• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.163-169
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• 2013

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the unique anatomical and physiological characteristics of the avian egg. Thus an alternative strategy for conservation of oviparous species of animals must be developed. Recent technological developments for producing germline chimeras by the transfer of primordial germ cells (PGCs) into recipient embryos has enabled the conservation and retrieval of chicken genetic resources in their complete form. In the present study, fertilized eggs were incubated for about 5.5 days to obtain embryos at stage 28. The whole embryo was collected from the germinal gonad using a fine glass micro pipette under a microscope. The PGCs were then purified using MACS method. Two commercially available cryoprotectants (A and B) were used to preserve the PGCs, and EG were used as a control. The average recovery rate of PGCs after thawing was 35.5% and 60.5% with the A and B treatments, respectively. There was no significant difference between B treatments and control, which showed an average recovery rate of 52.8%. However, the recovery rate obtained using A cryoprotectant (35.5%) was significantly lower than using treatment control and B. The average viability of the PGCs after thawing were 77.9% and 77.4% for cryoprotectants A and B, respectively, and the control were was 81.6%. There was no statistically significant difference between the two treatments and control. It was concluded that all of the available cryoprotectants examined in this study could be used for preservation of PGCs from embryos. Further experiments to produce germline chimera from PGCs preserved using this techniques are strongly recommended.

• Journal of Korean Society of Forest Science
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• v.108 no.3
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• pp.290-295
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• 2019

This study was carried out to investigate the possibility of establishing a reproductive system for the seed of Pedicularis hallaisanensis, which is in the endangered wild species II class in Korea. The seed of P. hallaisanensis is egg-shaped, and the seed coat is dark brown. The embryo was identified as a dwarf type by the seed section. The seed length was $0.47{\pm}0.07mm$, width $0.16{\pm}0.006mm$, and thickness $0.12{\pm}0.01mm$. The weight of one seed was $0.0003{\pm}0.0001mg$, and 1000 seeds weighed $4.59{\pm}0.02mg$. The degree of seed viability was 75.33% by the tetrazolium (TZ) assay. The highest germination rate of P. hallaisanensis seed was 71% after 4 weeks of storage at $4^{\circ}C$. However, the germination rate tended to decrease gradually over a longer storage period. The germination rates after 6 or 8 weeks of storage at $4^{\circ}C$ were 64% and 60%, respectively. We used two host plants, Artemisia princeps and Dendranthema zawadskii, to determine the effect of host plants on P. hallaisanensis seed germination. The germination of P. hallaisanensis mixed with A. princeps or D. zawadskii started at 53.5 and 62.5 days after sowing, respectively. We did not find any germination 164 days postsowing with both host plants. When A. princeps and D. zawadskii were used as host plants for P. hallaisanensis seed germination, P. hallaisanensis seed germination rates were 45.5% and 19.5%, respectively. The average time to germination was 70.2 days for A. princeps, and 46.8 days for D. zawadskii.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.141-148
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• 1999

This study was to test whether the viability of bovine hatched blastocysts (HBs) can be maintained after vitrification and thawing. The HBs were produced in vitro at Day 9 and Day 10 after IVF, and they were classified to small (S-HBs; ø$\leq$300 ${\mu}{\textrm}{m}$) and large(L-HBs; ø＞300 ${\mu}{\textrm}{m}$) on the basis of embryo diameter using eyepiece micrometer. As freezing solution, we used EFS35 which consisted of 35% ethylene glycol (EG), 18% ficoll, 0.3 M sucrose and 10% FBS added in mDPBS. Vitrification was taken by two-step method, the HBs were equilibrated in 10% EG for 5 minutes and then shortly exposed in EFS35 and plunged into L$N_2$for 30~45 sec. After thawing, the survival rates were assessed by the re-expansion of the blastocoel during 2 h and 16 h of culture. The results obtained in these experiments were summarized as follows; 1) When the blastocysts(40.8%) recovered at Day 8 after IVF were further cultured for 24 h(Day 9 after IVF) and 48 h(Day 10 after IVF), the rates of HBs were 20.5% and 6.7%, respectively. Also, the total cell number of HBs on Day 9 was significantly higher than that of HBs on Day 10 (p＜0.01). 2) When the effects of freezing solution to the survival of Day 9 L-HBs were examined, the rate of vitrified group (75.7%) was significantly lower than 100% of control and exposed group(p＜0.05). 3) When the survival rates of vitrified HBs according to size and developmental age were examined, the data of L-HBs (75.5%) and S-HBs(63.6%) on Day 9 were slightly higher than those of L-HBs(64.3%) and S-HBs(60.7%) on Day 10. 4) Also, when the in vitro survival of Day 9 HBs was evaluated under different culture condition after thawing, the result in culture medium only (79.3%) was significantly higher than 43.2% in co-culture group (p＜0.05). These results demonstrated that bovine HBs can be successfully cryopreserved by two-step vitrification method using EFS35.