Toward controlled breeding of the blackfin icefish Chaenocephalus aceratus (Lönnberg 1906): determination of spermatozoa concentration and evaluation of short- and long-term preservation of semen

Abstract

Efforts to study the effects of environmental change on early development of Antarctic fishes would benefit from the ability to control reproduction by in vitro fertilization. Here, we established a spectrophotometric method to determine spermatozoa concentration in the semen of the blackfin icefish Chaenocephalus aceratus and we evaluated the potential for short (refrigeration) and long-term (cryopreservation) storage. The former will maximize the use of semen over several hours and the latter alleviate the risk that semen is unavailable when a spawning female is available. Seminal plasma osmolality and pH of GnRH-injected male semen were 423.65 ± 35.6 mOsm kg−1 and 8.05 ± 0.11, respectively. The predictive power of regression models between spermatozoa concentration and absorbance was determined from 200 to 600 nm and revealed a best-fitted correlation at 505 nm. The standard curve was tested against measured concentrations and the average difference between measured and estimated concentrations was 0.27 ± 10.23%. Semen could be kept on ice for up to 6 h with minimal loss ( 90% post-thawing motility using a salt-based extender (pH 7.5–7.85 and ~ 280 mOsm kg−1) and 10% DMSO after 72 h immersed in liquid nitrogen. Spermatozoa morphometry revealed significant inter-individual variability. Average head length, head width, and flagella length were 3.78 ± 1.03, 2.87 ± 0.78, and 17.75 ± 7.15 µm, respectively. Spermatids composed 9.7 ± 0.8% of total spermatogenic cells, probably indicating active spermatogenesis, but perhaps resulting to some degree from the semen extraction technique used. Variations on these baseline methods may facilitate semen preservation and ultimately in vitro fertilization in this and other notothenioid species, thereby enhancing our understanding of the responses of early-life stages of Antarctic fishes to future warming of the Southern Ocean.