Sperm cryopreservation preserves genetic resources for animal breeding and for human patients who suffers from permanent testicular damage. Although the sperm cryopreservation has been used for many years, the addition of cryoprotective agent (CPA) during cryopreservation negatively affects sperm function and quality. Our previous study reported that the addition of CPA reduced bull sperm physiological functions. However, the sperm cells collected from individual bulls presented different sensitivity to the damage induced by CPA. In the present study, we examined if CPA affect sperm cells acquired from individual bulls. Individual bull spermatozoa were divided into two groups based on motility parameters; high CPA-tolerant sperm (HCS) and low CPA-tolerant sperm (LCS). Our results showed that the HCS group presented good physiological function after CPA exposure, whereas the LCS group showed a significant decrease in the sperm function. We also found differentially expressed five proteins between the HCS and LCS groups, which refer to cytosolic 5′-nucleotidase 1B (NT5C1B), fumarate hydratase (FH), F-actin-capping protein subunit beta (CAPZB), voltage-dependent anion-selective channel protein 2 (VDAC2), and cytochrome b-c1 complex subunit 1 (UQCRC1). NT5C1B and FH showed abundant expression in the HCS group, while the expression of CAPZB, VDAC2, and UQCRC1 was relatively lower in the HCS group than in the LCS group. The current results suggest that NT5C1B, FH, CAPZB, VDAC2, and UQCRC1 can be used as potential markers to predict CPA-tolerable spermatozoa. Those markers provide a reliable tool to select animals and breeds with CPA tolerance.

Cryoprotectant is a substance used to protect biological tissue from freezing injury. However, there was few research paper on application of cryoprotectant in food stuff although its benefits was approved from the biological cell tissues. The objective of this study was to investigate the effect of the sugar addition as a cryoprotectant on the properties of frozen soybean sprouts. Before freezing process, the samples were blanched at 100°C for 1 min to observe the influence of blanching treatment. The blanched or non-blanched soybean sprouts was immersed in sugar solution as cryoprotectant, and continuously, the samples were frozen at -18°C for 24 h. Their physicochemical properties such as drip loss, hardness, color and cellular tissue were analyzed after thawing in running water. In our study, the drip loss of blanched sample without sugar was 43%, and comparatively, blanched one with sugar was 20% which was the lowest value among all samples. There was no significant difference of hardness between sample with sugar and without sugar. From our results, it was supposed that sugar can protect the soybean sprouts during freezing process regardless blanching process.

The objective of this study was to evaluate the toxicities of permeable cryoprotectants and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Toxicities of permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), Glycerol, and 1,2-PROH were investigated using a murine embryo model. Female F-1 mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to among DMSO, EG, Glycerol, and 1,2-PROH. Embryo development was evaluated up to the blastocyst stage. The total cell count of blastocysts that were treated with DMSO and Glycerol at the 2-cell stage was significantly lower than that were treated with EG (81.1±15.1), 1,2-PROH (88.0±21.1) or the control (99.9±21.3) (p<0.001). On comparison of four cryoprotectant treated groups, the DMSO and Glycerol treated group showed a decreased cell count compared with the EG and 1,2-PROH treated group (p<0.05). Both DMSO (14.7±1.3), EG (12.1±1.1), Glycerol (15.2±1.8), and 1,2-PROH (11.5±1.3) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control (6.5±0.7, p<0.0001). In addition, the DMSO or Glycerol treated group showed more apoptotic cells than the EG or 1,2-PROH treated group (p<0.001). The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to among DMSO, EG, Glycerol, and 1,2-PROH at room temperature. When comparing four permeable cryoprotective agents, EG and 1,2-PROH appeared to be less toxic than DMSO and Glycerol at least in a murine embryo model.

This study was carried out to investigate the effects of cryoprotectants, warming solution and removal of lipid on open pulled straw vitrification (OPS) method of porcine embryos produced by nuclear transfer (NT) of fetal fibroblasts. All solutions used during vitrification were prepared with holding medium consisting of 25 mM Hepes buffered TCM199 medium containing 20% fetal bovine serum (FBS) at 38.5℃. The blastocysts derived from NT with or without lipid were vitrified in each medium of different concentrations of dimethyl sulfoxide (DMSO) and ethylene glycol (EG). Also, blastocysts after cryopreservation were warmed into different concentrations of sucrose in warming solution. The optimal concentrations of cryoprotectants in vitrification solution were 10% DMSO + 10% EG in vitrification solution 1 (VS1) and 20% DMSO + 20% EG in vitrification solution 2 (VS2). The optimal concentrations of sucrose were 0.3 M sucrose in warming solution 1 (WS1) and 0.15 M sucrose in warming solution 2 (WS2). Lipid removal from oocytes before NT enhanced the viability of NT embryos after vitrification. Our results show that use of the OPS method in conjunction with lipid removal provides effective cryopreservation of porcine nuclear transfer embryos.

Cryopreservation allows for the advances of the reproductive technique and livestock industry. However, cryopreservation inevitably causes various types of stress, such as cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. Although cryoprotectant agent (CPA) is added to protect spermatozoa from freezing damage during cryopreservation, it has intrinsic toxicity that can affect components of the sperm membrane. Moreover, the addition of CPA induces osmotic stress and excessive reactive oxygen species (ROS) generation, resulting in disruption of mitochondrial membrane potential, alteration of membrane permeability, and damage of sperm surface proteins. To identify the effects of CPA to spermatozoa, we analyzed the sperm movement, capacitation status, and viability using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, we performed two-dimensional electrophoresis to find protein markers related CPA addition in cryo processes. CPA addition reduced sperm motility (%), viability (%), and non-capacitated spermatozoa, whereas acrosome-reacted spermatozoa increased significantly (p<0.05). Following addition of CPA, a total of ten proteins were altered their expression (eight increased, two decreased) (>3 fold, p<0.05). Among these, four differentially expressed proteins were related to several canonical pathways, such as the ephrinR-actin, ROS metabolism, actin cytoskeleton assembly, actin cytoskeleton regulation, and respiratory chain and oxidative phosphorylation pathway (p<0.05). The present study suggests that CPA significantly alters the functions and proteome content of spermatozoa. Additionally, we anticipated that the differentially expressed proteins might consider as biomarker of CPA-induced stress.

동결건조보호제(glucose, maltose, lactose 및 sucrose)를 첨가한 캡슐형 분말김치를 -20, 0, 4 및 25°C에서 4개월간 저장하면서 생균(젖산균)수를 측정한 결과, -20°C에서 저장한 캡슐형 분말김치에서 4개월 후에도 젖산균이 7 log CFU/g 이상 유지되었으며, 동결 건조 보호제로 glucose를 첨가한 캡슐형 분말 김치의 젖산균은 대조군 보다 약 3log CFU/g 이상 더 높았다. 4 및 0°C에 저장된 캡슐형 분말김치에서도 저장 4개월째의 젖산균수는 7~8 log CFU/g으로 유지되는 것을 확인하였다. 25°C에 저장된 캡슐형 분말 김치에서는 저장 10주까지는 4~5 log CFU/g으로 젖산균수가 유지되었으나, 저장 4개월 후 젖산균이 확인되지 않았다. 분말김치 내의 생균수를 7~8 log CFU/g로 4개월 이상 유지하기 위해서는 동결건조보호제(maltose 또는 glucose) 첨가처리와 더불어 냉장 및 냉동 보관이 필수적임을 확인하였다