• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.171-178
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• 2013

The purpose of this study was to evaluate the sperm viability, normal acrosome and mitochondrial activity in the frozen-thawed fowl semen by different cryoprotectants. The experiment was carried out on 10 sexually adult roosters of Ogye. The semen was collected twice a week and pooled semen was diluted 1:1 EK extender containing no cryoprotectant at $5^{\circ}C$. After equilibration for 30 minutes, diluted chicken semen was diluted 1:1 extender containing either 7% dimethylacetamide (DMA), 7% dimethylformamide (DMF) or 7.5% methylacetamide (MA) at final concentration and was put in 0.5 mL plastic straws and frozen for 30 minutes by exposure to liquid nitrogen vapor 4 cm above the surface of liquid nitrogen, followed by plunging into liquid nitrogen. Frozen semen was thawed in water bath at $5^{\circ}C$ for 2 minutes. For cytometric analysis, the frozen-thawed semen was diluted with EK extender to a final concentration of 90 million spermatozoa per mL. Sperm membrane integrity was evaluated as SYBR-14 and propidium iodide (PI). Acrosome integrity was assessed with fluorescein isothiocyanate-labeled PSA and PI. The percentage of mitochondrial function was estimated by using Rhodamine123 (R123) and PI. In conclusion, freezing rooster semen by using 7% DMF as cryoprotectant was significantly highest in rates of survival and mitochondrial function while its rate of damage of acrosome was significantly lowest. As a result, DMF is the cryoprotectant that has the lowest influences on sperm membranes and acrosome integrity. Therefore it could be used for freezing method of animal genetic conservation method for poultry diversity.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.25 no.2
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• pp.79-92
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• 1992

This study was carried out to develop new techniques useful for cryopreservation, thawing and artificial insemination, and ultrastructural changes of cryopreserved spermatozoa in rainbow trout(Oncorhynchus mykiss) . Two extenders, such as Tyrode solution and Whittingham's $T_6$ solution, were used to preserve rainbow trout sperm in refrigerator $(-20,\;-40\;and\;-70^{\circ}C)$ or liquid nitrogen $%(-196^{\circ})$. Hand-stripped semen was diluted to 1:16 with two extenders, an then the semen were frozen after mixing semen and each extender containing 1M or 1.5M DMSO solution to 1:1. After 60 days cryopreserved semen was thawed in a $13^{\circ}$ water bath, and subsequently centrifugated. After centrifugation at 1,000 rpm for 5 min thawed semen was washed with extenders, and then fertilized with fresh eggs. The results obtained in these experiments were summarized as follows: After cryopreservation, over 75% of spermatozoa were appeared motile and the survival rate was high. Following cryopreservation by the addition of cryoprotectant such as DMSO, methanol and glycerol, the fertilization rate of the thawed spermatozoa appeared over $99\%$ compared with the control having $99\%$ of fertilization rate. There was no difference between the control and experimental groups such as $(-20^{\circ}C\;-40^{\circ}C\;and\;-70^{\circ}C)$ and $-196^{\circ}$ in fertilization rate. Following cryopreservation at $-196^{\circ}$ by the addition of 1M DMSO of cryoprotectant, each fertilization rate following 24 hours and hatching rate following 24 days showed $96\%$ and $8\%$ by the addition of BSA, but showed $98\%\;and\;10%$ by no addition of BSA. Following 2 months of cryopreservation by the addition of 1M DMSO of cryoprotectant, there were $10%$ of hatching rate at $-196^{\circ}\;and\;10\%\;and\;35\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C$. Following 2 months of cryopreservation by the addition of 1M methanol of cryoprotectant, there were $22\%$ of fertilization rate at $-20^{\circ}C,\;and\;28\%,\;at\;-70^{\circ}C$ Following 2 months of cryopreservation by the addition of 1M glycerol of cryoprotectant, there were $22\%$ of fertilization rate at $-20^{\circ}C$, and $33\%,\;at\;-70^{\circ}C$. pollowing 2 months of cryopreservation by the addition of 1.5M DMSO of cryoprotectant, there were $27\%$ of fertilization rate at $-20^{\circ}C,\;an\;36\%\;and \;35\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C$. Following 2 months of cryopreservation by the addition of 1.5M glycerol of cryoprotectant, there were $34\% \;of\;fertilization\;rate\;at\;-20^{\circ}C, \;and\;31\%\;and\;31\%,\;respectively,\;at \;-40^{\circ}C\;and\;-70^{\circ}$. Following 2 months of cryopreservation by the addition of 1.5M methanol of cryoprotectant, there were $28\%$ of fertilization rate at $-20^{\circ}C,\;and\;29\%\;and\;28\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C.$ From 10 days and 15 days following fertilization at $13^{\circ}C\;and\;10^{\circ}C$, respectively, the mortality rate of fertilized ova was markedly increased. The middle piece of spermatozoa had two set of central doublets, nine set of outer coarse fibres, and mitochondrial sheath. Spermatozoa went through morphological changes during storage, e.g. winding of flagella, detachment of the nuclear envelope and the plasma membrane from the nucleus of the sperm head. There were $1\%$ abnormal spermatozoa in fresh sperm and about $15\%$ during storage.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.189-197
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• 2009

Two-pore domain $K^+(K_{2P})$ channels contribute to setting the resting membrane potential in excitable and nonexcitable cells. However, the cellular or tissue distribution and function of $K_{2P}$ channels expressed in mammalian germ cells and reproductive organs have not yet been reviewed by researchers. In this review, we focus on expression, localization and expected properties of $K_{2P}$ channels in germ cells and reproductive organs. The $K_{2P}$ channels are expressed in human cytotrophoblast cells, myometrium, placental vascular system, uterine smooth muscle, and pregnant term tissue, suggesting that $K_{2P}$ channels might be involved in the processes of pregnance. The $K_{2P}$ channels are also expressed in mouse zygotes, monkey sperm, ovary, testis, germ cells, and embryos of Korean cattle. Interestingly, $K_{2P}$ channels are modulated by changes in temperature and oxygen concentration which play an important role in embryonic development. Also, $K_{2P}$ channels are responsible for $K^+$ efflux during apoptotic volume decreases in mouse zygotes. These expression patterns and properties of the $K_{2P}$ channels in reproductive organs and germ cells are likely to help the understanding of ion channel-related function in reproductive physiology.

• The Korean Journal of Pesticide Science
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• v.5 no.4
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• pp.11-19
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• 2001

This review discusses the effects and roles of insect hormones and insect growth regulators (IGRs) on vitellogenesis in adult insects. Insect vitellogenesis is regulated by hormones such as juvenile hormone (JH), ecdysteroids, and neurosecretory hormones (ovaryecdysteroidogenic hormone : OEH) released by neurosecretory cells, diet, and other elements(male specific protein of sperm fluid). In the fat bodies, the vitellogenins are synthesized by the stimulation of JH released by corpus allatum (CA) and ecdysteroids produced by follicle cells with the ovary in most insects. Furthermore, vitellogenins are released into the hemolymph, transported to the ovarioles by carrier protein, and incorporated into oocytes for the developing ovary. Of IGRs, juvenile hormone and its mimics such as methoprene and pyriproxifen appear to have pharmacological effects such as membrane lysis, destruction of salivary grand and midgut epithlial cells, fat body cells, and ovarian tissue, and also anti-juvenile hormone such as precocenes I and II appear to have specific cytotoxicity such as inhibition of corpus allatum and oocytes development. These results suggest that IGRs may be useful as agents for integrated pest management.

• Development and Reproduction
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• v.2 no.1
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• pp.89-99
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• 1998

This study was carried out to investigate the morphological changes of gonadotropes in pituitary gland and spermatogenic cells in testis, obtained from 150 of 3-year-old immature and mature male rainbow trout (Oncorhynchus mykiss) during the reproductive cycles from March to February in the following year. In the maturation cycle of the pituitary gonadotropes of cultured rainbow trout, three periods can be distinguished i.e. a period of resting(March-August), a period of full spermatogenesis (September-November), and a period of breeding (December-February). The ultrastructures of the gonadotropes largely parallel the cyclical changes in the tests. The seminiferous tubules contain all spermatogenetic stages and sperm cells in a period of early maturation. At first, the size of the nucleus and cytoplasm decrease gradually at every stages from spermatogonia to spermatids. In the secondary spermatocytes, the small mitochondria are located over the outer cytoplam. In spermatids, the cytoplasmic masses move toward the posterior part of the nucleus. In spermatids, the two large mitochondria are located over the cytoplasm. In spermatids, the cytoplasmic masses move towark the posterior part of the nucleus. In spermatids, the two large mitochondria are located over the cytoplasm and begin to elongate. In spermatozoa, the surface of the nucleus devreases in volume. Examination by TEM shows that the nuclear envelope and plasma membrane are slightlywrinkled and closely adhered to the nucleus of spermatozoa. Two oval mitochondria are quite separated and the flagellum is inserted into the base of the spermatozoa head.The axoneme in this fish has the typical pattern such as nine peripheral doublets and a central doublet(9+2). there are remarkable individual differences in the size and morphology of spermatozoa head as observed by transmission and scanning electron microscopy.

• Applied Microscopy
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• v.33 no.1
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• pp.25-39
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• 2003

Cell differentiation and ultrastructural characteristics in the seminiferous epithelium of Myotis macrodactylus was investigated with the light and electron microscopes. Spermatogenesis has begun at April and finished at September. The nuclei of A spermatogonia (dark and pale type of spermatogonia) were oval, applied to the basal lamina, and surrounded by Sertoli cells. By comparison with other types of spermatogonia, the cell and nucleus of B type of spermatogonium is globular and larger than A types of spermatogonia. The nucleolus appears as a coarse and touches the nuclear membrane. The cell and nucleus of spermatocytes was globular and larger, but primary spematocyte is larger than secondary spermatocyte. Spermiogenesis was divided according to the level of fine structural difference, into Golgi, cap, acrosomal, maturation and spermiation phases; Golgi, cap, acrosomal and spermiation phases were further subdivided into steps of early and late phase respectively, and maturation phase has only one step. Hence, the spermiogenesis has been divided into a total of nine phases. In the change of karyoplasm, the chromatin granules are condensed at late Golgi phase and completed at spermiation phase. The sperm tail began to develop in early Golgi phase and completed in spermiation phase. The process of degeneration of spermatogenic cells in the seminiferous tubules was continually observed from October, before the beginning of hibernation, to hibernation phase (November, December, January, February, March). Immatured spermatogenic cells in the seminiferous tubules have been engulfed by phagocytosis of Sertoli cells during period of degeneration. It is deduced that the adaptative strategy serves as the mechanism to regulate the effective use of energy to prepare for long hibernation and regulation of breeding cycle.

• Applied Microscopy
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• v.29 no.3
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• pp.343-351
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• 1999

The structures of the fertilized egg envelope from three species, three-spot gourami (Trichogaster trichopterus), pearl gourami (Trichogaster leeri) and marble gourami (Trichogaster trichopterus trichopterus) belong to Belontiidae were observed, utilizing light, scanning and transmission electron microscopes. In all three species, the fertilized eggs were the colorless, transparent, spherical, adhesive and pelagic type. A large oil droplet was located in vitelline membrane of the fertilized egg. The egg envelopes have a single micropyle, which is thought to the pathway of sperm in the area of the animal pole. Specially, the micropyle of three-spot gourami was similar to that of marble gourami which is subspecies of three-spot gourami. An outer surface of the fertilized egg envelope was arranged by grooves in all three species. The fertilized egg envelopes consists of two distinct layers; an adhesive outer layer and an inner layer with high electron density. In conclusion, the morphological similarity of the fertilized egg, micropyle, outer surface and transverse section of the fertilized e9g envelope seems to be an indication of the Belontiidae.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.8
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• pp.1119-1126
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• 2018

Objective: Present investigation was aimed to study the Single Nucleotide Variants of the luteinizing hormone beta ($LH{\beta}$) gene and to analyze their association with the semen quality (fresh and post-thawed frozen semen) and luteinizing hormone (LH) concentrations in Murrah buffalo bulls. Methods: Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and Sanger sequencing method is used to study genetic variability in $LH{\beta}$ gene. LH assay was carried out using enzyme-linked immunosorbent assay method. A fixed general linear model was used to analyze association of single nucleotide polymorphism (SNP) of $LH{\beta}$ gene with semen quality in 109 and LH concentrations in 80 Murrah bulls. Results: $LH{\beta}$ gene was found to be polymorphic. Total six SNPs were identified in $LH{\beta}$ gene g C356090A, g C356113T, g A356701G, g G355869A, g G356330C, and g G356606T. Single Stranded Conformational Polymorphism variants of pattern 2 of exon 1+pattern 2 of exon 2+pattern 1 of exon 3 had highly significant (p<0.01) effect on sperm concentration (million/mL), percent mass motility, acrosome integrity and membrane integrity in fresh and frozen semen whereas significant (p<0.05) effect was observed on percent live spermatozoa. SSCP variants of pattern 2 of exon 1+pattern 2 of exon 2+pattern 1 of exon 3 had highly significant (p<0.01) effect on luteinizing hormone concentrations too. Conclusion: The observed association between SSCP variants of $LH{\beta}$ gene with semen quality parameters and LH concentrations indicated the possibilities of using $LH{\beta}$ as a candidate gene for identification of markers for semen quality traits and LH concentrations in Murrah buffaloes.

• Applied Microscopy
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• v.35 no.2
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• pp.89-96
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• 2005

The ultrastructures of the fertilized egg envelope from Hyphessobrycon serpae belonging to Characidae was studied using scanning and transmission electron microscopes to get systematic fundamental data for classification of species and to confirm whether micropyle is a common trait of Characidae or not. The fertilized egg was of colorless, transparent, spherical, adhesive and demersal type. There were not oil droplets in vitelline membrane and attached structures in the outside of fertilize egg envelope. The egg envelope had a single micropyle resembling the pathway of sperm in the area of the animal pole. The micropyle was surrounded by 13 to 15 protruded lines of the egg envelope in a radiated form. The outer surface of fertilized egg envelope was covered by reticular adhesive fibrous structures and irregularly arranged by pore canals. The fertilized egg envelope consisted of three distinct layers an outer adhesive fibrous layer with high electron density, a middle layer with pore canals, and an inner layer consisting of 6 to 7 lamellae alternating layers with interlamellae of lower electron density. These ultrastructural characters of fertilized egg envelope form Hyphessobrycon serpae can be utilized in taxonomy of teleost, and as fundamental data for study on early development of fertilized egg. It seems that the morphology of micropyle is a common trait of Characidae

• Korean Journal of Fisheries and Aquatic Sciences
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• v.35 no.4
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• pp.424-430
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• 2002

Vas deferens is a long tube and could be divided into four regions as its morphological characters: a short and slender proximal region, a convoluted region, a straight and more thick distal region, and an ejaculatory duct, It is connected with the posterior outer horns of the testis and runs to the gonopores opened on the coxa of the fifth walking legs, The proximal region consists almost entirely of simple cuboidal epithelial cells, $12\~28{\mu}$m in height, surrounded by a thin basal membrane. A small aggregation of high cuboidal epithelium is obsened from one side of the proximal region. The convoluted and distal region is composed of two kinds of epithelial cells; high cuboidal epithelial cells, $40\~120{\mu}m$ in height located in dorsal portion and simple cuboidal epithelial cells of $12\~28{\mu}$m in height located in ventral and lateral portion. The ejaculatory duct is surrounded with two kinds of muscle layers, inner longitudinal and outer circular muscle fibers, The lumen is lined with high and simple cuboidal epithelium in almost equal proportions. The proximal region contains mature sperm and basophilic substances. The eosinophilic substances appeared newly going toward the convoluted region and are laid along simple cuboidal epithelial cell layer. Tube-like content (spermatophore) of the vas deferens is ejaculated from a pair of genital pores at mating. Two masses of the content fuse together side by side and are usually deposited on the female thorax between the second and fourth or fifth pereiopods, The spermatophore formed measures $2.7\~4.0$ mm in length and $1.5\~2.7$ mm in width.