Contents Flow cytometry has become an important technique in sperm evaluation and is increasingly used both for routine assessment and for research in veterinary science. We have revised the literature, describing fluorescent probes that have been used for analysing spermatozoa by flow cytometry, regarding: viability, acrosomal status, capacitation, mitochondrial status, apoptotic markers, oxidative stress markers, DNA damage, sperm counting and sperm sizing. Details and problems of some techniques are reviewed, with special attention to the occurrence of non‐sperm particles in the samples (‘debris’). New and promising aspects of flow cytometry, such as sperm sorting using viability markers as selection criteria, are highlighted. The relationship between flow cytometry analyses and fertility and their future improvements are considered.

Background The question of an optimal strategy and outcomes in COVID-19 tracheostomy has not been answered yet. The critical focus in our case study is to evaluate the outcomes of tracheostomy on intubated COVID-19 patients. Methods A multicentric prospective observational study of 1890 COVID-19 patients undergoing tracheostomy across 120 hospitals was conducted over 7 weeks in Spain (March 28 to May 15, 2020). Data were collected with an innovative approach: instant messaging via WhatsApp. Outcome measurements: complications, achieved weaning and decannulation and survival. Results We performed 1,461 surgical (81.3%) and 429 percutaneous tracheostomies. Median timing of tracheostomy was 12 days (4-42 days) since orotracheal intubation. A close follow-up of 1616/1890 (85.5%) patients at the cutoff time of 1-month follow-up showed that in 842 (52.1%) patients, weaning was achieved, while 391 (24.2%) were still under mechanical ventilation and 383 (23.7%) patients had died from COVID-19. Decannulation among those in whom weaning was successful (n = 842) was achieved in 683 (81%) patients. Conclusion To the best of our knowledge, this is the largest cohort of COVID-19 patients undergoing tracheostomy. The critical focus is the unprecedented amount of tracheostomies: 1890 in 7 weeks. Weaning could be achieved in over half of the patients with follow-up. Almost one out of four tracheotomized patients died from COVID-19.

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We have applied a statistical protocol based on principal component analysis, clustering methods, and discriminant analysis for the identification of sperm subpopulations in computer-assisted sperm analysis (CASA) data. Samples were obtained from the cauda epididymis of 11 Iberian red deer and cryopreserved following a standard protocol. Motility by CASA was analyzed just after sperm recovery, just before freezing, and after thawing, and eight motility descriptors for each individual spermatozoon were recorded. Sperm viability and acrosomal status were also assessed. Subpopulation analysis was performed in four sequential steps: principal component analysis using the eight motility descriptors; nonhierarchical clustering analysis (k-means) using the first two principal components; hierarchical clustering analysis (UPGMA); and selection of the final number of clusters. Three clusters were obtained for each motility analysis: slow and nonlinear; rapid and linear; and rapid, high ALH, nonlinear. We detected variations in the clusters between treatments (initial, prefreezing and postthawed). Indeed, motility increased and linearity decreased in the prefreezing analysis. A discriminant analysis isolated three descriptors that were used again in the same statistical analysis, giving four clusters that resembled the pattern found in the first classification. We also performed a clustering analysis of the males according to prefreezing/postthawed variation of total motility, viability, and acrosomal status. The proportion of the linear subpopulations in the prefreezing treatment, in both clustering analyses, correlated positively with postthawed viability recovery. Our results show that clustering analysis of CASA data gives useful and practical information that is not obtained by conventional sperm analysis.

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