This study was conducted to investigate the survival rate of frozen-thawed spermatozoa in equine by glycerol concentration and freezing speed Two stallions (1 Thoroughbred-13 year old and 1 Arab-7 year old) bred in Korea Racing authority was examined for 1 times in a couple of weeks. Semen was collected by condom method standing heated mare and were centrifuged 650 g for 15 min. and isolated the seminal plasma. Thick fraction of semen was diluted EDTA-Lactose-egg yolk diluents to 1:1 and contained in 0.5 ml straw as $6{\sim}14{\times}10^7\;cells/ml$. Final concentrations of glycerol were 3, 5 and 7% in cryopreseved diluents and added 4 times for 2 hours equilibration. For the freezing, equilibrated straws were located 3 or 5 em above $LN_2$ gas for 5 or 10 min. Survival rates of pre-frozen sperm were $65.0{\pm}13.2%$, $68.3{\pm}10.4%$, $66.7{\pm}11.5%$ and post-frozen were $53.3{\pm}23.1%$, $45.0{\pm}15.0%$, $50.0{\pm}18.0%$ in 3, 5, 7% glycerol concentration, respectively. There was no difference between glycerol concentrations. Survival rates of frozen-thawed sperm on freezing speed were $36.7{\pm}10.4%$, $40.0{\pm}7.1%$, $30.0{\pm}13.2%$ at 3 cm-5 min and $33.3{\pm}11.5%$, $31.7{\pm}2.9%$, $21.7{\pm}10.4%$ at 3 cm-10 min in 3, 5, 7% glycerol concentration, respectively. Survival rates of frozen-thawed sperm on freezing speed were $43.3{\pm}15.3%$, $32.0{\pm}17.9%$, $22.3{\pm}15.7%$ at 5cm-5 min and were $47.5{\pm}15.0%$, $43.3{\pm}12.6%$, $48.3{\pm}15.3%$ at 5cm-10 min in 3, 5, 7% glycerol concentration, respectively. There were significantly different between groups (p<0.05). These results suggest that glycerol concentration did not affect cryopreservation of stallion semen within 3~7% but freezing speed affects. In our experiment, the best cryopreservation condition was at 5 cm above $LN_2$ gas for 10 min for pre-freezing and 7% of glycerol concentration. These results lead to commercial AI with frozen-thawed stallion semen.
In this study, two epididymal spermatozoa recovery methods in relation to sperm number, motility, viability and acrosome reaction were examined. Seven bulls were castrated and 7 testicles with epididymides were transferred to the laboratoy. Epididymis in each bull was randomly used for flushing and mincing methods with semen extender (Optixcell, IMV, France). The recovered spermatozoa with adjusted sperm concentration to $40{\times}10^6cells/mL$ was diluted with optixcell and cryopreserved. In experiment 1, the difference in the total number of spermatozoa using flushing and mincing methods was insignificant (2570.0 and $2505.2{\times}10^6cells/mL$, respectively). For experiment 2, the percentage of motile spermatozoa and motility parameters between flushing and mincing methods were studied through the use of sperm class analyzer after frozen-thawing. The percentage of total motile sperm between flushing and mincing methods was almost the same with $89.5{\pm}12.8$ and $91.4{\pm}7.9%$, respectively. The same is the case with experiment 3 wherein the viability and acrosomal integrity of frozen-thawed epididymal spermatozoa by flushing and mincing was insignificantly different. The results from the study showed that both flushing and mincing methods can be used for epididymal spermatozoa recovery in bull.
This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing $500{\mu}l$ mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to $24^{\circ}C$ over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were esuspended in a lactose-egg yolk and N-acetyl-Dglucosamine (LEN) diluent to contain $1{\times}10^{9}$ sperm/ml and cooled to $5^{\circ}C$ over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with $2{\times}10^{7}$ sperm concentration. Oocytes were coincubated for 6 h in $500{\mu}l$ mTBM fertilization. At 6 h after IVF, oocytes were transferred into $500{\mu}l$ NCSU-23 culture medium for further culture of 6 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF and developmental ability of oocytes at 48 h after IVF were evaluated. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium.
This study aimed to examine the testis toxicities of metal compound, manganese (Mn), which may be generated as mist or fume in the industrial sites. As well as serum prolactin (PRL) concentration was analyzed because Mn accumulation in basal ganglia up-regulates serum PRL and hyperprolactinemia consecutively induces the testis toxicity. Male F344 rats were divided into the 4 groups (2 controls and 2 Mn treated groups, n=10) on the basis of the test condition (inhalation, Mn $1.5mg/m^3$ or not) and treatment period (for 4-weeks and 13-weeks). The treatment time was 6 hr. a day, 5 days a week for the whole body. Basic tests including changes in body weight, feed rate were observed. Blood and testis Mn concentration, and testis toxicity test such as the number and deformity test of sperm were also observed. Serum PRL level was analyzed by ELISA to certify the relationship between the Mn induced increase of the serum PRL level and sperm production. Blood and testis Mn concentrations were significantly and dose-dependently increased. Sperm count was decreased in Mn-treatment groups than control in a treatment time dependent manner. Morphological analysis of cauda epidydimal sperm showed that the frequencies of morphologically abnormal sperms such as bent tail and small head were increased in the both Mn-treatment groups than control. A significant increase in serum PRL levels was found in response to Mn treatment but it was not hyperprolactinemia range. These results suggest that treatment of Mn up-regulates the serum PRL concentration and induces the testis toxicity. The No Aversed Effect Level (NOAEL) of inhaled Mn on the male rat testis may be under the $1.5mg/m^3$.
A simplified dilutor for cock spermatozoa at ambient temperature was achieved by adjusting the 5% concentration of fructose in isotonic saline. Motility of cock spermatozoa was arrested completely for maximum 6 hrs without affection the survivability of spermatozoa by employing this sugar. To study the effect of high concentration of fructose on fertility, sperm were inseminated with or without fructose at different hrs. Fructose from semen samples was removed by centrifugation. High fertility obtained in the hens inseminated with fructose free sperm (washed). In addition, washed sperm maintained the 85.00% fertility for 6 hrs in winter season ($17-21^{\circ}C$) and 82.67% fertility for 3 hrs in summer season ($31-35^{\circ}C$). Whereas control groups showed 47.33 and 25.33% fertility in winter and summer season respectively. No significant difference was found in percent motility and live counts between the control and washed experimental groups during winter season. However, these measures differed significantly in summer. Washing of cock spermatozoa more than once, high speed centrifugation and more duration for centrifugation proved harmful to fertility. It may be concluded that fructose (5%) can be used as a motility or metabolic inhibitor of spermatozoa for short-term storage of cock semen at ambient temperatures.
Park, Mi Seon;Min, Byung Hwa;Lim, Hyun-Jeong;Hur, Young Baek;Do, Yong Hyun;Myeong, Jeong-In
Korean Journal of Fisheries and Aquatic Sciences
/
v.47
no.6
/
pp.853-859
/
2014
The goal of this study was to evaluate the effects of cryoprotectants [dimethyl sulfoxide (DMSO), methanol, polyethylene glycol, and propylene glycol], cryoprotectant concentrations (10% and 20%), equilibration time (3, 10, and 30 min), cooling rate ($3^{\circ}$, $5^{\circ}$, $7^{\circ}$, and $10^{\circ}/min$), and straw size (0.25 and 0.5 mL) for sperm cryopreservation of tetraploid Pacific oysters. There was a significant difference among the four cryoprotectants, with 10% DMSO yielding the highest post-thaw survival and activity index of sperm. A significant relationship was observed between the cryoprotectant and its concentration. The sperm with equilibration times of 30 min yielded higher post-thaw survival and activity indices than those with 3 and 10 min equilibration times. The sperm cooled at a rate of $5^{\circ}/min$ yielded the highest post-thaw survival and activity index, and the results were significantly different from those observed for cooling at $7^{\circ}$ and $10^{\circ}/min$. Post-thaw survival and activity indices of sperm using a 0.25-mL straw were significantly higher than those using a 0.5-mL straw.
Baek, Sun Young;Chung, Hak Jae;Hong, Joon Ki;Cho, Eun Seok;Choi, Inchul
Korean Journal of Agricultural Science
/
v.47
no.3
/
pp.657-665
/
2020
The objective of this study was to investigate whether supplementation of pentoxifylline (PTX; phosphodiesterase inhibitor) to thawed boar semen improves the post-thaw motility of sperm and affects the efficiency of artificial insemination (AI) and further development. To determine the concentration of PTX for AI, frozen-thawed semen was incubated with 0, 5, 10, and 20 mM PTX in an extender freezing medium, respectively, after thawing. Kinematic properties of sperm were examined with a computer-assisted semen analysis (CASA) system. In addition, viability and mitochondrial activity were also tested by LIVE/DEAD and a MitoTracker kit. There were no significant differences in the kinetic parameters of thawed sperm between control and treatment groups, but overall assessment parameters such as motility and rapid progressive were higher in the 10 mM PTX group. In the viability and mitochondrial assay, there were no significant differences observed in the PTX treatment, compared to the control. For further analysis, artificial inseminations were performed using frozen semen and 10 mM PTX treated cryopreserved semen, respectively. There were no differences in pregnancy rates and fetus weights among the groups until 30 and 40 days, but litter size was reduced and relatively low-birth weight was observed in the PTX group. In summary, our findings suggest that enhancement of in vitro sperm quality or non-toxicity supplemented by PTX may have detrimental effects on fetus development.
During storage, boar spermatozoa undergo several changes including diminished motility and viability and accumulated reactive oxygen species (ROS). In this study, we investigated the effects of green tea extract (GTE) supplementation in the Sui Dil extender on the sperm motility, viability, ROS and lipid peroxidation (LPO) of long-term preserved boar semen at $17^{\circ}C$. A total number of eight boars were used for this experiment. Pooled ejaculates were diluted to $20{\times}10^6sperm/ml$ in the Sui Dil extender containing 0 (control), 1, 10, 100 or 500 mg/l GTE and were preserved at $17^{\circ}C$ for 24, 72, 120 and 168 h, respectively. At each storage time, sperm motility and viability were estimated by microscopic examination and the fluorescent double stain $Fertilight^{(R)}$, respectively. Sperm ROS level and LPO were assessed using the 2', 7'-dichlorodihydrofluorescein diacetate ($H_2DCFDA$)/propidium iodide (PI) and C11-BODIPY581/591/PI with flow cytometry, respectively. Compared to that of the 500 mg group, there were higher sperm motility and viability in the 1, 10 and 100 mg GTE groups during the preservation from 24 to 168 h (p < 0.05). The ROS levels of the 10 and 100 mg groups during the 168 h preservation were lower than those of the 0, 1 and 500 mg groups (p < 0.05). There were no significant differences in LPO regardless of the preservation period or the GTE concentration. In conclusion, the optimal concentrations (10 and 100 mg/l) of GTE that led to lower ROS levels may be useful for liquid boar sperm preservation at $17^{\circ}C$ for a period of 168 h.
Kim, Su-Hee;Lee, Young-Jun;Kang, Tae-Woon;Kim, Yong-Jun
Journal of Veterinary Clinics
/
v.28
no.1
/
pp.13-19
/
2011
Semen cryopreservation induces the formation of reactive oxygen species (ROS), and the ROS cause sperm damage. We aimed to investigate the effects of the antioxidative enzyme catalase (CAT) on sperm quality and ROS during cryopreservation. Sperm rich fractions collected from five Duroc boars were cryopreserved in freezing extender with (200 or 400 U/mL) or without CAT (control). After thawing, sperm motility, viability, normal morphology, plasma membrane integrity, mitochondrial function and intracellular ROS were evaluated. CAT significantly improved total sperm motility at a concentration of 400 U/mL (P < 0.05), but didn't improve progressive sperm motility, viability, morphological defects, plasma membrane integrity and mitochondrial function in frozen-thawed boar sperm. In evaluation of ROS, CAT had no effect on reduction in ${\cdot}O_2$, but scavenged $H_2O_2$ in viable frozen-thawed boar sperm at concentrations of 200 and 400 U/mL (P < 0.05). In conclusion, CAT was not enough to improve quality of frozen-thawed sperm, but can reduce $H_2O_2$ generation in viable boar sperm during cryopreservation.
In male reproducible health, fertility and IVF (in-vitro fertilization), semen analysis has been most important. Semen analysis can be divided into concentration, motional and morphological analysis of sperm. The existing method which was developed earlier to analyze semen concentrated on the sperm motility analysis. To provide more useful and precise solutions for clinical problems such as infertility, semen analysis must include sperm morphological analysis. But the traditional tools for semen analysis are subjective, imprecise, inaccurate, difficult to standardize, and difficult to reproduce. Therefore, with the help of development of microcomputers and image processing techniques, we developed a new sperm morphology analyzer to overcome these problems. In this study the agreement on percent normal morphology was studied between different observers and a computerized sperm morphology analyzer on a slide-by-slide basis using strict criteria. Slides from 30 different patients from the SNUH andrology laboratory were selected randomly. Microscopic fields and sperm cells were chosen randomly and percent normal morphology was recorded. The ability of sperm morphology analyzer to repeat the same reading for normal and abnormal cells was studied. The results showed that there was no significant bias between two experienced observers. The limits of agreement were 4.1%${\sim}$-3.8%. The Pearson correlation coefficient between readers was 0.79. Between the manual and sperm morphology analyzer, the same findings were reported. In this experiments the slides were stained by two different methods, PAP and Diff-Quik staining methods. The limits of agreement were 7.2%${\sim}$-5.7% and 6.0%${\sim}$-6.3%, respectively. The Pearson correlation coefficients ware 0.76 and 0.91, respectively. The limits of agreement was tighter below 20% normal forms. In the experiments of repeatability, 52 cells stained by PAP and Diff-Quik staining methods were analyzed three times in succession. Estimating pairwise agreement, the kappa statistic for the pairs were 0.76, 0.81, 0.86, and 0.75, 0.88, 0.88 respectively. In this study it was shown that there was good agreement between manual and computerized assessment of normal and abnormal cells. The repeatability and agreement per slide of computerized sperm morphology analyzer was excellent. The computer's ability to classify normal morphology per slide is promising. Based on results obtained, this system can be of clinical value both in andrology laboratories and IVF units.
본 웹사이트에 게시된 이메일 주소가 전자우편 수집 프로그램이나
그 밖의 기술적 장치를 이용하여 무단으로 수집되는 것을 거부하며,
이를 위반시 정보통신망법에 의해 형사 처벌됨을 유념하시기 바랍니다.
[게시일 2004년 10월 1일]
이용약관
제 1 장 총칙
제 1 조 (목적)
이 이용약관은 KoreaScience 홈페이지(이하 “당 사이트”)에서 제공하는 인터넷 서비스(이하 '서비스')의 가입조건 및 이용에 관한 제반 사항과 기타 필요한 사항을 구체적으로 규정함을 목적으로 합니다.
제 2 조 (용어의 정의)
① "이용자"라 함은 당 사이트에 접속하여 이 약관에 따라 당 사이트가 제공하는 서비스를 받는 회원 및 비회원을
말합니다.
② "회원"이라 함은 서비스를 이용하기 위하여 당 사이트에 개인정보를 제공하여 아이디(ID)와 비밀번호를 부여
받은 자를 말합니다.
③ "회원 아이디(ID)"라 함은 회원의 식별 및 서비스 이용을 위하여 자신이 선정한 문자 및 숫자의 조합을
말합니다.
④ "비밀번호(패스워드)"라 함은 회원이 자신의 비밀보호를 위하여 선정한 문자 및 숫자의 조합을 말합니다.
제 3 조 (이용약관의 효력 및 변경)
① 이 약관은 당 사이트에 게시하거나 기타의 방법으로 회원에게 공지함으로써 효력이 발생합니다.
② 당 사이트는 이 약관을 개정할 경우에 적용일자 및 개정사유를 명시하여 현행 약관과 함께 당 사이트의
초기화면에 그 적용일자 7일 이전부터 적용일자 전일까지 공지합니다. 다만, 회원에게 불리하게 약관내용을
변경하는 경우에는 최소한 30일 이상의 사전 유예기간을 두고 공지합니다. 이 경우 당 사이트는 개정 전
내용과 개정 후 내용을 명확하게 비교하여 이용자가 알기 쉽도록 표시합니다.
제 4 조(약관 외 준칙)
① 이 약관은 당 사이트가 제공하는 서비스에 관한 이용안내와 함께 적용됩니다.
② 이 약관에 명시되지 아니한 사항은 관계법령의 규정이 적용됩니다.
제 2 장 이용계약의 체결
제 5 조 (이용계약의 성립 등)
① 이용계약은 이용고객이 당 사이트가 정한 약관에 「동의합니다」를 선택하고, 당 사이트가 정한
온라인신청양식을 작성하여 서비스 이용을 신청한 후, 당 사이트가 이를 승낙함으로써 성립합니다.
② 제1항의 승낙은 당 사이트가 제공하는 과학기술정보검색, 맞춤정보, 서지정보 등 다른 서비스의 이용승낙을
포함합니다.
제 6 조 (회원가입)
서비스를 이용하고자 하는 고객은 당 사이트에서 정한 회원가입양식에 개인정보를 기재하여 가입을 하여야 합니다.
제 7 조 (개인정보의 보호 및 사용)
당 사이트는 관계법령이 정하는 바에 따라 회원 등록정보를 포함한 회원의 개인정보를 보호하기 위해 노력합니다. 회원 개인정보의 보호 및 사용에 대해서는 관련법령 및 당 사이트의 개인정보 보호정책이 적용됩니다.
제 8 조 (이용 신청의 승낙과 제한)
① 당 사이트는 제6조의 규정에 의한 이용신청고객에 대하여 서비스 이용을 승낙합니다.
② 당 사이트는 아래사항에 해당하는 경우에 대해서 승낙하지 아니 합니다.
- 이용계약 신청서의 내용을 허위로 기재한 경우
- 기타 규정한 제반사항을 위반하며 신청하는 경우
제 9 조 (회원 ID 부여 및 변경 등)
① 당 사이트는 이용고객에 대하여 약관에 정하는 바에 따라 자신이 선정한 회원 ID를 부여합니다.
② 회원 ID는 원칙적으로 변경이 불가하며 부득이한 사유로 인하여 변경 하고자 하는 경우에는 해당 ID를
해지하고 재가입해야 합니다.
③ 기타 회원 개인정보 관리 및 변경 등에 관한 사항은 서비스별 안내에 정하는 바에 의합니다.
제 3 장 계약 당사자의 의무
제 10 조 (KISTI의 의무)
① 당 사이트는 이용고객이 희망한 서비스 제공 개시일에 특별한 사정이 없는 한 서비스를 이용할 수 있도록
하여야 합니다.
② 당 사이트는 개인정보 보호를 위해 보안시스템을 구축하며 개인정보 보호정책을 공시하고 준수합니다.
③ 당 사이트는 회원으로부터 제기되는 의견이나 불만이 정당하다고 객관적으로 인정될 경우에는 적절한 절차를
거쳐 즉시 처리하여야 합니다. 다만, 즉시 처리가 곤란한 경우는 회원에게 그 사유와 처리일정을 통보하여야
합니다.
제 11 조 (회원의 의무)
① 이용자는 회원가입 신청 또는 회원정보 변경 시 실명으로 모든 사항을 사실에 근거하여 작성하여야 하며,
허위 또는 타인의 정보를 등록할 경우 일체의 권리를 주장할 수 없습니다.
② 당 사이트가 관계법령 및 개인정보 보호정책에 의거하여 그 책임을 지는 경우를 제외하고 회원에게 부여된
ID의 비밀번호 관리소홀, 부정사용에 의하여 발생하는 모든 결과에 대한 책임은 회원에게 있습니다.
③ 회원은 당 사이트 및 제 3자의 지적 재산권을 침해해서는 안 됩니다.
제 4 장 서비스의 이용
제 12 조 (서비스 이용 시간)
① 서비스 이용은 당 사이트의 업무상 또는 기술상 특별한 지장이 없는 한 연중무휴, 1일 24시간 운영을
원칙으로 합니다. 단, 당 사이트는 시스템 정기점검, 증설 및 교체를 위해 당 사이트가 정한 날이나 시간에
서비스를 일시 중단할 수 있으며, 예정되어 있는 작업으로 인한 서비스 일시중단은 당 사이트 홈페이지를
통해 사전에 공지합니다.
② 당 사이트는 서비스를 특정범위로 분할하여 각 범위별로 이용가능시간을 별도로 지정할 수 있습니다. 다만
이 경우 그 내용을 공지합니다.
제 13 조 (홈페이지 저작권)
① NDSL에서 제공하는 모든 저작물의 저작권은 원저작자에게 있으며, KISTI는 복제/배포/전송권을 확보하고
있습니다.
② NDSL에서 제공하는 콘텐츠를 상업적 및 기타 영리목적으로 복제/배포/전송할 경우 사전에 KISTI의 허락을
받아야 합니다.
③ NDSL에서 제공하는 콘텐츠를 보도, 비평, 교육, 연구 등을 위하여 정당한 범위 안에서 공정한 관행에
합치되게 인용할 수 있습니다.
④ NDSL에서 제공하는 콘텐츠를 무단 복제, 전송, 배포 기타 저작권법에 위반되는 방법으로 이용할 경우
저작권법 제136조에 따라 5년 이하의 징역 또는 5천만 원 이하의 벌금에 처해질 수 있습니다.
제 14 조 (유료서비스)
① 당 사이트 및 협력기관이 정한 유료서비스(원문복사 등)는 별도로 정해진 바에 따르며, 변경사항은 시행 전에
당 사이트 홈페이지를 통하여 회원에게 공지합니다.
② 유료서비스를 이용하려는 회원은 정해진 요금체계에 따라 요금을 납부해야 합니다.
제 5 장 계약 해지 및 이용 제한
제 15 조 (계약 해지)
회원이 이용계약을 해지하고자 하는 때에는 [가입해지] 메뉴를 이용해 직접 해지해야 합니다.
제 16 조 (서비스 이용제한)
① 당 사이트는 회원이 서비스 이용내용에 있어서 본 약관 제 11조 내용을 위반하거나, 다음 각 호에 해당하는
경우 서비스 이용을 제한할 수 있습니다.
- 2년 이상 서비스를 이용한 적이 없는 경우
- 기타 정상적인 서비스 운영에 방해가 될 경우
② 상기 이용제한 규정에 따라 서비스를 이용하는 회원에게 서비스 이용에 대하여 별도 공지 없이 서비스 이용의
일시정지, 이용계약 해지 할 수 있습니다.
제 17 조 (전자우편주소 수집 금지)
회원은 전자우편주소 추출기 등을 이용하여 전자우편주소를 수집 또는 제3자에게 제공할 수 없습니다.
제 6 장 손해배상 및 기타사항
제 18 조 (손해배상)
당 사이트는 무료로 제공되는 서비스와 관련하여 회원에게 어떠한 손해가 발생하더라도 당 사이트가 고의 또는 과실로 인한 손해발생을 제외하고는 이에 대하여 책임을 부담하지 아니합니다.
제 19 조 (관할 법원)
서비스 이용으로 발생한 분쟁에 대해 소송이 제기되는 경우 민사 소송법상의 관할 법원에 제기합니다.
[부 칙]
1. (시행일) 이 약관은 2016년 9월 5일부터 적용되며, 종전 약관은 본 약관으로 대체되며, 개정된 약관의 적용일 이전 가입자도 개정된 약관의 적용을 받습니다.