Then, the data were subjected to analysis of variance followed by Tukey’s post hoc test. Each week, a tube was randomly chosen, thawed, and used to evaluate seminal parameters using computerized semen analysis (CASA). The fish semen (n=10) was collected and frozen using an ultra-freezer (−80☌), with a solution containing 10% DMSO diluted in 5% Beltsville thawing solution in the proportion of 1:4 (semen/diluent) in 0.5-mL straws. The aim of this study was to develop an alternative protocol for freezing streaked prochilod Prochilodus lineatus semen using an ultra-freezer. Indeed, ultra-freezers have an increased capacity to store semen, and they can be available in places where there are no other technologies. For this purpose, ultra-freezers could, among other advantages, facilitate the maintenance of semen compared to liquid nitrogen containers. 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.Although sperm cryopreservation is well developed, different methods can be used for freezing fish semen, mainly with the intention of reducing the use of liquid nitrogen. There was a loss of response to bicarbonate in part of the live sperm population but an increasing degree of instability in the rest. However, flow cytometric analysis of sperm responses to capacitating and control media revealed two opposing effects of storage. During storage, standard semen parameters remained on a high level. By this means, it was possible to determine specific effects of bicarbonate and calcium on sperm subpopulations over incubation time. 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. At each time, basic semen quality was characterized by sperm motility and viability. Ejaculates of 14 boars were diluted in Beltsville thawing solution, cooled to 17☌ and stored for 12, 24, 72, 120, and 168 h before investigation. Therefore, we have investigated changes in responsiveness of boar spermatozoa to bicarbonate during storage. Capacitation, the essential prefertilization step for spermatozoa in the female genital tract, is specifically induced in vitro by bicarbonate. Standard semen parameters barely reflect these changes in semen quality, and new approaches for assessment of functional changes in stored spermatozoa are needed. The fertility of liquid‐preserved boar semen declines during storage at 17☌, insemination trials even indicating early losses in fertilizing ability within the first 24–48 h of storage. © 2012 International Society for Advancement of Cytometry 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. Bivalent response to long‐term storage in liquid‐preserved boar semen: A flow cytometric analysis Bivalent response to long‐term storage in liquid‐preserved boar semen: A flow cytometric analysis
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