Sperm cryopreservation is a fundamental reproductive biotechnology, enabling the long-term storage of genetic material and supporting assisted reproduction programs. Despite its widespread application, the process is associated with significant limitations due to the cryo-induced cellular damage that occurs during freezing and thawing. These injuries primarily affect the plasma membrane, nuclear DNA, and motility, thereby compromising the fertilizing potential of spermatozoa. Furthermore, interspecies variability in terms of cryo-sensitivity poses a major challenge to the development of standardized cryopreservation protocols. Recent advances have focused on mitigating cryodamage through the use of various strategies. The inclusion of antioxidants in cryopreservation media has proven effective in reducing oxidative stress, thereby enhancing cellular protection. Similarly, the addition of lipid-based supplements contributes to membrane stabilization, improving post-thaw sperm viability and functionality. Moreover, the application of omics technologies, such as transcriptomics and proteomics, has facilitated a deeper understanding of molecular damage and protective responses, paving the way for the development of tailored, species-specific protocols. These integrated approaches optimize cryopreservation conditions, maximizing post-thaw survival and the fertilizing capacity of sperm. Enhancing cryopreservation techniques not only improves the outcomes of assisted reproductive technologies, but also plays a crucial role in the conservation of genetically valuable livestock species. In conclusion, the integration of biotechnological and molecular tools holds significant promise for overcoming the current limitations and advancing the efficacy of sperm cryopreservation.

Natural products (NPs) are crucial for the development of novel antibiotics, anticancer agents, and immunosuppressants. To highlight the ability of fungi to produce structurally diverse NPs, this article focuses on the impact of genome mining and CRISPR-Cas9 technology in uncovering and manipulating the biosynthetic gene clusters (BGCs) responsible for NP synthesis. The CRISPR-Cas9 system, originally identified as a bacterial adaptive immune mechanism, has been adapted for precise genome editing in fungi, enabling targeted modifications, such as gene deletions, insertions, and transcription modulation, without altering the genomic sequence. This review elaborates on various CRISPR-Cas9 systems used in fungi, notably the Streptococcus pyogenes type II Cas9 system, and explores advancements in different Cas proteins for fungal genome editing. This review discusses the methodologies employed in CRISPR-Cas9 genome editing of fungi, including guide RNA design, delivery methods, and verification of edited strains. The application of CRISPR-Cas9 has led to enhanced production of secondary metabolites in filamentous fungi, showcasing the potential of this system in biotechnology, medical mycology, and plant pathology. Moreover, this article emphasizes the integration of multi-omics data (genomics, transcriptomics, proteomics, and metabolomics) to validate CRISPR-Cas9 editing effects in fungi. This comprehensive approach aids in understanding molecular changes, identifying off-target effects, and optimizing the editing protocols. Statistical and machine learning techniques are also crucial for analyzing multi-omics data, enabling the development of predictive models and identification of key molecular pathways affected by CRISPR-Cas9 editing. In conclusion, CRISPR-Cas9 technology is a powerful tool for exploring fungal NPs with the potential to accelerate the discovery of novel bioactive compounds. The integration of CRISPR-Cas9 with multi-omics approaches significantly enhances our ability to understand and manipulate fungal genomes for the production of valuable secondary metabolites and for promising new applications in medicine and industry.

The growing threat of antimicrobial resistance has intensified the search for new bioactive compounds, particularly in extreme environments such as Antarctica. Streptomyces fildesensis So13.3, isolated from Antarctic soil, harbours a biosynthetic gene cluster (BGC) associated with actinomycin D production, an antibiotic with biomedical relevance. This study investigates the regulatory role of TetR/AcrR transcription factors encoded within this biosynthetic gene cluster (BGC), focusing on their structural features and expression under different nutritional conditions. Additionally, we propose that repressing an active pathway could lead to the activation of silent biosynthetic routes, and our in-silico analysis provides a foundation for selecting key mutations and experimentally validating this strategy. Expression analysis revealed that TetR-279, in particular, was upregulated in ISP4 and IMA media, suggesting its participation in nutrient-dependent BGC regulation. Structural modelling identified key differences between TetR-206 and TetR-279, with the latter containing a tetracycline-repressor-like domain. Molecular dynamics simulations confirmed TetR-279’s structural stability but showed that the S166P CRISPy-web-guided mutation considerably affected its flexibility, while V167A and V167I had modest effects. These results underscore the importance of integrating omics, structural prediction, and gene editing to evaluate and manipulate transcriptional regulation in non-model bacteria. Targeted disruption of TetR-279 may derepress actinomycin biosynthesis, enabling access to silent or cryptic secondary metabolites with potential pharmaceutical applications.

The growing threat of antimicrobial resistance has driven the search for new bioactive compounds in extreme environments such as Antarctica. Streptomyces fildesensis So13.3, isolated from Antarctic soil, has been shown to contain a biosynthetic gene cluster associated with producing actinomycin D, an antibiotic with therapeutic potential. In this study, we analysed the regulatory role of TetR/AcrR family transcription factors present within this BGC, focusing on their activation under different nutritional conditions and their structural scharacterisation using bioinformatics tools and molecular dynamics simulations. The results showed that TetR/AcrR expression increased significantly in ISP4 and IMA media, suggesting their involvement in nutrient-dependent regulation of the cluster. At the structural level, two TetR proteins (TetR-206 and TetR-279) were modelled, with the latter standing out due to a C-terminal tetracycline repressor-like domain. A 200 ns molecular dynamics simulation was performed in GROMACS to evaluate the stability and flexibility of TetR-279, including analysis of point mutations (S166P, V167A, V167I). The S166P mutation significantly impacted structural flexibility, while V167A and V167I caused only minor alterations. This work demonstrates the value of integrating omics approaches, structural modelling, and gene editing with CRISPR to study and potentially activate silent BGCs in non-model bacteria such as Antarctic Streptomyces. In particular, the targeted inhibition of TetR-279 may trigger metabolic rewiring and facilitate the expression of novel antibiotics encoded in cryptic biosynthetic gene clusters.

The routine prophylactic use of antibiotics in sperm preservation for reproductive biotechnologies poses increasing concerns due to its potential role in promoting antimicrobial resistance. Therefore, developing storage systems that maintain sperm quality without antibiotics is essential. In dogs, chilled sperm is vital in assisted reproduction for genetic improvement and breed conservation. Typically, a 20% Tris–egg yolk extender with broad-spectrum antibiotics supports semen preservation for up to 3 days. This study evaluated a Tris–egg yolk medium without antibiotics, stored at 5 °C, as an alternative for short-term preservation of canine sperm. Eight ejaculates from dogs of different breeds were divided into two aliquots: a control group diluted in Tris–egg yolk with ampicillin, and a treatment group without antibiotics. Samples were stored at 5 °C and analyzed at 0, 24, and 48 h to assess motility, plasma membrane and acrosome integrity, intracellular oxidative stress, and mitochondrial membrane potential. No significant differences were found between treatments in most quality parameters during storage. However, storage time negatively affected membrane and acrosome integrity, as well as mitochondrial function, while slightly increasing oxidative stress. These results indicate that, under controlled short-term refrigeration conditions, canine sperm diluted in a Tris–egg yolk extender without antibiotics maintains functional parameters comparable to antibiotic-supplemented media for up to 48 h, despite time-dependent sub lethal cellular alterations.