• Asian-Australasian Journal of Animal Sciences
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• 제28권12호
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• pp.1713-1720
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• 2015

The present work describes experiments undertaken to evaluate the usefulness of selected physicochemical indices of semen, cell membrane integrity and sperm chromatin structure for the assessment of boar semen sensitivity to processes connected with pre-insemination procedures. The experiments were carried out on 30 boars: including 15 regarded as providers of sensitive semen and 15 regarded as providers of semen that is little sensitive to laboratory processing. The selection of boars for both groups was based on sperm morphology analyses, assuming secondary morphological change incidence in spermatozoa as the criterion. Two ejaculates were manually collected from each boar at an interval of 3 to 4 months. The following analyses were carried out for each ejaculate: sperm motility assessment, sperm pH measurement, sperm morphology assessment, sperm chromatin structure evaluation and cell membrane integrity assessment. The analyses were performed three times. Semen storage did not cause an increase in the incidence of secondary morphological changes in the group of boars considered to provide sperm of low sensitivity. On the other hand, with continued storage there was a marked increase in the incidence of spermatozoa with secondary morphological changes in the group of boars regarded as producing more sensitive semen. Ejaculates of group I boars evaluated directly after collection had an approximately 6% smaller share of spermatozoa with undamaged cell membranes than the ejaculates of boars in group II ($p{\leq}0.05$). In the process of time the percentage of spermatozoa with undamaged cell membranes decreased. The sperm of group I boars was characterised with a lower sperm motility than the semen of group II boars. After 1 hour of storing diluted semen, the sperm motility of boars producing highly sensitive semen was already 4% lower ($p{\leq}0.05$), and after 24 hours of storage it was 6.33% lower than that of the boars that produced semen with a low sensitivity. Factors that confirm the accuracy of insemination male selection can include a low rate of sperm motility decrease during the storage of diluted semen, low and contained incidence of secondary morphological changes in spermatozoa during semen storage and a high frequency of spermatozoa with undamaged cell membranes.

• Clinical and Experimental Reproductive Medicine
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• 제30권2호
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• pp.119-126
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• 2003

Objective: This study was carried out to compare the clinical outcomes of intracytoplasmic sperm injection (ICSI) in patients with obstructive azoospermia according to sperm retrieval site and technique; microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA), testicular sperm extraction by open biopsy (TESE). Methods: The outcomes of ICSI and IVF-ET were evaluated and compared among 3 groups. Seventy three men suffering from infertility due to obstructive azoospermia had 107 ICSI cycles using MESA (21 cycles in 15 patients), PESA (26 cycles in 17 patients) and TESE (60 cycles in 41 patients). Results: In the clinical outcomes in patients undergoing ICSI with epididymal or testicular sperm, there were no significant differences in fertilization rate (66.1% vs. 60.5%), cleavage rate (94.9% vs. 97.6%), cumulative embryo score (CES) (51.3 vs. 58.8), implantation rate (7.9% vs. 6.1), and clinical pregnancy rate per ET (30.4% (14/46) vs. 25.4% (15/59)) between both groups. Also, in the clinical outcomes in ICSI patients using MESA, PESA, TESE, there were no significant differences in fertilization rate (61.8%, 69.4%, 60.5%), cleavage rate (92.1%, 97.3%, 97.6%), CES (38.1, 52.0, 58.8), implantation rate (9.5%, 6.6%, 6.1%), and clinical pregnancy rate per ET (35% (7/20), 26.9% (7/26), 25.4% (15/59)) among 3 groups. Conclusion: When compared with MESA or TESE, PESA, the clinical outcomes were similar in ICSI patients with obstructive azoospermia whatever the origin or the technique of sperm retrieval. However, we considered PESA is more time-saving and cost effective for ICSI in patients with obstructive azoospermia.

• Clinical and Experimental Reproductive Medicine
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• 제21권1호
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• pp.1-11
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• 1994

The occurrence and time course of capacitation, acrosomal loss, and hyperactivated motility require quantitative definition in order to characterize fertile human sperm. Recently the method has been developed to estimate the quality of spermatozoa by using kinematic parameters such as curvilinear velocity(VCL), average path velocity(VAP), linearity(LIN), straightness(STR), amplitude of lateral head displacement(ALH), and beat cross frequence(BCF) from Computer Assisted Sperm Analysis (CASA). In this study, using the Hamilton Thorn motility analyzer HTM 2030(Hamilton Thorn Research, Beverly, MA), we attempted to identify the spermatozoa with hyperactivated motility (HA) objectively and to monitor hyperactivation of human spermatozoa during incubation in capacitating media and after treatment of calcium ionophore as compared with acrosome status. And we examined whether HA are related to the result of SPA. Semen samples obtained from 16 healthy men were prepared by swim up technique and preincubated in a capacitating media(modified BWW medium) for 5 hours and treated with calcium ionophore solution. The acrosome reaction was detected with PSA-FITC labelling of the acrosome and in vitro sperm ferilizing capacity was assessed by the zona free hamster ovum penetration assay (SPA). The incidence of hyperactivated sperm was 2.6% in fresh semen, 14.3% of the swim up population, 13.7% after 5h of incubation. Significant increase of percentage of hyperactivated sperm was observed after the incubation (p<0.05) but after treatment, no significant changes of percentage of hyperactivated sperm(l1.8%) in contrast to significant rise in the percentage of acrosome reacted cells. Correlation analysis failed to show any significant relationship between the percentage of sperm with HA and SPA score. In conclusion, although no direct correlations were found between the results of SPA and HA, hyperactivation of sperm is associated with capacitation and monitoring hyperactivated sperm will be expected as a method of evaluating the functional quality of sperm such as SPA.

• 대한수의학회지
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• 제33권2호
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• pp.337-343
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• 1993

To determine the test conditions to enhance the use of hamster test in dogs, semen were collected from four dogs which had been proven to be fertile in the past and then preserved in BWW (Biggers, Whitten, Whittingham) medium for about 20 hours. The semen were given each different treatment according to the experimental design and coincubated with zona-free hamster ova for 5 hours. The ova were stained by lacmoid and examined under phase contrast microscope to investigate the rates of ova bound with sperm(sperm binding) and ova penetrated by sperm (penetration), and also numbers of both bound and penetrated sperm per ovum. In comparison of different concentrations of canine sperm, the rate of sperm binding was higher in $1.5{\times}10^8$, $1{\times}10^8$, and $5{\times}10^7$ sperm concentrations than $5{\times}10^5$ concentration(p<0.01), and also than $5{\times}10^6$ concentration(p<0.05), respectively. The number of bound sperm per ovum was considerably higher in $1.5{\times}10^8$ sperm concentration than $5{\times}10^7$, $1.5{\times}10^6$, and $5{\times}10^5$ concentrations(p<0.01). The rate of penetration was considerably higher in $1.5{\times}10^8$ and $1{\times}10^8$ sperm concentrations than $5{\times}10^5$ concentration,(p<0.01), and also the higher result of penetration was shown in $5{\times}10^7$ than $5{\times}10^5$ (p<0.05). The number of penetrated sperm per ovum was considerably higher in $1.5{\times}10^8$ sperm concentrations than $5{\times}10^5$(p<0.01), and also the higher number was shown in $1{\times}10^8$ than $5{\times}10^5$ (p<0.05). In comparison of the different preincubation period of canine spermatozoa, no difference was obtained in the results of hamster test among the preincubation periods of 4 hours, 18~24 hours and 48 hours. The canine spermatozoa in BWW medium with $Ca^{2+}$(1.3mM) and without FCS(fetal calf serum), with both $Ca^{2+}$(1.3mM) and FCS, with $Ca^{2+}$(2.6mM) and without FCS, and with both $Ca^{2+}$(2.6mM) and FCS showed no difference in the results of hamster test.These results indicated that the appropriate concentration of sperm should be given in hamster test for dog sperm.

• 한국수정란이식학회지
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• 제25권4호
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• pp.229-235
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• 2010

The MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at $37^{\circ}C$ using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.

• 한국가축번식학회지
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• 제15권1호
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• pp.1-6
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• 1991

본 實驗에서는 Ca, BSA, heparin과, 精液의 體外貯藏, 및 수소개체가 新鮮精子 및 卵乳液에 부유되어 5$^{\circ}C$에서 4시간 貯藏된 貯藏精子의 活力과 尖帽反應에 미치는 影響을 조사하였던 바, 그 結果는 다음과 같다. 1. Ca, BSA, Ca + BSA, heparin, heparin + Ca, heparin + BSA 및 heparin + Ca + BSA가 첨가된 SCS에서 15분간 培養한 精子의 活力은 처리간에 유의차가 인정되었으며, BSA가 다른 처리보다 유의하게 높았다. 한편 精子의 尖帽反應率은 처리간에 유의차가 인정되었으며, BSA와 Ca + BSA가 다른 처리보다 유의하게 높았다. 2. 精子의 活力은 新鮮精液과 貯藏精液 공히 KNC 1이 KNC 2, HOL 1과 2보다 유의하게 낮았으나, 尖帽反應率은 KNC 1이 KNC 2, HOL 1과 2보다 유의하게 높았다. 즉 精子의 活力과 尖帽反應率은 공히 수소개체간에 차이가 있었다. 3. KNC 1과 KNC 2의 新鮮精子를 SCS, SCS + Ca, SCS + BSA, SCS + Ca + BSA, SCS + heparin, SCS + heparin + Ca, SCS + heparin + BSA 및 SCS + heparin + Ca + BSA 용액에 각각 15분간 培養하였을 때, 精子의 活力은 수소개체간에 유의한 차이가 있었는데, KNC 1에서는 BSA가 다른 처리보다 유의하게 높았으나, KNC 2에서는 BSA, Ca + BSA 및 Ca이 다른 처리보다 유의하게 높았다. 한편 精子의 尖帽反應率은 역시 수소개체간에 유의한 차이가 있었으며, KNC 1에서는 Ca이, KNC 2에서는 Ca + BSA가 다른 처리보다 높았다.

• 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.

• 대한의생명과학회지
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• 제23권2호
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• pp.128-132
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• 2017

Streptozotocin (STZ), bisphenol A (BPA), and diethylstilbestrol (DES) are known as endocrine disruptors, occurs oxidative stress in animal cells. Generally, oxidative stress induces reactive oxygen species (ROS) and lipid peroxidation of sperm and lead to decreased viability and fertility in pigs. Therefore, we investigated the influence of STZ, BPA and DES on ROS production and lipid peroxidation on boar sperm. Collected sperm were incubated with semen extender containing $10{\mu}M\;STZ$, $10{\mu}M\;BPA$ and $20{\mu}M\;DES$ for 3, 6 and 9 hours. Intracellular ROS and lipid peroxidation of sperm were analyzed by 2', 7'-dichlorofluorescein diacetate and malondialdehyde methods. The results show that, intracellular ROS was not significantly different among the all treatments, but lipid peroxidation was significantly increased in STZ group at 3 hour after incubation with boar sperm (P<0.05). These results suggest that STZ stimulates lipid peroxidation more than ROS production and may exert a negative effect on sperm fertility.

• 한국발생생물학회지:발생과생식
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• 제17권2호
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• pp.121-132
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• 2013

Ultrastructural characteristics of the germ cells and accessory cells in testis during spermatogenesis and taxonomic values of mature sperm morphology of Ruditapes philippinarum were investigated by the transmission electron microscope and scanning electron microscope observations. The testis is the diffuse organ that consists of branching acini containing developing germ cells and accessory cells associated with spermatogenesis. The morphology of the spermatozoon is of the primitive type and is somewhat different to those of other bivalves. The morphologies of the sperm nucleus type and the acrosome shape of this species have a cylinderical type and a modified cone shape, respectively. As some ultrastructural characteristics of the acrosomal vesicle, the peripheral parts of two basal rings show electron opaque part, while the apex part of the acrosome shows electron lucent part. These characteristics of sperm belong to the family Veneridae in the subclass Heterodonta, unlike a characteristic of the subclass Pteriomorphia showing all part of the acrosome being composed of electron opaque part. In particular, a cylinder-like nucleus of the sperm is curved. The spermatozoon is approximately $48-51{\mu}m$ in length, including a long acrosome (about $2.4{\mu}m$ in length), a curved sperm nucleus (about $3.40{\mu}m$ in length), and a tail flagellum. The axoneme of the sperm tail shows a 9+2 structure.

• 한국발생생물학회지:발생과생식
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• 제21권3호
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• pp.229-235
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• 2017

In the field of reproductive medicine, assessment of sperm motility is a key factor for achieving successful artificial insemination, in vitro fertilization, or intracellular sperm injection. In this study, the motility of boar sperms was estimated using real-time imaging via confocal microscopy. To confirm this confocal imaging method, flagellar beats and whiplash-like movement angles were compared between fresh and low-temperature-preserved ($17^{\circ}C$ for 24 h) porcine sperms. Low-temperature preservation reduced the number of flagellar beats from $11.0{\pm}2.3beats/s$ (fresh sperm) to $5.7{\pm}1.8beats/s$ and increased the flagellar bending angle from $19.8^{\circ}{\pm}13.8^{\circ}$ (fresh) to $30.6^{\circ}{\pm}15.6^{\circ}$. These data suggest that sperm activity can be assessed using confocal microscopy. The observed motility patterns could be used to develop a sperm evaluation index and automated confocal microscopic sperm motility analysis techniques.