Choi, Sun-Ho
(National Institute of Animal Science, RDA)
,
Lee, Mi-Jin
(National Institute of Animal Science, RDA)
,
Lee, Kyung-Mi
(National Institute of Animal Science, RDA)
,
Sa, Soo-Jin
(National Institute of Animal Science, RDA)
,
Kim, Hyun-Jong
(National Institute of Animal Science, RDA)
,
Jin, Hyun-Ju
(National Institute of Animal Science, RDA)
,
Song, Yong-Sup
(National Institute of Animal Science, RDA)
,
Park, Jun-Cheol
(National Institute of Animal Science, RDA)
The boar sperm has more lipid droplets and specialty of seminal plasma compared with other species, causing difficulties of freezing sperm and decreases for the utilization of frozen semen into the artificial insemination. However, several studies reported significant results for the recovery of spe...
The boar sperm has more lipid droplets and specialty of seminal plasma compared with other species, causing difficulties of freezing sperm and decreases for the utilization of frozen semen into the artificial insemination. However, several studies reported significant results for the recovery of sperm motility and reproductive by addition of cryoprotectants and seminal plasma after thawing. This study was designed to investigate the effects of supplementation of trehalose or glycerol in the LEY (lactose and egg yolk in BTS) solution for the conventional freezing and vitrification process. Two boars aged 16 months were used to collect semen for 2 times in a week. The samples were allotted to 3 freezing solutions (LEY + glycerol 10.5% + OEP 1.5%, LEY + trehalose 1M + OEP 1.5%, and sucrose 1.5M + trehalose 1 M + OEP 1.5%) after centrifugation at 800 g for 10 minutes. Semen was equilibrated in freezing solutions for 10 minutes and injected into plastic straws with 2~3 air bubbles to minimize freezing damages. Vitrification was performed to locate sperm in 5 cm above $LN_2$ for 5 minutes, and the conventional freezing was conducted with an automatic freezer. Motility and survival rates were measured by CASA (Computer assisted sperm an alyzing system) and FITC (Fluorescein isothiocyanate), respectively after thawing semen at $50^{\circ}C$ for 12 seconds. The results were analyzed by ANOVA with STATVIEW statistical program. The vitrificatioin solution (LEY + 10.5% glycerol + 1.5% OEP) presented higher motility (20.9%) than other solutions while the solution (LEY + 1M trehalose + 1.5% OEP) showed the lowest (motility : 5.2%). However, survival rates of vitrified sperms detected by FITC showed 1~4% live sperms in almost of dead sperms at all vitrification solutions' groups, but survival rate of freezing solution of LEY + 1M trehalose + 1.5% OEP LEY and LEY + 10.5% glycerol + 1.5% OEP were showed 49%, and 79%, respectively. There were differences (P<0.05) survival rate of conventional freezing in LEY + 10.5% glycerol + 1.5% OEP and LEY + 1M trehalose + 1.5% OEP and the remaining showed no differences. The results suggested that vitrified boar semen was not enough to be utilized for the artificial insemination, but it showed possibility to utilize for ICSI and conventional freezing with glycerol would be useful method for artificial insemination in pig while we choose the outstanding semen against tolerance to freezing damages.
The boar sperm has more lipid droplets and specialty of seminal plasma compared with other species, causing difficulties of freezing sperm and decreases for the utilization of frozen semen into the artificial insemination. However, several studies reported significant results for the recovery of sperm motility and reproductive by addition of cryoprotectants and seminal plasma after thawing. This study was designed to investigate the effects of supplementation of trehalose or glycerol in the LEY (lactose and egg yolk in BTS) solution for the conventional freezing and vitrification process. Two boars aged 16 months were used to collect semen for 2 times in a week. The samples were allotted to 3 freezing solutions (LEY + glycerol 10.5% + OEP 1.5%, LEY + trehalose 1M + OEP 1.5%, and sucrose 1.5M + trehalose 1 M + OEP 1.5%) after centrifugation at 800 g for 10 minutes. Semen was equilibrated in freezing solutions for 10 minutes and injected into plastic straws with 2~3 air bubbles to minimize freezing damages. Vitrification was performed to locate sperm in 5 cm above $LN_2$ for 5 minutes, and the conventional freezing was conducted with an automatic freezer. Motility and survival rates were measured by CASA (Computer assisted sperm an alyzing system) and FITC (Fluorescein isothiocyanate), respectively after thawing semen at $50^{\circ}C$ for 12 seconds. The results were analyzed by ANOVA with STATVIEW statistical program. The vitrificatioin solution (LEY + 10.5% glycerol + 1.5% OEP) presented higher motility (20.9%) than other solutions while the solution (LEY + 1M trehalose + 1.5% OEP) showed the lowest (motility : 5.2%). However, survival rates of vitrified sperms detected by FITC showed 1~4% live sperms in almost of dead sperms at all vitrification solutions' groups, but survival rate of freezing solution of LEY + 1M trehalose + 1.5% OEP LEY and LEY + 10.5% glycerol + 1.5% OEP were showed 49%, and 79%, respectively. There were differences (P<0.05) survival rate of conventional freezing in LEY + 10.5% glycerol + 1.5% OEP and LEY + 1M trehalose + 1.5% OEP and the remaining showed no differences. The results suggested that vitrified boar semen was not enough to be utilized for the artificial insemination, but it showed possibility to utilize for ICSI and conventional freezing with glycerol would be useful method for artificial insemination in pig while we choose the outstanding semen against tolerance to freezing damages.
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문제 정의
But vitrification of animal sperm is not established yet to use for commercial fields and has no trial. In this regards, the objective of this study was to investigate the motility and survival rate on cryoprotective effects of trehalose, glycerol and Equex STM paste and adaptation of vitrification in boar spermatozoa.
제안 방법
The samples were diluted with lactose egg-yolk (LEY) solution contained with 20% fresh egg yolk separated from whole egg. Experimental groups were allotted to 3 freezing solutions (LEY + glycerol 10.5% + OEP 1.5%, LEY + trehalose 1 M + OEP 1.5%, and sucrose 1.5 M + trehalose 1 M + OEP 1.5%) after centrifugation at 800 g for 10 minutes. Semen was equilibrated in freezing solutions for 10 minutes and injected into 0.
데이터처리
The results of vitrified sperm were analyzed by ANOVA with STATVIEW statistical program.
이론/모형
Motility and survival rates were measured by CASA (Computer assisted sperm analyzing system) and FITC (Fluorescein isothiocyanate) staining methods, respectively.
성능/효과
However, survival rates of vitrified sperms detected by FITC showed 1∼4% live sperms of almost of vitrified sperms in all vitrification solutions’ groups, but survival rate of freezing solution of LEY + 1M trehalose + 1.5% OEP LEY and LEY + 10.5% glycerol + 1.5% OEP were showed 49%, and 79%, respectively.
Even though AI technique is developed as manipulating injection of sperms, it is essential to prepare live sperms. In conclusion, our results are not enough to use AI for reproducing pigs, but trial of vitrification can be used in assisted reproduction. And numerous reports have been indicated the diversity of cryopreservation on breeds (Nicolas et al.
There were differences (P<0.05) survival rate of conventional freezing in LEY + 10.5% glycerol + 1.5% OEP and LEY + 1M trehalose + 1.5% OEP and the remaining showed no differences.
They indicated the cryopreservation is normally achieved tertiary combination of cells, permeable cryoprotectants and a low temperature en- vironment and cryoprotectants has negative effects on spermatozoa including damaging the cytoplasm, functional destabilization and mutagenesis.
후속연구
In conclusion, our results are not enough to use AI for reproducing pigs, but trial of vitrification can be used in assisted reproduction. And numerous reports have been indicated the diversity of cryopreservation on breeds (Nicolas et al., 2012), further studies are needed to investigate the possible optimal cryopreservation of boar semen.
참고문헌 (28)
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