• Clinical and Experimental Reproductive Medicine
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• v.22 no.2
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• pp.143-148
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• 1995

To present and assess the efficacy of combination of microsurgical epididymal sperm aspiration(MESA) and intracytoplasmic sperm injection(ICSI) for the treatment of infertility due to unreconstructable obstructive azoospermia or congenital bilateral agenesis of vas deferens (CBAVD), MESA was performed in the 45 husbands ( 16 CBAVD, 29 unreconstructable genital tract obstruction), followed by ICSI of oocytes recovered from the wives hyperstimulated by GnRH agonist in combination with hMG and FSH. Cleaving embryos were transfered to the uterine cavity or follopian tube(ZIFT) 18 or 24 hours after ICSI procedure. In 45 cycles of MESA, 492 oocyte complexes were recovered. ICSI was carried out on 355 metaphase II oocytes and 226 oocytes (63.7%) showed normal two pronucleus fertilization. After 198 embryos were transferred in 43 cycles, an average of 5 per cycle, 20 patients presented a positive HCG and intrauterine pregnancy was confirmed by US. So, the clinical ongoing pregnancy rate per transfer was 46.5%. Until now, 8 patients have given birth to 9 babies, 5 male and 4 female, including 1 twin. The babies were all healthy except 1 twin female baby. There was 1 miscarriage at 7 weeks and chromosomal study of abortus revealed as 45X, monosomy. These results suggested that it was possible to achieve high normal fertilization and pregnancy rate by ICSI using epididymal sperm.

• Archives of Pharmacal Research
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• v.17 no.4
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• pp.213-217
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• 1994

The effect of ccocaine on reproductive system were studied in male ICR mice. daily injection of cocaine HCL (10, and 40 mg/kg s.c for 15 days) disrupted epididymal sperm levels and testicular morphology. These disruptions were manifested in a decreased number of sperum, an increased number of dead sperm as compared with control vehicle, and wre evidenced by vacuolations, multinucleated giant cell formations and coagulation necrosis in the testicular seminiferous tubules. Responses in the current study were trended in dose dependent pattem. These results suggest that thronic cocaine intoxication can perturb on male reproductive organ functioning.

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.19-27
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• 1999

This study was carried out to investigate the effect of intracytoplasmic injection of $Ca^{2＋}$ and human sperm extract on the parthenogenetic activation of mouse oocytes. The results obtained were as follows: 1. The mouse oocytes were injected with 10 pl of PBS medium containing 0, 1.7 and 5 mM calcium concentrations, respectively. Activation rate of the oocytes with formation of pronuc1ei and extrusion of the second polar bodies was 14.5, 9.8 and 14.9% at the above calcium concentrations, respectively. There were no significant differences in the activation rates among the calcium concentrations. 2. The mouse oocytes were injected with 10 pl of non-heated human sperm extract, and cultured for 12~15 h in the PBS media with the 0, 1.7 and 5 mM calcium concentrations, respectively. Activation rate(51.8%) of the oocytes at the 1.7 mM calcium concentration was significantly higher than those at the 0 and 5 mM calcium concentrations. 3. The mouse oocytes were injected with 10 pl of heated human sperm extract, and cultured for 12~15 h in the PBS media with the 0, 1. 7 and 5 mM calcium concentrations, respectively. No significant differences were found in the activation rates (11.8~17.0%) among the calcium concentrations. 4. The mouse oocytes were injected with 10 pl of PBS medium, non-heated sperm extract and heated sperm extract, and cultured for 12~15 h in the PBS media with 1.7 mM calcium concentrations, respectively. Activation rate (54.5%) of the oocytes injected with the non-heated sperm extract was highest. There were significant differences in the activation rates among the above injection materials (P＜0.05). 5. The mouse oocytes were injected with 10 pl of 1 and 6 days old non-heated sperm extracts, and cultured for 12＜15 h in the PBS media with 1.7 mM calcium concentrations, respectively. Activation rate(60.0%) of the oocytes injected with 1 days old sperm extract was significantly higher than that (11.1%) injected with 6 days old sperm extract. The results obtained in this study suggest that non-heated human sperm extract may contain sperm-associated oocyte-activating factor such as oscillin.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.55-59
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• 2002

The objective of this study is to determine the developmental competence of in vitro matured bovine oocytes after intracytoplasmic sperm injection(ICSI) with frozen-thawed epididymal spermatozoa. The ovaries were obtained from slaughtered Korean native cows. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with frozen-thawed epididymal spermatozoa. After ICSI, a group of oocytes was activated with 7% ethanol fur 5 min, and the other group was not activated. The oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24~30 hrs in a incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. The percentage of oocytes reaching M II after 24 hrs and 30 hrs of incubation were significantly higher(p<0.05) after culture with TCM-199 media(80.0% and 88.3%) than M I(8.3% and 6.7%). The rate of cleavaged embryos to blastocyst obtained by ICSI treated activation oocytes was significantly higher(p<0.05) than that of nonactivation oocytes(22/46, 47.8% vs 10/39, 25.6%). The rates of embryos development to blastocyst obtained by ICSI treated sperm of flesh, epididymal and frozen-thawed epididymal were 24/45(53.3%), 15/40(37.5%), 11/43(25.6%), respectively and these values of fresh sperm injection were higher than frozen-thawed epididymal sperm. We also concluded that embryos can be produced with ICSI of in vitro matured oocytes by ICSI using frozen-thawed epididymal semen.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.275-281
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• 2008

The ability to preselect the sex of piglets is advantageous in the pig industry. The objective of this study was to examine the feasibility of using intracytoplasmic sperm injection (ICSI) with sorted spermatozoa to produce piglets with a preselected sex. Pig embryos were produced by ICSI of frozen X- and Y-sperm that had been separated by flow cytometry. The developmental competence of the embryos was investigated in vitro and in vivo. The populations of X- and Y-spermatozoa were 52.7% and 47.3%, respectively in our samples. The in vitro development of ICSI embryos was enhanced by longer of in vitro maturation of oocytes ($44{\sim}48\;h$ vs. $40{\sim}43\;h$). Their cleavage ($65{\sim}70%$) and blastocyst formation ($9{\sim}12%$) rates were not significantly different between male and female ICSI embryos, or between sorted and unsorted sperm-derived embryos. One pregnancy was established in a recipient that was transferred with 110 female ICSI embryos, but the pregnancy was terminated on Day 89 of gestation. Our results suggest that the separation X- and Y-spermatozoa by flow cytometric sorting can be a useful tool in combination with ICSI for the production of pig embryos and piglets of preselected sex.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.1
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• pp.22-29
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• 2015

Objective: Sperm must be properly prepared in in vitro fertilization (IVF)-embryo transfer (ET) programs in order to control the fertilization rate and ensure that embryos are of high quality and have appropriate developmental abilities. The objective of this study was to determine the most optimal sperm preparation method for IVF. Methods: Patients less than 40 years of age who participated in a fresh IVF-ET cycle from November 2012 to March 2013 were included in this study. Poor responders with less than three mature oocytes were excluded. Ham's F-10 medium or sperm-washing medium (SWM) was used in combination with the density-gradient centrifugation/swim-up (DGC-SUP) or SUP methods for sperm preparation. A total of 429 fresh IVF-ET cycles were grouped according to the media and methods used for sperm preparation and retrospectively analyzed (DGC-SUP/Ham's F-10, n=82; DGC-SUP/SWM, n=43; SUP/Ham's F-10, n=181; SUP/SWM, n=123). Results: There were no significant differences among these four groups with respect to the mean age of the female partners, duration of infertility, number of previous IVF cycles, and retrieved oocytes. We determined that both the DGC-SUP and SUP methods for sperm preparation from whole semen, using either Ham's F-10 or SWM media, result in comparable clinical outcomes, including fertilization and pregnancy rates. Conclusion: We suggest that both media and both methods for sperm preparation can be used for selecting high-quality sperm for assistive reproductive technology programs.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.205-214
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• 2002

There are many methods to introduce exogenous DNA into embryo to produce transgenic animals. Exogenous gene can be integrated into oocyte by sperm vector. In this study, sperm was used as a vector for a transgene, which is encoding enhanced green fluorescent protein (EGFP). The objective of this study was to investigate the expression of exogenous gene in bovine embryos after injection of spermatozoa cocultured with EGFP DNA fragment. Spermatozoa were plunged into liquid nitrogen and thawed several times or shook in 0.2% Triton X-100 to remove sperm membrane followed by DTT treatment. The injected oocytes were co-cultured with vero cells in CR1aa, and expression of EGFP gene was observed under fluorescent microscope. Blastocyst formation rates of oocytes injected with sperm treated with DTT, DTT-freezing or DTT-Triton X-100 were 34.7, 39.4 and 31.9%, respectively. The rates of EGFP expression in oocytes injected with 54 ng DNA after DTT-treated, DTT-freezing and DTT-Triton X-100-treated sperm were 0, 19.1 and 13.9%. On the other hands, expression rate of oocytes injected with sperm cocultured with 13.5, 27 and 63.5 ng of EFGP DNA were 6.7, 9.0 and 5.1%, respectively. When intact sperm was mixed with 63.5 ng/${mu}ell$ EGFP DNA fragment, and then electroporated before injection, the expression rate of injected oocyte was 2%. Unexpectedly, electro-poration could not increase the expression rate. These results suggest that sperm can be used as a transgene vector, even if the efficiency was low (19.1%).

• Clinical and Experimental Reproductive Medicine
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• v.41 no.3
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• pp.137-139
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• 2014

The effect of artificial oocyte activation (AOA) with a calcium ionophore on intracytoplasmic morphologically selected sperm injection (IMSI) was examined in patients with histories of repeated failed implantation attempts. Four singleton pregnancies and one twin pregnancy were obtained after embryos transfer (5/14, 35.7%). Therefore, AOA combined with IMSI can be considered an option for cycles with a fertilization defect and recurrent implantation failures.

• Journal of Aquaculture
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• v.12 no.4
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• pp.275-281
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• 1999

The utility of RSV-LTR and carp beta-actin promoters was evaluated in a marine flatfish species, Limanda Yokohamae by examining successful expression of transgenic DNA in muscles (transfected by direct injection) and in early embryos (transformed by lipofected sperm). The expressed pattern of injected DNA in skeletal muscles was dependent on the DNA amount injected. The activity reached to maximal level at 48 hours post injection, and persisted up ot 1 month transiently. Gene transfer into early embryo of this species was successfully achieved using lipofected sperm with the efficiency ranging 36.8% to 48.1%. The expression of transgene during embryonic development was shown as stage-specific and transient.

• Journal of Animal Science and Technology
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• v.63 no.5
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• pp.1194-1203
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• 2021

Preparation of recipient stallions is critical step to produce donor spermatogonial stem cell (SSC) derived sperm using transplantation technique. This study was conducted to evaluate the effects of intravenous busulfan infusion on germ cell depletion, semen production, and libido in stallions. Six Thoroughbred stallions were separated into two treatment groups: 1) a multiple low-dose (2.5 mg/kg bw for the first 4 weeks and 5 mg/kg bw for the 5th week); and 2) control group treated with PBS. Testicular samples were obtained at 11 weeks and classified into three different patterns of spermatogenesis, such as normal, Sertoli cell only, and destroyed. Semen collection and libido experiments were performed 1 week before treatment, and 4 and 8 weeks after treatment. For the sperm analysis, total spermatozoa and motility were measured using a light microscope with a motility analyzing system. In the multiple low-dose group, the numbers of tubules categorized as Sertoli cell only were significantly higher than those in the control as well as the total population and total/progressive motility of sperm were significantly decreased 8 weeks after the start of the treatment. The sperm production and motility in the multiple low-dose group appears to be reduced, while libido was maintained. In conclusion, multiple administration of 2.5 mg/kg bw busulfan depletes endogenous germ cells in the stallion recipients for SSC transplantation.