• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.123-126
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• 2008

The objective of this study was to determine the potential hazardous effects of sorting process by flowcytometry on the quality of boar spermatozoa by flowcytometer. Freshly collected boar semen was diluted and divided into two groups; control none sorted and sorted. Sperms in sorted group were processed with flowcytometer for cell sorting with $100\;{\mu}M$ nozzle under the 20 psi pressure. Measurements on each parameter were made at two time points, 0hr (right after sorting) and 24 hr post sorting. Although there was a tendency of lower viability in sorted group than none sorted control group, the percentage of live cells in control ($75.83{\pm}6.92\;&\;59.53{\pm}10.34$) was not significantly different from sorted ($59.70{\pm}7.37\;&\;43.97{\pm}3.76$) at both 0 and 24 hr post sorting. However, sorted sperm showed significantly lower mitochondrial function compared to the control at both 0 h ($79.37{\pm}3.22\;vs.\;63.50{\pm}10.05$) and 24 hr ($67.27{\pm}3.22$ vs. $46.97{\pm}5.37$) time points (p<0.007). Sperm DNA fragmentation rate was significantly lower in control ($22.0{\pm}7.04$) than that of sorted ($32.27{\pm}7.49$) at 24 hr time point (p<0.0002). Taken together, these data suggested thatsorting process by flowcytometer may have influenced sperm motility rather than viability. Also high speed sperm sorting by flowcytometer has significant effects on DNA fragmentation on elapsed time after sorting.

• Journal of Ginseng Research
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• v.34 no.4
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• pp.327-335
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• 2010

We reported previously that the administration of Korean red ginseng water extract (KRG-WE) protected the guinea pig testis against damage induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (a potent endocrine disruptor). We also found that crude saponin from ginseng was the active ingredient responsible for this protection. Here, we examined the biological role of KRG-WE in an animal model of age-induced dysfunction of spermatogenesis. Twenty-four male Sprague-Dawley (six 2-month-old and eighteen 12-month-old) rats were used. The young and old control groups received only vehicle. The ginseng saponin (GS)- and KRG-WE-treated groups received GS (40 mg/kg body weight/day) and KRG-WE (200 mg/kg body weight/day), respectively, for 4 months. The number of cells, Sertoli cell index, Johnsen's score, and sex hormone levels decreased significantly with age. However, the administration of KRG-WE and GS markedly improved the number of germ cells, seminiferous tubular size, and Johnsen's score in the old rats. Ginseng produced a distinct testicular histological improvement in old rats. KRG-WE and GS elevated testosterone levels, while attenuating the aberrant increase in follicle stimulating hormone and luteinizing hormone levels. Sperm kinematics evaluated by a computer-assisted sperm analyzer demonstrated improvement in the percentage of motile sperm, progressive sperm motility, and curvilinear velocity associated with sperm quality, supporting the beneficial role of red ginseng in senile spermatogenesis. Overall, the total water extract had a more potent effect than the corresponding saponin fraction. In conclusion, Korean red ginseng rejuvenated age-induced testicular dysfunction. Additionally, the total water extract was more potent than the corresponding saponin fraction.

• 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.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.69-69
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• 2002

Transgenic animals production tools have been valuable for research and purpose. The current methods of gene transfer, microinjection and nuclear transfer, which are widely used in transgenic animal production, but all most methods has only had limited success in production of larger species. Here, we report the possibility of a sperm-mediated gene transfer method in porcine embryos. Oocytes were collected from ovaries harvested at a local slaughterhouse were matured in 500${mu}ell$ drops of TCM-199 under mineral oil at 38.5$^{\circ}C$ in a humidified atmosphere of 5%CO2 in air. After 42-43h of in vitro maturation oocytes were denuded. for sperm injection into the cytoplasm of the porcine oocytes, sperm suspension in NIM medium are subjected extraction with TritonX-100 before mixing with a green fluorescent gene (GFP). Sperm with Tritonx-100 were prepared by adding TritonX-100 to a final volume of 0.05% in the sperm suspension and mixing by trituration for 60s before two wishes in NIM medium at 2$^{\circ}C$. A(ter wishing, sperm were mixed with TritonX-100 at $25^{\circ}C$ followed by washes at 2$^{\circ}C$. Sperm were resuspended in ice cold NIM to a final volume of 400${mu}ell$ and 2-20ng/${mu}ell$ DNA were triturated on ice for 60s. All microinjection was performed in HEPES-buffered CZB medium at room temperature within 2h. After culture in NCSU-23 for 72h, percent of porcine embryos transfected GFP gene are 20.7%(6/29) in 20ng/${mu}ell$ sperm-DNA mixed group and other groups were 3.7 %(2/54)and 4.7%(3/67). These data suggests that sperm-mediated gene transfer method should be used to the production tool of transgenic pig efficiently.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.10
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• pp.1417-1425
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• 2014

The selection of morphologically normal spermatozoa is critical to obtain high breeding performances in boar breeding farms and artificial insemination (AI) centers. Parameters for the selection of semen mainly include total sperm motility, concentration, and morphology. However, these primary parameters are often not reliable for discriminating between normal and abnormal, non-fertilizable spermatozoa. The present study was designed to compare the motion characteristics, fertilization ability using in vitro fertilization (IVF), and acrosome formation of the semen from boars having low (boar number 2012) and normal (boar number 2004 and 2023) breeding performances. The ultimate goal was to identify additional simple and easy criteria for the selection of normal sperm. There was no significant difference between boar 2004 and boar 2023 sperm total motility in computer assisted sperm analysis. However, boar number 2012 semen presented a significantly reduced population of rapid moving spermatozoa and an increased population of slow moving spermatozoa compared to boar numbers 2004 and 2023. Analysis of detailed motion characteristics revealed that sperm from boar number 2012 had significantly reduced motility in progressiveness, average path velocity, straight-line velocity (VSL), curvilinear velocity (VCL), straightness, and linearity. The assessment of the fertilizing ability by IVF also showed that sperm from boar number 2012 showed a fertility rate of 3.4%, whereas sperm from boar number 2023 had a fertility rate of 75.45%. Interestingly, most of the sperm nuclei were found on the peripheral area of the oocytes, suggesting that the sperm from boar number 2012 lacked penetration ability into the oocyte zonapellucida. The acrosome formation analysis using Pisum sativum agglutinin staining demonstrated that the sperm from boar number 2012 had a defect in acrosome formation. Consequently, primary parameters for selecting semen before AI such as motility are not sufficient to select normal and fertilizable spermatozoa. In conclusion, the present study suggests that the acrosome staining and detailed motion characteristics such as progressiveness, VCL, and VSL should be included in determining semen quality together with primary parameters for successful AI and high breeding performance in the swine industry.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.1-7
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• 2002

The present study was carried out to investigate the effects of maturation media on penetrability of pig oocytes by liquid boar sperm coincubated with different sperm concentrations in a modified Tris­buffered medium (mTBM). Follicular oocytes collected from ovaries of prepubertal gilts were matured in a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. The sperm­ich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ far 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes in 5% $CO_2$in air at 38.5$^{\circ}C$, cumulus cells were removed and oocytes (30~40) were coincubated for 6 h in 0.5 $m\ell$ mTBM fertilization medium with five different (1$\times$10$^{6}$ , 2$\times$10$^{6}$ , 4$\times$10$^{6}$ , 6$\times$10$^{6}$, 10$\times$10$^{6}$ $m\ell$) sperm concentrations. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ NCSU-23 culture medium fur further culture of 6 h. At 12 h after IVF, sperm penetration, polyspermy and male pronuclear formation of oocytes were evaluated. Oocytes of NCSU-23 maturation medium decreased polyspermy and increased male pronuclear formation compared to those of mTCM­199 and mWaymouth MB 752/1 maturation media. Of oocytes matured in NCSU-23 medium and inseminated in mTBM medium with 2~4$\times$10$^{6}$ $m\ell$ sperm concentrations, 50.8~50.9% showed sperm penetration, 13.3~19.5% polyspermy and 43.9~45.4% male pronuclear formation. In conclusion, we found out that oocytes matured in NCSU­23 medium and inseminated in mTBM medium showed superior in­vitro fertilization compared to those matured in mTCM­199 and mWaymouth MB 752/1 maturation media and inseminated in mTBM medium. The optimum sperm concentrations for in-vitro fertilization of oocytes matured in NCSU-23 medium by liquid boar semen stored at 17$^{\circ}C$ for 5 days were 2~4$\times$10$^{6}$ $m\ell$.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.7
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• pp.925-937
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• 2016

In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive $F_1$ piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive $F_1$ boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive $F_1$ sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.3
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• pp.340-345
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• 2008

Oxidative stress is one of the major causes of failure of in vitro storage of boar semen. Reactive oxygen species (ROS) are one of the important mediators of oxidative stress during in vitro storage of boar semen. Our study examined the effects of taurine on sperm characteristic and on in vitro developmental embryos during in vitro storage of boar semen for 7 days. Semen was randomly aliquoted into 3 centrifuge tubes and treated with different concentrations of taurine (25-100 mM). The characteristics of boar sperm were analyzed for motility by light microscopy, viability by using a Makler counting chamber and membrane integrity by a hypoosmotic swelling test (HOST). The percentages of motile spermatozoa in taurine groups after 5 days were significantly higher compared to the control. Sperm viability in the control was lower than in taurine groups after 7 days irrespective of different taurine concentration. In the hyoosmotic swelling test (HOST), significantly higher results were obtained in taurine groups after 3 days. Also, the developmental rates of IVM/IVF porcine embryos from semen treated with pyruvate and taurine were significantly increased when compared with the control (p<0.05). These results indicate that supplementation of taurine as an antioxidant in boar semen extender can improve the semen quality.

• Reproductive and Developmental Biology
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• v.37 no.2
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• pp.85-89
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• 2013

For more than two decades, the intracytoplasmic sperm injection (ICSI) technique has been used as a valuable tool to provide opportunities for studying fertilization, treating human infertility, and producing transgenic animals. Not only in facilitating fertilization but also in propagating mammalian species, ICSI has enhanced the potential of assisted reproductive technologies in human. Polyspermic fertilization has been one of major problems in pig reproduction, but the ICSI helped to solve the problem, and used widely to generate transgenic piglets. Although the ICSI technique is considered to be a very useful tool in assisted reproductive technologies, including generation of transgenic animals, there are some disadvantages using the technique. In this review, we describe the ICSI technique and its application in animal production and human infertility, and discuss advantage and disadvantage of the technique in mammals.

• Korean Journal of Animal Reproduction
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• v.27 no.1
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• pp.77-85
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• 2003

This study was undertaken to evaluate the effects of catalase using xanthine (X)-xanthine oxidase (XO) system on in vitro maturation and fertilization in the pig. When follicular oocytes were cultured with X or XO, the maturation rates were not significantly different between in medium with and without catalase despite of different culture periods. However, significantly (P<0.05) higher maturation rates were obtained in culture with X-XO-catalase system. The rates of degenerated oocytes were increased with culture periods prolonged, and were significantly (P<0.05) higher in medium without that than with catalase at 120 h of culture. On the other hand, the parthenogenetic oocytes were observed with high proportions at 72 h of culture, but were not different between the medium with and without catalase at various times of culture. In another experiment, the frozen-thawed boar spermatozoa treated with X-XO system for in vitro fertilization. The penetration rates were higher in medium with that than without catalase during the in vitro fertilization with none (P<0.05), XO and X+XO. On the other hand, when sperm were treated with none, X, XO and X+XO, lipid peroxidation were produced with higher rates in medium without that than with catalase, and consequently the changes in sperm penetration and lipid peroxidation showed opposite patterns. Under the above all conditions, however, sperm-SH group were higher detected by catalase. When the activity of sperm binding to zona pellucida was evaluated through binding to salt-stored porcine oocytes, sperm binding to zona pellucida in control group were higher than in medium with X, XO and X+XO groups. No significant differences, however, were observed between medium with and without catalase. In conclusion, the exposure of follicular oocytes and spermatozoa to X-XO-catalase system may be caused stimulating in vitro maturation and fertilization in the pig.