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Felipe Martínez-Pastor, Elsa Cabrita, Florbela Soares, Luis Anel, and Maria Teresa Dinis

Computer-assisted sperm analysis (CASA) and clustering analysis have enabled to study sperm subpopulations in mammals, but their use in fish sperm has been limited. We have used spermatozoa from Senegalese sole (Solea senegalensis) as a model for subpopulation analysis in teleostei using two different activating solutions. Semen from six males was activated using 1100 mOsm/kg solutions: artificial seawater (ASW) or sucrose solution (SUC). Motility was acquired at 15, 30, 45, and 60 s post-activation. CASA parameters were combined into two principal components, which were used in a non-hierarchical clustering analysis, obtaining four subpopulations (CL): CL1 (slow/non-linear), CL2 (slow/linear), CL3 (fast/non-linear), and CL4 (fast/linear). We detected spermatozoa lysis, especially in ASW. Sperm motility was higher for SUC and decreased with time. The subpopulation proportions varied with time and activating treatment, showing both an increase in CL1 and CL2 and a decrease in CL3 and CL4 with time. Both CL3 and CL4 were higher in samples activated with SUC, at least in early post-activation. Proportions of CL3 and CL4 at 15 s were associated with higher quality at 60 s and with lower lysis. A second clustering analysis was conducted, classifying the males accordingly to their motility subpopulations. This analysis showed a high heterogeneity between samples. Subpopulation analysis of CASA data can be applied to Solea spermatozoa, allowing identification of potentially interesting sperm subpopulations. Future studies might benefit from these techniques to establish the relationship of these subpopulations with fish sperm quality and fertility, helping to characterize males according to their reproductive potential.

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Ana Filipa Ferreira, Maria Soares, Sandra Almeida Reis, João Ramalho-Santos, Ana Paula Sousa, and Teresa Almeida-Santos

Mitochondrial supplementation was proposed as a complementary treatment to assisted reproductive technologies to improve oocyte competence and support post-fertilization development. This strategy is based on the fact that poor-quality/aged oocytes contain lower and dysfunctional mitochondria. However, the efficacy and safety of mitochondrial supplementation are still controversial. Therefore, this review summarizes the clinical/biological outcomes of mitochondrial supplementation, aiming to improve oocyte competence or explore the safety of this technique, and was based on an online search using PubMed and Web of Science, until September 2019. The studies included reported outcomes related to the efficacy and safety of mitochondrial supplementation either in human or animal models (bovine, porcine and mouse). Extracted data were organized according to study objective, the mitochondrial source and the main outcomes: fertilization/pregnancy rates, embryo development and adverse outcomes. Clinical pregnancy was not improved in the only randomized controlled trial published, although an increase was demonstrated in other non-randomized studies. Fertilization rate and embryo development were not different from control groups in the majority of studies, although performed in different contexts and using diverse sources of mitochondria. The safety of mitochondria transfer is still a concern, however, the euploid rate and the absence of reported congenital malformation from the clinical studies are reassuring. In summary, mitochondrial supplementation does not seem to cause harm although the benefit of improving oocyte competence is still unclear due to the diversity of methodological approaches and low-quality of the data available. Analyzed data support the need to investigate further, in both pre-clinical and clinical contexts.