• Journal of Aquaculture
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• v.17 no.1
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• pp.46-50
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• 2004

In the present study, attempts were made to cryopreserve sea urchin, Anthocidaris crassispina sperm in liquid nitrogen, to evaluate the effects of various cryoprotectants and freezing rates on motility, survival rate and fertilization rate of the post-thawing sperm, and the ultrastructural changes of sperm after cryopreservation were observed. The highest values of sperm motility (motility index: 3.3$\pm$0.37) and survival rate (72$\pm$3.5%) were obtained with 15% dimethyl sulfoxide (DMSO), and these values were significantly higher than those of sperm preserved with glycerol. Comparisons of motilities and survival rates between treatments of difference freezing rates showed that there was no difference between procedures (a) 5$0^{\circ}C$/min to -8$0^{\circ}C$ (motility index: 3.3$\pm$0.31 ; survival late 70$\pm$2.7%) and (b) 3$0^{\circ}C$/min to -8$0^{\circ}C$ (motility index: 3.1$\pm$0.29; survival rate 69$\pm$3.7%), while the results of (c) 1$0^{\circ}C$/min to -8$0^{\circ}C$ were significantly lower than the others (motility index: 2.2$\pm$0.33 ; survival rate 42$\pm$4.6%). There was no significant difference in fertilization rate between fresh sperm and sperm preserved with 15% DMSO as cryoprotectant and freezing rate (3$0^{\circ}C$/min to -8$0^{\circ}C$). Some ultrastructural changes of sperm, such as the detachment of plasma membrane, the destruction of mitochondria, and the flagellum rolling up head, were observed after cryopreservation. Morphological normality of the sperm in 15% DMSO frozen at the ratio of 5$0^{\circ}C$/min to -8$0^{\circ}C$ was better than the others.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.2
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• pp.181-189
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• 2011

Cryopreservation protocols induce partially irreversible damage to mammalian sperm plasma membranes. Previous studies have indicated that adding cholesterol to the plasma membrane, as cholesterol-loaded-cyclodextrins, improves cryosurvival of sperm. Therefore, the purpose of this study was to determine if treating sperm of Markhoz bucks with cholesterol-loaded-cyclodextrins (CLC) (0, 0.75, 1.5, 2.25 and 3 mg/ml diluted $240{\times}10^6$ sperm/ml) in Tris-citric acid-glucose diluents with and without egg yolk (containing 5% glycerol) would improve the post-thaw sperm quality. The motion characteristics were evaluated with a Computer Assisted System Analyzer (CASA); acrosome integrity and vitality were measured with the triple-stain technique. Samples were recovered before and after freezing by means of putting straws into $37^{\circ}C$ water for 30 sec and then parameters were assessed. The results showed that the treatments significantly affected motility, progressive motility, recovery rate, curvilinear velocity, beat cross frequency, live sperm with reacted acrosome, live sperm with unreacted acrosome, dead sperm with reacted acrosorne, and dead sperm with unreacted acrosome during freezing (p<0.05). However; no significant differences were found for average path velocity, straight line velocity, amplitude of lateral head displacement, straightness and linearity (p>0.05). The best results were observed for extender containing 2.25 mg/ml ($240{\times}10^6$ sperm/ml) CLC supplemented with 2.6% egg yolk. In conclusion, the findings of this study indicate improved Markhoz sperm viability and motility following treatment in the presence of egg yolk.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.2
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• pp.67-75
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• 2019

Objective: Sperm cryopreservation has been widely used in assisted reproductive technology, as it offers great potential for the treatment of some types of male infertility. However, cryopreservation may result in changes in membrane lipid composition and acrosome status, as well as reductions in sperm motility and viability. This study aimed to evaluate sperm DNA fragmentation damage caused by conventional freezing using the sperm chromatin dispersion test. Methods: In total, 120 fresh human semen samples were frozen by conventional methods, using SpermFreeze Solution as a cryoprotectant. Routine semen analysis and a Halosperm test (using the Halosperm kit) were performed on each sample before freezing and after thawing. Semen parameters and sperm DNA fragmentation were compared between these groups. Results: There was a significant decrease in sperm progressive motility, viability, and normal morphology after conventional freezing (32.78%, 79.58%, and 3.87% vs. 16%, 55.99%, and 2.55%, respectively). The sperm head, midpiece, and tail defect rate increased slightly after freezing. Furthermore, the DNA fragmentation index (DFI) was significantly higher after thawing than before freezing (19.21% prior to freezing vs. 22.23% after thawing). Significant increases in the DFI after cryopreservation were observed in samples with both normal and abnormal motility and morphology, as well as in those with normal viability. Conclusion: Conventional freezing seems to damage some sperm parameters, in particular causing a reduction in sperm DNA integrity.

• Journal of Embryo Transfer
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• v.25 no.2
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• pp.97-101
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• 2010

Intracytoplasmic sperm injection (ICSI) is one of the artificial fertilization methods when only a few sperm are available for insemination, and an important tool for the preservation of genetic materials of endangered animal species, especially the male is infertile. Different from other species such as mice and pigs, the conventional ICSI method which uses spiked pipette for injection (Spike-ICSI) is exhibited low success rates in cattle because the bovinesperm head membrane is hard to break during injection procedure. We chose piezo-assisted ICSI (Piezo-ICSI) for the improvement of the injection procedure including sperm head membrane rupture and efficient puncture of the plasma membrane of the oocytes. In this experiment, we compared the efficacy of the bovine ICSI embryo production between the Piezo-ICSI and Spike-ICSI. The second polar body extrusion, pronuclear formation, cleavage and blastocyst formation were evaluated after implementation of two different ICSI techniques. The Piezo-ICSI tended to show comparably higher rates of the second polar body extrusion (41.7%), the pronuclei formation (42.9%) and the two-cell cleavage (41.4%) than Spike-ICSI does (33.3%, 28.6% and 23.5%, respectively) although there is no statistic significance between two groups. In addition, the blastocysts were only obtained from the Piezo-ICSI group (10.3%). Our finding shows that the Piezo-ICSI may be used as an artificial fertilization method in cattle when in vitro fertilization is not applicable.

• Clinical and Experimental Reproductive Medicine
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• v.47 no.3
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• pp.186-193
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• 2020

Objective: The effectiveness of Staphylococcus protein A (SPA) in improving the penetration ability of sperm and reducing antisperm antibody (ASA) titers in immunologically infertile males was evaluated. Methods: Seminal fluid samples were obtained from 15 infertile men, and ASA titers were assessed with the latex agglutination test. Identification of immunoglobulin (Ig) classes and characterization of the antigens involved in the immune response were performed using indirect immunofluorescence. Local ASAs typically present as a mixture of IgG and IgA classes. The capillary tube penetration method was used to assess the capability of spermatozoa to penetrate the cervical mucus (CM). Results: ASAs associated with the neck region of sperm showed a significantly lower migration distance in the CM of infertile females than ASAs associated with the head or tail segments. ASA-positive seminal fluid exhibited significant increases in the mean migration distance (2.6 ± 1.4 cm vs. 1.54 ± 1.1 cm, respectively; p< 0.001) and sperm concentration (174 ± 121.0 × 10³/mL vs. 101 ± 93.7 × 10³/mL, respectively; p= 0.033) after treatment with SPA compared to pre-treated samples. A significant reduction (p< 0.01) in the recorded ASA titer was detected. Conclusion: These results indicate that SPA can be used as a sorting regimen for insemination programs. However, further studies are warranted to assess its influence on pregnancy rate.

• Journal of Biomedical Engineering Research
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• v.17 no.1
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• pp.25-32
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• 1996

A new analytic method has been developed for the analysis of sperm morphology using Hough transform. This method is based on the characteristic that sperm heads have elliptic shape in addition to the density difference with the background Sperm heads are represented in elliptic form with five parameter, and the optimal parameters are estimated by iterative Hough transform. To reduce processing time practically, we restricted the transformed space in minimum volume and moved the searching volume to the maximum gradient for the estimated error. Morphological parameters were calculated from estimated sperm head boundaries without further processing.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.151-157
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• 2000

Objective: The purpose of this study was to evaluate the effect of polycystic ovarian follicular fluid on sperm motility in human in vitro fertilization (IVF). Methods: From May, 1998 to July, 1999, 55 patients who complained of infertility were involved in this study. We obtained ovarian follicular fluids from the patients by ultrasono-guided aspiration. Subjects were divided into two groups. 20 patients who had polycystic ovarian disease were belong to study group, and 25 patients who had normal ovarian follicular fluid were belong to control group. The follicular fluid dilution was done with Ham's fluid as 10%, 20%, 50%, 100%. The sperm motility was analyzed by CASA at 6hr and 12hr after incubation in follicular fluids. Results: The levels of average path velocity (VAP) in all concentration fluid didn't show significant difference between study and control group. The other parameters including curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), and linerity (LIN) were didn't show any significant difference between both groups. Conclusion: PCOD fluid had seemed to have an adverse effect on the sperm biological function. But, this study showed that PCOD fluid had no different effect on sperm motility with normal follicular fluid.

• Korean Journal of Animal Reproduction
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• v.10 no.1
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• pp.19-26
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• 1986

This experiment was carried out to investigate the effect of bovine serum ablumin (BSA), sugars, glycerol equilibration time, straw size and thawing method on the survival index and the morphology of frozen boar spermatozoa. The results obtained were summarized as follow: 1. When the semen frozen in BF5 dilutor as pellet form was thawed in BTS at 37$^{\circ}$and 50$^{\circ}C$, BF5 dilutor with fructose showed higher sperm survival index than that with dextrose, however, when the semen was thawed on dry test tube at 37$^{\circ}C$, BF5 dilutor with sucrose showed higher sperm survival index than with other sugars. 2 When the semen forzen in BF5 dilutor with straw and thawed at 37$^{\circ}C$, BF5 dilutor with dextrose showed higher sperm survival index than those with other sugars, and there was no difference in sperm survival index between 0.5 and 1.0 ml straws. 3. The sperm survival index of frozen sperm was significantly (P<0.05) improved due to addition of BSA (0.05%) to BF5 dilutor. 4. When the extended semen with BF5 dilutor contatining 0.01 to 0.05% of BSA was frozen in the straw, the semen without glycerol equilibration showed significantly (P<0.05) higher sperm survival index than those with 2, 4 and 6 hrs glycerol equilibration time. 5. The sperm frozen in BF5 dilutor with dextrose or fructose, sucrose and raffinose showed 77 to 88% in normal acrosome rate and no difference among sugars. 6. The frozen semen showed lower normal acrosome rate than the first and second diluted semen, whereas the frozen semen showed higher swollen, damaged and missing acrosome rate than the first and second diluted semen. 7. Damaged and missing acrosome rate of sperm head due to freezing was somewhat inhibited by addition of BSA (0.01 to 0.05) to the BF5 dilutor.

• Biomedical Science Letters
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• v.2 no.1
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• pp.57-69
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• 1996

To examine the process of spermiogenesis in the Korean red-backed vole, Clethrionomys rufocanus regulus, the seminiferous epithelium in the testis, was studied by trasmission electron microscopy, and the following results were obtained based on the morphological characteristics of cell differentiation. 1. According to the fine structural differentiation, spermiogenesis was divided into Golgi, cap, acrosome, maturation and spermiation phases. Besides, these phases were sub-divided into two steps : early and late phases respectively, Thus, the spermiogenesis of Clethrionomys rufocanus regulus was divided into a total of ten steps. 2. In the changes of the chromatin, the chromatin granules began to be condenced in the late Golgi phase, regularizated at maturation phases, and a perfect nucleus of sperm was formed at the spermiation phases. 3. The sperm head had the falciform, and the formative period of sperm tail began to be develop in the early Golgi phase and completed at the spermiation phases. 4. In the morphological features of spermiation phases, the spermatid of early spermiation phase was divided into three types : (1) A-type spermatid contained cytoplasmic droplets in the neck region and middle piece, and the mitochondria was irregular, and arranged around the axoneme. (2) B-type contained cytoplasmic droplet in the middle piece only, and the mitochondria are arranged the center of axoneme regularly, and (3) In the C-type spermatid, the arrangement of mitochondria was regular, and was contained cytoplasmic droplet in the neck region only. In the late spermiation phase, only the sperm head was surrounded by cytoplasm of Sertoli cell or the matured sperm just before the spermiation from the cytoplasm of Sertoli cell.

• Fisheries and Aquatic Sciences
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• v.10 no.3
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• pp.143-149
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• 2007

This study describes spermatogenesis and sperm ultrastructure of the granular ark, Tegillarca granosa using light and electron microscopy. In the active spermatogenic season, the testis comprises many spermatogenic follicles that contain germ cells in different developmental stages. Primary spermatocytes in the pachytene stage are characterized by synaptonemal complexes. The early spermatids are characterized by the appearance of several Golgi bodies, increased karyoplasmic electron density, and tubular mitochondria. The mass of proacrosomal granules consists of numerous heterogeneous granules with high electron density that are about 20 nm in diameter. From the midstage of spermiogenesis, the well-developed mitochondria in the cytoplasm aggregate posterior to the nucleus and surround the proximal and distal centrioles. The proacrosomal granules condense and form a single acrosome with a thin envelope. During late spermiogenesis, the acrosome begins to elongate becoming conical. The sperm is approximately $35.0{\mu}m$ long and consists of a head, midpiece, and tail. The head comprises a round nucleus and a conical acrosome. A micro fibrous axial rod is observed between the nucleus and acrosome. The midpiece has a calyx-like structure with five mitochondria, and the tail, which has the typical "9+2" microtubular system, originates from the distal centriole.