• 한국가축번식학회지
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• 제20권2호
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• pp.155-161
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• 1996

For a large sclase production of genetically identical or cloned animals, the effect of cryopreservation by vitrification on the post-thaw viability of nuclear transplant rabbit embryos were investigated. The embryos of 16-cell stage were collected from the mated does at 48 hours post-hCG injection, and they were synchronized to G1 phase of 32-cell stage were injected into enucleated recipient cytoplasms by micromanipulation. After culture until 20h post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation. After in vitro culture for 48h, the nuclear transplant embryos developed to morula stage were cryoperserved with EFS solution by vitrification method. The forzen nuclear transplant embryos were thawed and cultured for 72h and the nuclear transplant of blastomeres under a fluorescence microscopy. The in vitro development to blastocyst of intact-fresh and intact-frozen 16-cell embryos was found to be 96.9 and 63.9%, respectively. The in vitro development to blastocyst of nuclear transplant and frozen-thawed nuclear transplant embryos was found to be 74.5 and 42.9%, respectively. Also, their mean blastomere numbers and mean cell cycles/day was 153 and 105, 145 and 1.34, respectively. From the above results it was concluded that the present cryopreservation by vitrification of nuclear transplant rabbit embryos might be useful though was decreased significantly.

• Clinical and Experimental Reproductive Medicine
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• 제27권3호
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• pp.267-273
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• 2000

Objectives: We have previous reported that thawed testicular sperm and sperm extracted from seminiferous tubule could achieved optimal fertilization and pregnancy in azoospermic patients. However, thawed testicular sperm did not show motility in many cases. Therefore we studied viability of immotile sperm extracted from frozen-thawed seminiferous tubule using hypo-osmotic swelling (HOS) test and eosin-Y test. Materials and Methods: After sperm extraction using for ICSI, the remained sections of seminiferous tubules were frozen with a computerized freezer. For thawing and preparation of testicular sperm, the seminiferous tubules were thawed by removing from $LN_2$ and letting them at room temperature for 10 min followed by %37^{\circ}C$ water bath for 10 min. The prepared samples were washed for free of preservation medium and sperm preparation method described previous. Sperm was suspended in 0.1 ml hypoosmotic solution. After 30 minutes, the type of distally coiled sperm were assessed. Results: In 44 cases of cryopreservation of seminiferous tubules in obstructive azoospennic patients, the fertilization rates with 2PN were 71.4% and pregnancy rates were 34.1%. The presence of motile spermatozoa on subsequent post-thaw testicular sperm remarked 15.1% and were increased to 77.3% just before ICSI. After sperm extracted from frozen-thawed seminiferous tubule, 3 hrs later in in vitro culture, the cases of presence of motile sperm, reaction of hypo-osmotic swelling test and viable sperm were 63.6% (28/44), 93.2% (41/44), and 77.3% (34/44), respectively. Conclusions: Just after post-thawed testicular sperm did not showed motility. Although motility was gained after in vitro culture, many cases showed non-motile sperm until optimal insemination time. However, HOS test showed positive reaction in non-motile sperm. Therefore, HOS test is an alternative method for the selection of viable sperm for ICSI.

• 한국수정란이식학회지
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• 제9권1호
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• pp.7-14
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• 1994

The present experiments on cryopreservation were designed to examine the effects of solution toxicity, equilibration time and cell stages on the post-thaw survival of bovine IVF embryos. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g /ml FSH, 10 $\mu$g /ml LH, 1 $\mu$g /ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro(IVC) with bovine oviductal epithelial cells for 7 to 9 days. The bovine IVF embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen, and thawed rapidly. 1. after the bovine blastocysts were exposed to EFS solution for 2 min. at room temperature and then they were washed in 0.5 M sucrose solution and TCM-199, they were cultured to examined cryoprotectant induced injury during exposure, Most of the embryos(95.0%) developed to reexpanded blastocoels. However, when the exposure time was extended to 5 and 10 min, these development rates dropped dramatically in 5 min. (69.5%) and 10 min. (47.4%), respectively, 2. When the bovine IVF embryos were vitrified in EFS solution after the equilibration for 1 and 2 min. exposure, The embryos to have reexpanded blastocoels following thawing, washing and culture processes were found to he 82.6 and 73.9%, respectively. However, when the exposure time was extended to 3 min, this survival rate dropped to 18.2%. The optimal time for equilibration of bovine IVF blastocysts in EFS solution seemed to he 1~2 min. 3. When the bovine IVF embryos were equilibrated for 1 min. the significantly (P<0. 05) higher post-thaw survival rates were obtained from the embryos of blastocyst stage(81.3%) than morulae stage(5. 1%). The optimal cell stage for viterification with EFS solution proven to he blastocyst stage in bovine IVF embryos. 4. The number of blastomeres of blastocyst stage was examined with nuclear staining with Hoechst 33342 during 7 to 9 days post-insemination. The cell counts of frozen bovine IVF embryos were found significantly(P$\geq$7.5 and those of the fresh embryos 76.6$\geq$7. 1, which were cultured in the sarne period and conditions as frozen embryos.

• Journal of Animal Science and Technology
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• 제66권5호
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• pp.891-904
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• 2024

The primary cause of sperm quality decline during the freeze-thaw pathway is the peroxidation hazard caused by reactive oxygen species produced by the biological molecules of sperm. Ascorbic acid (Vitamin C) and lycopene are two potent antioxidants that operate to prevent oxidation processes. This study aimed to analyse the effects of ascorbic acid and lycopene on the motility, viability, abnormality and plasma membrane integrity of post-thawed Sapudi rams. Sperm samples were obtained and pooled from six sexually mature Sapudi rams, separated into ten equal proportions and diluted with Tris-egg yolk-glycerol (TEY) extender. Semen was supplemented with 0 (C0; L0), 1 (C1; L1), 2 (C2; L2), 3 (C3; L3) and 4 (C4; L4) mg/100 mL (1%-4%) diluent each of ascorbic acid and lycopene, respectively. Total sperm motility, viability, abnormalities and semen membrane plasma (%) were analysed after thawing. C3 and L3 extenders resulted in higher total motility (p < 0.05) compared to the other extenders, with all treatments higher than that of the control. The extender C3 (p < 0.05) exhibited the highest semen quality. Finally, the current findings show that C3 and L3 can increase the quality of post-thawed Sapudi ram spermatozoa.

• 한국동물생명공학회지
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• 제36권2호
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• pp.69-75
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• 2021

Cryopreservation is a widely-used efficient means of long-term sperm preservation. However, unlike other types of semen, cryopreserved boar semen has reduced fertility and the efforts continue to optimize post-thawing sperm recovery. In this study, we evaluated the effects of various washing solutions (Hulsen solution, lab-made DPBS and commercial DPBS) on post-thawing porcine sperm kinematics (CASA system), viability (SYBR-14/PI) and acrosome integrity (PSA/FITC). We also examined the effect of washing-centrifugation on frozen-thawed semen kinematics. The results indicate that type of washing solution and post-thawing centrifugation alters parameters linked to sperm quality (total motility, progressive motility, viability and acrosome integrity). Significantly higher (p < 0.05) motility and progressive motility were obtained when cryopreserved semen was processed with Hulsen solution. The post-thaw percentage of live and intact acrosomal sperm was significantly higher in group 1 (Hulsen solution) as compared to other groups. Following thawing-centrifugation, the results showed significantly higher motility and progressive motility in group 1 than other groups. However, the latter two DPBS groups did not differ statistically. Taken together, Frozen-thawed spermatozoa motility, acrosome integrity and viability can be affected by the type of washing solution used. Moreover, centrifugation of frozen-thawed semen has an unfavorable effect on total motility and progressive motility.

• 한국축산식품학회지
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• 제34권6호
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• pp.742-748
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• 2014

This study was conducted to evaluate the activities of antioxidant enzyme (glutathione peroxidase (GSH-Px)) and lysosomal enzymes (alpha-glucopyranosidase (AGP) and beta-N-acetyl-glucosaminidase (BNAG)) of the longissimus dorsi (LD) muscle from Hanwoo (Korean cattle) in three freezing conditions. Following freezing at -20, -60, and $-196^{\circ}C$ (liquid nitrogen), LD samples (48 h post-slaughter) were treated as follows: 1) freezing for 14 d, 2) 1 to 4 freeze-thaw cycles (2 d of freezing in each cycle), and 3) refrigeration ($4^{\circ}C$) for 7 d after 7 d of freezing. The control was the fresh (non-frozen) LD. Freezing treatment at all temperatures significantly (p<0.05) increased the activities of GSH-Px, AGP, and BNAG. The $-196^{\circ}C$ freezing had similar effects to the $-20^{\circ}C$ and $-60^{\circ}C$ freezing. Higher (p<0.05) enzymes activities were sustained in frozen LD even after 4 freeze-thaw cycles and even for 7 d of refrigeration after freezing. These findings suggest that freezing has remarkable effects on the activities of antioxidant enzyme and lysosomal enzymes of Hanwoo beef in any condition.

• The Journal of Advanced Prosthodontics
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• 제15권1호
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• pp.1-10
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• 2023

PURPOSE. Accuracy of image matching between resting and smiling facial models is affected by the stability of the reference surfaces. This study aimed to investigate the morphometric variations in subdivided facial units during resting, posed and spontaneous smiling. MATERIALS AND METHODS. The posed and spontaneous smiling faces of 33 adults were digitized and registered to the resting faces. The morphological changes of subdivided facial units at the forehead (upper and lower central, upper and lower lateral, and temple), nasal (dorsum, tip, lateral wall, and alar lobules), and chin (central and lateral) regions were assessed by measuring the 3D mesh deviations between the smiling and resting facial models. The one-way analysis of variance, Duncan post hoc tests, and Student's t-test were used to determine the differences among the groups (α = .05). RESULTS. The smallest morphometric changes were observed at the upper and central forehead and nasal dorsum; meanwhile, the largest deviation was found at the nasal alar lobules in both the posed and spontaneous smiles (P < .001). The spontaneous smile generally resulted in larger facial unit changes than the posed smile, and significant difference was observed at the alar lobules, central chin, and lateral chin units (P < .001). CONCLUSION. The upper and central forehead and nasal dorsum are reliable areas for image matching between resting and smiling 3D facial images. The central chin area can be considered an additional reference area for posed smiles; however, special cautions should be taken when selecting this area as references for spontaneous smiles.

• Animal Bioscience
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• 제37권7호
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• pp.1177-1184
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• 2024

Objective: Semen cryopreservation is an effective method of preserving genetic material, particularly in native chicken breeds facing a substantial decline. In this study, we evaluated the quality of frozen/thawed rooster semen treated with different concentrations of oral administrations of black ginger (Kaempferia parviflora: KP) extract and determined its fertility. Methods: Thirty-two Thai native roosters (Pradu Hang Dum, 42 weeks old) were used in this study. The treatments were classified into four groups according to the concentration of KP extract administered to the roosters: 0, 100, 150, and 200 mg/kg body weight. The quality of fresh semen was analyzed before cryopreservation. Post-thaw sperm quality and fertility potential were determined. Also, lipid peroxidation was determined. Results: The results showed that sperm concentration and movement increased in roosters treated with 200 mg/kg of KP extract (p<0.05). The malondialdehyde (MDA) in the roosters receiving 200 mg/kg KP extract was lower than that in the other but had an insignificant difference within the KP treatment groups (p>0.05). The highest MDA levels were observed in the control group (p<0.05). The percentage of motile sperm (total motility and progressive motility) after semen thawing was higher in roosters that received 150 and 200 mg/kg KP extract than in those that received 100 mg/kg KP extract and the control (p<0.05). MDA levels decreased significantly in roosters that received 150 and 200 mg/kg KP extract than in those that received 100 mg/kg KP extract and the control (p<0.05). Fertility and hatchability were greater in the KP150 and KP200 groups than in the KP100 and control groups (p<0.05). Conclusion: The optimal amount of KP extract influencing initial sperm quality was determined to be 200 mg/kg. However, 150 mg/kg was the optimal low dosage of KP extract administration that maintained sperm quality and fertility following semen cryopreservation.

• ALGAE
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• 제21권1호
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• pp.125-131
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• 2006

Long-term preservation of bloom-forming cyanobacteria was evaluated using cryopreservation and freeze-drying of nine strains belonging to four genera and seven species. All test strains, except Aphanizomenon flos-aquae NIER- 10028, showed partial or complete survival following cryopreservation and freeze-drying. Frozen and freeze-dried strains were preserved for more than two years and were revived monthly. Most strains showed higher post-thaw viability after cryopreservation, especially without cryoprotectant compared to freeze-drying. Microcystis aeruginosa NIER-10010, M. viridis NIER-10020, M. ichthyoblabe NIER-10023, M. novacekii NIER-10029 and Oscillatoria sancta NIER-10027 were revived after 2.5 years of cryopreservation. These results suggest that cryopreservation may be an easy and timesaving long-term preservation method for bloom-forming cyanobacteria.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.3-7
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• 2002

In vitro embryo culture techniques provide significant contributions not only for a basic research of fertilization and early embryogenesis, but also for a low cost mass production of bovine embryos for transfer, embryo diagnosis, nuclear cloning and the production of transgenic cows. This presentation introduces newly developed serum-free media (IVD101 and IVMD101) that are effective far high yields of transferable embryos of excellent quality from in vitro-matured and fertilized oocytes. Both serum-free media are superior to a conventional serum-containing medium on the increased rates of blastocyst formation, post-thaw embryo viability, and pregnancy after transfer. Furthermore, reduced risks of calf mortality and large calf syndrome are also observed for the serum-free-derived embryos. Serum-derived embryos contain a large number of lipid droplets and immature mitochondria in their cytoplasm that may account for the lower production of transferable embryos and poor embryo quality.