• 한국동물위생학회지
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• 제31권4호
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• pp.547-554
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• 2008

Bacteriospermia is a frequent finding in fresh boar semen and can result in detrimental effects on semen quality and longevity. The objectives of this study was to evaluate types of bacterial contaminants in porcine fresh semen and the reducing effect of antibiotic and density gradient with percoll on the bacterial contaminants. Fresh semen was collected by gloved-hand method into a pre-warmed($37^{\circ}C$) thermostable bottle, and was inoculated onto blood agar and MacConkey agar, respectively. After incubated for 48 hour, 7.5% $CO_2$ at $37^{\circ}C$, bacterial colonies were selected and identified by Gram staining, oxidase test, catalase test and finally identified using API kits and Vitek system. Aerobic culture yielded a variety of bacteria from different genera. The most prevalent contaminant of fresh semen were Leclecia adecarboxylata, Acineobacter banmanni, Staphylococcus epidermidis, Staphylococcus cohni spp urealyticus, Proteus mirabilis. Most of identified bacteria were Gram(-) and non-pathogenic bacteria. It seems that bacterial contaminants in fresh semen were seem originated from multiple sources at the stud/farm, and were from animal and non-animal origins. Gentamicin treatment did not eliminate the bacterial contaminants completely but 3 step-density gradient with percoll completely removed the bacterial contaminants in fresh semen. Therefore, future study is necessary to prove that density gradient method with percoll can eliminate bacteria in fresh semen without significantly affecting sperm viability or function.

• 한국수정란이식학회지
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• 제31권3호
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• pp.281-285
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• 2016

Cluster-of-differentiation antigen 9 (CD9) gene expressed in the male germ line stem cells is crucial for sperm-egg fusion, and was therefore selected as a candidate gene to investigate Duroc boar semen motility and kinematic characteristics. This study was performed to investigatetheir association with semen motility and kinematic characteristics. DNA samples from 96 Duroc pigs with records of sperm motility and kinematic characteristics [Total motile spermatozoa (MOT, $82.27{\pm}5.58$), Curvilinear velocity(VCL, $68.37{\pm}14.58$), Straight-line velocity(VSL, $29.06{\pm}6.58$), the ratio between VSL and VCL(LIN, $47.36{\pm}8.42$), Amplitude of Lateral Head displacement(ALH, $2.88{\pm}0.70$)] were used in present study. A single nucleotide polymorphism (g.358A>T) in intron 6 was associated with MOT, VCL, VAP and ALH in Duroc population (p<0.05). Therefore, we suggest that the porcine CD9 may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not clear yet. These results will improve the understanding of the functions of the CD9 in spermatogenesis within the reproductive tracts, and will shed light on CD9 as a candidate gene in the selection of good sperm quality boars.

• 한국임상수의학회지
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• 제33권6호
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• pp.356-361
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• 2016

During storage, boar spermatozoa undergo several changes including diminished motility and viability and accumulated reactive oxygen species (ROS). In this study, we investigated the effects of green tea extract (GTE) supplementation in the Sui Dil extender on the sperm motility, viability, ROS and lipid peroxidation (LPO) of long-term preserved boar semen at $17^{\circ}C$. A total number of eight boars were used for this experiment. Pooled ejaculates were diluted to $20{\times}10^6sperm/ml$ in the Sui Dil extender containing 0 (control), 1, 10, 100 or 500 mg/l GTE and were preserved at $17^{\circ}C$ for 24, 72, 120 and 168 h, respectively. At each storage time, sperm motility and viability were estimated by microscopic examination and the fluorescent double stain $Fertilight^{(R)}$, respectively. Sperm ROS level and LPO were assessed using the 2', 7'-dichlorodihydrofluorescein diacetate ($H_2DCFDA$)/propidium iodide (PI) and C11-BODIPY581/591/PI with flow cytometry, respectively. Compared to that of the 500 mg group, there were higher sperm motility and viability in the 1, 10 and 100 mg GTE groups during the preservation from 24 to 168 h (p < 0.05). The ROS levels of the 10 and 100 mg groups during the 168 h preservation were lower than those of the 0, 1 and 500 mg groups (p < 0.05). There were no significant differences in LPO regardless of the preservation period or the GTE concentration. In conclusion, the optimal concentrations (10 and 100 mg/l) of GTE that led to lower ROS levels may be useful for liquid boar sperm preservation at $17^{\circ}C$ for a period of 168 h.

• 한국수정란이식학회지
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• 제21권4호
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• pp.345-351
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• 2006

본 연구는 항생제가 첨가되지 않은 돼지 혼합액상 정액을 17$^{\circ}C$ 정액 보관고에 보관하면서 보관일수의 증가가 따라 정자의 운동성, 정액 내 세균의 증식 여부 및 체외 수정란 생산 효율에 미치는 영향을 조사하고자 하였다. 정자의 운동성은 1일 (78.7$\pm$2.4%)에 비하여 3일(78.7$\pm$2.4%)과 5일째(64.8$\pm$2.4%)는 유의적으로(p<0.05) 낮은 운동성을 나타내었다. 보관일수에 따른 정액 내 세균수의 변화는 보관 5일이 $57.8\pm105.2\times10^4$ Cfu로 0일과 3일의 $32.1\pm76.8\times10^4$ Cfu와 $26.9\pm46.6\times10^4$ Cfu에 비하여 유의적인(p<0.05)증가를 나타내었다. 보관된 정액을 이용하여 체외 수정한 결과, 정상적인 수정(2PN)은 1일과 3일째의 66.0$\pm$2.7%와 64.0$\pm$2.7%에 비하여 5일째에는 56.0$\pm$2.6%로 유의적인(p<0.05) 차이를 나타내었다. 체외 수정란의 발달율에서 난할율은 1일째의 75.0$\pm$l.4%에 비하여 3일과 5일째는 70.0$\pm$0.3%와 71.0$\pm$0.3%로 유의적으로(p<0.05) 낮게 나타났으며, 상실배로의 발달율에 있어서 1일째는 32.0$\pm$1.4%의 발달율을 보였으나 3일과 5일째에는 28.0$\pm$1.3%와 24.0$\pm$1.3%로 유의적인(p<0.05) 감소치를 나타내었고, 배반포로의 발달율에 있어서도 1일에 15.0$\pm$1.0%에 비하여 3일과 5일은 11.0$\pm$0.9%와 8.0$\pm$0.9%로 유의적으로(p<0.05) 낮은 발달율을 나타내었다. 이상의 결과를 종합할 때 항생제가 첨가되지 않은 돼지 혼합 정액은 보관일수 3일째부터 정자의 운동성이 감소하고 세균수는 증가하였다. 또한 보존된 정액을 이용하여 체외 수정을 실시할 경우, 보관일수가 증가할수록 정상 수정율과 체외 발달율이 감소함으로 항생제를 첨가하지 않는 경우 3일 이상 정액을 보관하여 사용하지 않는 것이 바람직 하다고 사료된다.

• 한국수정란이식학회지
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• 제25권4호
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• pp.229-235
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• 2010

The MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at $37^{\circ}C$ using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.

• Reproductive and Developmental Biology
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• 제36권3호
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• pp.163-166
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• 2012

This study was conducted to determine the relationship between elapsed time after semen preservation on the changes of bacteria and semen quality. Semen was diluted with BTS(Beltsville Thawing Solution) extender without antibiotic for 7 days and sperm parameter and fertility were measured. Sperm motility was measured by CASA and total bacteria number was counted after 22~24 hr incubation from counting agar plate in which sperm dilute to $10{\sim}10^6$ in 0.9% saline solution and inoculate to agar. Acrosomal integrity was measured by Chlortetracycline (CTC) staining. CTC patterns were uniform fluorescence over the whole head (pattern F), characteristic of incapacitated acrosome-intact spermatozoa; fluorescence-free band in the post-acrosomal region (pattern B), characteristic of capacitated acrosome-intact spermatozoa; and almost no fluorescence over the whole head except for a thin band in the equatorial segment (pattern AR), characteristic of acrosome reacted spermatozoa. Total number of bacteria was significantly increased (p<0.0001) 3 days after preservation. Sperm motility, viability, and morphological abnormality on elapsed time after preservation were lower from 5 ($77.24{\pm}6.47$, p<0.001) and 7 days ($77.24{\pm}6.47$, p<0.001) after preservation compared to 1 ($15.71{\pm}7.18$) and 3 days($18.39{\pm}7.22$) after preservation, respectively. Sperm viability was significantly lower ($53.25{\pm}35.03$, p<0.0001) at 7 days after preservation. Morphological abnormality of sperm was lower (p<0.001) at 1 ($15.71{\pm}7.18$) and 3 ($18.39{\pm}7.22$) days compared to 5 ($21.84{\pm}7.91$) and 7 ($22.59{\pm}9.93$) days after preservation. Acrosomal integrity and capacitation rate (pattern F) were significantly lower (p<0.001) from 5 days after preservation. Based on the data we obtained from this study suggested that semen preserved more than 5 days without antibiotic would not recommend use for artificial insemination.

• Asian-Australasian Journal of Animal Sciences
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• 제17권10호
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• pp.1369-1373
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• 2004

The percentages of sperm motility and normal acrosome on the liquid boar semen diluted and preserved at $4^{\circ}C$ with lactose hydrate, egg yolk and N-acetyl-D-glucosamine (LEN) diluent were significant differences according to preservation day and incubation time, respectively. The sperm motility steadily declined from 96.9% at 0.5 h incubation to 78.8% at 6 h incubation at 1 day of preservation. However, the sperm motility rapidly declined after 4 day of preservation during incubation. The normal acrosome steadily declined from 93.3% at 0.5 h incubation to 73.8% at 6 h incubation at 1 day of preservation. However, the normal acrosome rapidly declined after 3 day of preservation during incubation. 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. Mean numbers of sperm in penetrated oocyte were highest in $10{\times}10^6$ sperm/ml compared with other sperm concentrations. The rates of blastocysts from the cleaved oocytes (2-4 cell stage) were 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 $1{\times}10^6$sperm/ml concentration for in vitro fertilization of pig oocytes.

• 농업과학연구
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• 제47권3호
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• pp.657-665
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• 2020

The objective of this study was to investigate whether supplementation of pentoxifylline (PTX; phosphodiesterase inhibitor) to thawed boar semen improves the post-thaw motility of sperm and affects the efficiency of artificial insemination (AI) and further development. To determine the concentration of PTX for AI, frozen-thawed semen was incubated with 0, 5, 10, and 20 mM PTX in an extender freezing medium, respectively, after thawing. Kinematic properties of sperm were examined with a computer-assisted semen analysis (CASA) system. In addition, viability and mitochondrial activity were also tested by LIVE/DEAD and a MitoTracker kit. There were no significant differences in the kinetic parameters of thawed sperm between control and treatment groups, but overall assessment parameters such as motility and rapid progressive were higher in the 10 mM PTX group. In the viability and mitochondrial assay, there were no significant differences observed in the PTX treatment, compared to the control. For further analysis, artificial inseminations were performed using frozen semen and 10 mM PTX treated cryopreserved semen, respectively. There were no differences in pregnancy rates and fetus weights among the groups until 30 and 40 days, but litter size was reduced and relatively low-birth weight was observed in the PTX group. In summary, our findings suggest that enhancement of in vitro sperm quality or non-toxicity supplemented by PTX may have detrimental effects on fetus development.

• 한국임상수의학회지
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• 제28권1호
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• pp.13-19
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• 2011

요 약: 정액 동결 과정은 활성산소종의 생성을 유발하며, 생성된 활성산소종은 정자의 손상을 일으키는 것으로 알려져 있다. 따라서 본 연구의 목적은 동결 과정 중 항산화 효소 중 하나인 catalase (CAT)를 첨가함으로써 융해 후 정자의 기능과 활성산소종의 수준에 미치는 효과를 알아보고자 하였다. 5마리 돼지에서 채취한 정액은 0 (대조군), 200, 400 U/mL CAT가 첨가되어 있는 동결 희석액으로 각각 동결하였다. 융해 후, 정자 운동성, 생존성, 정상 형태율, 형질막 온전성, 미토콘드리아 기능, 세포내 ROS를 평가하였다. CAT는 400 U/mL의 농도에서 전체 정자 운동성을 향상시켰지만 (P < 0.05), 전진 운동성, 생존성, 기형율, 형질막 온전성, 미토콘드리아 기능의 향상을 나타내지 않았다. 활성산소종의 평가에서, CAT는 융해된 생존 정자의 ${\cdot}O_2$의 감소에는 효과를 나타내지 않은 반면 $H_2O_2$를 감소시켰다(P < 0.05). 결론으로 CAT는 동결 및 융해된 정자의 질을 향상시키는 데 큰 효과를 나타내진 않았지만 생존 정자에서 $H_2O_2$을 제거함로써 생존정자의 산화적 손상을 감소시킬 수 있으리라 판단된다.

• 한국수정란이식학회지
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• 제30권1호
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• pp.7-15
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• 2015

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM ($61.1{\pm}1.5%$,$64.7{\pm}2.0%$) of nicotinic acid than other groups (0 mM, $52.1{\pm}2.3%$; 20 mM, $47.8{\pm}5.1%$, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid ($26.1{\pm}1.8%$, $24.9{\pm}1.5%$) than other groups (0 mM, $35.3{\pm}0.8%$; 20 mM, $36.5{\pm}1.9%$, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM ($84.2{\pm}3.6%$, $88.4{\pm}2.3%$) of nicotinic acid than other groups (0 mM, $77.3{\pm}4.4%$; 20 mM, $73.3{\pm}3.6%$, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM ($17.0{\pm}1.3%$) of nicotinic acid than other groups (0 mM, $9.4{\pm}0.5%$; 5mM, $12.6{\pm}0.8%$; 20 mM, $5.0{\pm}1.0%$, P<0.05). Moreover, total cell number was higher in 5 and 10 mM ($53.6{\pm}2.9%$, $57.9{\pm}2.8%$) of nicotinic acid than other groups (0 mM, $41.0{\pm}1.4%$; 20 mM, $23.2{\pm}2.8%$, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid ($0.7{\pm}0.1%$) than other groups (0 mM, $1.0{\pm}0.1%$; 10mM, $0.9{\pm}0.0%$; 20 mM, $1.4{\pm}1.0%$, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.