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Title: FLOW CYTOMETRIC ASSESSMENT OF THE MEMBRANE INTEGRITY OF FRESH AND STORED TURKEY SPERM USING A COMBINATION OF HYPO-OSMOTIC STRESS AND FLUORESCENT STAINING

Authors
item Donoghue, Ann
item Garner, D - UNIVERSITY OF NEVADA-RENO
item Donoghue, D - FDA
item Johnson, Lawrence

Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 6, 1995
Publication Date: N/A

Interpretive Summary: Almost all commercial turkeys are produced using artificial insemination. When semen is inseminated fresh or within six hours of collection, fertility of eggs is high. However, when sperm is stored at refrigeration temperatures for 24 hours or longer, a progressive decline in fertility over the course of egg production is observed. This limits the efficiency of artificial insemination programs. One of the problems in attempts to improve fetility after 24 hr storage of semen is that most semen evaluation procedures show no difference in the quality of fresh or stored semen. The objective of this study was to develop an assay which would stress the sperm (by placing them in water) and then evaluating their viability using fluorescent stains. Using this method we were able to determine that after 24 h storage the percentage of viable sperm was lower than fresh sperm. This procedure provides a precise way of analysing sperm individually and will benefit scientists studying ways to improve semen preservation procedures to increase the efficiency of artificial insemination in turkeys.

Technical Abstract: A study was conducted to evaluate the integrity of turkey sperm membranes subjected to various hypo-osmotic conditions, and to develop a test to determine the % viable sperm capable of withstanding hypo-osmotic stress after in vitro storage. For Trial I, sperm were subjected to varying osmotic solutions by suspension in PBS, 297-19 mosm/kg H20 and stained to assess membrane integrity with Calcein AM and propidium iodide. Viable sperm, as determined by flow cytometric analysis, was not different from 100% PBS, to 20% PBS. Fewer viable sperm, however, were detected in 0% PBS (P<0.05). Swollen tails observed for viable sperm were 0, 4.5, 6.5, 24.3, 50.5 and 100% for 100, 80, 60, 40, 20 and 0% PBS, respectively. Semen was also evaluated fresh or after 24 h in vitro storage at 5 degrees C in PBS or H2O (Trial II). The % viable sperm was not different for fresh or in vitro stored sperm in PBS. For sperm stored 24 h in vitro, viable sperm was lower in H20 than in PBS (P<0.05). Subjecting in vitro stored sperm t hypo-osmotic stress before fluorescent staining resulted in detection of labile sperm not accounted for by staining alone indicating that the turkey sperm membrane is more susceptible to damage after cold storage.

   
 
 
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