanus, Foot and Mouth Disease, Peste des Petits Ruminants) and, husbandry management like; feeding, grazing, watering and housing remained as routinely done in Sheep Research Farm (Jha et al, 2018). A factorial design (2×3) was used to observe the effects of freezing methods and diluents types, as pooling effects (freezing method or diluents) on post-thaw sperm parameters. Eight ejaculates were used for each treatment combination. Two semen straws from each experimental batch were tested for sperm quality. Semen was collected routinely twice a week. An artificial vagina, specially designed for sheep and goats (Minitube, Germany) was used for semen collection (Jha et al, 2018). Semen was evaluated at 35°C for semen volume, sperm motility (SM), concentration (SC; 106/mL), viability (SV), plasma membrane integrity (SPMI), and acrosome integrity (SAI) (Jha et al, 2019). Briefly, SC was calculated by fixing the semen sample (sperm cells) in buffered formal saline (1:200) and counting onto haemocytometer (Neubauer counting chamber) at 400×. The SM was estimated by placing 5 µL semen and coverslip at 100×). The SV was estimated using nigrosine-eosin staining technique at 400×.Sperm cells counted as alive that ignored stain and dead that stain eosin. The SPMI was estimated using the hypo-osmotic swelling test (HOST) technique at 400×. Sperm with swollen and coiled tails were recorded as intact plasma membrane integrity. Acrosomal integrity was evaluated by fixing the sperm cells in buffered formal saline (1:10) and observed the presence or absence of a normal apical ridge of the sperm head at 1000×. At least 200 spermatozoa were examined from each smear. Three diluents, two hand-made (Tris-based) and one commercial (Triladyl®, Minitube, Germany) were used for semen dilution. The hand-made tris-based diluents were prepared in two fraction procedures: Fraction- A (without glycerol) and Fraction-B (with glycerol) according to Jha et al. (2019). Briefly, two hand-made tris- based diluents: D1 (7% glycerol) and D2 (5% glycerol) consists of tris 3.63g, fructose 0.5g, citric acid 1.99g, yolk 20 mL, deionized water, penicillin G (sodium salt) 100,000 IU and dihydrostreptomycin sulphate 100 mg to prepare 100 mL final volume. Similarly, the commercial diluent; (Triladyl®; D3) was prepared by adding 1 volume Triladyl®, 3 volumes of deionized water and 1 volume of egg yolk. The diluents were prepared on the day of semen collection at room temperature and stored at 5°C. Only ejaculates with volume ≥ 0.5 mL, SM ≥ 80%, SV ≥ 90%, SC ≥ 2500×106 spermatozoa/mL, SPMI ≥ 85% and SAI ≥ 90% were considered for processing. Semen samples were diluted using two steps for D1 and D2, and one step for D3 to obtain a final sperm concentration of 800×106 spermatozoa/mL (Jha et al, 2019). In two-steps dilution, the calculated volume of diluents fraction-A was added to the semen at 35°C. The diluted semen sample (semen + Fraction-A) was allowed to cool down to 5°C for 120 min (-0.25°C/min). The second dilution was carried out with the remaining calculated volume of diluents Fraction-B (previously cooled to 5°C). The diluted semen sample (semen + Fraction-A + Fraction-B) was further maintained at 5°C for 120 min for temperature equilibration. In one-step dilution, the calculated volume of diluents was added to the semen sample as a whole fraction at 35°C and maintained at 5°C for 240 min for temperature equilibration. Finally, the diluted semen sample was aspirated into 0.25 mL French mini straws and sealed with polyvinyl alcohol. The semen straws were frozen in liquid nitrogen (LN2) vapour using two methods: F1 [manually in Styrofoam box, using three-steps method (Jha et al, 2019); where the semen straws were placed 9 cm above the surface of LN2 for 7.5 min (+5°C to -80°C at 11.33°C/min), 7 cm for 1.5 min (-80°C to -120°C at 26.66°C/min), and 5 cm for 1.5 min (-120°C to -140°C at 13.33°C/min)] and F2 [programmable bio-freezer (Freeze Control CL-3300 System; Cryologic Pty. Ltd., Australia) using two-steps method (García-Álvarez et al., 2010); where the semen straws were left for 5.25 min (+5°C to -100°C at -20°C/min) and for 4 min (-100°C to -140°C at -10°C/min)]. Finally, the straws were plunged into the LN2 (-196°C). Two semen straws from each batch were evaluated (37°C for 20 sec) for sperm parameters. All data obtained from the study were analyzed using SPSS (20.0 Version) statistical software. Analysis of variance (ANOVA) was carried to observe the pooling effects (freezing method or diluents) on sperm parameters. When ANOVA revealed a significant effect, the values were compared by Duncan’s multiple range test (DMRT). To compare the pooling effects between the freezing methods on sperm parameters, independent samples t-test was applied. Differences were considered to be statistically significant at P < 0.05.