Using skin mucus for the identification of ovulation biomarkers in North American Atlantic salmon (Salmo salar)

Authors: Legacki, Erin; Peterson, Brian; BOGGS, ASHLEY - National Institute Of Standards And Technology

Submitted to: Aquaculture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/30/2023
Publication Date: 5/30/2023
Citation: Legacki, E.L., Peterson, B.C., Boggs, A. 2023. Using skin mucus for the identification of ovulation biomarkers in North American Atlantic salmon (Salmo salar). Aquaculture. https://doi.org/10.1016/j.aquaculture.2023.739717.
DOI: https://doi.org/10.1016/j.aquaculture.2023.739717

Interpretive Summary: Reproductive studies on fish are fundamental for the ever-growing aquaculture industry by improving broodstock programs through increased egg production and enhanced viability of those eggs. In Maine, USA two of the largest facilities for North American Atlantic salmon production have seen a 20% decrease in the viability (eye-up %) of embryos. Several studies have focused on the maternal endocrine environment as being an important factor for viable embryos. However, there are still gaps in the body of knowledge of the maternal endocrine pathways in North American Atlantic salmon which lead to ovulation and production of viable eggs. Combining new matrices (Fish skin mucus) and advanced technologies (liquid chromatography tandem mass spectrometry (LC-MS/MS) will advance the knowledge of the endocrine milieu surrounding oocytes at the time of ovulation in North American Atlantic salmon. The objectives of this study were to 1) identify mucus as a viable matrix to measure steroid hormones, 2) quantify steroid hormone profile during ovulation of North American Atlantic salmon and 3) compare hormone profiles of salmon with high hatch versus low hatch or eye-up rate. Mucus and plasma were collected from female sexually mature North American Atlantic salmon (n= 29) through absorption onto filter paper. Females were sampled before ovulation (n= 9) and after ovulation (n= 20). All samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). For further analysis the fish collected after ovulation were divided into groups based on embryo viability, fish with greater than 70% eye-up rates (n=4) and fish with less than 70% eye up rates (n=14). Of the 23 compounds measured 6 pregnanes, all 5 glucocorticoids and all 6 androgens were detected in salmon mucus. In mucus allopregnanolone, 11-deoxycorticosterone and 11-deoxycortisol were significant higher after ovulation. Comparatively, in plasma allopregnanolone was significantly lower after ovulation and 17a, 20ß-hydroxyprogesterone was significantly elevated after ovulation. Circulating plasma concentrations of pregnenolone were significantly correlated with mucus concentrations of 17a-hydroxyprogesterone and 17a, 20ß-hydroxyprogesterone before ovulation and with mucus concentrations of cortisol and cortisone after ovulation. Fish with an eye-up rate less than 70% (reduced embryo viability) had significantly elevated mucus concentrations of corticosterone. While fish with an eye-up rate greater than 70% had elevated plasma and testosterone concentrations. Circulating androgens (androstenedione and testosterone) were significantly correlated with eye-up rate. The results of this study support the use of mucus as a matrix for the measurement of steroid hormones. Mucosal concentrations of steroid hormone did correlate with circulating concentrations of related steroids, and they reflected changes in reproductive physiology. The significant changes in mucosal and circulating steroid concentrations of the 5a reduced pregnane, allopregnanolone suggests a role of 5a-reduced pregnanes during ovulation in North American Atlantic salmon. To our knowledge this is the first time any 5a-reduced pregnane has been reported in teleosts. Mucosal concentrations of precursor glucocorticoids significantly elevated after ovulation combined with significant correlation with hydroxylated pregnanes suggesting a synergistic mechanism between the two classes of steroid hormones is needed for ovulation. Additionally maternal glucocorticoids and androgens appear to influence the viability of fertilized eggs adding need for further research into the roles glucocorticoids play in both oocyte maturation and embryo viability.

Technical Abstract: In Maine, facilities for North American Atlantic salmon production have seen a 20% decrease in the viability (eye-up %) of embryos. The maternal endocrine environment is suggested to be an important factor for ovulation and viable embryos but needs to be further investigated. Combining new matrices (skin mucus) and advanced technologies (liquid chromatography tandem mass spectrometry (LC-MS/MS)) will advance the knowledge of the endocrine status in reproductively mature North American Atlantic salmon. The objectives of this study were to 1) identify mucus as a viable matrix to measure steroid hormones, 2) quantify steroid hormone profile during ovulation of North American Atlantic salmon and 3) compare hormone profiles of salmon with high and low embryo viability. Mucus and plasma were collected from female sexually mature North American Atlantic salmon (n= 29) through absorption onto filter paper. Females were sampled before ovulation (n= 9) and after ovulation (n= 20) and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Fish collected after ovulation were slotted into groups based on embryo viability, fish with high embryo viability (= 70% eye-up rate, n=4) and fish with low embryo viability (= 70% eye up rate, (n=14). In mucus allopregnanolone, 11-deoxycorticosterone and 11-deoxycortisol were significantly higher after ovulation (p = 0.05). Comparatively, in plasma allopregnanolone was significantly lower after ovulation and 17a, 20ß-hydroxyprogesterone was significantly elevated after ovulation (p = 0.05). Fish with an eye-up rate = 70% had elevated mucus concentrations of corticosterone (p = 0.05). While fish with an eye-up rate = 70% had elevated plasma testosterone concentrations (p = 0.05). Circulating androstenedione (R2= 0.46, p = 0.05) and testosterone (R2= 0.47, p = 0.05) were significantly correlated with eye-up rate. Mucosal concentrations of steroid hormone correlated with circulating concentrations of related steroids, and they reflected changes in reproductive physiology. The significant changes in mucosal and circulating steroid concentrations of the 5a reduced pregnane, allopregnanolone suggests a role of 5a-reduced pregnanes during ovulation in North American Atlantic salmon. To our knowledge this is the first time any 5a-reduced pregnane has been reported in teleosts. Mucosal concentrations of precursor glucocorticoids significantly elevated after ovulation combined with significant correlation with hydroxylated pregnanes suggests a synergistic mechanism between glucocorticoids and pregnanes is needed for ovulation. Additionally, glucocorticoids and androgens appear to influence the viability of fertilized eggs adding need for further research into the synergistic roles hormones play in both oocyte maturation and embryo viability.