• 한국수정란이식학회지
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• 제30권3호
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• pp.121-128
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• 2015

The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed ($37^{\circ}C$) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at $38.5^{\circ}C$ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

• 한국패류학회지
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• 제27권2호
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• pp.149-157
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• 2011

Some characteristics of the formations of acrosomal vesicles during the late stage of spermatids during spermiogenesis and taxonomical charateristics of sperm morphology in male two species (Saxidomus purpurata and Meretrix petechialis) in the family Veneridae were investigated by electron microscope observations. In two species, the morphologies of the spermatozoa have the primitive type and are similar to those of other bivalves in that it contains a short midpiece with five mitochondria surrounding the centrioles. The morphologies of the sperm nuclear types of S. purpurata and M. petechialis in Veneridae have the curved cylindrical and cylinderical type, respectively. And the acrosome shapes of two species are the same cap-shape type. In particular, the axial filament is not found in the lumen of the acrosome of two species, however, subacrosomal material are observed in the subacrosomal spaces between the anterior nuclear fossa and the acrosomal vesicle of two species. The spermatozoon of S. purpurata is approximately 46-$52{\mu}m$ in length, including a curved sperm nucleus (about $3.75{\mu}m$ in length), a long acrosome (about $0.40{\mu}m$ in length),and a tail flagellum (about 45-$47{\mu}m$ long). And the spermatozoon of M. petechialis is approximately 47-$50{\mu}m$ in length including a slightly curved sperm nucleus (about $1.50{\mu}m$ in length), an acrosome (about $0.56{\mu}m$ in length) and tail flagellum (44-$48{\mu}m$ in length). In two species, the axoneme of the sperm tail flagellum of each species consists of nine pairs of microtubules at the periphery and a pair of cental doublets at the center. Therefore, the axoneme of the sperm tail flagellum shows a 9 + 2 structure. In particular, taxonomically important some charateristics of sperm morphologies of two species in the family Veneridae are acrosomal morphology of the sperm, The axial filament is not found in the acrosome as seen in a few species of the family Veneridae in the subclass Heterodonta. The acrosomal vesicle is composed of right, left basal rings and the apex part of the acrosomal vesicle. In particular, right and left basal rings show electron opaque part (region), while the apex part of the acrosomal vesicle shows electron lucent part (region). These charateristics belong to the subclass Heterodonta, unlikely a characteristic of the subclass Pteriomorphia showing all part of the acrosome being composed of electron opaque part (region). Therefore, it is easy to distinguish the families or the subclasses by the acrosomal structures. The number of mitochondria in the midpiece of the sperm of S. purpurata and M. petechialis in Veneridae are five. However, the number of mitochondria in the midpiece of the sperm in most species of Veneridae in the subclass Heterodonta are four. Therefore, the number of mitochondria of the sperm midpiece of two species are exceptionally 5, and it is only exceptional case in the species in Veneridae in the subclass Heterodonta. Except these cases, the number of mitochondria in the sperm midpiece in all families in the subclass Heterodontaare are 4, and now widely used in taxonomic analyses.

• Journal of Animal Science and Technology
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• 제49권5호
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• pp.585-592
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• 2007

본 연구는 풍산개 동결정액 제조기술을 정립하기 위하여 정액성상과 정액 동결 시 희석액에 첨가되는 Glycerol 농도, 동결속도, 융해온도와 시간에 따라 정액의 운동성과 생존율 및 CASA를 이용한 운동성 등에 대하여 조사하여 최적의 동결조건을 확립하기 위해 실시하였다.1.풍산개의 평균 정액량 5.9ml, 정액농도 116.3 ×106sperm/ml 총정자수 789.3×106sperm, 운동성 88.7±1.77% 및 생존율 87.6±1.85% 였다. 2.1차 희석액을 상온에서 희석 후 상온에서 4°C까지 하강시킨 후 glycerol 농도가 3%, 5% 및 7% 첨가된 2차 희석액을 희석 후 6일간 운동성을 측정한 결과 5일째 3%일 때 46.2±9.3%, 5% glycerol에서는 48.1±8.5% 및 7%일 때 52.7±8.2%로 glycerol 7%가 유의적으로 높은 운동성을 나타냈다.3. 각기 다른 glycerol 농도를 함유한 동결보존액에서 동결보존 후 융해하였을 때 7%의 glycerol 농도에서 각각 52.7%와 57.7±10.3%로서 다른 처리구에 비해 유의적인(P<0.01) 운동성 및 생존율을 나타냈다.4.정액을 동결함에 있어 예비동결시 57 및 10cm의 높이에서 정치시킨 후 동결을 실시하였을 때 액체질소의 표면 7cm의 높이에서 동결을 실시한 처리구에서 전체적으로 유의한 운동성과 생존율을 나타냈다.

• 한국패류학회지
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• 제27권3호
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• pp.273-282
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• 2011

The ultrastructural characteristics of germ cells during spermatogenesis and mature sperm morphology in male Pinctada martensii were investigated by transmission electron microscope observation. The morphologies of the sperm nucleus and the acrosome of this species are the oval shape and cone shape, respectively. Spermatozoa are approximately $47-50{\mu}m$ in length including a sperm nucleus (about $1.24{\mu}m$ in length), an acrosome (about $0.60{\mu}m$ in length), and tail flagellum (about $45-47{\mu}m$). The axoneme of the sperm tail shows a 9+2 structure. In P. martensii in Pteriidae, a special substructure showing a thick and wide triangular shape which is composed of electron-dense opaque material (occupied about 50% of all, the upper part of the acrosomal vesicle), appeared in the upper region (part) of the acrosomal vesicle, while the lower region (part) of the acrosomal vesicle is composed of electron-lucent material. Thus, this special structure, which exist in the upper part of the acrosomal vesicle in P. martensii, is somewhat different from those of other subacrosomal vesicle in other families in subacrosomal vesicles. Therefore, we assume that the existence of a special substructure showing a thick and wide triangular shape in the acrosomal vesicle of the spermatozoon can be used as a key characteristic for identification of P. martensii or other species in Pteriidae in subclass Pteriomorphia. The number of mitochondria in the midpiece of the sperm of this species are five (exceptionally sometimes four), as one of common characteristics appear the same number of mitochondria in the same families of superfamilyies. This species in Pteriidae does not contain the axial rod and satellite fibres which appear in the species in Ostreidae in subclass Pteriomorphia. These characteristics can be used for the taxonomic analysis of the family or superfamily levels as a systematic key or tools.

• 대한수의학회지
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• 제40권1호
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• pp.187-195
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• 2000

The ejaculates from 67 HanWoo prove bull, bred in Livestock Improvement Main center of NLCF, were used to determine the correlation between the sperm motional characteristics and the pregnancy rate of artificial insemination(AI). The motional characteristics of sperm were analysed by Computer-assisted sperm analyser(CASA), thereafter inseminated equally 1,256 heads of cow regarding to parity, age, and live weight. There were no significant difference(p>0.05) in the pregnancy rate according to year from 1996 to 1998, but the LIN, ALH, STR, BCF, MAD and WOB of sperm in the year 1997, were highest pregnancy rate, were higher than those of sperm in the year 1998, were lowest pregnancy rate(p<0.05). The semen had no significant effect on pregnancy rate according to season(p>0.05). However spring, had a little higher pregnancy rate than that of autumn, were higher than autumn in VSL, VAP, LIN, ALH, BCF, MAD and WOB, but in DNM. The pregnancy rates of spring in the year 1996 and 1997 were higher than that of autumn in the year 1998(p<0.05). The spring in the year 1997, highest in pregnancy rate, were higher than the autumn in the year 1998 in VSL, VAP, LIN, STR, BCF, MAD and WOB, but in DNM(p<0.05). There were no the motion characteristic of sperm that was significant correlate with pregnancy rate of AI as the semen were analysed before artificial insemination and those, had some degree characteristics in motility, viability and abnormality, were used to AI. However there were a tendency that the higher the VSL, VAP, ALH, LIN, STR, BCF, MAD and WOB and the lower the DNM were, the higher the pregnancy rate of AI were.

• Asian-Australasian Journal of Animal Sciences
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• 제19권11호
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• pp.1561-1565
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• 2006

The objective of this study was to investigate the anti-oxidative effects of taurine on sperm characteristics for in vitro storage of boar semen. Semen was randomly divided into 10 groups in conical tubes and treated with different concentrations of taurine (25-100 mM) with or without $250{\mu}M$ $H_2O_2$. The percentage of motile spermatozoa in taurine groups after 6 and 9 h were significantly higher at >94% and 87%, respectively, compared to the control group ($85.1{\pm}0.5$ and $72.4{\pm}0.3$, p<0.05). The sperm motility in taurine with $H_2O_2$ after 6 h incubation was slightly decreased compared to the taurine alone treatment, but after 9 and 12 h incubation % sperm motility dropped sharply in taurine with $H_2O_2$ ($75.3{\pm}0.3$ and $69.6{\pm}2.9$, p<0.05). For 3, 9 and 12 h incubation, sperm viability in the control was lower than in taurine groups, irrespective of taurine concentration. In eosin Y and nigrosin staining (ENS), the sperm survival rates (%) for 6 h incubation were significantly higher in 25 mM ($76.0{\pm}0.6$) and 50 mM taurine groups ($78.0{\pm}0.7$), respectively. Sperm survival rates for 9 and 12 h incubation were higher in taurine groups (${\geq}48%$ in 9 h and ${\geq}42%$ in 12 h) compared to controls ($43.0{\pm}2.1$ and $31.0{\pm}0.6$, respectively). In the hyoosmotic swelling test (HOST), sperm membrane integrity was similar to the results of sperm survival. These experiments indicate that supplementation of taurine to the semen extender can increase the sperm characteristics(motility, viability, survival and membrane integrity).

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.385-390
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• 2011

The purpose of this study was undertaken to evaluate of cryopreservation efficiency in ${\alpha}$ 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen ($LN_2$) vapours for 10 min before placing them into $LN_2$ for cryopreservation. After thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.

• Clinical and Experimental Reproductive Medicine
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• 제38권1호
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• pp.47-52
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• 2011

Objective: To determine whether characteristics of sperm motility obtained by computer-assisted sperm analysis (CASA) could predict pregnancy after intrauterine insemination (IUI) in couples with unexplained infertility. Methods: Three hundred eighty-three cycles of intrauterine insemination with superovulation were retrospectively analyzed. Semen analysis was performed with CASA before and after swim-up and the parameters were compared between pregnant and non-pregnant women. Results: The pregnancy rate per cycle was 14.1%. Pregnant and non-pregnant women were comparable in terms of age, infertility duration, the number of dominant follicles. While sperm concentration, motility, and parameters such as average path velocity (VAP) and percentage rapid (RAPID) before semen preparation were significantly different between the pregnancy and non-pregnancy groups, there were no differences in sperm parameters when comparing the two groups after preparation. Using a receiver operating characteristic curve to measure sensitivity and specificity, the optimal threshold value for the predictors of pregnancy was revealed to be a concentration of ${\geq}111{\times}10^6/mL$, a motility of ${\geq}$ 51.4%, and RAPID ${\geq}$ 30.1% before preparation for IUI. Conclusion: Sperm parameters including concentration, motility, and RAPID before sperm preparation could have predictive value for pregnancy outcome after intrauterine insemination with superovulation in couples with unexplained infertility, and would be helpful when counseling patients before they make the decision to proceed with IVF/ICSI-ET.

• 한국수정란이식학회지
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• 제26권4호
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• pp.257-263
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• 2011

BPA, a diphenyl compound containing groups, that make it structurally similar to synthetic estrogen and is considered as one of the major endocrine disruptors. Silymarin has extensively been used to prevent and/or alleviate some human disease, especially for the treatment of adverse liver conditions. It has an antioxidative efficacy and cancer preventive efficacy. Therefore, we examined the hypothesis that silymarin can inhibit BPA-induced toxicity in boar sperm duing in vitro storage. Sperm characteristics (motility, viability, membrane integrity and mitochondrion activity) in semen exposed to BPA (10~200 uM) were sharply lowered, while it increase in a dose and time dependent manner due to silymarin addition (50~200 uM) into semen extender in the presence of BPA (100 uM). All of the evaluated characteristics were gradually improved in the groups that were treated with silymarin (50~200 uM) in the presence of BPA (100 uM) in comparison to BPA 100 uM alone group, irrespective of incubation periods (3 and 6 h). These results demonstrate that silymarin can ameliorate the toxicity of BPA on boar sperm characteristics during in vitro storage, suggesting that silymarin indirectly act as an antioxidant.

• 한국가축번식학회지
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• 제26권3호
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• pp.253-261
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• 2002

본 연구는 진도개 동결정액 제조기술을 정립하기 위하여 진도개 정액성상과 동결 전후 정액의 활력과 생존율 및 CASAs를 이용한 운동성 등에 대하여 조사하였고, 백구와 황구간, 개체간의 정액성상과 내동성을 비교, 조사하였다. 이상의 연구결과는 다음과 같다. 1. 총 63회 정액채취 후 신선정액의 평균 정액량 3.8 $m\ell$, 농도 145.6$\times$$10^{6}$$m\ell$, 총정자수 396.2$\times$08/$m\ell$, 전진운동율 79.7% 및 생존율 89.5% 였다. 황구와 백구간에는 황구가, 개체간에는 황구 2호가 정자농도, 총정자수, 전진운동정자율 및 생존율 등의 정액성상에서 유의적으로 우수하였다(P<0.05). 2. 동결전.후 정자의 전진운동율과 생존율을 46회 조사한 결과 동결전 73.5%와 82.3%를, 동결후 51.1%와 64.9%를 나타내 동결과정이 전진운동율과 생존율에 영향이 있었으며, 역시 황구와 백구간에는 황구가, 개체간에는 황구 2호가 동결전후 전진운동율과 생존율에서 유의적인 차이가 인정되었다 (P<0.05). 3. 동결.융해정자의 보다 객관적인 평가를 위하여 CASA system을 이용한 총 44회 평가한 결과, 생존율 65.6%, 전진운동율 54.8%, VAP 75.3 $\mu\textrm{m}$/sec, VCL 90.0 $\mu\textrm{m}$/sec, VSL 69.4 $\mu\textrm{m}$/sec 및 ALH 4.4 $\mu\textrm{m}$로 동결 융해 정액의 운동성은 양호하였고, 황구와 백구간의 운동성에는 유의적인 차이가 없었으나, 개체간에는 역시 황구 2호가 운동성이 우수하여 유의적인 차이가 인정되었다 (P<0.05). 4. 채취된 정액중 백구는 46%(13/28), 황구는 94%(33/35)가 동결정액 제조가 가능해 황구가 내동성이 좋았으며, 전체적으로 73%(46/63) 동결정액 제조가 가능하였다. 결론적으로 진도개의 동결정액을 제조하기 위하여 정액성상 및 동결 전후 운동성을 조사한 결과 동결정액 제조와 생산체계의 구축이 가능하였으며, 황구와 백구간, 개체간의 정액성상과 동결전후의 운동성에 차이가 인정되므로 정액성상과 내동성을 고려한 종견선발 체계가 필요하다고 사려되었다.