• 한국가금학회지
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• 제25권3호
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• pp.113-118
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• 1998

These experiments were conducted to develope the artificial insemination methods of wild duct (Anas platyhychis platylyachos) . The characteristics of erect phallus and semen collected by the abdominal massage method were investigated in wild duck. The erection and withdrawal time of phallus were 50.70${\pm}$18.66 and 92.58${\pm}$51.95 sec. , respectively, in wild duck. The length, long and short diameter of erect phallus were 3.98${\pm}$0.49crn, 1.53${\pm}$0.15cm and 1.05${\pm}$0.04 cm, respectively, and the spiral grooves of erect phallus were 4 in wild duck. The volume of semen, concentration of spermatozoa and total sperm of an ejaculate were 0.18${\pm}$0.06 ml, 2.84${\pm}$0.03 x 10 9 /ml and 0.52${\pm}$0.21 x 10 cells, respectively, in wild duck. The motility of sperm and pH of semen were 49.06${\pm}$14. 35 and 7.6${\pm}$0.14 in wild duck.

• 한국가축번식학회지
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• 제27권3호
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• pp.215-223
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• 2003

본 연구는 체외성숙된 돼지난포란을 액상정액으로 수정시 수정시간과 배양배지가 난포란의 발달에 미치는 영향을 조사하기 위하여 실시하였다. 정자농후정액 (30∼60 ml)을 채취하여 실온에서 2시간 정도 서서히 냉각시킨 후, 정액을 15 ml 튜브에 담아 800${\times}$g로 10분간 원심분리하였다. 상층액은 버리고 하부의 정자는 5 ml LEN 희석액으로 1${\times}$$10^{9}$ 전자/ml가 되도록 재희석하였다. 희석된 정액은 4$^{\circ}C$ 냉장고에 보존하였다. 미성숙 난모세포의 성숙에 사용된 배지는 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin B$_{12}$ , 25 mM HEPES, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml PMSG, 10 IU/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate그리고 10% pFF를 첨가한 TCM-199 배지였다. 22시간 성숙 배양한 후 난모세포는 cysteamine과 hormone들을 배제한 후 38.5$^{\circ}C$, 5% $CO_2$ incubator에서 22시간 더 성숙시켰다. 성숙된 난모세포는 채취 후 2일간 4$^{\circ}C$에 보존된 액상정액으로 수정되었다. 난모세포는 500 $\mu$l mTBM 수정 배지에서 1${\times}$$10^{6}$ 정자/ml의 농도로 1, 3, 6 그리고 9시간 동안 수정시켰다. 그 후 난모세포는 500 $\mu$l NCSU-23, Hopes buffered NCSU-23, PZM-3 그리고 PZM-4 배양배지에 옮겨서 6, 48 그리고 144시간을 더 배양하였다. 정자침투율, 웅성전핵형성율 그리고 난모세포의 난할율은 6 및 9시간 수정시간에서 1 및 3시간 수정시간 보다 높았다. 6시간 수정시 배반포형성율 (33.6%)은 1, 3 그리고 9시간 수정시 배반포형성율 (11.4, 23.0 그리고 29.6%) 보다 높았다. 배반포의 평균세포수는 6, 9, 3 그리고 1시간 수정시 각각 32.9, 27.6, 26.3 그리고 24.4개 였다. 분할된 난모세포의 배반포형성율 그리고 배반포의 평균세포수는 NCSU-23, PZM-3 그리고 PZM-4 배양배지보다 HEPES buffered NCSU-23 배양배지가 우수하였다. 결론적으로 4$^{\circ}C$ 보존 돼지액상정액은 체외성숙된 돼지 난모세포의 체외수정에 사용될 수 있음이 입증되었다. 또한 체외성숙된 돼지 난모세포는 500 $\mu$l mTBM 수정배지에서 1${\times}$$10^{6}$ 정자/ml로 6시간 공배양시키는 것이 바람직하며, HEPES buffered NCSU-23 배양배지에서 배양하는 것이 좋다는 결과를 얻었다.

• 한국수정란이식학회지
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• 제28권3호
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• pp.297-302
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• 2013

Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of genetically elite populations. Castration, catastrophic injury, sudden death or any other event that makes semen collection or mating impossible may prematurely terminate a stallion reproduction. Stallion epididymal spermatozoa vary widely in the loss of progressive motility, acrosomal integrity, and viability during freezing and thawing. The objective of this work was to investigate the effect of (1) freezing package types on cryopreservation efficiency, (2) thawing temperatures (37, 56 or $70^{\circ}C$) on Computer Assisted Sperm Analysis (CASA) parameters and (3) post-thawing incubation time (0, 1, 2 or 4h) on castrated stallion epididymis. Post-thawed sperm motility ranged between 59.69% and 64.28% ($56^{\circ}C$ and $37^{\circ}C$) in various thawing temperatures. When stallion epididymis sperm was frozen, straw was better than freezing tube on VCL (Velocity of Curvilinear Line) and VAP (Velocity of Average Path) parameter. Higher percentage of motility was observed at $37^{\circ}C$ thawing temperature even though no significant difference was observed among various temperatures. The motility, VCL, ALH (Amplitude of Lateral Head displacement), VAP, BCF (Beat-Cross Frequency) and STR (Straightness index) parameter of post-thawed sperm were significantly decreased by increasing the incubation time for all thawing temperatures. The present study showed that type of freezing package (Straw vs. Freezing tube) was not significantly different on cryopreservation efficiency. Furthermore, stallion epididymal spermatozoa frozen-thawed at $37^{\circ}C$ for 1 min resulted the highest proportion of motility and velocity movement. In addition, motility and viability of frozen-thawed stallion epididymal spermatozoa were also decreased by incubation.

• Asian-Australasian Journal of Animal Sciences
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• 제19권2호
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• pp.160-164
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• 2006

Currently, boars selected for commercial use as AI sires are evaluated on grow-finish performance and carcass characteristics. If AI sires were also evaluated and selected on semen production, it may be possible to reduce the number of boars required to service sows, thereby improving the productivity and profitability of the boar stud. The objective of this study was to estimate genetic correlations between production and semen traits in the boar: average daily gain (ADG), backfat thickness (BF) and muscle depth (MD) as production traits, and total sperm cells (TSC), total concentration (TC), volume collected (SV), number of extended doses (ND), and acceptance rate of ejaculates (AR) as semen traits. Semen collection records and performance data for 843 boars and two generations of pedigree data were provided by Smithfield Premium Genetics. Backfat thickness and MD were measured by real-time ultrasound. Genetic parameters were estimated from five four-trait and one five-trait animal models using MTDFREML. Average heritability estimates were 0.39 for ADG, 0.32 for BF, 0.15 for MD, and repeatability estimates were 0.38 for SV, 0.37 for TSC, 0.09 for TC, 0.39 for ND, and 0.16 for AR. Semen traits showed a strong negative genetic correlation with MD and positive genetic correlation with BF. Genetic correlations between semen traits and ADG were low. Therefore, current AI boar selection practices may be having a detrimental effect on semen production.

• Clinical and Experimental Reproductive Medicine
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• 제30권4호
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• pp.333-340
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• 2003

Objective: This study was performed to examine the maturation and the development to the blastocyst stage of immature oocytes collected from patients with high risk of ovarian hyperstimulation syndrome (OHSS). Materials and Methods: Cumulus-oocyte complexes (COCs) were collected following only HCGpriming for non stimulated IVF-ET cycles of the patients. At the time of oocyte collection, COCs were classified into three groups in accordance with their appearance (Group I: oocytes with dispersed cumulus cells; Group II: oocytes with compacted cumulus cells; Group III: oocytes with sparse cumulus cells). The in vitro maturation and blastocyst development rates of the COCs were compared among these groups. From August 2001 to June 2002, 48 IVM/IVF-ET cycles from 42 patients (mean age: $32.4{\pm}3.8$ years) were performed. To prevent the occurrence of OHSS, the patients were primed with 10, 000 IU HCG alone 36 h before oocyte collection without gonadotropin stimulation. Oocytes were aspirated on cycle days from 7 to 13. The normal COCs were classified into three groups according to their appearance. The aspirated immature oocytes were cultured in YS maturation medium containing 30% (v/v) human follicular fluid (HFF), 1 IU/ml FSH, 10 IU/ml HCG and 10 ng/ml rhEGF. Fertilization was induced by intracytoplasmic sperm injection (ICSI). All zygotes were co-cultured with cumulus cells in $10{\mu}l$ YS medium containing 10% HFF until day 7 after oocyte collection. Blastocyst transfer was performed on day 5 after ICSI. Results: Th e mean number of oocytes cultured in the IVM/IVF cycles was $24.7{\pm}10.6$. Of 1185 COCs, those assigned to Group I, II and III were 470 (39.7%), 414 (35.0%) and 301 (25.4%), respectively. The maturation rate (94.5%, 444/470, p<0.05) in Group I was significantly higher than those of Group II (62.8%, 260/414) and Group III (73.1%, 220/301). Especially, 30.9% of COCs in Group I (145/470) was matured on the day of oocyte aspiration. There were no differences in the rates of fertilization and cleavage among the three groups. The development rate to the blastocyst stage in Group I (54.6%, 206/377, p<0.05) was also significantly higher than those in Group II (33.0%, 68/206) and Group III (30.1%, 52/173). Twenty-four clinical pregnancies (50.0%) was obtained and 22 pregnancies (45.8%) are ongoing. Implantation rate in the present study was 24.6%. Conclusion: These results suggest that there is a positive correlation between the appearance of COCs and the developmental competence of the immature oocytes in non stimulated IVM/IVF cycles.

• 한국수정란이식학회지
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• 제24권3호
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• pp.145-150
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• 2009

This study was conducted to examine the effects of human follicular fluid and gonadotropin (FSH+HCG+rhEGF) on in vitro maturation, fertilization and development of human immature oocytes. Cumulus-oocyte complexes (COCs) were collected following for in vitro fertilization and embryo transfer (IVF-ET) cycles of the patients. At the time of oocytes collection, oocytes were classified into MII, MI and GV in accordance with their appearance (MII: Fully mature oocyte at metaphase II of meiosis; MI: Nearly mature oocytes at metaphase I of meiosis; GV: Immature oocytes at prophase I of meiosis). After controlled ovarian stimulation using gonadotropin(FSH) and human chorionic gonadotropin (HCG) in 70 ICSI cycles, 158 MI to MII matured oocytes were intracytoplasmic sperm injection (ICSI) ${\sim}4$ h after in vitro culture and 553 MII oocytes were ICSI after denudation. The aspirated MI and GV oocytes were cultured in culture medium containing 10% (v/v) serum protein substitute (SPS), 10% (v/v) human follicular fluid (hFF) and 10% (v/v) serum protein substitute (SPS)+1 IU/ml FSH+10 IU/ml HCG+10 ng/ml recombinant human epidermal growth factor (rhEGF). The maturation rate of immature oocytes was similar among the three group. When maturation medium was supplemented with 10% SPS, 10% hFF or gonadotropins, the fertilization rate of in vitro matured oocytes was higher in 10% SPS (80.0%), but there was no statistical significance (78.2%; hFF, 76.9%; gonadotropin, p>0.05). The development rate of human embryos developed to $6{\sim}8$ cells were not significant difference in the medium containing SPS, hFF and gonadotropins (65.6%, 65.9% and 66.7%). The results of these study suggest that human follicular fluid and gonadotropins supplemented in the culture medium was not effected on the in vitro maturation, fertilization and development of human immature oocytes.

• 한국수정란이식학회지
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• 제33권4호
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• pp.265-270
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• 2018

Up-to-date artificial insemination (AI) using frozen sperm consider as the most widely using technology for improvement of Korean Native Cow (Hanwoo) embryo production. However, it is time consuming, required at least 15~20 years to make more than 6 generations, and their offspring number is limited. To overcome such limitations, superovulation and in vitro fertilization have been developed. For superovulation, the number of produced embryos are not enough for commercialization and donor cows need rest period. This led to use of slaughterhouse ovary for in vitro fertilization, but it is impossible to repeat the collection from the same individual and it only can improve the genetic merits of offspring for one generation. Production of embryos using Ovum Pick-Up (OPU) technique, where oocytes can be repeatedly collected from living elite donor, might overcome these limitations. In this study, we investigated the possibility of using OPU technique from donors at different age and different session periods for mass-embryo-production. Oocytes were collected from 26 donor cows twice per week, 3 - 4 months per year, between 2013 and 2016. Results showed that, the average number of embryo produced in first year used donor was significantly higher than that in second year used donor ($3.89{\pm}2.85$ vs $3.29{\pm}2.70$), however, there was no significant difference between third year used donor ($3.51{\pm}3.32$) and other groups. Taken together, our data showed that repeated using of donor up to three years is possible for in vitro embryo mass-production. Moreover, OPU can be used as suitable embryo producing technique for livestock breed improvement.

• 한국가축번식학회지
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• 제23권2호
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• pp.165-174
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• 1999

본 연구는 동결융해 후 정자의 생존성에 영향을 미치는 요인을 찾기 위하여 수행하였다. 동결융해 후 생존성에 대한 요인으로써 동결보존액, 동해보호제, 예비동결법 및 동결소요시간을 비교하였다. 동결과정중 정액의 질을 평가하기 위하여 활력, NAR 및 생존율을 조사한 결과는 다음과 같다. 돼지정액을 BF5, LYE, Soejima 및 modified Soejima 보존액으로 동결하였을 때 동결융해 후 정자활력은 M-Soejima 보존액이 44.5$\pm$6.4%로 다소 높았다. M-Soejima 보존액의 2차 회석액에 caffeine(2mM), heparin(l00 ${\mu}\ell$/$m\ell$) 및 caffeine＋heparin 를 첨가하였을 때 동결융해 후 활력은 caffeine 첨가구가 61.7% 로 가장 높았으며, 단독 혹은 혼합첨가가 첨가하지 않은 대조구보다 유의적으로 높은 활력을 나타내었다 (p＜0.05). M-Soejima 보존액에 동해보호제로서 glycerol(Gly), ethylene glycol(EG), propylene glycol(GP), Gly＋EG 및 Gly＋PG을 첨가하였을 때 동결융해 후 활력 및 NAR 율은 Gly＋PG의 혼합첨가시 (31.3%/39.5%) 가 다른 첨가구보다 다소 높았으며 생존율은 Gly＋EG 첨가구가 21.2% 로 다른 첨가구보다 다소 높았다. BE5와 M-Soejima 보존액으로 straw 및 pellet 동결법으로 동결하는 동안 dry ice-pellet, dry ice-straw 및 L$N_2$vapor-straw 법으로 예비동결하였을 때 각각 22.8, 47.5, 52.5% 및 42.5, 47.5, 57.5% 의 활력을 나타내었다. 또한 M-Soejima 보존액의 straw 법으로 l차 희석부터 동결완료까지 소요되는 시간을 2, 5 및 7시간으로 하였을 때 동결융해 후 활력 및 생존율은 처리간에 큰 차이가 없었으나, NAR 율은 처리시간이 길어질수록 다소 높은 경향을 나타내었다. 이러한 결과로 미루어 볼 때 동결융해 후 활력, NAR 및 생존율을 높이기 위해서는 caffeine 이 첨가된 M-Soejima 보존액에 동해보호제로 glycerol과 propylene glycol 또는 ehtylene glycol 을 사용하여 2시간 동안 빠르게 동결처리된 정액이 다소 유리할 것으로 사료되었다.