Evaluating barramundi (Lates calcarifer) sperm quality using high throughput advanced reproductive tools
Marc, A., Guppy, J., Jerry, D., Mulvey, P., Bauer, P., and Paris, D. (2019) Evaluating barramundi (Lates calcarifer) sperm quality using high throughput advanced reproductive tools. In: [Abstracts from Aquaculture Europe]. From: Aquaculture Europe 19, 8-10 October 2019, Berlin, Germany.
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Abstract
Introduction: Barramundi, or Asian sea bass (Lates calcarifer), is an emerging species for aquaculture worldwide (ABARES, 2013). The growing demand for barramundi fillets necessitates intensification of barramundi production. Breeding practices in barramundi farming have not evolved since the 1980s with the introduction of hormonal therapy to trigger group-spawning events. This group mating strategy often leads to highly skewed paternity among offspring, often resulting in a loss of valuable genetic diversity (Frost et al., 2006; Loughnan et al., 2013). With few studies available on male fertility and a lack of technology to quantify sperm quality in barramundi, efforts to develop reproductive tools to obtain such knowledge for this species is warranted. Thus, the aim of this study was to develop high throughput techniques to evaluate barramundi sperm quality in order to provide preliminary information on basic sperm biology that allows future investigation of male fertility.
Materials and methods: Spermatozoa were collected by catheterization on multiple occasions from n=15 mature male broodstock maintained in breeding condition at 30 °C in 28,000 l tanks, with salinity at 30 ppt on a 16 h light: 8 h dark cycle. Samples were diluted in marine Ringer’s solution, adjusted to 400 mOsm to mimic osmolality of barramundi seminal plasma, then held on ice. Sperm morphology was measured by ImageJ on SpermBlue-stained smears, and this data was used to calibrate and validate automated sperm counting and motility assessment by computer-assisted sperm analysis (CASA; Androvision, Minitube). Factors such as optimal sample dilution, the minimum number of fields, and the effect of motility were examined to determine sperm detection accuracy compared to manual haemocytometer methods. A Hoechst/propidium iodide (PI) viability assay was validated using 70 °C heat-treated controls and a 5-point intact:damaged dilution curve of 0:100, 25:75, 50:50, 75:25 and 100:0% spermatozoa assessed by flow cytometry (≥100 000 cells; FACS Canto II, BD Biosciences). Lastly, a FITC/PI TUNEL DNA fragmentation assay (In situ cell death detection kit - fluorescein, Roche) was validated using DNase-treated sperm controls assessed by flow cytometry (≥100 000 cells). Once validated, the optimised assays were used to characterise baseline barramundi sperm quality.
Results: Barramundi spermatozoon has a primitive structure, consisting of an ovoid head (length 2.44 ± 0.03 µm; width 2.23 ± 0.03 µm; ratio 1.11 ± 0.01), and a single flagellum (length 30.99 ± 0.49 µm) connected by a short midpiece. Based on these measurements, a detection profile was created on the Androvision CASA system to identify barramundi spermatozoa. Accuracy of the automated sperm count was highly correlated with the manual counting method (r = 0.99, P < 0.001). Sample dilution at 1:1000 (across 5 dilutions ranging from 1:250, 1:500; 1:1000, 1:2500 and 1:5000) gave the most accurate automated sperm concentration when compared to manual haemocytometer (r = 0.87, P = 0.001, ICC = 0.99). Automated sperm concentration determined using three or more CASA fields of view have similar or improved precision (coefficient of variation ≤ 8.7%) when compared to manual haemocytometer (coefficient of variation = 8.0%). Moreover, detection accuracy was not affected by sperm motility since automated sperm concentration was highly correlated when using motile or immotile cells (r= 0.99, P < 0.001). Approximately 99.9 ± 0.07% of heat-treated spermatozoa were detected as dead Hoechst+/PI+ cells by flow cytometry. Moreover, sperm viability detected by Hoechst/PI assay was highly correlated with predicted viability using the 5-point dilution curve of intact:damaged spermatozoa (r= 0.98, P < 0.001). Lastly, 71.9 ± 4.4% of DNase-treated spermatozoa were detected as DNA damaged FITC+/PI+ cells by flow cytometry. Based on the validated assays and optimized conditions above, male barramundi broodstock exhibited baseline sperm quality of 15.1 ± 3.6 x 109 sperm/ml concentration, 52.8 ± 9.6% total and 13.1 ± 4.2% progressive motility, 64.2 ± 3.5% live, and 43.5 ± 6.0% DNA damaged spermatozoa.
Discussion and conclusion: Through several technical trials, we have validated the use of CASA, Hoechst/PI staining, TUNEL and flow cytometry for reliably assessing the concentration, motility, viability and DNA integrity of barramundi spermatozoa. Importantly, these assays permitted a rapid and accurate assessment of up to 100 000 sperm per fish; providing a comprehensive assessment of barramundi semen characteristics. Preliminary sperm quality data obtained in this study exhibited levels of cellular damage similar to that of frozen-thawed spermatozoa of other species (Zilli et al., 2003; Pérez-Cerezales et al., 2010). Further research is necessary to identify the cause of such damage to determine whether it was artificially induced by sperm handling procedures (e.g. sub-optimal extender, prolonged storage), or was the result of poor quality spermatozoa in some individuals that could potentially explain the underlying mechanism for highly skewed paternity previously observed in offspring from captive bred barramundi.
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