Premises for fowl sperm preservation based on applied bioenergetics1

Abstract

The primary goal of this work was to test whether the sperm mobility assay could be used to derive mathematical relationships from which predictions could be made about sperm cell function. A precondition was random sampling from a pool of sperm. This precondition was met by centrifuging mobile sperm through 12% (wt/vol) Accudenz containing the Ca(2+) chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and then holding washed sperm at 20°C within buffered potassium chloride. These 2 conditions rendered washed sperm immobile at 20°C. Resumption of sperm mobility was independent of time (P > 0.8558) when sperm were reactivated at body temperature with 2 mM Ca(2+) in isotonic sodium chloride at pH 7.4. Reactivated sperm mobility was 93% of the prewash control. Subsequent experiments served to define a dose response, predict optimal conditions for in vitro sperm mobility, and show how sperm can recover from an imposed non-physiological condition. Thus, functions were derived from which predictions were made. Whereas the utility of BAPTA treatment was confirmed in a new context, such utility did not address the question of whole-cell Ca(2+) flux during sperm cell manipulation. This issue is pivotal for the application of bioenergetics to fowl sperm preservation. Therefore, the secondary goal of this research was to investigate sperm cell Ca(2+) flux using a simulation of conditions encountered by sperm during centrifugation through 12% (wt/vol) Accudenz. These conditions included a temperature of 30°C, a Ca(2+) sink, and no exogenous substrate. Sperm motion was measured with a Hobson SpermTracker. Data points conformed to parabolic functions when motile concentration and velocity were plotted as functions of time. In each case, maximums were observed, e.g., 26 min for motile concentration. The upswing was attributed to a redistribution of intracellular Ca(2+) whereas the downswing was attributed to sperm cell Ca(2+) depletion. A pronounced isothermal increase was observed for each variable when the Ca(2+) sink was overcome with exogenous Ca(2+). Experimental outcomes supported four testable premises applicable to fowl sperm preservation research: 1) the importance of sperm mobility phenotype, 2) the relationship between mitochondrial Ca(2+) cycling and sperm mobility, 3) the utility of the sperm mobility assay for predicting experimental outcomes, and 4) understanding mitochondrial Ca(2+) cycling in terms of whole-cell Ca(2+) flux.

Evaluation of a semen extender from a bioenergetic perspective1

“…Experiment 3 also served to test the premise that the sperm mobility assay can predict the status of sperm cell mitochondria in response to sperm manipulation. In this case, the sperm cell environment affected mitochondrial well-being as evidenced by the inability of sperm to respond to extracellular Ca 2+ under physiological conditions (Froman, 2014). This conclusion is valid because fowl sperm propulsion, as measured by either the sperm mobility assay or computer-assisted sperm motion analysis, is absolutely dependent on mitochondrial Ca 2+ cycling.…”

“…Sperm concentration was measured with a spectrophotometer as outlined by Froman and Rhoads (2012). Sperm mobility was measured as described by Froman (2014) at an osmolality of 290 mmol/kg. Next, sperm were diluted to 2 × 10 9 sperm/mL with TES-buffered saline.…”

“…Most recently, the author has demonstrated how the sperm mobility assay can be used to predict mathematical relationships that characterize fowl sperm function (Froman, 2014). Four premises for sperm preservation were derived from this effort.…”

“…Roosters in a random bred flock of New Hampshire chickens (n = 454) were phenotyped at 27 wk of age with the sperm mobility assay as outlined by Froman (2014). Data were assigned to frequencies in increments of 10 percentage units.…”