한우 정자의 성 분리 시 HEPES를 첨가한 Sheath Fluid가 생존율에 미치는 영향

초록

Spermatozoa sorted by flow cytometry have been successfully used to produce offspring in domestic animals and are commercially available for cattle. Also sheath fluid is the important environment for viability of sex-sorted sperm in flow cytometry. The aim of this study was to investigate whether or not HEPES (N-2-hydroxyethylpiperazine-N’-2- Ethanesulfonic acid) has any effect on the viability in sex-sorted Hanwoo (Korean native cattle) sperm. In this study, the semen was collected from Hanwoo of Hoengseong Livestock Cooperation by artificial vagina method then pooled and subjected to cryopreservation in straws. Sperm were cultured for 0, 30, 60 and 120 min with 0, 2.5, 5, 7.5 and 10 mM of HEPES added to the sheath fluid and incubated at 4, 20 and 38℃, respectively. For the cytometric analysis the frozen-thawed semen was extended with 5 mM HEPES extender to final concentration (2×10^7 spermatozoa) at 4, 20 and 37℃. Sperm viability was assessed with SYBR-14 and propidium iodide (PI) staining. This study shows that the viability of sperm was decreased with prolongation of incubation time in all of test. But the viability of sperm which were treated with 38℃ was gently decreased than that of treated with other temperature. The viability of the control was sharply decreased (p<0.05) than all of the HEPES treatment group at 60 to 120 min in 38℃. X-sexed sperm was more sensitive than Y-sexed sperm to temperature during flow cytometry (p<0.05). In conclusion, the results of this study suggest that the sheath fluid with 5 mM HEPES has effect on maintenance of viability after sperm sexing at 37℃ in Hanwoo.

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