Study area: The work was conducted at the Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202 during the period from May to August 2012.
Selection and Management of rams: Nine indigenous rams bought from the local market were used for this study. They were purchased by observing the age, body weight and body appearance. The age of the rams ranged from 24 to 48 months. The body weight ranged from 12 to 22 kg. All rams were apparently healthy with the absence of any clinical signs of abnormality. They were acclimatized for 21 days and dewormed once before starting of the work. The rams were provided feed at the rate of 1.5 to 2.0% of the total body weight on dry matter basis. Total required feed included 25% concentrates and 75% roughages. The concentrate feed included wheat bran, rice polish, maize bran, dry fish meal, DCP powder and table salt. The rams were provided with green grass as a source of roughages. They were allowed to free grazing 7-8 hours daily. Water was provided ad libitum.
Determining the factors affecting quality of local ram semenAge Two different age groups of rams, Group I: 24 to 36 months (n=4) and Group II: 37 to 48 months (n=4) months were categorized to observe the effects on ram semen with respect to volume, color, density, mass activity, fresh motility, sperm concentration and morphology of spermatozoa. Semen was collected at 7 days interval for evaluating of eight ejaculate from each ram. Body weight: The body weight was categorized into two groups, Group I: 12 to 18 kg (n=4) and Group II: 18 to 24 kg(n=4) to evaluate the effects of body weight on indigenous ram semen with respect to volume, color, density, mass activity, fresh motility, sperm concentration and morphology of spermatozoa. All rams were collected at 7 days interval for evaluation of semen for eight ejaculates from each ram.
Semen collection interval
To determine the effects of collection interval on semen quality two collection interval groups were designed, Group I (n=4): Once in a week and Group II (n=4): Twice in a week. The parameters were evaluated on eight ejaculates from each ram. Temperature: To determine the influence of temperature on semen quality with respect to volume, density, mass activity, fresh motility, sperm concentration and morphology all rams were divided into two groups on the basis of temperature on the day of collection. Group I: 25 to 29°C and Group II: 29 to 32°C temperature. All rams were collected at 7 days interval for evaluation of semen for eight ejaculates from each ram.
Semen collection: Semen was collected once in a week during the first period of study (May-June, 2012), followed by twice in a week during next period of study (July-August, 2012). Two ejaculates were collected from each ram during each collection time. Collection was always performed in the morning between 7-9 AM. Semen was collected aseptically. After collection, semen was kept at 37°C in water-bath until the media were added with it. Each collected sample diluted with semen extender at 1:6/8 ratios depending upon the concentration. Then the sample was evaluated. The semen extender was prepared with Tris 3.603 gm, fructose 1.488 gm, citric acid 2.024 gm, penicillin 1 lac, and streptomycin 500 mg in a 100 ml volume. Final diluents were made by mixing stock solution with 10% egg yolk.
Preparation of artificial vaginaSemen was collected by Artificial Vagina (AV) method (Miller, 1986). The AV consists of outer rubber cylinder, inner rubber line, rubber band, cone and collecting tube. Semen was collected with the help of a teaser ram. All the apparatus used for semen collection was sterilized before collection using autoclave machine. The inner liner temperature of AV was maintained at 42-43°C temperature by loading two-third area of jacket with water of 52-54°C temperature. The rest of one-third area of water jacket was filled with air. Before collection of semen some sterile non spermicidal Vaselinewas given into the inner side of artificial vagina by a glass rod. The AV was attached to the rubber cone attached with graduated collection tube.
Evaluation of collected semen: Individual fresh ejaculates were evaluated for volume, color, density, mass activity, sperm concentration, motility and morphology according to factors included in the study. The volume of fresh semen was recorded from the graduated mark of the semen collecting tube. The color was recorded with a necked eye as slightly creamy-milky white. The density of the fresh ejaculate was recorded and scored in 5 scales, 1=watery, 2=milky, 3 =thin creamy, 4= creamy, 5= creamy to grainy (Coulter, 1992). A small drop of fresh undiluted semen was placed on a pre-warmed (37°C) geese free slide and observed under microscope at 10X without coverslip and mass activity (0-5) score was recorded following the criteria; 1= no perceptible motion, 2= few spermatozoa were moving without forming any waves, 3= small slow-moving waves, 4= various movement with moderately rapid waves and eddies and 5= dense vary rapidly moving waves and eddies. Sperm concentration was determined by using haemocytometer under a 40X magnification. For sperm motility, a small drop (5μl) of diluted semen was placed on a clean pre-warmed glass slide and covered with a coverslip. Then motility was determined by eye estimation observing the proportion of spermatozoa actively moving forward at medium magnification (40X) and expressed as percentage. The proportion of normal spermatozoa with respect to Head, midpiece and tail was evaluated in buffered formol saline-fixed semen.
Statistical analysis: All data were entered in Excel program. They were expressed as mean ± standard deviation. A significant difference between the two groups of each factor was determined by using t-test. The difference between groups was regarded as significant when the P value was less than at least 0.05 (P<0.05).