Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

Effect of exogenous lipids on cryotolerance of Atlantic salmon (Salmo salar) spermatozoa.

The development of semen cryopreservation strategies is necessary to improve the semen storage technologies of species of great commercial interest for aquaculture. Recent studies demonstrate that lipids play an important role in the fertility and cryotolerance of fish gametes. This study investigated the effect of exogenous lipids in the freezing medium on the post-thaw functional parameters of Salmo salar spermatozoa. Semen samples (n = 12) were incubated in standard extender supplemented with different concentrations of oleic acid (OA, C18:1n9), linoleic acid (LA, C18:2n6), arachidonic acid (ARA, C20:4n6) and cholesterol-loaded cyclodextrin (CLC). Post-thaw motility, membrane integrity, mitochondrial membrane potential (ΔΨm), superoxide anion (O2•-) and fertility rates were analyzed. The results revealed that the semen incubated with 0.003 mmol/L OA increased the motility (~7%) and ΔΨm (~2%) (P < 0.05), but membrane integrity and fertility were not increased. The addition of 0.003 mmol/L LA increased the motility (~4%) and all LA extenders increased the ΔΨm (P < 0.05); however, LA increased the O2•- levels and decreased the membrane integrity and fertility (P < 0.05). Semen incubated with ARA improved sperm motility (~5%), membrane integrity (~10.5%) and fertility rates (~11%) (P < 0.05). The maximum improvement in post-thaw sperm functionality was observed by adding 0.003 mmol/L ARA. In contrast, sperm quality parameters and fertility were decreased by the CLC addition (P < 0.05). This study showed that ARA could be considered as an additive for semen cryopreservation and could be relevant in the reproductive process and reproductive management of Salmo salar.

Effects of cryopreservation on cAMP-dependent protein kinase and AMP-activated protein kinase in Atlantic salmon (Salmo salar) spermatozoa: Relation with post-thaw motility.

Sperm motility in fish with external fertilization is critical for reproductive efficiency in aquaculture, especially in salmonids. Gamete preservation techniques, such as cryopreservation, however, reduce sperm motility and fertilizing capacity. Very few studies have addressed cryodamage from energetic and cell signalling approaches. In this study, cAMP-dependent protein kinase (PKA) and AMP-activated kinase (AMPK) activities were quantified in fresh and cryopreserved spermatozoa of Atlantic salmon (Salmo salar); and the relation with motility was analysed. Results indicate there was a decrease in membrane integrity and motility in post-thawed spermatozoa compared to fresh samples, however, there was about 30% of cells with intact plasma membrane but incapable of motility. The PKA and AMPK activities were less after cryopreservation, indicating that loss of motility may be related to alteration of these key enzymes. Furthermore, PKA and AMPK activities were positively correlated with each other and with motility; and inhibition decreased motility, indicating there is a functional relationship between PKA and AMPK. The PKA inhibition also decreased AMPK activity, but results from protein-protein docking analyses indicated AMPK activation directly by PKA is unlikely, thus an indirect mechanism may exist. There have been no previous reports of these kinase actions in fish spermatozoa, making these findings worthy of assessment when there are future studies being planned, and may serve as base knowledge for optimization of cryopreservation procedures and development of biotechnologies to improve reproduction efficiency in the aquaculture industry.

Mitochondria in teleost spermatozoa.

There is an extraordinary diversity of reproductive modes in teleost and this variability is related to the phylogenetic relationships and adaption to very different biotopes. As in all vertebrates, sperm is produced as the end product of the process of spermatogenesis, and regarding teleost the spermatozoa lack an acrosome in almost all species and motility is activated as a response to osmolarity and ion content of the aquatic medium where the sperm is released. In this context, mitochondria possess a fundamental role for fish spermatozoa motility and integrity, hence, fertilizing potential; they are the energy supplier that allows flagellar movement and their dysfunction could play a main role in structural and functional damage to the spermatozoa. The ATP production through oxidative phosphorylation provides not only energy for cell activities, which includes Na+/K+ ATPase pump, endocytosis, protein synthesis and many other cell processes; but also produces reactive oxygen species, that under mitochondrial dysfunction causes oxidative stress. The assessment of mitochondrial function (e.g. through measurement of mitochondrial membrane potential) as well as ATP content (mostly supplied by mitochondrial respiration) can be useful as quality markers of fish spermatozoa. Also quantification of ROS and antioxidant status, strongly influenced by mitochondria, are used as complementary measurements. There is much information about sperm mitochondria and their function but studies of these aspects on fish reproduction are still required for applications in aquaculture. The real role of fish sperm mitochondria under short and long term storage and in vitro manipulation is not fully understood yet. Thus future research should focus on these matters.