Effect of processing and freezing procedures on the acrosome morphology of buck spermatozoa

Abstract

Six pooled semen samples (two ejaculates) of good quality from five Malabari crossbred bucks were processed and frozen in two different protocols to evaluate the effect of processing and freezing procedures on the acrosome morphology of buck spermatozoa. In protocol I, the samples were diluted 10 fold in Tris buffer before centrifuging twice and the final pellet was re-suspended in the non glycerolated fraction of Tris yolk diluent. The sample was glycerolated (six per cent), equilibrated (four hours), frozen (eight minutes), and thawed (250 C for 30 seconds). In protocol 11, centrifugation was done only once, after 15 fold dilution in Tris buffer. The re suspended pellet was glycerolated (seven per cent), equilibrated (three hours), frozen (10 minutes) and thawed (60° C for 10 seconds). The semen characters such as motility, live sperm, sperm abnormalities and acrosome abnormalities were evaluated at the end of washing and initial extension (stage I), cooling to 5° C (stage II), glycerolisation and equilibration (stage Ill) and freezing and thawing (stage IV). The results were compiled to evaluate the effect of different processing and freezing procedures on the semen characters in general and acrosome morphology in particular.

The semen sample used for split sample dilution had a mean volume of 1.3282± 0.067 ml, creamy in colour, DDDD density, ++++ mass activity, pH of 7.275 2± 0.040 and a concentration of 2972 2± 293 millions per ml. No significant difference in the above semen characters were found between bucks. The initial sperm motility of 82.000 2± 0.606 was found to drop significantly during processing and freezing and the final post thaw motility obtained was 44.000 2± 0.790 in protocol I. Similarly in protocol II the initial motility dropped from 81.375 2± 1.089 to 44.750 2± 1.075 at the end of stage IV. Even though there was significant drop in motility between stages in both the protocols, there was no significant difference in the corresponding stages of the two protocols. It could be inferred that good post thaw motility was obtained in both the protocols. The fact that a single washing and centrifugation was only adopted in protocol II makes it a more acceptable procedure for buck semen freezing. The mean live sperm percentage of fresh semen was evaluated using both NE and NEG staining technique. The percentage of live sperms of 90.050 2± 0.801 was found to decrease to 54.250 2± 0.593 after freezing and thawing in protocol by NE staining. Similarly in protocol 11, the mean percentage of live sperms was found to reduce to 53.125 2± 0.793 with the same staining. Even though there was significant difference in the live sperm percentage between stages within protocol I and II no significant

difference in the live sperm percentage between the corresponding stages of protocol I and I I . With NEG staining the initial live sperm percentage of 80.850 ± 1.494 was found to drop to 54.875 ± 0.677 in protocol I as against 53.400 ± 0.730 in protocol II. While there was significant difference in the live sperm percentage between stages within protocol I and II there was no variation between corresponding stages of the two protocols. A significantly lower percentage of live sperms was recorded with NEG staining when compared with NE staining probably on account of the fact that the differentiation of live and dead sperm was difficult in the former staining method as live sperms were stained light blue instead of colourless. The mean percentage of abnormal sperms of 3.050 ± 0.245 in fresh semen did not register any significant increase during processing. However, there was significant increase in the percentage of sperm abnormalities during freezing and thawing with the final abnormality percentage of 7.125± 0.706 in protocol I and 6.300± 0.36 in protocol II. The initial acrosomal abnormality of 8.825 in the fresh semen steadily rose to 23.375 in protocol I as against 19.825 in protocol II at the end of stage IV. There was no significant difference in the percentage of various acrosomal abnormalities between corresponding stages of the two protocols. However, there was significant increase in the acrosomal abnormalities during glycerolisation, equilibration, freezing and thawing

under both the protocols. It was concluded that the processing and freezing under two different protocols did not significantly alter the post thaw motility, percentage abnormal and dead sperms and acrosomal abnormalities. A good post thaw motility and low acrosomal abnormality was obtained with a single washing of buck semen with 15 fold Tris buffer which was comparable with double washing with 10 fold Tris buffer.