Optimization of a non-activating medium for short-term chilled storage of barramundi (Lates calcarifer) testicular spermatozoa

Abstract

Reliable short-term chilled sperm storage is a critical prerequisite to using advanced reproductive techniques for captive breeding of barramundi (Asian sea bass; Lates calcarifer). Marine Ringer's solution (MRS) is a common non-activating medium (NAM) and has previously been used to store sperm from wild-caught barramundi. However, MRS-stored spermatozoa from captive-bred barramundi were observed to lyse within 30 min incubation. Therefore, this study aimed to optimize the composition of NAM for short-term chilled storage by characterizing and mimicking the biochemical profile of seminal and blood plasma of captive-bred barramundi. To further understand the effect of each component, osmolality was first examined to determine its effect on sperm viability. Thereafter, the effects of NaHCO₃, pH, and Na⁺ and K⁺ concentrations on sperm motility were investigated. Optimization of the NAM formula was achieved through iterative adaptions. The increase in NAM osmolality from 260 to 400 mOsm/kg led to a significant improvement in sperm viability. Moreover, using HEPES instead of NaHCO₃ as buffering agent significantly enhanced sperm motility and velocity. As a result, sperm samples diluted with optimized NAM (185 mM NaCl, 5.1 mM KCl, 1.6 mM CaCl₂·2H₂O, 1.1 mM MgSO₄·7H₂O, 10.0 mM HEPES, 5.6 mM D⁺ glucose, 400 mOsm/kg, pH 7.4) and stored at 4 °C showed no significant loss in total motility for up to 48 h and retained progressive motility for up to 72 h. The optimized NAM developed in this study significantly extended the functional lifespan of spermatozoa during chilled storage, permitting the ongoing development of advanced reproductive technologies for barramundi.

Keywords: Biochemical; Computer-assisted sperm analysis; Extender; Fish; Flow cytometry; Sperm quality.

Fig. 1

Effect of NaHCO₃-buffered non-activating medium (NAM) osmolality on sperm viability. Percentage of live barramundi (Lates calcarifer) spermatozoa was analyzed using Hoechst 33,342/Propidium Iodine (PI) dual nuclear stains and flow cytometry 1 h post-incubation at 4 °C in NaHCO₃-buffered NAM with an osmolality of 260 to 450 mOsm/kg (n = 5). Osmolality of NAM (Marine Ringer's solution consisted of 124.1 mM NaCl, 5.1 mM KCl, 1.6 mM CaCl₂·2H₂O, 1.1 mM MgSO₄·7H₂O, 0.1 mM NaHCO₃, 2.6 mM NaH₂PO₄·2H₂O, and 5.6 mM D⁺ glucose at pH 7.4 and osmolality 260 mOsm/kg) was modified using NaCl. Data are mean ± SEM. Different lowercase letters indicate a significant difference between treatments (P < 0.05)

Fig. 2

Effect of NaHCO₃-buffered non-activating medium (NAM) pH on sperm motility. Total motility of barramundi (Lates calcarifer) spermatozoa was analyzed at 1 and 24 h post-incubation at 4 °C in NaHCO₃-buffered NAM with different pH (n = 10). Undiluted sperm was used as a control. pH of NAM (Marine Ringer's solution consisted of 182.4 mM NaCl, 5.1 mM KCl, 1.6 mM CaCl₂·2H₂O, 1.1 mM MgSO₄·7H₂O, 0.1 mM NaHCO₃, 2.6 mM NaH₂PO₄·2H₂O, and 5.6 mM D⁺ glucose, osmolality 400 mOsm/kg) was modified using NaOH. Data are mean ± SEM. Different lowercase letters indicate a significant difference between pH treatments within an incubation period, and different capital letters indicate a significant difference between incubation periods at the same pH (P < 0.05)

Fig. 3

Effect of HEPES-buffered non-activating medium (NAM) pH on sperm motility. Total motility of barramundi (Lates calcarifer) spermatozoa was analyzed at 1 and 24 h post-incubation at 4 °C in HEPES-buffered NAM with different pH (n = 7). Undiluted sperm was used as a control as well as sperm diluted in the NaHCO₃-buffered NAM pH 6.5 for comparison. pH of NAM (Marine Ringer's solution consisted of 182.4 mM NaCl, 5.1 mM KCl, 1.6 mM CaCl₂·2H₂O, 1.1 mM MgSO₄·7H₂O, 10.0 mM HEPES, and 5.6 mM D⁺ glucose, osmolality 400 mOsm/kg) was modified using NaOH. Data are mean ± SEM. Different lowercase letters indicate a significant difference between pH treatments within an incubation period, and different capital letters indicate a significant difference between incubation periods at the same pH (P < 0.05)

Fig. 4

Effect of HEPES-buffered non-activating medium (NAM) NaCl and KCl concentration on sperm motility. Total motility of barramundi (Lates calcarifer) spermatozoa was analyzed at 1 and 24 h post-incubation at 4 °C in HEPES-buffered NAM with different NaCl and KCl concentrations (n = 6). Undiluted sperm was used as a control. NaCl and KCl concentrations of NAM (Marine Ringer's solution consisted of 0–190 mM NaCl, 0–190 mM KCl, 1.6 mM CaCl₂·2H₂O, 1.1 mM MgSO₄·7H₂O, 10.0 mM HEPES, and 5.6 mM D⁺ glucose at pH 7.4 and osmolality 400 mOsm/kg) were modified accordingly to maintain osmolality of 400 mOsm/kg. Data are mean ± SEM. Different lowercase letters indicate a significant difference between concentrations within an incubation period, and different capital letters indicate a significant difference between incubation periods at the same concentration (P < 0.05)

Fig. 5

Evaluation of the optimized NAM post-short-term chilled storage on sperm motility. (a) Total motility and (b) VCL of barramundi (Lates calcarifer) spermatozoa were analyzed for up to 96 h at 4 °C in an optimized HEPES-buffered non-activating medium (n = 6). Undiluted sperm was used as a control. Optimized NAM consisted of 185 mM NaCl, 5.0 mM KCl, 1.6 mM CaCl₂·2H₂O, 1.1 mM MgSO₄·7H₂O, 10 mM HEPES and 5.6 mM D⁺ glucose at pH 7.4 and osmolality 400 mOsm/kg. Data are mean ± SEM. Different lowercase letters indicate a significant difference between extender treatments within an incubation period, and different capital letters indicate a significant difference between incubation periods for the same medium (P < 0.05)