• 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.

• Development and Reproduction
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• v.14 no.4
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• pp.269-279
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• 2010

Some characteristics of germ cell differntiations and the function of accessory cells during spermatogenesis, and mature sperm ultrastructure in male Protothaca (N.) jedoensis were investigated by transmission electron microscope observations. The morphology of the spermatozoa of this species has a primitive type and is similar to those of other species in the subclass Heterodonta. Accessory cells, which are connected to adjacent germ cells, are involved in the supplying of the nutrients for germ cell development. The morphologies of the sperm nucleus and the acrosome of this species are the cylindrical type and cap shape, respectively. Spermatozoa are approximately $46{\sim}50{\mu}m$ in length including a long sperm nucleus (about $2.44{\mu}m$ in length), an acrosome (about $0.45{\mu}m$ in length), and tail flagellum (about $42{\sim}46{\mu}m$). The axoneme of the sperm tail shows a 9+2 structure. As some characteristics of the acrosomal vesicle structures, the basal and lateral parts of basal rings show electron opaque part (region), while the anterior apex part of the acrosomal vesicle shows electron lucent part (region). These characteristics of the acrosomal vesicle were found in the family Veneridae and other several families in the subclass Heterodonta. These common characteristics of the acrosomal vesicle in the subclass Heterodonta can be used for phylogenetic and systematic analysis as a taxonomic key or a significant tool. The number of mitochondria in the midpiece of the sperm of this species are four, as one of common characteristics appear in most species in the family Veneridae and other families in the subclass Heterodonta. However, exceptionally, only three species in Veneridae of the subclass Heterodonta contain 5 mitochondria. The number of mitochondria in the sperm midpiece can be used for the taxonomic analysis of the family or superfamily levels as a systematic key or an important tool.

• Development and Reproduction
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• v.13 no.2
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• pp.115-122
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• 2009

Spermatogenesis and ultrastructural characteristics of sperm of brackish water diploid Corbicula japonica were investigated by electron microscope observations. Based on the cytological studies, the spermatozoon of this species (brackish water diploid) C japonica is approximately 55 ${\mu}m$ in length. The sperm head (about 12 ${\mu}m$ long) is elongated and tapers with a slight curve. Sperm nucleus is about 7.90 ${\mu}m$ long, and the acrosome is about 2.70 ${\mu}m$ long: The morphologies of the sperm nucleus type and the acrosome shape of this species are a long arrow-like type and long cone-like shape, respectively. The sperm head of this species (external fertilization, dioecious and oviparous species) is partially modified from that of the primitive type, as seen in triploid Corbicula species (internal fertilization, hermaphrodite and ovoviparous species), reported by some authors. However, this species produces uniflagellate spermatozoa, unlike freshwater triploid hermaphroditic clams being possessed of partially modified biflagellate spermatozoa. Diploid C japonica is similar to those of other bivalves being possessed of a short midpiece containing four mitochondria surrounding the centrioles. The axoneme of the sperm tail flagellum consists of nine pairs of microtubules at the periphery and a pair at the center. The axoneme of the sperm tail shows a 9+2 structure, and from uniflagellate sperm cross sectioned, in particular, wing-like axonernal lateral fins are observed, as seen in external fertilization fishes.

• Korean Journal of Ecology and Environment
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• v.33 no.4 s.92
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• pp.405-412
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• 2000

The fine structure of spermatozoa of Leiocassis ussuriensis was examined with scanning and transmission electron microscopies. The spermatozoon of L. ussuriensis is approximately $68.8\;{\mu}m$ in length and a relatively simple cell with a spherical nucleus, a short midpiece and a tail. The ultrastructure of spermatozoa of L. ussuriensis is characterized by the following features. The nuclear fossa, the length of which is about two-thirds of the nuclear diameter, contains two centrioles. The centrioles are orientated approximately $180^{\circ}$ to each other. The mitochondria are arranged in two layers and their number is 12 or more. The axoneme is the 9+2microtubular pattern and has inner but no outer dynein arms as in other bagrids. The two axonemal fins are in the same plane with the two central microtubules, the doublets 3 and 8. The axonemal fins and the inner dynein arm are shared in Bagridae and the deep nuclear fossa is shared in Siluriformes. The axonemal finsobse observed in Bagridae and Amblycipitidae of Siluriformes might be the apomorphic character in Ostariophysi. They are not reported in Cyprinidae and Characiformes.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.4
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• pp.272-279
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• 2019

The study was conducted to evaluate the seminal attributes and cryobanking of Achhami (Bos indicus) bull semen. Of two Achhami bulls, 8 ejaculates from each bull were evaluated for seminal attributes. For semen freezing and cryo-banking, 4 ejaculates (having ≥2 mL semen volume, ≥75% of sperm motility and ≥1,000 × 10⁶ cells/mL of sperm concentration) from each bull were used. Semen samples were diluted in egg-yolk-tris-citrate extender using a two-step dilution protocol, and were frozen in liquid nitrogen (LN₂) vapour in a styrofoam box. The mean semen volume, colour, sperm mass activity, motility, viability, concentration, abnormal acrosome, midpiece and tail and, abnormal head of two Achhami bulls were 4.4 ± 0.5 mL vs. 2.5 ± 0.2 mL, 2.5 ± 0.1 vs. 2.4 ± 0.1, 3.5 ± 0.1 vs. 3.5 ± 0.1, 77.0 ± 1.1% vs. 78.3 ± 1.3%, 94.4 ± 0.5% vs. 91.0 ± 0.6%, 1137.7 ± 73.7 × 10⁶ cells/mL vs. 1060.0 ± 44.3 × 10⁶ cells/mL, 10.2 ± 0.5% vs. 10.3 ± 0.5% and 6.7 ± 0.5% vs. 8.2 ± 0.3%, respectively. The post-thawed sperm motility and viability were 53.0 ± 2.0% vs. 50.0 ± 0.0% and 80.2 ± 0.4% vs. 73.2 ± 0.7%, while evaluating by computer-assisted sperm analysis (CASA) system, the percentage of the progressive motility, fast motility, slow motility, local motility and immotile sperm were 75%, 68%, 7.4%, 16.6% and 8.6%, respectively. A total number of 620 doses semen straw were cryo-banked. Due to the acceptable post-thawed sperm motility and viability recorded, cryopreservation of Achhami semen is hereby recommended so as to preserve the Achhami breed. For further validation, the fertility will be observed from the produced frozen semen.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.127-131
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• 2001

The Jeju horse has been raised for centuries in Jeju island. Recently, as the number of this indigenous horse has been dropped dramatically, this breed became Natural Monument #347 to conserve and multiply this endangered breed. To provide the basic information for AI, sexual activity and semen characteristics in Jeju horse were investigated. Jeju horse semen was collected using Missouri style artificial vagina from fertile stallion.\\`she number of mount per ejaculation was 2..3$\pm$1.8, and the ejaculation time was 27.0$\pm$12.5 seconds. The total volume and gel-free volume of semen was 47.8$\pm$26.7 ml and 42.7$\pm$27.4ml, respectively, and the concentration of sperm and the total number of spermatozoa per ejaculation was 200.7$\pm$112.9$\times$10$^{6}$ ml and 7.6$\pm$3.9$\times$10$^{9}$ ml, respectively. The percentage of motile sperm and the number of live spermatozoa per ejaculation was 75.0$\pm$18.2% and 6.1$\pm$3.4$\times$10$^{9}$ ml, respectively, and the pH of gel-free semen was 7.3$\pm$0.2. The total percentage of abnormal sperm was 31.5%, and the percentage of sperm with abnormal head, midpiece and tail was 9.5$\pm$11.7%, 7.0$\pm$4.0% and 15.0$\pm$15.0%, respectively.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.69-75
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• 2006

The objective of the study was to assess the general characteristics and motility characteristics with Computer Assisted Sperm Analyzer (CASA) system in Jeju horse semen. Semen was collected from 5 fertile Jeju horse by use of a Missouri type artificial vagina. Semen volume and pH were recorded, and sperm concentration was determined with a hematocytometer and motional characteristics of sperm were analysed by CASA. The viability and morphological abnormalities were assessed by a vital staining. The average volume of ejaculates was 42.5 ml and the average of sperm concentration was $198.5x10^6/m1$. The motional characteristics in Jeju horse semen was showed $70.4{\pm}28.7{\mu}m/s\;for\;VAP,\;69.6{\pm}28.9{\mu}m/s\;for\;VSL,\;94.1{\pm}30.0{\mu}m/s\;fo\;VCL,\;2.3{\pm}0.7{\mu}m/s\;for\;ALH,\;7.6{\pm}1.1Hz\;for\;BCF,\;99.1{\pm}1.2%\;for\;STR,\;and\;77.1{\pm}12.7%\;for\;LIN$. The percentage of sperm with abnormal head, midpiece and tail was 4.2%, 20.6%, 4.6% respectively.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.301-309
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• 1997

The objective of this study was to determine the microtubule assembly and chromatin configuration during the first cell cycle in bovine oocytes following injection of spermatozoon, sperm head and tail. The microtubule and chromatin configuration was imaged with fluorescent labeled monoclonal ${\alpha}$-tubulin antibody and propidium iodide under laser scanning confocal microscope. Microtubule and chromatin dynamics in bovine oocytes following intracytoplasmic sperm injection (ICSI) were not different from those observed during in vitro fertilization (IVF). Following ICSI, the microtubular aster was observed around sperm midpiece. During pronuclear formation, the sperm aster was enlarged and seen around male and female pronuclei. At mitotic metaphase, the microtubular spindle assemble astral poles and chromosomes were aligned on the spindle equator. At mitosis, asters were concentrated to each spindle pole and they filled the cytoplasm. After injection of the isolated sperm head, the microtubular aster was not seen around sperm head in any cases (0/18). Instead, microtubules were organized from the cytoplasm, which filled the whole cytoplasm during pronuclear apposition. These microtubules seem to move male and female pronuclei. These results suggest that isolated sperm head can develop into normal pronucleus in mature bovine oocytes, and competent to participate syngamy with the ootid chromatin. The functional microtubules following isolated sperm head injection in bovine oocytes appeared to be organized solely from maternal stores.

• Applied Microscopy
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• v.29 no.2
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• pp.177-187
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• 1999

Ultrastructure of gametes in the three-spine stickleback, Gasterosteus aculeatus aculeatus was observed, utilizing light, scanning and transmission electron microscopes. The egg of three-spine stickleback is spherical and demersal type. The eggs are highly adhesived to each other but not to substrates. There are many oil droplets in vitelline membrane. The outer surface of egg envelope is arranged by mushroom-like structures and pore canals. The egg have a micropyle, sperm entry site, in the area of the animal pole. The egg envelope consists of three layers, an outer layer with high electron density, a middle layer consisting two layers and an inner layer consisting of 16 to 20 layers. In the fertilized egg envelope, the molecular weights of these components ranged from 14 kDa to 205 kDa. The molecular weights of nam protein bands are 19.4 kDa, 36.7 KDa, 39.4 kDa, 42.9 kDa, 46.1 kDa and 53.0 kDa. The head of spermatozoa is spherical shape and the acrosome is absent. The mitochondria in midpiece are arranged from one to three layers and separated from the axoneme by the cytoplasmic canal. The tail has two lateral fins and the axoneme is of the 9+2 structure.