• Journal of Embryo Transfer
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• v.18 no.2
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• pp.143-149
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• 2003

This study was carried out to cryopreserve and to investigate characteristics of semen in Hanwoo. Semen was obtained from bulls selected by Daekwanryeong Branch station. Semen was collected each morning of the experiment, placed in water jacketed tubes at 37$^{\circ}C$, and trans-ported to the research laboratory within 10 minutes. Semen was extended with Egg yolk-glycerol extender to contain 50${\times}$10$^{6}$ sperm/ml. Semen was cooled over a 6h period in water jacketed tubes from about 25 to 5$^{\circ}C$, Egg yolk-glycerol extender was added in one step at 5$^{\circ}C$. Semen was aspirated into 0.5ml straws, which were sealed with powder. Egg yolk-glycerol extender, which is used in Hanwoo sperm frozen and stored, semen from 13 Hanwoo bulls collected, the postthawed percentages of motile sperm were 65.7%. In semen characteristics of Hanwoo bulls, number of bulls volume are 5.7 ml and total cell count are 975${\times}$10$^{6}$ m1 ejaculate.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.63-63
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• 2003

This study was carried out to investigate the effects of liquid boar sperm stored at 4$^{\circ}C$ on sperm motility, normal acrosome, and in-vitro fertilization and culture of pig oocytes matured in-vitro. The sperm-rich fraction (30~60 ml) of ejaculate was collected into an insulated vacuum bottle. Semen was slowly cooled to room temperature (20~23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min at 800$\times$g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of lactose, egg yolk and N-acetyl-D-glucosamine (LEN) diluent to provide 1.0$\times$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$ and preserved for 5 days to examine sperm motility and normal acrosome. The medium used for oocyte maturation was modified tissue culture medium (TCM) 199. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 6 h in 500 ${mu}ell$ mTBM fertilization media with 0.2, 1, 5 and 10$\times$10$^{6}$ /ml sperm concentration, respectively. At 6 h after IVF, oocytes were transferred into 500 ${mu}ell$ Hepes-buffered NCSU-23 culture medium for further culture of 6, 48 and 144 h. There were significant differences in sperm motility and normal acrosome among preservation days and incubation times, respectively. The rates of sperm penetration and polyspermy were higher in 5 and 10$\times$10$^{6}$ sperm/ml than in 0.2 and 1$\times$10$^{6}$ sperm/ml. Male pronuclear formation was lower in 0.2$\times$10$^{6}$ sperm/ml than in 1, 5 and 10$\times$10$^{6}$ sperm/ml. Mean numbers of sperm in penetrated oocyte were highest in 10$\times$10$^{6}$ sperm/ml compared with other sperm concentrations. The rate of blastocysts from the cleaved oocytes (2~4 cell stage) was highest in 1$\times$10$^{6}$ sperm/ml compared with other sperm concentrations. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend 1$\times$10$^{6}$ ml sperm concentration for in-vitro fertilization of pig oocytes.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.167-172
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• 2004

This study was performed to examine the correlations among dog sperm viabilities evaluated by flow cytometry, by microscopic evaluation (ME), by carbo-xifluorescein diacetate and propidium iodide (CFDA/PI) and by hypoosmotic swelling (HOS) test. Semen were collected from 5 dogs ranging in age from 2 to 4 years. Each ejaculate was divided into 3 aliquots and different proportions of freeze-killed cells were added to each aliquot (1:0, 1:1 and 1:3). In the other experiment, semen was extended with Sweden extender containing 5% glycerol and equex STM paste, and frozen using liquid nitrogen vapor. Fresh and frozen-thawed dog sperm viability were assessed by flow cytometry using PI staining method. The accuracy of flow cytometry was evaluated by comparing with other classic assessments, microscopic evaluation, epifluorescence microscopic analysis using CFDA/PI, and HOS test. High correlations of sperm viabilities were found among flow cytometry, epifluorescence evaluation, HOS test (p<0.01) in fresh semen. Especially, sperm viability assessed by HOS test was highly correlated with viability by flow cytometry in all the ratios of live and dead spermatozoa, 1:0, 1:1 and 1:3 (p<0.01). The viability evaluated by ME were significantly correlated with that by flow cytometry in ratios of 1:0 and 1:3 (p<0.05) however, there was no significance in ratio of 1:1. The viability evaluated by C/p were highly correlated with that by flow cytometry in ratio of 1:0 and 1:1 (p<0.01) and significantly correlated in ratio of 1:3 (p<0.05). In frozen-thawed spermatozoa, the viability determined by HOS test was considerably correlated with that by flow cytometry (p<0.01). There was significant correlation between the viabilities by ME and by flow cytometry (p<0.05). But the viability evaluated by CFDA/PI was not correlated with viability by flow cytometry. The result from this study validate the use of flow cytometry as a precise method for assessing the viability of fresh and frozen-thawed dog spermatozoa.

• 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.

• Korean Journal of Biological Psychiatry
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• v.7 no.1
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• pp.85-98
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• 2000

Objective : There are four possible explanations for the sexual dysfunction of schizophrenics. The first is the possibility of a real structural aspect. The second possibility is that sexual function changes secondary to the illness. The third possibility is that there are medical and sociocultural barriers to sexual expression for chronic schizophrenics. The fourth possibility is that sexual dysfunction due to antipsychotic medication. However, we didn't know the precise cause of sexual dysfunction in schizophrenics. Therefore, the purpose of this study was to explore the mechanism of illness itself and antipsychotics on sexual dysfunction in male schizophrenics. Methods : The serum prolactin(PRL), testosterone(TST), and the plasma serotonin(5-HT) concentrations were measured by radioimmunoassay and high performance liquid chromatography method for 100 healthy male schizophrenics according to the DSM-IV. Concomitantly, the severity of psychotic symptoms using Clinical Global Impression(CGI), Brief Psychiatric Rating Scale(BPRS), Positive and Negative Syndrome Scale(PANSS), and the severity of side effects for antipsychotics using Extrapyramidal Side Effects Scale(EPSE), Anticholinergic Side Effects Scale(ACSE), the cognitive function using PANSS-Cognitive Function(PANSS-CF), Mini Mental State Exam-Korean(MMSE-K), and the sexual dysfunction using Sexual Functioning Questionnaire(SFQ), Questionnaire for Sexual Dysfunction in Men were assessed. The PRL, TST, and 5-HT levels of 50 healthy male controls who had no medical, neurological, and psychiatric illnesses were evaluated. The sexual function using SFQ(items FGa, FNa) were also assessed. Furthermore, the correlation with age, education, religion, economic status, age at onset, duration of illnesses, duration of admission, levels of PRL, TST, 5-HT, antipsychotic dosages, potency, benztropine, total duration of medication, EPSE, ACSE, CGI, BPRS, PANSS, PANSS-CF, MMSE-K and sexual dysfunctions were identified in male schizophrenics. Results : 1) The frequencies of sexual dysfunctions for schizophrenics(80%) were significantly(p<0.001) higher than those for controls(42%). The sexual dysfunctions according to sexual response cycle were 'low sexual desire' 76%, 'impairment of achieving erection' 75%, 'impairment of maintaining erection' 75%, 'impairment of obtaining orgasm' 32%, 'impairment in the quality of orgasm' 61%, 'impairment in quantity of ejaculate' 44%, 'premature ejaculation' 15%, and 'delayed ejaculation' 50%. 2) The PRL, 5-HT levels of schizophrenics($28.5{\pm}20.6ng/ml$, $298.5{\pm}89.1ng/ml$) were significantly(p<0.001) higher than those of controls($10{\pm}5.6ng/ml$, $169.2{\pm}37.8ng/ml$), while the TST levels of schizophrenics($4.3{\pm}1.5ng/ml$) and controls($4.5{\pm}1.2ng/ml$) were not significantly different. The sexual dysfunctions of schizophrenics who had abnormal 5-HT levels($4.7{\pm}1.3$ scores) were significantly(p<0.05) higher than those of who had normal 5-HT levels($3.8{\pm}1.6$ scores) on item D7. 3) The sexual dysfunctions of unmarried schizophrenics were significantly(p<0.01 : p<0.05) higher than those of married schizophrenics($6.1{\pm}2.8$ scores, $4.7{\pm}1.3$ scores on item FGa : ${\beta}$=-0.211 on item FNa). The sexual dysfunctions were positively correlated with the rise of 5-HT levels(r=0.209, p<0.05 on item D4 and r=0.241, p<0.05 on item D7), the higher age at onset(r=0.275, p<0.01 on item FNa : r=-0.202, p<0.05 on item FDa), the longer duration of illnesses(r=0.237, p<0.05 on item D6), the longer duration of admission(r=0.234, p<0.05 on item D4 : r=0.328, p<0.05 on item D6), the longer total duration of medication(r=0.237, p<0.05 on item D6). However, age, education, religion, economic status, PRL, TST levels, antipsychotics dosage, potency, benztropine, ACSE, CGI, BPRS, PANSS, PANSS-CF, MMSE-K scores were not correlated with increased sexual dysfunctions. Conclusions : Male schizophrenics have significantly more sexual dysfunction to compare with controls. The higher frequencies of sexual dysfunctions were low sexual desire and erectile disorder. The unmarried, higher age at onset, and longer duration of diseases were positively correlated with increased sexual dysfunctions. Also high 5-HT levels were positively correlated with increased sexual dysfunctions. This means that studies of plasma 5-HT levels, albeit questionable indicators of central 5-HT function, offer some additional support for the association of sexual dysfunction with excess 5-HT activity as primary pathology of schizophrenia. Our findings suggest that excess 5-HT activity seems to affect the patient's sexual function.

• 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%).