The choice of the culture medium is an important factor for the mass production of mycelia in submerged cultures. The influence of liquid medium on the mycelial dry weight of Paecilomyces tenuipes was investigated in this study. The regression equation is expressed as Y=-1292.94187+17.78612X1+18.92425X2+2.11464X3-0.019375X1X2-0.006276X1X3+0.008177X2X3- 0.070169X1 2-0.292175X2 2-0.008818X3 2, where Y represents the value of the mycelial dry weight (g/L), X1 is the particle size of wood sawdust in liquid medium (mesh), X2 is the concentration of the wood sawdust in liquid medium, and X3 is incubation time (h). The medium was optimized using a response surface methodology, and the optimal medium contained 30 g of wood sawdust (140 mesh), 20 g of glucose, and 10 g/L of peptone. Under these conditions, the mycelial dry weight reached 38.1 g/L (actual value). The culture medium containing wood sawdust is simple and easy to use, highly efficient, and eco-friendly, and its effectiveness in large preparations of P. tenuipes mycelia with low material costs has been demonstrated.

In the present study, we evaluated the effect of glucose-fructose and sucrose supplementation in glycerol-free tris (GFT) on sperm motility, viability, ROS level, apoptosis (BAX and BCL2) and motility (SMCP) related gene expression of dog sperm according to different post-thaw incubation time. The spermatozoa collected from five dogs were resuspended (5×107 cell/ml) with GFT containing 86 mM glucose and 86 mM fructose (GF-GFT) or 100 mM sucrose (S-GFT). The sperm (500 μl) were loaded in straws, cooled for 50 min at 4℃, frozen using liquid nitrogen (LN2) vapor for 20 min and plunged in LN2. The progressive motility, viability, ROS (H2O2) level and mRNA expression of spermatozoa were evaluated according to post-thaw incubation time (0 h, 3 h and 6 h) at 24℃. ROS was assessed using H2DCFDA stain by flow cytometry. The relative abundances of BAX, BCL2 and SMCP were assessed using quantitative real-time polymerase chain reaction (RT-PCR). The motility of spermatozoa cryopreserved in GF-GFT was increased throughout the post-thaw incubation time. The motility of spermatozoa cryopreserved in S-GFT was increased at 3 h of post-thaw incubation. Whereas, the sperm ROS level in GF-GFT group was decreased at 6 h of post-thaw incubation. However, the ROS level in the group S-GFT was gradually increased with the progress of post-thaw incubation period. The post-thaw incubation had no substantial effect on mRNA expression of BAX, BCL2 and SMCP genes of dog spermatozoa in both the GF-GFT and S-GFT groups. These results indicate that GF supplementation in GFT improves the progressive sperm motility during the 6 h of post-thaw incubation with maintaining similar sperm viability and is more efficient in reducing ROS after 3 h of post-thaw incubation. The addition of GF in GFT for the cryopreservation of dog spermatozoa and post-thaw incubation would open an option to achieve more functioning spermatozoa for future assisted reproduction practices.

The objective of the present study was to evaluate the effect of disaccharides supplementation in glycerol-free tris (GFT) on dog sperm cryopreservation with respect to pH adjustment of extender and post-thaw incubation. The spermatozoa collected from five dogs were resuspended (5×107 cell/ml) with GFT containing 100 mM of lactose (L), trehalose (T) or sucrose (S) or pH adjusted (6.85) 100 mM of lactose (LP), trehalose (TP) or sucrose (SP). The sperm (500 μl) were loaded in straws, cooled for 50 min at 4℃, frozen using liquid nitrogen (LN2) vapor for 20 min and plunged in LN2. After thawing at 37℃ for 25 s in a water bath, the spermatozoa were incubated at 24℃ for 30 min. The progressive motility, viability, mitochondrial membrane potential (MMP) and mRNA expression of SMCP gene were then assessed. The MMP was evaluated by combined JC-1 plus PI staining. The relative abundance of SMCP was assessed using quantitative real-time polymerase chain reaction (RT-PCR). Adjustment of pH in GFT extender supplemented with disaccharides did not improve sperrm motility and viability. In general, post-thaw incubation increased the progressive motility of spermatozoa. The sperm motility in the group S was significantly (P<0.05) higher than other groups regardless of post-thaw incubation time. Similarly, the sperm viability in the group S was significantly (P<0.05) higher following post-thaw incubation. The higher sperm motility in the group S was also supported with the significantly (P<0.05) higher live sperm having high MMP. There was no significant difference in mRNA expression of SMCP gene among the experimental groups. These results indicate that cryopreservation of dog sperm in GFT supplemented with S and 30 min post-thaw incubation at 24℃ could provide better freezability of dog spermatozoa with improved motility and higher MMP.

Presently, the effect of 0.5 mM dibutyryl cAMP (dbcAMP)-supplemented maturation medium during different incubation time on meiotic arrest (germinal vesicle) and resumption (metaphase II) of porcine oocytes and embryonic development of porcine oocytes following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Porcine cumulus oocyte complexes (COCs) were cultured in 0.5 mM dbcAMP for 17, 22, 27, or 42 h, and an additional 22 h without 0.5 mM dbcAMP. The nuclear status was examined at each time point. Oocytes cultured from 39~49 h displayed more than 80% meiotic resumption. More than 85 % of meiotic arrest was presented at 17~22 h. Oocytes were cultured for 22 h with 0.5 mM dbcAMP and additional 22 h without dbcAMP to assess developmental potential following IVF or PA. There were no significant differences in blastocyst rates among the dbcAMPIVF, IVF, dbcAMP-PA, and PA groups, although cleavage rate of IVF group was significantly higher than those of dbcAMP-PA, and PA groups. In conclusion, 0.5 mM dbcAMP influenced meiotic maturation of porcine oocytes depending on incubation time of oocyte, although embryonic development was not improved in both IVF and PA.

동결 과정 중 필수적인 단계중 하나인 냉각(cooling)과 냉각 후 배양시간이 생쥐 난자의 방추체의 형태와 염색체의 배열에 미치는 영향을 알아봄으로서 냉각 후 손상되었던 난자의 방추체와 염색체가 정상적으로 회복하는데 필요한 최적의 배양시간을 알아보기 위해 본 실험을 실시하였다. 생후 4-6주령의 암컷 B6C3Fl 생쥐를 과배란 처리하여 metaphase II상태의 난자를 회수하여 다음과 같이 처리하였다. 대조군은 난자를 냉각처리하지 않았으며 실험군은

본 연구는 개의 불임해결과 체외수정란을 생산할 목적으로 난소의 보존 및 난구세포의 부착 여부가 신선 및 동결 개 정자를 이용한 투명대 반응에 미치는 영향을 조사하였다. 1. 적출한 난소를 4 와 salt에 각각 48시간 보존 후 회수한 난구세포 부착 난자와 나화난자의 정자침입율은 각각 62.5%, 37.5% 및 42.5% 및 22.4%로서 난소를 적출 후 곧 바로 회수한 난구세포 부착 및 나화난자 내 정자침입율인 93.3%와 56.7%에 비해 현저히 낮

본 연구는 소형 개의 불임 해결과 체외수정란을 생산하기 위한 방안의 하나로써 난자의 형태, 번식주기, 배양시간 및 활성화 처리가 난포란의 체외성숙 및 체외발생에 미치는 영향을 조사하였다. 1. 신선, salt 및 4에 보존한 난소로부터 채취한 난구세포부착 난자와 나화 난자로 각각 체외수정시켰을 때 16세포기로의 발생율은 14.3%, 5.0% 및 7.5%, 2.8%, 5.7% 및 0.0%로써 난구세포 부착난자군의 체외발생율이 나화 난자군에 비해 높게 나타