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Ice binding proteins (IBPs) have been attracting significant interest on account of their characteristic of inhibiting ice growth and recrystallization. Owing to their unique characteristics, IBPs have been studied for applications in food, pharmaceuticals, and medicine, as well as from a general scientific point of view. In this study, we have used differential scanning calorimetry (DSC) and Raman spectroscopy as tools to understand the ice binding activity of the Arctic-yeast-originating extracellular ice binding glycoprotein (LeIBP) isolated from Leucosporidium sp. AY30. From the DSC results, an increase in the specific heat capacity was confirmed for 1 mg/mL LeIBP, which suggested that additional heat flow was required for the change in temperature. In addition, the temperature corresponding to the phase change of the solution was measured, and Raman spectroscopy was carried out on the frozen and molten phases, respectively. From the results of Raman analysis, we confirmed that the helical vibrations related to the ice binding sites on LeIBP were dramatically suppressed when the LeIBP solution was frozen. Furthermore, principal component analysis (PCA) of the Raman spectra yielded the contrast factor between the freezing and melting states. Both DSC and Raman spectroscopy are widely used to study the ice binding activity and the structural changes associated with molecular vibrations in cryobiology., (Copyright © 2018. Published by Elsevier Inc.)

We examined the effect of ice‐binding protein derived from Leucosporidium (LeIBP) on the cryopreservation of bull semen and compared it with that derived from previously reported Antifreeze Protein III (AFPIII). Six concentrations of LeIBP (10–1 ~ 104 μg/ml) and AFPIII (10–1 ~ 104 μg/ml) were added to the bull semen extender, respectively. Sperm kinematic parameters were measured to examine sperm toxicity and cryopreserved sperm quality. Measures of antioxidant activity such as superoxide dismutase (SOD), reduced glutathione/oxidative glutathione (GSH/GSSG), and total antioxidant capacity (TAC) were analysed to identify the effect of LeIBP on sperm quality. In addition, sperm viability was analysed using a flow cytometer and fluorescence microscope by SYBR14/PI staining. The results showed that the LeIBP groups (0.1, 1 and 10 μg/ml) were less toxic, and the quality of the sperm were dramatically improved in the extenders containing 0.1 μg/ml LeIBP among concentrations of LeIBP and AFPIII. The SOD activity of LeIBP was greater than that of AFPIII and control. In addition, sperm viability was enhanced in the LeIBP‐treated group. In summary, LeIBP is a useful cryoprotective adjuvant for bull sperm cryopreservation, and the most efficient concentration of LeIBP is 0.1 μg/ml. [ABSTRACT FROM AUTHOR]