BBA Mahmuda
Department of Surgery and Obstetrics, Bangladesh Agriculture University , Mymensingh 2202
Azizunnesa
Department of Medicine and Surgery, Faculty of Veterinary Medicine, Chittagong Veterinary and Animal Science University, Khulsi 4202, Chittagong , Bangladesh
BF Zohara
Department of Surgery and Obstetrics, Bangladesh Agriculture University , Mymensingh 2202
MGS Alam
Department of Surgery and Obstetrics, Bangladesh Agriculture University , Mymensingh 2202
FY Bari
Department of Surgery and Obstetrics, Bangladesh Agriculture University , Mymensingh 2202
Frozen semen, Preservation time, Quality, Ram
Laboratory of the Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University
Postharvest and Agro-processing
The work was conducted at the Reproduction Laboratory of the Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University during the period from November 2013 -October 2014 . Four rams were selected from parent flock of BAS -USDA -PAULS funded research project. Age, body weight, and scrotal circumference of the ram were 4 years, 20 to 22 kg and 20 to 24 cm, respectively. They were allowed 6 to 7 hours natural grazing . Each ram was fed approximately 300gm (Maize grit, wheat bran, wheat polish with salt) daily. The rams were dewormed routinely . Stock solution for tris -fructose-citrate diluent was prepared by dissolving tris (3.63 gm), fructose (0.5 g) and citric acid (1.99 g) in up to 76 ml distilled water. The stock solution was preserved at 4- 7°C for maximum 2 weeks. At the day of collection, fresh well churned egg yolk (10%) and glycerol (7%) were added with the stock solution to make 10 ml of complete medium that was part B diluent and part A containing egg yolk without glycerol. In part of A diluent, distilled water of 7% was added instead of glycerol. Semen was collected once in a week using AV method. After collection, semen was kept at 37°C in water -bath until the media and reagents were added with it. The individual ejaculate was evaluated for volume, colour, density, mass activity, concentration, motility and morphology. Two-step dilution method was used to freeze the semen in this experiment. Before semen collection, the final diluent was prepared and divided into two parts (part A and part B) and placed in the water bath at 37°C. After collection, individual ejaculate was diluted with calculated amount of diluent part A. One drop of semen was placed in a warm slide and the sperm motility was recorded. After that, diluted semen and diluent B part were transferred to refrigerator for two hours. After two hours, calculated amount of diluent B was poured into the previous diluted semen in three divided parts. Then , the semen was filled into the straws using micropipette. After filling, the straws were sealed by sealer. For equilibration, the sealed straws were placed in the refrigerator at 4°C for further 2 hours. After two hours, the motility was recorded by using one drop of semen placed on the previously warmed slide under the microscope. Liquid nitrogen was poured in a special box where rack was placed in the box keeping a gap of 5-6 cm above the surface of the liquid nitrogen and kept for 30 minutes to stable bubbling of liquid nitrogen. The freezing was done in liquid nitrogen vapour (temperature -80°C) in a special box for 5-6 minutes. After 5-6 minutes, the straws were plunged into liquid nitrogen (temperature -196°C). After that straws were transferred into cryocan at -196°C. Before added extender, fresh semen was evaluated for volume, density, mass motility, concentration and observed motility, viability and normal morphology . The volume of the ejaculate was measured by reading the value on graduated tube and color and density were observed by naked eye. To evaluate the mass activity, a drop (5µl) of semen was placed on a pre-warmed slide (37 ºC) without any cover slip and examined under microscope equipped with phase-contrast optics (10X). The mass activity was scored into 5 scales The wave motion was scored 0 = no motility, 1 = few sperm with weak movement (<20%), 2 = some motile spermatozoa (20– 40%) without wave movement, 3 = slow wave movement (40–60%) with motile spermatozoa, 4 = rapid wave movement without whirlpool (60– 80%) with motile spermatozoa and 5 = very rapid wave movement with clear whirlpools (>8 0%) motile spermatozoa. The concentration of spermatozoa was determined by using hemocytometer. The concentration of sperm was measured in billion. For study of motility, a drop (5µl) of semen diluted at 1:4 ratio with tris was placed on a clean pre -warmed slide (+37°C) and covered with a cover slip. The motility was determined by eye-estimation of the proportion of spermatozoa moving progressively straight forward at 40X. Viability was studied using eosin -nigrosin stain . A drop of sperm was placed on a glass slide. About 5 µl eosin-nigrosin stain was added. The semen sample and stain were mixed with a clean stick, and a homogenous thin smear was prepared. The smear was observed at 40X. Live spermatozoa appear unstained and dead spermatozoa stained pink against a brownish purple background. At least 200 spermatozoa were examined from each smear. Morphology of sperm was studied using formal saline solution. A drop (5µl) of semen was fixed with 2 ml formal saline solution. The morphology was observed at high magnification (100X). At least 500 spermatozoa were individually examined. The percentages of normal morphology were recorded. After freezing, semen was thawed on 24 hrs 7th day , 15th day and 30th All values relating to semen evaluation parameters were expressed as Mean±SD the statistical analyses were done using SPSS 17.0 . One way analysis of variance was done to find out significant differences in semen parameters among the different duration of preservation. day of storage according to Salamon and Maxwell. T hawed semen was evaluated for motility, viability and normal morphology.
Bang. J. Anim. Sci. 2015. 44 (1): 10- 15
Journal