• Korean Journal of Animal Reproduction
• /
• v.26 no.1
• /
• pp.17-23
• /
• 2002

The present study was carried out to investigate the effects of the maturation media such as a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium on penetrability of pig oocytes by liquid boar sperm. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. When immature pig oocytes were cultured in mTCM-199, mWaymouth MB 752/1 and NCSU-23 maturation media for 44 h in 5% $CO_2$, in air at 38.5$^{\circ}C$, the germinal vesicle breakdown (CVBD) rates of the oocytes were 95.6, 94.1 and 94.9%, respectively, and the maturation rates (metaphase II) of oocytes were 92.5, 90.1 and 91.1%, respectively. No differences were observed among the maturation media. The spermrich 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$^{\circ}C$ for 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, cumulus cells were removed and oocytes (30~40) coincubated far 6 h in 0.5 $m\ell$ mTCM-199 and mTBM fertilization media with 2$\times$1061$m\ell$ sperm concentration. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ mTCM-199 and NCSU-23 culture media for further culture 6 or 42 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF, and developmental ability of oocytes at 48 h after IVF were evaluated. The oocytes in combination with NCSU-23 medium for maturation and mTBM medium for IVF increased male pronuclear formation (48.0%) compared to those in combination with mTCM-199 media for maturation and IVF, and mWaymouth MB 752il medium for maturation and mTCM-199 medium far IVF. The rates of cleaved embryos (2~4 cell stage) at 48 h after IVF were 24.1% in combination with mTCM-199 media for maturation, IVF and culture, 43.6% in combination with mWaymouth MB 75211 medium fur maturation and mTCM-199 media for IVF and culture, and 71.2% in combination with NCSU-23 medium for maturation, mTBM medium for IVF and NCSU-23 medium for culture. In conclusion, we found out the oocytes matured in vitro were fertilized by liquid boar sperm stored in BTS extender at 17$^{\circ}C$ for 5 days. We recommend the simple defined NCSU-23 medium for nuclear maturation, mTBM medium and liquid boar sperm for IVF, and NCSU-23 medium for embryo culture.

• Journal of Embryo Transfer
• /
• v.25 no.4
• /
• pp.263-266
• /
• 2010

Prediction of semen's fertilizing ability used in artificial insemination (AI) is one of very important factors on pig reproductive performance. In vitro fertilization (IVF) has been used for indirect evaluation of sperm's fertilizing ability and it has been showed as highly correlated index. In swine industry, increasing interest in preservation of boar semen raises questions on the sperm motility from semen used in commercial AI centers. Mitochondria in sperm mid-piece generate the energy to support motility and could be an explanation of impaired fertility. Objective of this study was to suggest usable sperm motility to farms in measuring the effect of sperm motility and sperm abnormality on in vitro production of embryo in which sperm's fertilizing ability can be determined indirectly. Semen samples were provided from local AI center and used within 3 days after collection. Semen samples were divided by 4 different motile groups (>70%; 61~70%; 51~60%; <50%) using CASA (computer-assisted sperm analysis) on the days of IVF. Developmental rate to the blastocyst stage from over 61% motile sperm group showed significantly higher rate than below 60% motile sperm group ($16.5{\pm}0.7{\sim}18.4{\pm}0.8%$ vs $6.3{\pm}0.8{\sim}11.5{\pm}0.7%$, p<0.05). In experiment to determine the relationship between sperm motility and viability and abnormality, over 61% motile sperm groups showed significantly higher viability rate compared to below 60% motile sperm groups ($84.8{\pm}4.0{\sim}88.1{\pm}4.0%$ vs $69.1{\pm}4.0{\sim}74.2{\pm}4.0%$, p<0.05). On the other hand, morphological sperm abnormality showed significantly higher in over 70% motile sperm group ($10.2{\pm}2.2$ vs $16.0{\pm}2.2{\sim}21.0{\pm}2.2%$, p<0.05). In experiment to find the correlation between sperm motility of 4 different motile groups and amount of mitochondria, lower motility group also showed lower level of mitochondria (p<0.05). The mitochondria parameter used in this study showed another possibility to differentiate the sperm motility. Taken together, because below 60% motile semen used in AI reduce the fertility, AI centers should provide the over 60% motile sperm to the farms at the time of AI.

• Journal of Embryo Transfer
• /
• v.26 no.1
• /
• pp.71-78
• /
• 2011

This study was undertaken to find out the effect of cholesterol and serum albumin on sperm ability and lipid peroxidation levels period to the liquid storage of miniature pig sperm. Ejaculated semen from miniature pigs was collected by gloved-hand method into a pre-warmed ($37^{\circ}C$) thermos bottle, and extended with Modena solution {with and without BSA, methyl-beta-cyclodextrin (-cholesterol) and cholesterol loaded cyclodextrin (+cholesterol)}. Each semen was assessed for viability (SYBR-14/PI staining) and acrosome intactness, intensity and capacitation status by chlorotetracycline (CTC) staining at 1, 3, 5, 7 and 10 days of storage. At for the effects of cholesterol and serum albumin on lipid peroxidation, semen were incubated with $H_2O_2$ ($10\;{\mu}M$), and lipid peroxidation level were measured by flow cytometry using the lipid peroxidation reporter probe $C_{11}-BODIPY^{581/591}$. The result, lipid peroxidation level in sperm added with cholesterol were lower in $10\;{\mu}M$ $H_2O_2$ compared to the added sperm with serum albumin. Also, added cholesterol to sperm had significant (p<0.05) higher viability when storage for 7 and 10 days and lower when 10 days of storage percentage of acrosome-reacted sperm (AR pattern) in acrosome state as say result compared to other treated groups. In conclusion, role of cholesterol during lipid storage in miniature pig spermatozoa was protected boar spermatozoa from lipid peroxidation prior to lipid storage. Addition serum albumin during lipid storage in sperm may be induce sperm membrane damage by lipid peroxidation. Therefore, addition of cholesterol to miniature pig sperm will be lead to extension of liquid storage periods.

• Journal of the Korea Academia-Industrial cooperation Society
• /
• v.21 no.9
• /
• pp.251-258
• /
• 2020

This study investigated the effect of Zardaverine supplementation in freezing extender, on kinetic characteristics of post-thawed boar sperm. Cryopreservation of boar sperm is an important technique of assisted reproductive technology and genetic resource banking. Although this technique is particularly useful, freeze-thaw cycles associated with sperm cryopreservation significantly reduce sperm quality. Semen from mature Duroc boars were collected and cryopreserved in freezing extenders (LEY) treated with varying concentrations of Zardaverine (0, 20, 50, 75, 100 𝜇M). The time-dependent kinetic characteristics of post-thawed spermatozoa were determined after thawing by applying computer-assisted sperm analysis (CASA). We observed that the motility immediately after thawing was significantly higher in 20 𝜇M stocks than in control (0 𝜇M) and the other treatments (p<0.05). Curvilinear velocity (VCL) in 0 𝜇M and 20 𝜇M stocks were significantly higher than the other treatment groups, except 75 𝜇M (p<0.05). Higher average path velocity (VAP) was obtained at 20 𝜇M as compared to 100 𝜇M, whereas amplitude of head lateral displacement (ALH) was significantly higher at 20 𝜇M than 50 𝜇M and 100 𝜇M (p<0.05). No differences were obtained for Straight-line velocity (VSL) and Linearity (LIN). In conclusion, our results indicate that Zardaverine improves the motility, VCL, VAP, and ALH of post-thawed boar sperm.

• Journal of Embryo Transfer
• /
• v.30 no.3
• /
• pp.121-128
• /
• 2015

The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed ($37^{\circ}C$) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at $38.5^{\circ}C$ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

• ANNALS OF ANIMAL RESOURCE SCIENCES
• /
• v.29 no.4
• /
• pp.158-165
• /
• 2018

The present study was undertaken to investigate the reproductive performance of the female breeding pigs after artificial insemination (AI) using the frozen boar semen imported from Canada, thereby finding insights into improving the efficiency of AI using the frozen semen (FSAI). Analyzed in the present study were the records of a total of 626 FSAI in a great grandparent (GGP) farm beginning from May through November of the year of 2016 (Farm A) and 2,024 FSAI beginning from 2015 through 2017 from a second GGP farm (Farm B). Both the total number of piglets born (TNB) and the number born alive (NBA) were greater during May than during September within FSAI (p<0.05) in Farm A (p<0.01 for the effect of the month). In Farm B, no difference was detected between the years in any of the farrowing rate, TNB, and NBA. When the records from Farm A and Farm B were pooled, the farrowing rate was greater for Farm A vs. Farm B (p<0.01), with no difference between the two farms in TNB and NBA. Moreover, TNB and NBA were less for FSAI than for AI using the liquid semen (LSAI; $10.9{\pm}0.3$ vs. $13.4{\pm}0.1$ and $10.0{\pm}0.3$ vs. $12.0{\pm}0.1$ piglets, respectively, for FSAI vs. LSAI in TNB and NBA, respectively; p<0.01). In conclusion, these results suggest that the reproduction efficiency for FSAI, which is lower than that for LSAI, could be improved by selecting an optimal period of the year for the use of the former.

• Journal of Life Science
• /
• v.27 no.5
• /
• pp.517-523
• /
• 2017

This study was to investigate the role of antioxidants on the characteristics of arsenite-damaged boar semen. Collected sperm was diluted with semen extender, and $100{\mu}M$ arsenite was used for sperm damage. Then melatonin, silymarin, curcumin, and vitamin E were applied for 3, 6, and 9 hr in arsenite-treated boar sperm. Sperm characteristics were then analyzed for motility, plasma membrane integrity, mitochondrial activity, and lipid peroxidation. In the results, sperm motility (control, $77.3{\pm}1.8%$) was decreased by arsenite ($33.3{\pm}1.5%$), while the antioxidant treatment groups (100 nM melatonin, $55.8{\pm}3.4%$; $2{\mu}M$ silymarin, $48.8{pm}3.4%$; $10{\mu}M$ curcumin, $53.9{\pm}2.8%$; and $500{\mu}M$ vitamin E, $54.5{\pm}3.1%$) showed increases compared to the arsenite group (p<0.05). $100{\mu}M$ arsenite decreased the sperm plasma membrane integrity ($24.5{\pm}1.6%$) and mitochondrial activity ($58.2{\pm}2.6%$), and increased lipid peroxidation ($5.3{\pm}0.2%$) at 3 hr (p<0.05). However, arsenite-treated samples with 100 nM melatonin, $2{\mu}M$ silymarin, $10{\mu}M$ curcumin, and $500{\mu}M$ vitamin E increased the plasma membrane integrity and mitochondria activity, and decreased lipid peroxidation compared to the arsenite-treated samples. In summary, arsenite may induce sperm damage and oxidation stress, while antioxidants such as melatonin, silymarin, curcumin, and vitamin E are useful for maintaining sperm characteristics. Therefore, antioxidants can protect sperm against damage by arsenite in fresh boar semen.

• Journal of Animal Science and Technology
• /
• v.48 no.6
• /
• pp.777-784
• /
• 2006

This study was designed to investigate between the semen characteristics and sperm chromatin structure in boar with different farrowing rates and relationship between fertility by AI and results of sperm chromatin structure assay (SCSA). The CASA (computer-aided sperm analysis) and SCSA were performed with liquid semen in boars. The all SCSA parameters based on the farrowing rates by AI were significantly differ (P<0.05). The significant negative correlations (P<0.05) were observed between all SCSA parameters and farrowing rate obtained by AI in the field. In conclusion, these results suggest that the sperm parameters evaluated in these studies may be useful indicators to predict the fertility by AI.

• Asian-Australasian Journal of Animal Sciences
• /
• v.31 no.6
• /
• pp.842-850
• /
• 2018

Objective: Several studies have reported the development of new molecular methods for the prognosis and diagnosis of male fertility based on biomarkers aimed at overcoming the limitations of conventional male fertility analysis tools. However, further studies are needed for the field application of these methods. Therefore, alternative methods based on existing semen analysis methods are required to improve production efficiency in the animal industry. Methods: we examined the possibility of improving litter size in various pig breeds using combined Hoechst 33258/chlortetracycline fluorescence (H33258/CTC) staining. The correlation between field fertility and capacitation status by combined H33258/CTC staining in different ejaculates spermatozoa (n = 3) from an individual boar (20 Landrace, 20 Yorkshire, and 20 Duroc) was evaluated as well as overall accuracy. Results: The acrosome reacted (AR) pattern after capacitation (%) was positively correlated with the litter size of Landrace, Yorkshire, and Duroc pigs and the overall accuracy was 75%, 75%, and 70% in Landrace, Yorkshire, and Duroc pigs, respectively. The difference (${\Delta}$) in AR pattern before and after capacitation was positively correlated with the litter size of Landrace, Yorkshire, and Duroc pigs and the overall accuracy was 80%, 65%, and 55% in Landrace, Yorkshire, and Duroc pigs, respectively. However, the difference (${\Delta}$) in capacitated (B) pattern before and after capacitation was negatively correlated with the litter size of Landrace pigs and the overall accuracy was 75%. Moreover, average litter size was significantly altered according to different combined H33258/CTC staining parameters. Conclusion: These results show that combined H33258/CTC staining may be used to predict male fertility in various breeds. However, the selection of specific efficiency combined H33258/CTC staining parameters requires further consideration. Taken together, these findings suggest that combined H33258/CTC staining may constitute an alternative method for predicting male fertility until such time as fertility-related biomarkers are further validated.

• Journal of Embryo Transfer
• /
• v.14 no.1
• /
• pp.69-77
• /
• 1999

본 연구는 돼지 정액의 동결과정동안 flow cytometric 분석에 의한 정액내 생존정자의 비율을 조사하여 주관적으로 평가되는 활력 및 정상첨체율(normal apical ridge ; NAR)과 비교하여 정자의 손상과 생존성에 대한 적절한 평가법을 찾기 위하여 실시하였다. 동결과정 중 정액채취, 냉각, 예비동결 및 동결융해 후에 flow cytometric 분석에 의한 정자 생존율은 각각 93.0$\pm$3.6, 85.1$\pm$3.9, 28.9$\pm$6.8 및 26.1$\pm$5.9%이었다. 동결처리동안에 생존율은 예비동결 및 동결융해 후 가장 많은 정자사멸로 동결상태 이전의 생존율보다 유의적으로 낮게 나타났다. (p<0.05). 평가기법으로 정액 채취시 활력, NAR율 및 생존율을 조사한 결과 각각 91.0$\pm$4.2, 96.8$\pm$2.5 및 92.2$\pm$3.2%로 NAR율이 생존성 및 활력보다 높게 평가되었으며, 생존율이 활력보다 다소 높게 평가되었다. 그러나 동결융해 후에는 각각 44.0$\pm$8.9, 49.0$\pm$7.9 및 35.6$\pm$9.7%로 활력이 생존율보다 다소 높게 평가되었다. 전체적으로 NAR율은 활력은 생존율보다 높게 평가되었으며, SYBR-14 / PI(propidium iodide) 이중형광염색법에 의한 flow cytometric 평가법으로 생존율은 동결되지 않은 정액에서의 활력 및 NAR 평가보다 다소 민감하게 나타났다. 이러한 결과로 미루어보아 SYBR-14 / PI 형광염색에 의한 flow cytometry의 생존성 평가는 동결되지 않은 정액의 평가방법으로는 적절하지만 동결된 정액의 생존성 평가는 부적절한 것으로 사료되었다.