Selection of breeding bulls:
100% Holstein Friesian (HF) and Sahiwal (SW) pure breeding bulls have been imported and maintained in American dairy Limited, Vagnahati, Sreepur, Gazipur. Among them, three Holstein Friesian and three Sahiwal breeding bulls were selected on the basis of age, body weight and scrotal circumference (SC) and used for this research. The ages of these breeding bulls ranged from 4 to 6 years, bodyweight from 1048 to 1134 kg and scrotal circumference from 34 to 37 cm for HF bulls. In case of SW bulls, age ranged from 4 to 5 years, body weight 600 to 645 kg and SC was 34.5 to 37.0 cm, respectively. Bulls were maintained in intensive farming conditions and supplied with a balanced ration comprising concentrate and roughage depending on their body weight. Fresh water was made available all the time.
Preparation of diluter:
Egg yolk-citrate (EYC) diluter was prepared at previous night of collection of semen. It was a solution of 2.94% sodium citrate in 100 ml of distilled water and in which 100000 IU of penicillin and 100000 μg of streptomycin were added. One parts of egg by volume was added with three parts of the sodium citrate solution. It was stored in proper temperature and used after overnight preservation.
Semen collection and evaluation of fresh and diluted semen:
Semen was collected by means of the artificial vagina (AV) method once in a week. Ejaculate volume was measured using a graduated collection vial tube in milliliter. The concentration of spermatozoa was determined by Density Spectrophotometer (SDM-6, Minitübe, Germany). Fresh semen was diluted in cuvettes with 0.9% sodium chloride solution at the ratio of 1:100. The reading was recorded from the Density Spectrophotometer in million/ml. After that fresh semen was diluted with prepared EYC diluter. Progressive motility, fast motility, slow motility, local motility and immotility of diluted semen were measured by using Computer Assisted Semen Analysis (CASA) system (Andro Vision AX10, Minitübe, Germany).
Processing for frozen semen production:
Diluted semen was filled with an automatic filling, sealing and printing machine (MPP Quattro, Minitube, Germany) that could fill 4 straws at a time. For equilibration, semen straws were decorated in the racks and placed in the cold handling cabinets named cooling castle maintained at 4-5º c for 3-4 hours. Semen straw was transferred from cold handling cabinets to Bio freezer (TurboFreezer L, Minitube, Germany). After keeping in the chamber of the device, the temperature was controlled by programmable software. Temperature curves and data of each freezing cycle could be stored, displayed and transferred into excel for further analysis. Subsequent freezing cycles started immediately after the previous freezing. The unidirectional nitrogen gas flow in the freezer ensured that all straws passed through identical freezing curves. After freezing one (1) or two (2) straw were taken per batch and analyzed to know the status of progressive, fast, slow, local motility and immotility of the spermatozoa by using CASA system and then transferred to large cryocan maintained at -196º c for preservation.
Artificial insemination and fertility measurement:
Artificial insemination (AI) was performed in different areas of Mymensingh districts using the frozen-thawed semen of pure Holstein Friesian and Sahiwal breeding bulls. Bull fertility was measured in terms of Non-return rate (NRR). NRR was recorded by the percentage of cows that were not returned to estrus at 60 days after AI and calculated in percentage using the following formula. Non-return rate (%) = {(Cows not returned to estrus within 60 days) / (Total number of cows inseminated)} x 100.
Statistical analysis:
The data obtained from the experiment were entered in Microsoft Excel worksheet, organized and processed for further analysis. Analysis was performed with the help of the Statistical Analysis System (SAS, 2009) computer package.