Changes in responsivenessto bicarbonate under capacitating conditions in liquid preserved boar spermatozoa in vitro

Liquid-stored boar semen is commonly used for artificial insemination (Al) up to 72 h after dilution. Insemination with semen stored for longer periods generally results in reduced fertility. Standard semen parameters, i.e. motility and membrane integrity, usually give no indication of this reduction. Therefore, more sensitive methods are needed for detection of storage-induced changes in sperm quality. Capacitation has long been known to be an essential step in fertilization. In a number of studies bicarbonate hasbeen shown to be the key capacitating agent in boar sperm in vitro (reviewed in Harrison & Gadella 2005). The ability of sperm to respond to bicarbonate in vitro by undergoing capacitatory changes can be measured as a sperm property crucial to fertilization (Petrunkina et al. 2005a; Silva & Gadella 2006). In this study we used calcium influx as a parameter to investigate the responsiveness of stored semen samples to bicarbonate. This parameter has been shown to be sensitive with respect to evaluating detrimental effect of cooling during liquid storage of boar sperm (Petrunkina et al. 2005b). Three ejaculates from each of 14 boars of proven fertility were diluted in Beltsville Thawing Solution (BTS)extender to a concentration of 20 x 106sperm / ml and stored at 17°C. After 12, 24, 72, 120 and 168 h of storage, motility was assessedin the diluted semen with a CASA-system, and membrane integrity was checked with propidium iodide (PI)and FITC-conjugated peanut agglutinin (FITC-PNA) using a flow cytometer. Samples were then washed through Percoll, loaded with the calcium probe Fluo-3-AM and PI, and incubated at 38°C in parallel in two variants of a Tyrode's medium. Medium A contained 15 mM bicarbonate aswell as 2 mM Ca2+,whereas bicarbonate was omitted from medium B; incubation in medium A was performed under 5°/c.CO2. Changes in Ca2+ influx were assessedon a flow cytometer at 3, 20, 40, 60, 90, 120, 150 and 180 min. The resulting kinetics of cell sub-populations were compared between media and storage time points, based on analyses of the non-agglutinated population. During storage, motility declined only from 89.0 ± 3.2 to 74.4 ± 10.4°/c.(p —0.001) and membrane integrity from 83.1 ± 4.2 to 71.0 ± 20.9°/s (p —0.001). However, bicarbonate induced marked changes in membrane permeability, asmeasured by increases in the population of Ca2+-positiveand Pl-negative (live) cells as well as by increases in the population of PI-positive (dead) cells. After 12 and 24 h of storage, the population of Ca2+-positiveand Pknegative cells reached a maximum within 90 min of incubation in medium A, but as storage was prolonged the increase lessened although it reached its maximum more rapidly (after 40-60 min). A time point of 60 min was chosen for comparisons between storage periods and media. Values for the total % Ca2*-positive / PI-negative cells in medium A declined significantly from 21.6 ± 6.4% at 12 h to 15.7 ± 2.78% at 72 h of storage (p < 0.01) after which they stayedat a constant level. At 3 min of incubation proportions of Pl-positive (dead) cells in medium A varied between 9.8 ± 4.9% at 12 h of storage and 14.7 ± 3.3% at 168 h, whereas after 60 min of incubation their percentage had risen to around 46% regardless of storage period. In contrast, in medium B, 60 min values of both populations (i.e. Ca2+-positive / Pl-negative and Pl-positive cells), though initially much lower than in Medium A, increased significantly through-