A novel approach for human sperm cryopreservation with AFPIII.

Sperm cryopreservation causes different stresses including thermal shock, osmotic damage, and ice crystal formation, thereby reducing sperm quality. Few studies have evaluated the application of AFPs in cryopreservation. The effects of antifreeze protein III (AFP III) on human sperm cryopreservation is not fully understood therefore, we conducted this study to investigate the effects of AFPIII treatment on human sperm parameters following cryopreservation. First, for 20 semen samples the effects of various concentrations of AFPIII (0, 0.01, 0.1, 1, 5, 10 µg/ml) were evaluated. Sperm parameters, such as motility and viability were assessed in order to identify an optimal dose. Next, liquefied 20 semen samples were divided into three aliquots and diluted in glycerol-egg-yolk-citrate (GEYC) cryopreserved without AFPIII (control), with optimal dose of AFPIII, as well as fresh groups. After thawing, samples were evaluated for plasma membrane integrity (PMI), DNA fragmentation index (DFI), reactive oxygen species (ROS), and total antioxidant capacity (TAC) levels. Spermatozoa treatment with 0.01, 0.1 and 1 µg/ml AFPIII increased the sperm motility and viability compared to the control group, but the highest concentrations were ineffective. In conclusion, the results showed that the addition of AFPIII to GEYC at 1 µg/ml improved motility, PMI, viability and TAC, and decreased ROS and DNA fragmentation of cryopreserved human semen compared to the control group.

Improvement of sperm cryo-survival of cynomolgus macaque (Macaca fascicularis) by commercial egg-yolk-free freezing medium with type III antifreeze protein.

When nonhuman primate sperm undergoes cryopreservation in an egg yolk medium there is an increased risk that the egg yolk might adversely affect the sperm due to containing of avian pathogens. Although commercial egg-yolk-free medium for human sperm cryopreservation has been used for macaque sperm, the cryo-survival remains less than optimal. The present study, therefore, was conducted to determine the optimal concentration of antifreeze protein (AFP) III supplemented in a commercial egg-yolk-free medium for cynomolgus macaque (Macaca fascicularis) sperm cryo-survival. The function of frozen-thawed sperm was evaluated by post-thaw sperm motility, acrosome integrity, and mitochondrial function. Results indicate that the sperm motilities were greater when 0.1, 1, and 10 µg/ml of AFP III were supplemented into the sperm freezing medium (P < 0.05). In addition, the mitochondrial membrane potential was greater in the sperm cryopreserved with the medium that was supplemented with 0.1 µg/ml of AFP III (P < 0.05). The addition of AFP III at any of the concentrations, however, did not have any cryoprotection effect on the sperm acrosome, and the greatest concentrations of AFP III at 100 and 200 µg/ml had detrimental effects on acrosomal integrity (P < 0.05). Results of the present study indicated the methods used are effective for the cryopreservation of cynomolgus monkey sperm while reducing associated health risks due to avian pathogens being present in egg yolk-based extenders.

Safety evaluation of ice-structuring protein (ISP) type III HPLC 12 preparation. Lack of genotoxicity and subchronic toxicity.

Ice-structuring proteins (ISPs) naturally occur in a range of species (including edible plants and fish) that need to protect themselves against freeze damage. ISPs have potential applications in a number of areas including cryopreservation and frozen foods manufacture. However, these materials are not currently generally available for commercial use. ISP type III HPLC 12 is of particular interest and although it is likely to be consumed naturally, its toxicological safety has not previously been assessed. This paper presents data from a set of in vitro and in vivo genotoxicity assays (bacterial mutation, chromosome aberration, mammalian cell gene mutation and rat bone marrow micronucleus) and a 3-month repeat-dose gavage study in the rat using high levels of ISP type III HPLC 12 preparation produced by recombinant baker's yeast. No evidence was seen of a genotoxic potential (using levels accepted as limit concentrations for the assays used) or notable subchronic toxicity following oral administration for 3 months in the rat at up to 580 mg ISP type III HPLC 12/kg/day, the highest dose tested (which was considered to be a NOAEL).