The success of cryopreservation is influenced by cryoprotectant and extender. Some methodologies, development and application of cryopreservation of fish spermatozoa were reported some for species: Osphronemus goramy (Abinawanto et al., 2011; 2012a; 2012b), Barbonymus gonionotus (Abinawanto et al. 2013, carp (Withler, 1982; Harvey, 1983; Horvath et al., 2003), and other salmonids (Harvey and Ashwood-Smith, 1982).
The objective of present study is to investigate the effect of skimmed milk in various concentration of 0%, 5%, 10%, 15%, 20%, and 25%, respectively, on spermatozoa quality of Barbonymus gonionotus Bleeker, 1850 cryopreserved for 24 hours.
Materials and Methods
Mature male of Barbonymus gonionotus obtained from a private commercial hatchery were brought into laboratory. The ejaculates from a total of four males were collected by hand stripping.The ejaculated semen were diluted with the solvent (fish ringer extender + 5% methanol + skim milk; 1 : 5) according to Harvey (1983). Skim milk concentration used in this study were: 0%, 5%, 10%, 15%, 20%, and 25%, respectively. Samples were then equilibrated at 4-5°C for 20 minutes according to Sunarma et al. (2007), and were freezed at -34°C for 24 hours according to Huang et al. (2004). Thawing was
carried out at 40°C for 13 seconds according to Horvath et al. (2003). Each sample was then evaluated for the following parameters using a light microscope with the aid of a digital eye-piece connected to the computer (image driving software; Scopephoto 2.0.4).
Results
Based on Kruskal-Wallis test, there were significant effect (P<0.05) of various concentration of skim milk on post-thaw sperm viability and abnormality, but not on post-thaw motility (P>0.05), compared to control-1; C1 (0% of methanol ; 0% of skim milk) and also compared to control-2; C2 (0% of skim milk only). According to the Dunnet test, the concentration of 20% of skim milk showed significant difference (P<0.05) on post-thaw viability and abnormality, respectively (Fig. 1).
Discussion
Cryoprotectant, extender, and different species, are some factors cause the differences of spermatozoa quality after subzero freezing.The effect of 20% of skim milk + 5% methanol on the percentage of spermatozoa motility 24 hours postcryopreservation was higher (83.23%) than those observed in other fish species such as Osphronemus goramy (80.98%; Abinawanto et al., 2012b), Brachydanio rerio (51%; Harvey et al., 1982), Oreochromis mossambicus (70%; Harvey, 1983),
tilapian’s fish (40-80%; Chao et al., 1987), Cyprinus carpio (55%; Akcay et al., 2004), and Osteochiius hasseltii (63.33%; Sunarma et al., 2007).
The combination of 20% of skim milk + 5% methanol was also maintained the percentage of
spermatozoa viability (81.75%). This result (spermatozoa viability) was higher than other species such as Cyprinus carpio (20%; Withle, 1982; 58%; Horton & Otto, 1976), but lower than Osphronemus goramy (84%; Abinawanto et al., 2012b) . Furthermore, the combination of 20% skim milk + 5% methanol was also maintained the percentage of spermatozoa abnormality (26.25%). The spermatozoa abnormality in this study (26.25%) was also lower compared to other species like, Osphronemus goramy (29%; Abinawanto et al., 2011). But, relatively higher compared to another our previous study
using 0.5% of sucrose + 10% of methanol which was shown 12.5% of abnormality (Abinawanto et al., 2012a) or 14% of abnormality if using 15% skim milk-fish ringer + 10% methanol (Abinawanto et al., 2012b). In general, this result showed the highest percentage of either motility or viability and the lowest abnormality after sub-zero freezing compared to other species or different cryoprotectant and extender.
Conclusion
The combination of 20% of skim milk + 5% methanol showed the highest post-thaw sperm viability (81.75 ± 8.22)%, and the lowest post-thaw abnormality (26.25 ± 1.89)%.
September 2013
