Bivalent response to long‐term storage in liquid‐preserved boar semen: A flow cytometric analysis

Abstract

The fertility of liquid-preserved boar semen declines during storage at 178C, insemination trials even indicating early losses in fertilizing ability within the first 24-48 h of storage. Standard semen parameters barely reflect these changes in semen quality, and new approaches for assessment of functional changes in stored spermatozoa are needed. Capacitation, the essential prefertilization step for spermatozoa in the female genital tract, is specifically induced in vitro by bicarbonate. Therefore, we have investigated changes in responsiveness of boar spermatozoa to bicarbonate during storage. Ejaculates of 14 boars were diluted in Beltsville thawing solution, cooled to 178C and stored for 12, 24, 72, 120, and 168 h before investigation. At each time, basic semen quality was characterized by sperm motility and viability. Subsequently, washed subsamples were incubated in variants of an in vitro fertilization (IVF) medium and assessed for kinetic changes of viability (plasma membrane integrity) and intracellular calcium concentration using flow cytometry in combination with propidium iodide and Fluo-3. By this means, it was possible to determine specific effects of bicarbonate and calcium on sperm subpopulations over incubation time. During storage, standard semen parameters remained on a high level. However, flow cytometric analysis of sperm responses to capacitating and control media revealed two opposing effects of storage. There was a loss of response to bicarbonate in part of the live sperm population but an increasing degree of instability in the rest. Assessment of response to capacitating media by flow cytometry appears a markedly more sensitive way of monitoring sperm functionality during storage than the standard semen parameters of motility and viability. ' 2012 International Society for Advancement of Cytometry

The Effect of Resveratrol on the Quality of Extended Boar Semen During Storage at 17ºC

“…Moreover, conventional sperm parameters used to test the quality of boar semen doses mostly are too insensitive to assess the fertilization potential of liquid stored semen (Waberski et al, 2011). In recent studies, effort has been focused to test whether spermatozoa membranes retain their ability to respond to oviductal signals, considering dynamic responses in spermatozoa under experimentally mimicked fertilizing conditions (Henning et al, 2012;Petrunkina et al, 2005a). Several studies have indicated that a certain subpopulation of boar spermatozoa may loose their responsiveness to the capacitating stimulus bicarbonate during hypothermic storage (Green & Watson, 2001;Guthrie & Welch, 2005;Harrison et al, 1996;Petrunkina et al, 2005b).…”

“…Several studies have indicated that a certain subpopulation of boar spermatozoa may loose their responsiveness to the capacitating stimulus bicarbonate during hypothermic storage (Green & Watson, 2001;Guthrie & Welch, 2005;Harrison et al, 1996;Petrunkina et al, 2005b). Henning et al (2012) showed that the comparison of calcium-dependent spermatozoa responses between three different media, capacitating Tyrode's medium, (including calcium and bicarbonate), bicarbonate-free Tyrode's medium and calcium-and bicarbonate-free medium, sensitively detected storage-related changes in the spermatozoa population. Our results show that previous incubation in the presence of RSV 33 µM is unable to revert the decline in the response to bicarbonate and therefore the increasing inherent instability brought about by liquid storage time of boar semen.…”

“…Signals for PI distinguished between death cells with defective plasma membranes (PI-positive) and live cells with intact plasma membranes (PI-negative), whereas Fluo-3 signal subdivided the PI-negative spermatozoa population into cells with a low Fluo-3 fluorescence signal (live, low-Ca 2+ sperm cells; Fluo-3-negative) and those with a higher Fluo-3 fluorescence signal (live, high-Ca 2+ sperm cells; Fluo-3-positive). The change in the amount of a spermatozoa subpopulation between 3 and 60 min of incubation in TyrBicCa, TyrCa or TyrControl medium indicates the responsiveness of a sperm sample to capacitating conditions (Henning et al, 2012;Schmid et al, 2013). Responsiveness was calculated as changes in the live, low-Ca 2+ subpopulation (PI-negative/ Fluo-3-negative; ∆ = 60 min-3 min).…”

“…Examinations at the University for Veterinary Medicine in Hannover were carried out with SpermVision ® program as described in Henning et al (2012). A 0.63 camera adapter (U-PMTVC tv-0.63, Olympus, Hamburg, Germany) was used.…”

“…Calcium influx and the specific response to bicarbonate in liquid preserved boar spermatozoa were assessed as described in Henning et al (2012) with minor modifications. Three types of a Tyrode's medium were used for exposing spermatozoa to capacitating or non-capacitating conditions.…”