The cryopreservation of sperm has become the subject of research for successful artificial insemination technologies. Antifreeze proteins (AFPs), one of the factors necessary for effective cryopreservation, are derived from certain Antarctic organisms. These proteins decrease the freezing point of water within these organisms to below the temperature of the surrounding seawater to protect the organism from cold shock. Accordingly, a recent study found that AFPs can increase the motility and viability of spermatozoa during cryopreservation.To evaluate this relationship, we performed cryopreservation of boar sperm with AFPs produced in the Arctic yeast Leucosporidium sp. AFP expression system at four concentrations (0, 0.01, 0.1, and 1 μg/ml) and evaluated motility using computer assisted sperm analysis. DNA damage to boar spermatozoa was measured by the comet assay, and sperm membrane integrity and acrosome integrity were evaluated by flow cytometry. The results showed that motility was positively affected by the addition of AFP at each concentration except 1 μg/ml (p<0.001).Although cryopreservation with AFP decreased the viability of the boar sperm using, the tail DNA analyses showed that there was no significant difference between the control and the addition of 0.1 or 0.01 μg/ml AFP. In addition, the percentage of live sperm with intact acrosomes showed the least significant difference between the control and 0.1 μg/ml AFP (p<0.05), but increased with 1 μg/ml AFP (p<0.001). Our results indicate that the addition of AFP during boar sperm cryopreservation can improve viability and acrosome integrity after thawing.

The aim of this study was to analyze the effect of antifreeze proteins (AFPs) on vitrification of mouse mature (MII) oocytes. We studied about 3 types of AFPs from different origins (FfIBP, LeIBPand Type III AFP). The MII oocytes were obtained from 4-week-old BD-F1 mice. Vitrification of oocyte was performed by 2 steps using the Cryotop (equilibration: 7.5% EG + 7.5% PROH for 5 min, vitrification: 15% EG + 15% PROH + 0.5M sucrose for 1 min). The concentrations of AFPs added to these solutions were 0.05 mg/ml for FfIBP and 0.1 mg/ml for LeIBP and Type III AFP. After fertilization, embryo development was assessed up to 5 days. Through immunostaining of vitrified-warmed oocytes, we assessed the normal meiotic spindle. Also, intracellular ROS and mitochondrial activity was analyzed. In the developmental stages, FfIBP and LeIBP groups showed significantly higher survival rates. In the blastocyst and apoptotic blastomere rates were significant differences in AFPs treated groups. AFPs treated groups were significantly higher in blastocyst cell numbers than control group. Among the AFPs treated groups, FfIBP, LeIBP groups were significantly higher rates. And, in cleavage rates, FfIBP group was significantly higher rates than the other groups. In vitrified-warmed MII oocytes, the normal meiotic spindle organization and chromosome alignment rate was significantly higher in FfIBP and LeIBP groups. And in intracellular ROS levels, control group was significantly increased than AFPs treated groups. However, in the mitochondrial activity, LeIBP group was significantly higher than control, FfIBP and LeIBP groups. AFPs treated groups were significant differences in development, meiotic spindle organization and intracellular ROS levels. And in the AFPs treated groups, FfIBP and LeIBP groups were significantly higher rates in normal meiotic spindle and mitochondrial activity than Type III AFP group respectively. In conclusion, FfIBP and LeIBP can be thought to improve oocyte cryopreservation efficiency.

In this study, we present different physiological responses to cold acclimation between the freezing tolerant barley landrace, Jeonnamjaerae, and the freezing sensitive line, PI283398, chosen by the previous field test. We tried to identify some key facto

In order to measure an antifreezing tolerance, antifreeze proteins accumulated upon cold acclimation in apoplast were analyzed. As Dongborilho were cold-acclimated for 3 to 74 days there was an abrupt increase in apoplastic proteins up to 30 days and then decrease to the similar levels. Among the known antifreeze proteins, CLP produced in E. coli. and TLP purified from apoplast were used to generate antisera that allow to measure and localize the proteins in leaves of barley. The CLP of 27.7 kDa and TLPs of 6, 26, 27, 30, and 31 kDa were increased in their amounts in apoplast as cold treatment being longer. There was a correlation among the amounts of those proteins accumulated in apoplast and freezing tolerance as shown in field and ion leakage tests for five cultivars. The deposit of CLP was localized in the marginal area and the area adjacent to leaf vescular bundle cells in an increasing manner according to duration of cold acclimation but no variation was observed in terms of it's distribution. Based on the close correlation between levels of antifreeze proteins and degrees of freezing tolerance, the immunological methods was to develop to estimate a freezing tolerance in barley

Freezing-resistant plants can survive subzero temperatures by withstanding extracellular ice formation. During cold acclimation, their leaves accumulate antifreeze proteins (AFPs) that are secreted into the apoplast and have the ability to modify the normal growth of ice crystals. Three barley, two wheat and two rye cultivars were grown under two different temperature regimes (20/16~circC and 5/2~circC , day/night). Apoplastic proteins from winter cereals were separated by SDS-PAGE and detected with antisera to AFPs from winter rye. Apoplastic proteins accumulated to much higher levels in cold-acclimated (CA) leaves compared with nonacclimated (NA) ones in winter cereals. After cold acclimation, the protein concentration of apoplastic extracts increased significantly from 0.088 mgmL-1 to 0.448 mgmL-1 , with about 5-fold increment. Also, the apoplastic protein content per gram leaf fresh weight in CA leaves ranged from 31 ~mu~textrmg (gFW)-1 to 120 ~mu~textrmg (gFW)-1 with an averaged value of 77 ~mu~textrmg (gFW)-1 , and coefficients of variation of 54.9%. The CA leaves in Musketeer (a Canadian winter rye cultivar) showed the greatest AFPs and antifreeze activity followed by 'Geurumil' (a Korean winter wheat cultivar), and 'Dongbori l' (Korean facultative barley cultivar). The proteins secreted into the wheat leaf apoplast at CA condition were more numerous than those observed in winter rye, where two β -1,3-glucanase-like proteins (GLPs), two chitinase-like proteins (CLPs) and two thaumatin-like proteins (TLPs) accumulated during cold acclimation. The proteins in barley leaf apoplast at CA conditions were a little different from those in wheat leaves. The AFPs were various among and within species. More freezing-resistant cultivars had more clear and numerous bands than less freezing-resistant ones. The high determination coefficient (R2 =91 %) between freezing resistance and AFPs per gram leaf fresh weight indicated that the amount of AFPs was highly related to freezing resistance in winter cereal crops.