2.1Study area and period The experiment was conducted at the Community-based Dairy Veterinary Foundation (CDVF) Laboratory, Department of Surgery and Obstetrics and the Poultry Farm, Bangladesh Agricultural University, Mymensingh during the period from January to May, 2013.
2.2 Experimental cocks Three cocks from each of 4 different 4th generation lines (total 12 cocks) that have been developed by selective line breeding namely Sasso, Synthetic, White Rock and Assel RIR as shown in figure 1A, 1B, 1C,1D were selected as semen donors for this experiment. The cocks of all lines were 40 weeks old. The body weight of Sasso cocks was 3.0 to 3.5 kg, Synthetic cocks was 5.0 to 5.5 kg, White Rock cocks was 2.0 to 3.5 kg and Assel RIR cocks was 2.2 to 2.5 kg. The selected cocks were matured, aparently healthy and free from any physical defects.
2.3 Housing and feeding of the cocks The cocks were managed intensively in a battery cage system and each cock was kept in an individual cage (52x45x38cm). All males received 16 h light/day throughout the experiment. Each cock was fed daily with 110-160 gram nourish layer commercial feed of Nourish poultry feed at 40 weeks age.
2.4 Semen collection The cocks were allowed a week of adaptation prior to the onset of semen collection and were trained to respond to the abdominal massage technique prior to semen. Single ejaculate of semen was collected from each cock twice a week between 9:00 and 11:00 AM by abdominal massage method. Five replicates of semen samples were collected from each of 12 cocks across the days. Semen collection and evaluation was performed at room temperature.
2.5 Semen evaluation The ejaculate volume and color of semen were macroscopically evaluated immediately after collection and recorded directly from the semen collection tube. The volume of semen was expressed as microlitre (µl). To evaluate mass activity, a drop of undiluted semen was placed on a slide without cover slip and examined under compound microscope (100x) and scored into 1-5 scales (1+=no perceptible motion, 2+=few spermatozoa move without forming any waves, 3+=small slow moving waves, 4+=vigorous movement with moderately rapid wave and eddies and 5+=dense,rapidly moving waves and eddies). Semen was then diluted 1:100 (semen: extender) using modified Ringer’s solution (sodium chloride: 68 g, potassium chloride: 17.33 g, calcium chloride: 6.42 g, magnesium sulphate: 2.5 g, sodium bicarbonate: 24.5 g, distilled water: 1000 ml) according to[12].For evaluation of motility, one drop of the diluted semen was placed on the slide and covered with glass cover slip. The sperm motility was estimated by microscopic observation at 400x magnification. Motility was expressed as the percentage of motile spermatozoa with moderate to rapid progressive movement. At least 3 microscopic fields were examined for each sample.
The sperm concentration of an ejaculate was determined by using a hemocytometer chamber after dilution with distilled water at 1:400 ratio and expressed as billion (109 ) per ml.To determine the percentage of live sperm, eosin-nigrosin stain was used. The stain was prepared by dissolving 1.67g of eosin, 10g nigrosin (water soluble) and 2.9g sodium citrate with two molecules H2O in 100ml distilled water. The stain was filtered before use. Briefly, a 10μl drop of fresh semen was mixed with 200μl of eosin-nigrosin stain on a glass slide followed by making a thin smear of it. The spermatozoa were examined at 400x magnification under a phase contrast microscope. At least 100 spermatozoa were counted to determine the percentage of live sperm spermatozoa. The spermatozoa which were appeared with pink color (stained with eosin) were regarded as dead as shown in figure 2and spermatozoa which were appeared without any color (no penetration of eosin stain) were regarded as live.
2.6 Statistical Analyses Data were analyzed by using statistical program (SPSS, version 17). One way analysis of variance (ANOVA) was done to find out significant difference among different level of lines of cocks. Pair t-test was done to find the differences in semen quality with respect to volume, mass activity, motility, concentration, viability and morphological defects among four lines of cocks. The data were presented as Mean±SEM. The variations in parameters among individual cocks and line of cocks were regarded as significant when the P value was <0.05.