Validation of qPCR reference genes in the endangered annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions.

The annual killifish Austrolebias charrua is an endangered species, endemic to the southern region of South America, which inhabits temporary ponds that emerges in the rainy season. The main anthropogenic threat driving the extinction of A. charrua stems from extensive agriculture, primarily due to the widrespread use of glyphosate-based herbicides near their habitats. Annual killifishes have been used as models for ecotoxicological studies but, up to now, there are no studies about reference genes in any Austrolebias species. This represents an obstacle to the use of qPCR-based technologies, the standard method for gene expression quantification. The present study aimed to select and validate potential reference genes for qPCR normalization in the annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions. The candidate reference genes 18 s, actb, gapdh, ef1a, shox, eif3g, and the control gene atp1a1 were evaluated in male and female individuals in three different tissues (brain, liver, and gills) under two experimental conditions (control and acute exposition to Roundup Transorb®). The collected tissues were submitted to RNA extraction, followed by cDNA synthesis, cloning, sequencing, and qPCR. Overall, 18 s was the most stable reference gene, and 18 s and ef1a were the most stable combination. Otherwise, considering all variables, gapdh and shox were the least stable candidate genes. Foremost, suitable reference genes were validated in A. charrua, facilitating accurate mRNA quantification in this species, which might be useful for developing molecular tools of ecotoxicological assessment based on gene expression analysis for environmental monitoring of annual killifish.

Expression of sperm microRNAs related to bull fertility: A systematic review.

In this study we proposed to address the following question: "Are there differentially expressed sperm microRNAs related to fertility in bulls?". A systematic review of scientific literature until November 2022 was performed, in accordance with PRISMA guidelines. The main outcome was differentially expressed sperm microRNA from bulls with low versus high fertility profiles identified by using different methods such as field fertility evaluation and sperm laboratory analysis. Were identified 786 documents, of which 13 were selected for qualitative analysis. A total of 182 unique differentially expressed miRNAs were identified, among these, 49 miRNAs were found in common between at least two studies. It is believed that from these 49 miRNAs, it is possible that miRNAs such as miR-10a, -10b, -103, -15b, -122, -125b, -126-5p, -151-5p, -193a-5p, -196a, -27a-5p and -99b could be potential universal biomarkers to assess the reproductive potential of males.