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Cryopreservation of Silver Barb Spermatozoa Using Skimmed Milk as a Cryoprotectant

09 September 2013

Cryopreservation is a process to maintain genetic material (such as spermatozoa) in subzero freezing. Abinawanto Zuraidah and Retno Lestari, University of Indonesia, disscussed the effect of skimmed milk on spermatozoa quality of silver barb (Barbonymus gonionotus) during their presentation at Aquaculture Europe 2013.

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).


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).


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.


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

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