Cryopreservation is a technique used to preserve cells for long-time storage. It is widely used in agriculture to store male gametes in liquid nitrogen. The aim of this study was to determine the optimum thawing temperature and time for samples subjected to annexin V magnetic-activated cell sorting (AnMACS) as the sperm preparation technique. Pooled semen samples from three ejaculates were divided into two groups. The treatment group was subjected both to AnMACS and to being cryopreserved, whilst the control group was cryopreserved directly without MACS. Post-thaw analysis was carried out for samples thawed at either 20°C for 13 s, 37°C for 30 s, 40°C for 7 s, 60°C for 6 s or 80°C for 5 s. Sperm kinematics, viability and capacitation status were determined for samples subjected to all thawing temperatures described. Results showed that thawing at 37°C for 13 s for MACS-processed samples was a superior option compared with other thawing procedures; there was a significant difference in P < 0.05 values for curvilinear velocity (VCL μm/s) and sperm straightness (STR %) when samples were thawed at 40°C for 7 s, with fewer capacitated spermatozoa (P < 0.05) when samples were thawed at 37°C for 30 s, 40°C for 7 s or 60°C for 6 s. Hence, we can speculate that the use of AnMACS as the sperm preparation technique can somehow enhance sperm cryosurvival rate after cryopreservation, however the fertilization potential of these cells has yet to be determined.

54 strains of viable green algae and 26 strains of viable cyanobacteria were recovered from 128 and 56 samples collected from Siberian and Antarctic permafrost, respectively, with ages from modern to a few million years old. Although species of unicellular green algae belonged to Chlorococcales were subdominant inside permafrost, green algae Pedinomonas sp. were observed in Antarctic permafrost. Filamentous cyanobacteria of Oscillatoriales, Nostocales were just found in Siberian permafrost. Algal biomass in the permanently frozen sediments, expressed as concentration of chlorophyll a, was 0.06–0.46 μg g−1. The number of viable algal cells varied between <102 and 9×103 cfu g−1, but the number of viable bacterial cells was usually higher from 102 to 9.2×105 cfu g−1. Frozen but viable permafrost algae have preserved their morphological characteristics and photosynthetic apparatus in the dark permafrost. In the laboratory, they restored their photosynthetic activity, growth and development in favourable conditions at positive temperatures and with the availability of water and light. The discovery of ancient viable algae within permafrost reflects their ability to tolerate long-term freezing. In this study, the tolerance of algae and cyanobacteria to freezing, thawing and freezing–drying stresses was evaluated by short-term (days to months) low-temperature experiments. Results indicate that viable permafrost microorganisms demonstrate resistance to such stresses. Apart from their ecological importance, the bacterial and algal species found in permafrost have become the focus for novel biotechnology, as well as being considered proxies for possible life forms on cryogenic extraterrestrial bodies.