Assessment of chilling injury in hypothermic stored boar spermatozoa by multicolor flow cytometry

Abstract

Hypothermic storage of boar semen may allow antibiotic‐free semen preservation but is limited due to chilling sensitivity of boar spermatozoa. Progress in this area requires sensitive tools to detect chilling injury. Therefore, multiparameter flow cytometry panels were evaluated to ascertain whether they are useful tools for identifying sublethal damage of sperm function at a single cell level, thus considering the high intrinsic sperm heterogeneity in a sample. The first fluorochrome panel consisted of Hoechst 33342 to identify DNA‐containing events, Yo‐Pro 1 to detect viability, merocyanine 540 to describe membrane fluidity, and PNA‐Alexa Fluor™ 647 to identify acrosomic integrity. The second fluorochrome panel consisted of SiR700‐DNA to identify DNA‐containing events, JC‐1 to characterize the mitochondrial transmembrane potential (MMP), and Calbryte 630 to assess the intracellular calcium level. Extended boar semen was stored either at 17°C (control) or 5°C (chilled). It is shown that chilling increased membrane fluidity in the viable (Yo‐Pro 1 negative) sperm population at 24 h (p < 0.05). At 144 h, the viable, acrosomic intact sperm population with low membrane fluidity was similar for both storage temperatures. Moreover, chilling reduced the main sperm population with high MMP, medium fluorescence for JC‐1 monomer and low intracellular calcium level (p < 0.05). However, after in vitro sperm capacitation, this population did not differ between the two storage temperatures. Exemplary computational data visualization in t‐distributed stochastic neighbor embedding (t‐SNE) maps and moving radar plots revealed similar subpopulations as identified by three‐dimensional stacked bar charts. In conclusion, sperm surviving an initial chilling injury withstand long‐term storage and respond in a similar manner to capacitation conditions as sperm stored conventionally at 17°C. Multicolor flow cytometry is a valuable tool for detecting chilling‐induced alterations of cell function in sperm subpopulations.

La citometría de flujo, un universo de posibilidades en el ámbito veterinario. Revisión

“…Los gráficos que se obtienen en ambos casos y su interpretación son similares independientemente del tipo de reactivo que se haya utilizado. La determinación de la viabilidad de los espermatozoides antes y después del proceso de criopreservación es un ejemplo del uso rutinario de esta aplicación de la CF y constituye una evaluación indispensable en inseminación artificial en diferentes especies animales (51,52) .…”

An Exploration of Current and Perspective Semen Analysis and Sperm Selection for Livestock Artificial Insemination

“…One is to make use of hypothermic storage as alluded to in the Horse section. In boar semen storage, hypothermic storage consists of storing extended semen at 4–5 °C [ 23 , 69 ]. This brings about added stress to sperm cells and the inclusion of additional compounds that allow the cells to survive at this temperature.…”

“…Horse semen is routinely stored at regular refrigeration temperature (4 °C) compared with swine semen (17 °C), but must be used within 24 h after which the quality of the spermatozoa is greatly diminished [ 11 ]. The swine industry is starting to adapt to this hypothermic storage application in the face of eliminating antibiotics with long term semen storage metrics remaining acceptable well beyond 24 h [ 23 ].…”

Temperature limits for storage of extended boar semen from the perspective of the sperm's energy status

“…Beyond the increase in antibiotic multi-drug resistance, storage at lower temperature is desired to inhibit bacterial growth. Recently, the suggested lower temperature limits have been successfully shifted toward 5 • C without comprising semen quality or fertility (11)(12)(13)(14). It is not only that chilling may corrupt the cellular integrity but also the resumption of metabolic activity in viable sperm during rewarming to body temperature after insemination.…”