• Journal of Embryo Transfer
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• v.19 no.3
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• pp.265-273
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• 2004

This study was carried out to examine the effects of fertilization media and sperm concentration on in vitro fertilization (IVF) and development (IVD) of porcine oocytes matured in vitro. Cumulus-oocyte complexes (COCs) were collected from antral follicles of porcine ovaries collected from abattoir, and were matured in vitro in modified NCSU-23 (mNCSU-23) supplemented with 10％ porcine follicular fluid (pFF). After the fertilization by experimental scheme, putative embryos were developed in vitro in NCSU-23. The results are as follows. When the oocytes were fertilized in vitro in modified TBM or modified TLP-PVA by 1 ${\times}$10$^{5}$ sperm/$m\ell$, all of the fertilization parameters were not significantly different between two media. Subsequently, as these putative embryos were developed in vitro in NCSU-23, the percentage of oocytes cleaved and of blastocysts were not different between two media, either. When the oocytes were fertilized in vitro in mTBM by 5${\times}$10$^4$, 1${\times}$10$^{5}$ or 5${\times}$10$^{5}$ sperm/$m\ell$, all of the fertilization parameters were significantly (P<0.05 or P<0.01) increased as sperm concentration was elevated. Subsequently, as these putative embryos were developed in vitro in NCSU-23, the percentage of oocytes cleaved and of blastocysts were significantly boosted (P<0.01) as sperm concentration at fertilization was elevated from 5${\times}$10$^4$ to 1${\times}$10$^{5}$ sperm/$m\ell$, but were not different between 1${\times}$10$^{5}$ and 5${\times}$10$^{5}$ sperm/$m\ell$.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.2
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• pp.238-241
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• 1999

Experiments were performed to find out the physico-chemical properties of milt and the sperm motilities in various conditions using the grey mullet, Mugil cephalus. The average concentration of sperm in the milt was $1,11 \pm0.36\times10^{10}/ml$. Spermatocrit was 96.7$\pm$2.6. pH and osmolality of seminal fluid were 7.8$\pm$0.1, 370$\pm$6 mOsm/kg, respectively, Total protein concentration of sperm was higher than that of seminal fluid, but total lipid concentration of seminal fluid was higher than that of sperm. The sperm motility was high in the diluent of milt : artificial seawater (1:10, by volume) and in 822 mOsm/kg and 983 mOsm/kg similar to seawater osmolality, but it decreased after 20 minutes. But activity of sperm was highly maintained in 482 mOsm/kg which was a little higher than osmolality of seminal fluid, and was high in pH 7$\~$9.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.1
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• pp.25-31
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• 2001

연구목적: $Isolate^{(R)}$ gradient와 swim-up 방법이 정자의 형상 및 정밀정자형태 (strict morphology)에 미치는 영창을 비교분석하고, 이러한 정자처리방법이 정자의 동결-융해과정에 미치는 영향을 비교하고자 하였다. 연구재료 및 방법: 20명의 정상 정자를 대상으로 하였으며 각각의 정자는 두 가지 정자처리방법으로 나누어 정자의 형상과 정밀정자형태를 컴퓨터를 이용한 정자자동분석기를 통하여 측정하였고, 동결보호제의는 TYB 용액을 사용하였으며, 동결 및 융해는 cryo Magic사의 기계를 사용하였다. 통계는 SPSS PC+(version7.0)를 이용하였으며 통계학적인 유의성은 p<0.05로 하였다. 결과: 정자의 농도는 $Isolate^{(R)}$ gradient 처리군이 swim-up 처리군보다 유의성 있게 높았으나 ($51.2{\pm}40.1,\;156.6{\pm}64.3$), 운동성 VCL, VSL, VAP, Linearity, 및 ALH는 swim-up 처리군에서 유의성 있게 높았다. 정밀 정자형태는 swim-up 처리군과 $Isolate^{(R)}$ gradient 처리군에서 차이가 없었다 ($53.7{\pm}6.8$ vs $50.3{\pm}9.1%$). 동결-융해과정 중 두 가지 정자 처리군에서 정자의 형상들은 swim-up 처리군에서 전반적으로 높은 양상을 보였으나, 정밀정자형태는 $Isolate^{(R)}$ gradient 처리군이 swim-up 처리군 보다 감소율이 컸지만 두 군간에 유의한 차이는 없었다 ($12.8{\pm}8.5$ vs $8.6{\pm}6.6$). 결론: 정상 정자에서 swim-up 방법이 $Isolate^{(R)}$ gradient 방법보다 정자 회수율은 우수하였으나, 동결-융해과정 중 정밀정자형태에는 차이가 없어 두 방법을 상호보완적으로 사용할 수 있을 것으로 사료된다.

• Korean Journal of Animal Reproduction
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• v.25 no.3
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• pp.269-275
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• 2001

This study was carried out to investigate the general characteristics such as volume, sperm concentration, sperm motility, sperm abnormality on whole semen, RSP-S and RSP-T semen and fractional semen of small size dogs, and the effect of temperature and preservation time and cryoproservation on motility of whole and RSP-S and RSP-T semen. Multiple ejaculates were collected from small dogs by the digital manipulation of penis. 1. The volume per ejaculate semen, sperm of concentration and motility and abnormal sperm rate of 1st fractional semen were 0.65$\pm$0.09 $m\ell$, 4.52$\pm$0.35$\times$10$^{6}$ cells/$m\ell$, 15.64$\pm$3.85% and 5.50$\pm$0.62%. Also, 2nd fractional semen were 1.25$\pm$0.20$m\ell$, 3.35$\pm$0.48$\times$10$^{6}$ cells/$m\ell$, 96.25$\pm$4.65% and 4.24$\pm$0.46%. And 3rd fractional semen were 1.45$\pm$0.21$m\ell$, 3.55$\pm$0.52$\times$10$^{6}$ cells/$m\ell$, 92.82$\pm$4.24% and 4.66 $\pm$0.58%, respectively. 2. The sperm of concentration and motility and abnormal sperm rates of whole, RSP-S and RSP-T semen were 5.45$\pm$0.82$\times$10$^{6}$ cells/$m\ell$, 95.55 $\pm$4.65%, 4.58$\pm$0.45% and 4.82$\pm$0.36$\times$10$^{6}$ cells/$m\ell$, 90.10$\pm$3.42%, 6.48$\pm$0.68% and 4.55$\pm$0.45$\times$10$^{6}$ cells/$m\ell$, 93.25$\pm$3.85%, 4.82$\pm$0.58%, respectively. 3. The motility of whole, RSP-S and RSP-T semen were higher at 4$^{\circ}C$ than at 38$^{\circ}C$. When preservation temperature was at 4$^{\circ}C$, survival rates of RSP-S and RSP-T sperm were 97.54~6.25% at 1~72 hrs, 97.40~5.62% at 1~100 hrs, respectively. 4. The survival rates of slow and rapid frozen 2nd fraction, RSP-S and RSP-T semen were 67.3$\pm$4.45%, 88.8$\pm$4.46% and 46.4$\pm$3.84%, 74.4$\pm$4.20%, respectively. Survival rates was significantly higher in frozen RSP-S and RSP-T semen than that in control group(8.5$\pm$2.12%).

• Clinical and Experimental Reproductive Medicine
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• v.26 no.1
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• pp.89-96
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• 1999

Some of the information concerning sexual function in the male diabetes has been focused upon the problems of endocrine or semen parameters. However, the characteristics of acrosome reaction and spermatozoa concentration at the epididymis and vas deferens have scarcely been studied, and the causes of the infertility has not been critically identified. So, we designed to inspect the spermatozoa concentration and the characteristics of acrosome reaction at epididymis and vas deferens of diabetic Wistar rat induced by streptozotocin (STZ, 70 mg/kg, ip). Experimental animal was sacrificed at 3 days and 14 days after the STZ injection. In the diabetes-induced rat, the levels of insulin and glucose had a pattern of inverse proportion. The spermatozoa concentrations in caput and corpus epididymis were significantly decreased in all diabetic condition. In cauda epididymis, however, there was significant decrease in sperm concentration at 14 days onward. In diabetic rat, the spontaneous reaction rate of spermatozoa of cauda and vas deferens were significantly higher than the control group. The ARIC (acrosome reaction to ionophore challenge) value of caudal sperm was 28.7 at control, 22.1 at 3 days, and 8.3 at 14 days. In the present study the spermatozoa concentration was decreased and the spontaneous reaction rate was increased by diabetes. In ARIC-test, it is revealed that the fertility of spermatozoa of 14 days group was lower than control or 3 days group. Diabetes mellitus may be provoke the decreased fertilization rate and subsequent infertility and subsequent infertility.

• Journal of Aquaculture
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• v.12 no.1
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• pp.1-5
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• 1999

To obtain fundamental data for sperm cryopreservation in river puffer (Takifugu obscurus), the proper conditions of cryopreservation were investigated. In the sperm cryopreservation of river puffer, marine fish Rinser's solution (MFRS) was found to be good diluent and dimethyl sulfoxide (DMSO) was proved to be superior to glycerol as a cryoprotectant. The highest fertilization rate was achieved when river puffer sperms were cryopreserved with MFRS adding 5％ DMSO.

• Development and Reproduction
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• v.16 no.2
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• pp.77-85
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• 2012

Various cryoprotective agents (CPA) were tested to establish the best conditions for the cryopreservation of sperm from black porgy Acanthopagrus schlegeli acclimated and raised in freshwater (BFW). Survival rates of frozen/thawed sperm from BFW were higher in the order of dimethy sulfoxide (DMSO), glycerol, ethylene glycol (EG) and methanol. Sperm motility was higher in the order of glycerol, DMSO, EG and methanol. These effects were the same in thawed sperm from black porgy raised in seawater (BSW). Thus, optimum CPA for sperm cryopreservation of BFW and BSW were DMSO and glycerol where the highest survival rates and sperm motility were found at the concentration of 10%. In particular, the survival rates and motility of thawed sperm from BFW and BSW after cryopreservation using 10% DMSO were better than when cryopreserved using 10% glycerol. On the other hand, for the thawed sperm from both BFW and BSW, the longer the preservation period was, the lower the survival rates and sperm motility were. Notably, the higher the concentration of CPA was, the lower the survival rates and sperm motility were.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.3
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• pp.243-246
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• 1999

To obtain the basic data for the preservation of river puffer (Takifugu obscurus) sperm, experiments were carried out on the physico-chemical properties and cold storage of milt. The average number of sperm and spermatocrit in milt stripped were $1.13\pm0.34\times10^{10}/ml$ and 64.8$\pm$1.4, respectively. Osmolality of seminal fluid was 266$\pm$2 mOsm/kg, Total protein and total lipid from sperm were higer than that from seminal fluid. $Ca^{2+}$ and $Na^{+}$ concentrations were higher in the seminal fluid than in the sperm, while $Mg^{2+}$ and $K^{+}$ concentrations were lower in the seminal fluid. When sperm of river puffer were preserved in $0\pm0.5^{\circ}C$ with various diluents for 16 days, fertilization rate was $0\~0.7\%$. It suggested that cold storage of river puffer sperm was detrimental to sperm fertility.

• Journal of Aquaculture
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• v.20 no.3
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• pp.173-177
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• 2007

An experiment was performed to obtain cryopreservation techniques of starry flounder (Platichthys stellatus) sperm. Milt obtained from 24 males were cryopreserved using two diluents, artificial seminal plasma (ASP) and Stein's solution (SS) with three cryoprotectants, dimethyl sulfoxide (DMSO), methanol, and glycerol concentrated from 5% to 20%. Post-thaw sperm activity (motility and/or speed) revealed the highest in 10% DMSO and 15% methanol in ASP and SS as diluent. Motility and speed of cryopreserved sperm were decreased according to increase of glycerol concentration. To conclude, DMSO was a better cryoprotectant than methanol or glycerol for cryopreservation of starry flounder sperm. Glycerol was incongruent cryoprotectant because of toxic to starry flounder sperm. Most cryopreserved spermatozoa without cryoprotectant showed the enlarged head with granulated chromatin and ruptured plasma membrane by freezing and thawing injuries compared with unfrozen normal spermatozoa.

• Journal of Embryo Transfer
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• v.21 no.4
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• pp.323-330
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• 2006

본 연구의 목적은 3주 동안 장기간 냉각상태로 보관된 개 정자의 기능적 특성을 알아보고자 두 종류의 정자 희석액, Glucose-BSA(G-BSA), Dimitropoulos-II(BIMI)가 정자의 기능적 특성에 미치는 영향을 알아보고자 한다. 개 정액을 수지법에 의해 채취하여 원심분리한 후 정장은 제거하였다. 정자를 G-BSA나 DIMI으로 희석하여 최종 농도를 $100\times10^6$ sperm/ml로 하였다. 희석된 정자를 정자 운송 시스템을 이용하여 루지애나 주립 대학에서 뉴올리언즈 실험실까지 운송하였다. 정자 운송 시스템은 $20^{\circ}C$에서 $9^{\circ}C$까지 분당 $0.08^{\circ}C$ 냉각율로 설정하였다. 희석된 정자는 $4^{\circ}C$에서 3주간 보관하였다. 보관 1일부터, 매주 단위로 정자의 활력, 정자 원형질막 고유성(생존성), 정자 첨체의 고유성을 검사하였다. 또한 체외조건하에서의 정자 번식 능력을 평가하기 위해 zona binding assay를 실시하였다. 실험 결과 냉각 상태로 3주 동안 보관된 개 정자는 여전히 정자의 기능적 특성을 나타냈다. 냉각기간이 길어질수록 정자의 활력 및 생존성은 감소하였으며(P<0.01), 첨체의 고유성은 냉각기간에 따라 감소하였으나 유의성은 없었다. 정자의 특성 중 활력이 다른 특성에 비해 냉각처리에 민감한 반응을 보였다. G-BSA배지에 보관된 정자의 활력 및 생존성이 DIMI에 비해 높게 나타났으며, 1 주 동안 보관된 정자의 생존성의 비교시 유의성이 높게 나타났다(P<0.05). 그러나 첨체의 고유성은 1일 동안 DIMI에 보관된 정자에서 높게 나타났다(P<0.05). 3주 동안 냉각상태로 보관된 개 정자는 여전히 난자의 투명대에 결합하는 능력을 보였으며, G-BSA에 보관된 정자의 경우 난자에 부착되는 평균 정자 수가 보관일수에 따라 유의적으로 감소하였다(P<0.05). 그러나 희석액 종류에 따른 난자에 부착되는 평균 정자 수의 유의적 차이는 없었다. 정자의 활력 및 생존성과 난자의 투명대에 결합하는 정자 수와의 상관관계를 본 결과, 활력 및 생존성이 높은 정자일수록 난자의 투명대에 많이 결합하여 정자의 잠재적 번식력이 증가함을 보였다. 결론적으로 G-BSA나 DIMI의 희석액으로 희석된 후 $4^{\circ}C$에서 냉각 보관된 정자는 3주 동안 정자의 기능적 특성을 유지하였으며 냉각 보관 2주까지 좋은 성적의 정자 특성을 나타냈다. 개 정자 냉각 보존에 대안적인 희석액으로 G-BSA도 사용 가능한 것으로 사료된다.