Fourteen ram lambs 4-5 months old, selected and purchased from the local market on the basis of indigenous or local characters and kept under semi-intensive condition at the Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh. After acclimatization, rams were dewormed and vaccinated against rabies and tetanus. Rams were maintained on natural grazing. Lump some amount of concentrates consisted of wheat bran (50%), crushed maize (25%), soy bean meal (20%), fish meal (1%), dicalcium phosphate (DCP) powder (2%), vitamin mineral premix (0.5%), and salt (1.5%) was supplied to the rams along with grazing. Rams were supplied ad libitum drinking water. 2.3. Experimental design Body weight (kg), scrotal circumference (cm) and their growth rate were measured weekly. Body condition score (BCS) was measured by palpating lumber region as like other small ruminants and recorded in a score (1-5 with 0.5 increment) [9]. Scrotal circumference (cm) was measured by passing a flexible tape around the scrotum (both testes at the same level) at the point of maximum circumference. Age (months), BCS, body weight (kg) and scrotal circumference (cm) at puberty were calculated. Puberty was determined as the age when the ejaculate contained >50x106 spermatozoa, sufficient to accomplish fertilization [10]. Libido index was graded from 0 to 3; 0 no desire to move towards a teaser ewe. 1 very reluctant to reach the teaser. 2 willingly moved towards the teaser. 3 moved towards the ewe in an uncontrolled manner. Other sexual behaviours were recorded during the whole experimental period.
2.4 Semen Collection, preservation and evaluation Semen was collected by artificial vagina once a week. All glassware for collection and handling were cleaned and sterilized using high-pressure steam, dried and warmed at 35°C. After ejaculation, the tube was immediately placed in a bath at 37°C. The volume of semen was measured directly from the graduated collecting tube. Colour was estimated by visual inspection and density was scored by making the tubes slant with score range 0-5. Microscopic examination was performed under phase contrast microscope (Gallenhamp, No. 82TT8, Cat No. M/6-200-H HZ 60, England). Mass motility was estimated by assessment of wave motion of fresh undiluted semen under microscope 10x with 0-5 score. The concentration of spermatozoa was counted by placing a drop of diluted (1:400) semen on haemocytometer. To evaluate sperm motility, 5 µl of diluted semen was placed on a warmed (37°C) slide, covered by a cover slip and examined (400x). Semen was diluted (1:10) with tris based diluents and was stored at chilling (5-8°C) for up to 48h and freezing method accordingly.
2.5. Morphological evaluation Eosin-nigrosin stain was used to determine the viability of spermatozoa. Small drop of semen and one drop of eosin-nigrosin stain were placed on a clean slide and mixed with a clean stick, a thin smear was made, dried in air and examined under microscope (400x). At least 200 spermatozoa were examined from each smear to calculate the percentages of live spermatozoa. Hypo-osmotic swelling (HOST) test was used to measure the proportion of spermatozoa that swelled, giving an estimate of the proportion with functional integrated spermatozoa. Normal (acrosome, midpiece and tail) rate of spermatozoa were evaluated using Spermac stain® (Minitube, Box 152, Wellington, 7654, South Africa) (400-1000x). Before evaluation, semen was taken to warm up and thawed at 39 to 40 for 14 second for chilled and frozen, respectively.
2.6. Statistical analysis Data generated and statistical analyses were carried out for mean ±SEM in each particular parameter. Data were analyzed using SPSS 17.0 computer program package (SPSS, USA).