Flow cytometric evaluation of ram semen freezability treated with α- tocopherol

Year 2020, Vetexpo-2019, KHVD-2019, 40 - 40, 31.03.2020

Ongun Uysal Muhammed Enes İnanç Ayhan Şükrü Güngör Ayhan Ata

Abstract

The aim of this study was evaluation of ram semen freezability treated with different rate of α-tocopherol
in non-breeding season. In this study, ejaculates were collected from four Merino rams (2-3 ages were
used and belonging to the special sheep farm in Bademli, Burdur/Turkey). The ejaculates containing
spermatozoa with >80% motility and concentrations higher than 1.5x109 spermatozoa/ml
(normozoospermic) were mixed and used in the study. The mixed ejaculates were divided into five equal
aliquots and samples were extended with tris base medium (tris, citric acid, glucose) containing 800 μM
group, 400 μM group, 200 μM group, 100 μM group or 0 μM group (control) α-tocopherol and they were
equilibrated to 4°C for 2 hours, frozen in mini straws then stored in liquid nitrogen. Straws were thawed at
37°C for 30s in water bath for plasma membrane acrosome integrity (PMAI %) and high mitochondrial
membrane potential (HMMP %) with flow cytometric analyses. Flow cytometry analyses were performed
with a Cytoflex Flow Cytometer (Beckman Coulter, Fullerton, CA, USA). Assessments of semen samples
were made with a laser beam at 488 nm (50 mW laser output), with 525 ± 40, and 585 ± 42 and 610 ± 20
nm emission filters. Data were collected from 10.000 events. The highest PMAI (24.39±2.11 %) and
HMMP (39.90±3.13 %) were in 100 μM compared to other groups (p<0.05). Also, the lowest PMAI and
HMMP values were obtained in 800 μM and 400 μM groups respectively (p<0.05). In conclusion, 100 μM
α-tocopherol should be supplemented to ram semen freezing extender for membrane & acrosome integrity
and high mitochondrial membrane potential.

Keywords

α-tocopherol, flow cytometry

References

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