Assessment of bacterial load in chilled and frozen buck semen

Abstract

With the object of assessing the 'bacterial load of buck semen during processing and preservation by freezing and chilling, a study was carried out at Artificial Insemination Centre, College of Veterinary And Animal Science, Mannuthy, Thrissur, using 72 ejaculates from six Malabari cross bred bucks. The average volume of semen from two pooled ejaculates was l.23 ± 0.03 millilitre. Semen samples with creamy colour, BB mass activity and DDDD density were only used for processing and preservation. The samples were diluted 10 fold in phosphate buffered .. saline before centrifuging twice and the pellet was reconstituted to the original volume with PBS. These were then split into two portions, one for chilling and other for freezing. The sample for chilling was diluted ten fold with Tris-citric acid- fructose egg yolk diluent and preserved under refrigerated conditions for 48 hours. The sample for freezing was diluted five fold in nonglycerolated fraction of Tris- citric acid-fructose-egg yolk-glycerol diluent, cooled to 50 centigrade, glycerolated, equilibrated for 4 hours, frozen in liquid nitrogen and preserved upto 30 days. The initial live sperm percentage was 94.69 ± 0.67 which dropped to 57.83 ± 0.90 after freezing and storage for 30 days. Similarly, the initial sperm motility of75.14 ± 1.42 after washing and reconstitution dropped significantly to 33.17 ±

1 . 14 during the same period. There was an increase in the percentage of sperm abnormalities from 1.31 ± 0.67 to 7.42 ± 0.45 and that of acrosomal abnormalities from 0.70 ± 0.15 to 14.76 ± 0.77 during the same period. The bacterial load of neat semen was 1166.67 ± 348.64 organisms per millilitre which increased on washing and reconstitution to 3493.05 ± 734.90 organisms per millilitre. Further there was a significant increase on initial extension to 27272.22 ± 4012.70 organisms per millilitre. The declining trend started after glycerolisation with a reduction of bacterial load to '24466.67 ± 3682.40 organisms per millilitre. But on equilibration, reduction in the bacterial load was much more faster and significant and reduced to 2691.11' ± 664.81 organisms per millilitre. This further reduced significantly to 221.81 ± 129.77, 161.00 ± 19.94 and 162.78 ± 29.03 organisms per millilitre on storage at zero, 15 and 30 days of freeze preservation. With respect to preservation by chilling the live sperm percentage at zero, 24 and 48 hours were 88.24 ± 0.56, 80.82 ± 0.53 and 72.72 ± 1.70 respectively. The sperm motility also reduced from 73.47 ± 4.53 to 70.55 ± 0.17 and 62.50 ± 1.27 during the same period. There was a slight increase in the percentage of sperm abnormalities from 2.97 ± 0.37 at zero hour to 3.68 ± 0.51 and 4.74 ± 0.48 respectively at 24 and 48 hours of preservation. The percentage of acrosomal abnormalities were 7.20 ± 0.58, 8.58 ± 0.60 and 9.31 ± 0.66 respectively at zero, 24 and 48 hours of preservation.