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ARS Home » Research » Publications at this Location » Publication #31847
Title: VIABILITY ASSESSMENT OF TURKEY SPERM USING FLUORESCENT STAINING AND FLOW CYTOMETRY

Author
item Donoghue, Ann - Annie
item GARNER DUANE L - UNIVERSITY OF NEVADA
item DONOGHUE DAN J - FDA
item Johnson, Lawrence

Submitted to: Poultry Science Association
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/28/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: Current procedures for assessing viability of fresh and stored turkey sperm are often time consuming, subjective in nature and evaluate few sperm within a particular sample. In addition, the methods for determining sperm senescence during storage or predicting subsequent fertilization are limited. This study evaluated the efficacy of several fluorescent stains and flow cytometric analysis in determining the functional status of turkey sperm. Dilution curves and several extenders were used to determine optimal stain concentrations. Staining combinations were effective in estimating the viability of turkey sperm and could be useful for monitoring sperm viability before and after storage. This research effort is continuing.

Technical Abstract: Current procedures for assessing viability of fresh and stored turkey sperm are often time consuming, subjective in nature and evaluate few sperm within a particular sample. In addition, the methods for determining sperm senescence during storage or predicting subsequent fertilization are limited. This study evaluated the efficacy of several fluorescent stains and flow cytometric analysis in determining the functional status of turkey sperm. Dilution curves and several extenders were used to determine optimal stain concentrations of CMFDA, CEFDA, CAL, SYBR-14, R-123, PI, EthD-1 and EthD-2 for staining turkey sperm. SYBR-14, which requires membrane potential for fluorescence, or Calcein AM (CAL), which assesses the membrane integrity of cells (both green fluorescence), in combination with propidium iodide (PI) to stain the dead or degenerating cells (red fluorescence) provided optimal results. Semen from 30 individual toms was collected on 2 separate days and examined in replicate using these staining combinations. The proportion of viable sperm, as indicated by uptake of SYBR-14 or CAL stain, ranged from 55.8 to 86.7 and 38.0 to 86.1, respectively. Staining combinations were effective in estimating the viability of turkey sperm and could be useful for monitoring sperm viability before and after storage. This research effort is continuing.