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. 2024 Apr 26;14:9651. doi: 10.1038/s41598-024-60118-2

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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(A) Gene expression profile of N-acetylneuraminic acid synthase a (nansa) (means ± sem) in different rainbow trout tissues. Samples were obtained from three different fish. The nansa mRNA abundance was determined by qPCR and normalized to the abundance of 18S. Abundance was set to 1 for the maximal gene expression and data are expressed as a percentage of transcript abundance in the post-ovulatory (Ovary post ov). (B) Ovarian expression profile of N-acetylneuraminic acid synthase a (nansa) gene in the ovary of rainbow trout during the post-ovulatory period (means ± sem). Ovaries were samples at ovulation, then 6 and 20 days after ovulation. The nansa mRNA abundance was determined by qPCR and normalized to the abundance of 18S. Abundance is pressed as percentage of mRNA levels at ovulation. For each ovary stage, ovaries were sampled from 3 separate females. Different letters indicate significant differences (Welch t-test, p < 0.05). (C) Representative cropped western blot analysis of N-acetylneuraminic acid synthase a (Nansa) expression in the rainbow trout ovary. Ovarian samples were analyzed at 0, 6, and 20 days after ovulation. For each ovary stage, ovaries were sampled from 3 separate females. Band intensity was normalized by setting the maximal signal obtained to 100%. Upper panel Graphs represent means ± sd of 3 independent western blots performed on 3 biological replicates (Suppl Fig. S1A). Pre-immune serum (PiP) was used as a negative control (Lower panel). (D) Representative cropped western blot analysis of N-acetylneuraminic acid synthase a (Nansa) expression in the rainbow trout coelomic fluid. CF samples were analyzed at 0, 6, and 20 days after ovulation. CF series were collected from 3 separate females. Band intensity was normalized by setting the maximal signal obtained to 100% (Upper panel). Graphs represent means ± sd of 3 independent western blots performed on 3 biological replicates (Suppl Fig. S1B). Pre-immune serum for antibody sialic acid synthase-like was represented (PiP) (Low panel). (E) Percentage of rainbow trout eggs reaching eyed stage after 3 days of in vitro storage in mineral medium (MM), mineral medium complemented with 2 different concentrations of N-acetylneuraminic acid synthase a (Nansa) (SA1, 4 µg/mL; SA2 40 µg/mL), a CF fraction exhibiting a low egg preservation potential (Fraction), the same fraction complemented with 2 different concentrations of N-acetylneuraminic acid synthase a (Nansa) (SA1, 4 µg/mL; SA2 40 µg/mL), and in CF (positive control). BSA was used as a negative control for mineral medium and fraction complementation at the same concentrations as sialic acid synthase-like (BSA1, 4 µg/mL; BSA2 40 µg/mL). All bars represent the means ± sem of 3 independent experiments conducted on the eggs of 3 different females.