ELUCIDATING THE EFFICACY OF SODIUM ALGINATE AS AN ANTIBACTERIAL ALTERNATIVE IN CRYOPRESERVATION OF SAHIWAL BULL SEMEN
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Date
2022
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ICAR-NDRI, KARNAL
Abstract
The present study was conducted to investigate the efficacy of sodium alginate (S.A.) as an
antibacterial alternative in cryopreservation of Sahiwal bull semen. In 1st objective, each of
the pre-dominant Gram –ve (Proteus spp.) & Gram +ve (Bacillus spp.) bacterial isolates
from Sahiwal bulls were collected and the antibacterial potency of sodium alginate was
determined. Minimum inhibitory concentration (MIC) was determined by the broth
microdilution method, in which two-fold serial dilutions of S.A. were done from 10 to 0.078
mg/ml concentration. It was found that the MIC of S.A. against Proteus spp. & Bacillus
spp. was 0.625 and 1.25 mg/ml respectively. Similarly, the minimum bactericidal
concentration (MBC) of S.A. against Proteus spp. & Bacillus spp. was found to be 0.625
and 2.5 mg/ml respectively. In 2nd objective, effect of S.A. as an antibacterial alternative on
microbial load and freezability of Sahiwal bull semen was studied. Dose tolerance test in
semen, as a pilot study was done by using six different S.A. concentrations (without streptopenicillin)
based on MIC doses keeping one group as negative control (without streptopenicillin).
Among these treated groups, three best doses (i.e. 1.25, 0.625 & 0.313 mg/ml)
were selected on the basis of progressive motility & CFU/ml count for the standardization
experiment. In standardization experiment, extended ejaculates were split into 3 treatment
groups (1.25 mg/ml T1; 0.625 mg/ml T2; and 0.353 mg/ml T3- no antibiotics) and one
control (Penicillin-1000IU/ml; Streptomycin-1mg/ml) group. Post-dilution, pre-freeze,
post-thaw seminal parameters & bacterial load (CFU/ml) were evaluated, and T2 (0.625
mg/ml S.A.) was selected as the best dose for the final experiment. In the final experiment,
ejaculates were split into 3 groups; one was kept as control (Penicillin-1000IU/ml;
Streptomycin-1mg/ml), T1 (0.625 mg/ml S.A., no antibiotics) and T2 (0.625 mg/ml S.A. +
antibiotics) groups. It was found that progressive motility, viability, HOST and acrosomal
integrity of spermatozoa in the post-thaw stages was found to be significantly higher
(p<0.05) in the T2 group as compared to the control & T1 group. Bacterial load (CFU/ml)
at all the cryopreservation stages was found to be significantly lower (p<0.05) in the T2
group as compared to the control. Cryo-capacitation were significantly (p<0.05) reduced
in the T2 group as compared to the control and T1 group at the post-thaw stages. No
significant difference (p<0.05) was recorded in the protamine deficiency of the
spermatozoa between the control, T1 & T2 groups at post-thaw stages. In the CASA study,
total motility (TM), and progressive motility (PM) was found to be significantly higher
(p<0.05) in T2 as compared to the control and T1 group. While there was no significant
difference recorded in curvilinear velocity (VCL), straight linear velocity (VSL), average
path velocity (VAP), and beat cross frequency (BCF) in the control and T2 groups. It can
be concluded that S.A. has limited anti-bacterial activity as compared to strepto-penicillin,
however it potentiates the antibacterial activity of strepto-penicillin if used in the
supplementation. Also, S.A. in supplementation with antibiotics, enhances progressive
motility, viability, plasma membrane integrity and acrosomal integrity of the sperm, as it
protects the sperm from the adverse effects of cryopreservation.