• Journal of Embryo Transfer
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• v.16 no.1
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• pp.35-40
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• 2001

Artificial insemination (AI) with frozen or cooled semen is widely used in commercial fields of cattle and pig. Little is known about characteristics of canine sperm after freezing or cooling. For both practical and commercial goal, the canine semen treated with cooling and freezing should be carried out to exam the fundamentals, including sperm motility, survivability and fertilizing capacity. The aim of this study, thus, was to identify the effects of extended exposure to 4$0^{\circ}C$ on canine semen by motility, survivability, acrosomal changes following different duration. Fifteen ejaculates collected by digital manipulation twice per week from 3 dogs (Shih-Tzu) were divided to 16 aliquots after adding Tris-egg yolk (TE) buffer formulated by our laboratory, and cooled from 37 to 4$^{\circ}C$, by ramp rate of 0.6$^{\circ}C$/min. Each sample was evaluated by their motility, survivability and the acrosomal status at 0h (control), 2h, 12h and 1 d~10 d, respectively. The motility of spermatozoa was graded to 6 levels using the modified method of Seager. The survivability of sperm was assessed using an epifluorescence microscope after Fert/Light (Mole-cular Probes Inc.) staining. To estimate the proportion of the spermatozoa of intact acrosome, 200 spermatozoa were assessed in randomly selected fields, using epifluorescence microscope after FITC/PSA (Sigma) staining. At 2 h after cooling, the motility of most spermatozoa were assessed to be grade 0 and 1. At 12 h, high number of sperm were in grade 0 to 1, however, it was significantly (P<0.05) lower than that of 2 h. From 1 d to 4 d, ~50% of sperm was assessed to grade 0 to 1. On day 7, a little sperm were in grade 0 to 1. No sperm showed motility on day 10. Sperm motility was rapidly reduced by the percent of 10% of grade 0 to 1. From 2 h to 6 h, the number of live sperm was 90% and the sperm chilled for 10 days lived>50%. Acrosomal intact of spermatozoa exposed to 4$^{\circ}C$ for 2 h was 51%, supposed the sperm of control was 100%. Our results suggest that 1) this is easy to transfer and preservation for short periods 2) AI can be used by semen chilled for 6-Day.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.181-189
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• 2001

The objective of this study was to evaluate the development of porcine follicular oocytes fertilized by intracytoplasmic sperm injection (ICSI). Cumulus-oocyte-complexes (COCs) were collected by aspiration from follicles of 2~7 mm in diameter from a local slaughterhouse. Oocytes were matured in vitro for 40~44 h, and spermatozoa were prepared by swim-up in the presence or absence of 5 mM dithiothreitol (DTT) and then M II stages of the oocyte were either centrifuged or not centrifuged for the following injection of ooplasm. Injected oocytes were cultured in NCSU 23 medium for 6 to 8 days. The results obtained were as follows: 1. The rates of cleavage and development rates into blastocyst by ICSI were not significantly different between the with (53.0% and 19.7%) or without (48.3% and 23.8%) centrifugation, respectively (P＜0.05). 2. The cleavage and developmental rates to blastocyst after ICSI with or without 5 mM DTT treated-sperm were not significantly different (60.4% vs 16.4% and 45.5% vs 22.2%), respectively (P＜0.05). 3. The cleavage and the developmental rates to btastocyst were not significantly different between the zygotes obtained by IVF (51.8% vs. 22.4%) and ICSI (51.4% vs. 21.6%) (P＜0.05). 4. The number of blastomere in blastocyst stages after IVF or ICSI was not significantly different (46.7$\pm$2.9 and 41.9$\pm$4.6).

• Development and Reproduction
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• v.11 no.3
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• pp.227-233
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• 2007

The milt properties of filefish(Thamnaconus modestus) included physical properties of sperm and biochemical properties of seminal plasma. The physical properties of milt were $0.3{\pm}0.1\;mL{\cdot}fish^{-1}$ in sperm volume, $2.6{\pm}0.1{\times}10^7\;spermatozoa{\cdotg}mL^{-1}$ in sperm concentration and $73.3{\pm}6.7$ in spermatocrit. The biochemical properties of seminal plasma contained $9.8{\pm}0.9\;mmol{\cdot}L^{-1}$ potassium, $164.0{\pm}4.0\;mmol{\cdot}L^{-1}$ sodium, $151.0{\pm}1.2\;mmol{\cdot}L^{-1}$ chloride, $14.9{\pm}0.6\;mg{\cdot}dL^{-1}$ calcium, $7.2{\pm}0.1\;mg{\cdot}dL^{-1}$ magnesium, $1.0\;mg{\cdot}dL^{-1}$ glucose, $0.1\;g{\cdot}dL^{-1}$ total protein and $1.0\;mg{\cdot}dL^{-1}$ total lipid. The osmolality and pH of seminal plasma were $322.8{\pm}2.8\;mOsmol{\cdot}kg^{-1}$ and $7.7{\pm}0.1$, respectively. The spermatozoon of filefish consisted of three parts: head without acrosome, mid-piece with five mitochondria and flagellum with "9+2" pattern. The head of spermatozoon in longitudinal section was horseshoe-shaped, and $1.3{\sim}1.6\;{\mu}m$ long and $1.0{\sim}1.3\;{\mu}m$ wide.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.59-64
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• 2006

This study was conducted to establish a convenient freezing method of boar semen. Boar semen was cooled until $5^{\circ}C$ for 3 hrs using cell freezer and loaded into straws. Semen straws were frozen in different steps in strofoam box filled with $LN_2$. Highest sperm viability (54.0%) was obtained by 1-step freezing(holding at 10 cm height from the surface of $LN_2$ for 10 min). Sperm viability increased by holding at $-102^{\circ}C$ for 10min (74.0%, P<0.05). In thawing regime, sperm viability was significantly higher in $37^{\circ}C$ group than in $52^{\circ}C$ group. The sperm characteristics did not differ between 1-step and 3-step. After IVF using frozen-thawed boar semen, developmental rate of embryos to the morula+blastocyst stage was in 1-step freezing group than that of 3-step freezing group (27.5 vs 14.7%, P<0.05). The result shows that the 1-step freezing with holding at $-102^{\circ}C$ for 10min before plunging into $LN_2$ is a convenient and easy freezing method for boar semen.

• Applied Microscopy
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• v.28 no.1
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• pp.39-48
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• 1998

The spermatozoa of bagrid catfish, Pseudobagrus fulvidraco are approximately $76{\mu}m$ in length, and a relatively simple and elongated cell composed of a spherical head, a short middle piece and a tail. The ultrastructure of spermatozoa of P. fulvidraco is characterized by the following features. The acrosome is absent as in most teleost. The round nucleus measuring about $1.67{\mu}m$ in length and diameter is depressed with a deep nuclear fossa. The nuclear fossa, the length of which is about three-fifths of the nuclear diameter, contains the proximal and distal contrioles. The two centrioles are oriented approximately $160^{\circ}$ to each other. The filamentous materials give rise to satellite appendages arranged tangentially from the triplets of the distal centriole and the doublets of the anterior end of the axoneme toward the nuclear envelope. The mitochondria are not fused and their number is 20 or more. They are arranged in two or three layers and two rings within the cytoplasmic collar and surround the axoneme. They are separated from the axoneme by the cytoplasmic canal. The axoneme is of the 9+2 microtubular pattern and has inner but no outer dynein arms. The two lateral fins are in the same plane with the two central microtubules, the doublets 3 and 8, which are ultrastructural characteristics of the sperm tail unlike other siluroids lacking the lateral fins.

• Applied Microscopy
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• v.42 no.2
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• pp.61-66
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• 2012

Ultrastructural characteristics of blacktip grouper, Epinephelus fasciatus spermatozoa were investigated using transmission and scanning electron microscopy. The spermatozoa of E. fasciatus consisted of a spherical head part, a midpiece with cytoplasmic canal entrance and a flagellum with lateral fins. Internal ultrastructurally, the nucleus contains high electron dense chromatin having granular particles and has no acrosome. The centriolar complex lies outside of the nuclear fossa and it is connected by the osmophilic filaments. Also the osmophilic filaments connect between the centriolar complex and the nuclear membrane. The midpiece contains eight to nine spherical mitochondria, cytoplasmic canal and necklaces. The flagellum has a typical 9+2 axonemal structure. The lateral fins contain vesicles and a typical 9+2 axonemal structure. Consequently this study contributes to comparative grouper spermatology and provide useful systematic taxonomic characters.

• Applied Microscopy
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• v.32 no.2
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• pp.107-119
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• 2002

Early spermatocytes of U. unicinctus are found in cluster floating in the coelomic fluid. The spermatocytes in a cluster form a syncytium or cytoplasmic mass, but there are no indications that the cytoplasmic mass is a component of a somatic cell. This work suggested that this type of spermatogenesis can be subordinated to solitary spermatogenesis in the sense excluding structural and functional support of a somatic cell for sperm developments. The solitary spermatogenesis in U. unicinctus is different in appearances and developmental details of sperm organelles and stage distributions from that of localized spermatogenesis. The acrosomal rudiments and centrioles can be observed in the early single cells of spermatogonia and clearly disclosed in the primary spermatocyte. In the stage of secondary spermatocyte, the acrosomal precursor and the centrioles begin to move to each cytoplasmic poles. The polarities of the organelles are attained at stage of spermatids. The spermatocytes and spermatids are arranged circumferentially along the cytoplasmic mass in which some amorphological cytoplasmic components are included. The spermatids reveal to be detached from the cytoplasmic mass into coelomic fluid. It suggests that the spermatogenesis are progressed in support of coelomic fluid, and the fact take into consideration that the spermatogenic cells can be in vitro cultured without somatic cells and with supplements of coelomic fluid.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.2
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• pp.198-203
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• 1999

Gonadal development and reproductive cycle off Gomphina melanaegis collected in the coastal waters of Chumunjin, Korea were investigated monthly from April 1996 to April 1997. G. melanaegis was dioecious, The gonads were located between the digestive diverticula and muscle tissues of the foot, The ovary was composed of a number of ovarian sacs, and the testis was composed of several testicular tubules. The flesh weight rate was reached the maximum in August ($23.0\%$), and then decreased to $19.8\%$ in September. In March, the value was reached the minimum ($17.8\%$) and then increased, The size of mature oocyte was ranged $50\~60\mu$m in diameter and had a germinal vesicle with a nucleolus. Mature oocyte contained a large number of yolk granules and lipid granules in its cytoplasm. The spermatozoon was consisted of a conical nucleus with acrosome, a middle piece containing four mitochondria and proximal and distal centrioles, and a flagellum, Sex ratio (male/female) and minimum size for sexual maturation of G. melanaegis were 0.79 and about 25 mm in shell length, respectively. The reproductive cycle could be classified into five succesive stages: multiplicative (December to March), growing (April and May), mature(June), sprawning (July and August), and degenerative and resting (September to November) stages.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.9-14
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• 2000

Objective: The sperm acrosome reaction is a $Ca^{2+}$-dependent exocytotic event that is triggered by adhesion to the mammalian egg's zona pellucida. Previous studies suggested a role of $Ca^{2+}$ channels in acrosome reactions. This study was conducted to investigate the T-type calcium channel is operated in acrosome reaction of human spermatozoa. Method: Human semen samples were obtained from healthy donors with normal criteria. The spermatozoa were divided into five groups: Group 1 were non-treated as a control; Group 2 where spermatozoa were exposed to 5 ${\mu}M$ $Ca^{2+}$ A23187 $(Ca^{2+}i)$; Group 3 where spermatozoa were exposed 5 ${\mu}M$ $Ca^{2+}i$ and mibefradil; Group 4 where spermatozoa were exposed 5 ${\mu}M$ $Ca^{2+}i$ and nifedipine, and Group 5 where spermatozoa were treated with 5 ${\mu}M$ $Ca^{2+}i$ and both of mibefradil and nifedipine. Spermatozoa in all groups were retrieved after incubation for 15 and 30 minutes at $37^{\circ}C$. After staining with PSA-FITC, fluorescence was observed under a fluorescence microscope, and AR was evaluated on a total>100 spermatozoa/side. Result and Conclusion: We observed on acrosome reaction inhibition rate in human spermatozoa the various of concentration of mibefradil, nifedipine. Maximum response was noted with 1.0 ${\mu}M$ mibefradil and the decrease of acrosome reaction inhibition rate 45%. Nifedipine in acrosome reaction inhibition rate was only about 25%. The $Ca^{2+}i$-induced AR of spermatozoa was significantly suppressed by mibefradil. Incidence of the suppression was depending on concentration of mibefradil. Results from the present study suggest that the human spermatozoa possess T-type channel. The observation that reversible inhibitor of T channels in male germ cells provides a new mechanism of contraceptive action.

• Applied Microscopy
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• v.39 no.3
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• pp.227-236
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• 2009

The ultrastructure of spermatogenesis and sperm in Coreoleuciscus splendidus, belonging to Gobioninae, Cyprinidae was investigated by light and electron microscopes. The testis was located between intestine and air bladder. The size of testis was major axis 1.8 cm, minor axis 3 mm. The testis of C. splendidus contained numerous testicular cysts, and spermatogenesis was non-synchronized in these testicular cysts. In May, the upper area of testis contained with other germ cells and sperm but the lower area of testis contained with matured sperm only. In case of spermatogonia, the nucleus was comparatively large spherical, and mitochondria showed a marked development. The size of primary spermatocyte was smaller than that of spermatogonia, and that of secondary spermatocyte was smaller than that of primary spermatocyte. The chromatin of spermatocyte was highly condensed according to their development. The nucleus with electron-dense was round shape. In spermiogenesis, flagella started to be formed and chromatin was more condensed. The mitochondria were rearranged in a middle piece. The head of matured sperm was a spherical shape and had not acrosome. The microtubules of flagella were arranged 9+2 structure. Also, the tail of sperm had not lateral fins and 7 outer coarse fibers.