Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
REVIEW

The future of equine semen analysis

Fernando J. Peña https://orcid.org/0000-0002-1311-2947 A * , Francisco Eduardo Martín-Cano A , Laura Becerro-Rey A , Cristina Ortega-Ferrusola A , Gemma Gaitskell-Phillips A , Eva da Silva-Álvarez A and María Cruz Gil A
+ Author Affiliations
- Author Affiliations

A Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.

* Correspondence to: fjuanpvega@unex.es

Handling Editor: Marc Yeste

Reproduction, Fertility and Development 36, RD23212 https://doi.org/10.1071/RD23212
Submitted: 9 December 2023  Accepted: 15 February 2024  Published online: 12 March 2024

© 2024 The Author(s) (or their employer(s)). Published by CSIRO Publishing

Abstract

We are currently experiencing a period of rapid advancement in various areas of science and technology. The integration of high throughput ‘omics’ techniques with advanced biostatistics, and the help of artificial intelligence, is significantly impacting our understanding of sperm biology. These advances will have an appreciable impact on the practice of reproductive medicine in horses. This article provides a brief overview of recent advances in the field of spermatology and how they are changing assessment of sperm quality. This article is written from the authors’ perspective, using the stallion as a model. We aim to portray a brief overview of the changes occurring in the assessment of sperm motility and kinematics, advances in flow cytometry, implementation of ‘omics’ technologies, and the use of artificial intelligence/self-learning in data analysis. We also briefly discuss how some of the advances can be readily available to the practitioner, through the implementation of ‘on-farm’ devices and telemedicine.

Keywords: artificial intelligence, CASA, computer-assisted sperm analysis, flow cytometry, metabolomics, proteomics, self-learning, spermatozoa, stallion.

References

Aitken RJ, Lambourne S, Medica AJ (2023) Predicting the outcome of thoroughbred stallion matings on the basis of dismount semen sample analyses. Reproduction 165(3), 281-288.
| Crossref | Google Scholar | PubMed |

Argelaguet R, Clark SJ, Mohammed H, Stapel LC, Krueger C, Kapourani C-A, Imaz-Rosshandler I, Lohoff T, Xiang Y, Hanna CW, Smallwood S, Ibarra-Soria X, Buettner F, Sanguinetti G, Xie W, Krueger F, Gottgens B, Rugg-Gunn PJ, Kelsey G, Dean W, Nichols J, Stegle O, Marioni JC, Reik W (2019) Multi-omics profiling of mouse gastrulation at single-cell resolution. Nature 576(7787), 487-491.
| Crossref | Google Scholar | PubMed |

Arutyunyan A, Roberts K, Troule K, Wong FCK, Sheridan MA, Kats I, Garcia-Alonso L, Velten B, Hoo R, Ruiz-Morales ER, Sancho-Serra C, Shilts J, Handfield L-F, Marconato L, Tuck E, Gardner L, Mazzeo CI, Li Q, Kelava I, Wright GJ, Prigmore E, Teichmann SA, Bayraktar OA, Moffett A, Stegle O, Turco MY, Vento-Tormo R (2023) Spatial multiomics map of trophoblast development in early pregnancy. Nature 616(7955), 143-151.
| Crossref | Google Scholar | PubMed |

Aurich C, Ortega Ferrusola C, Pena Vega FJ, Schrammel N, Morcuende D, Aurich J (2018) Seasonal changes in the sperm fatty acid composition of Shetland pony stallions. Theriogenology 107, 149-153.
| Crossref | Google Scholar | PubMed |

Barratt CLR, Wang C, Baldi E, Toskin I, Kiarie J, Lamb DJ, other Editorial Board Members of the WHO Laboratory Manual for the Examination and Processing of Human Semen (2022) What advances may the future bring to the diagnosis, treatment, and care of male sexual and reproductive health? Fertility and Sterility 117(2), 258-267.
| Crossref | Google Scholar | PubMed |

Becht E, McInnes L, Healy J, Dutertre CA, Kwok IWH, Ng LG, Ginhoux F, Newell EW (2018) Dimensionality reduction for visualizing single-cell data using UMAP. Nature Biotechnology 37, 38-44.
| Crossref | Google Scholar |

Bompart D, Garcia-Molina A, Valverde A, Caldeira C, Yaniz J, Nunez de Murga M, Soler C (2018) CASA-Mot technology: how results are affected by the frame rate and counting chamber. Reproduction, Fertility and Development 30(6), 810-819.
| Crossref | Google Scholar | PubMed |

Bray M-A, Singh S, Han H, Davis CT, Borgeson B, Hartland C, Kost-Alimova M, Gustafsdottir SM, Gibson CC, Carpenter AE (2016) Cell painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes. Nature Protocols 11(9), 1757-1774.
| Crossref | Google Scholar | PubMed |

Buss T, Aurich J, Aurich C (2019) Evaluation of a portable device for assessment of motility in stallion semen. Reproduction in Domestic Animals 54(3), 514-519.
| Crossref | Google Scholar | PubMed |

Cannarella R, Condorelli RA, Calogero AE, La Vignera S (2020) Novel insights on the role of the human sperm proteome. Protein & Peptide Letters 27(12), 1181-1185.
| Crossref | Google Scholar | PubMed |

Carracedo S, Briand-Amirat L, Dordas-Perpinya M, Ramos Escuredo Y, Delcombel R, Sergeant N, Delehedde M (2022) ProAKAP4 protein marker: towards a functional approach to male fertility. Animal Reproduction Science 247, 107074.
| Crossref | Google Scholar | PubMed |

Castaneda C, Juras R, Kjollerstrom J, Hernandez Aviles C, Teague SR, Love CC, Cothran EG, Varner DD, Raudsepp T (2021) Thoroughbred stallion fertility is significantly associated with FKBP6 genotype but not with inbreeding or the contribution of a leading sire. Animal Genetics 52(6), 813-823.
| Crossref | Google Scholar | PubMed |

Castelvecchi D (2019) Black hole pictured for first time – in spectacular detail. Nature 568(7752), 284-285.
| Crossref | Google Scholar | PubMed |

Castillo J, Jodar M, Oliva R (2018) The contribution of human sperm proteins to the development and epigenome of the preimplantation embryo. Human Reproduction Update 24(5), 535-555.
| Crossref | Google Scholar | PubMed |

Castillo J, de la Iglesia A, Leiva M, Jodar M, Oliva R (2023) Proteomics of human spermatozoa. Human Reproduction 38, 2312-2320.
| Crossref | Google Scholar |

Chu DS, Liu H, Nix P, Wu TF, Ralston EJ, Yates JR, III, Meyer BJ (2006) Sperm chromatin proteomics identifies evolutionarily conserved fertility factors. Nature 443(7107), 101-105.
| Crossref | Google Scholar | PubMed |

Davila MP, Munoz PM, Bolanos JMG, Stout TAE, Gadella BM, Tapia JA, da Silva CB, Ferrusola CO, Pena FJ (2016) Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation. Reproduction 152(6), 683-694.
| Crossref | Google Scholar | PubMed |

Dini P, Troch L, Lemahieu I, Deblende P, Daels P (2019) Validation of a portable device (iSperm®) for the assessment of stallion sperm motility and concentration. Reproduction in Domestic Animals 54(8), 1113-1120.
| Crossref | Google Scholar | PubMed |

Duetz C, Bachas C, Westers TM, van de Loosdrecht AA (2020) Computational analysis of flow cytometry data in hematological malignancies: future clinical practice? Current Opinion in Oncology 32(2), 162-169.
| Crossref | Google Scholar | PubMed |

Duetz C, Van Gassen S, Westers TM, van Spronsen MF, Bachas C, Saeys Y, van de Loosdrecht AA (2021) Computational flow cytometry as a diagnostic tool in suspected-myelodysplastic syndromes. Cytometry Part A 99(8), 814-824.
| Crossref | Google Scholar | PubMed |

Escada-Rebelo S, Mora FG, Sousa AP, Almeida-Santos T, Paiva A, Ramalho-Santos J (2020) Fluorescent probes for the detection of reactive oxygen species in human spermatozoa. Asian Journal of Andrology 22(5), 465-471.
| Crossref | Google Scholar | PubMed |

Escoffier J, Krapf D, Navarrete F, Darszon A, Visconti PE (2012) Flow cytometry analysis reveals a decrease in intracellular sodium during sperm capacitation. Journal of Cell Science 125(2), 473-485.
| Crossref | Google Scholar |

Escoffier J, Navarrete F, Haddad D, Santi CM, Darszon A, Visconti PE (2015) Flow cytometry analysis reveals that only a subpopulation of mouse sperm undergoes hyperpolarization during capacitation. Biology of Reproduction 92(5), 121.
| Crossref | Google Scholar | PubMed |

ESHRE Andrology Special Interest Group (1998) Guidelines on the application of CASA technology in the analysis of spermatozoa. European Society for Human Reproduction and Embryology. Human Reproduction 13(1), 142-145.
| Crossref | Google Scholar |

Evenson DP (2013) Sperm chromatin structure assay (SCSA®). Methods in Molecular Biology 927, 147-164.
| Crossref | Google Scholar | PubMed |

Evenson DP (2016) The sperm chromatin structure assay (SCSA®) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility. Animal Reproduction Science 169, 56-75.
| Crossref | Google Scholar | PubMed |

Evenson DP (2022) Sperm chromatin structure assay (SCSA®) for fertility assessment. Current Protocols 2(8), e508.
| Crossref | Google Scholar | PubMed |

Evenson D, Jost L (2001) Sperm chromatin structure assay for fertility assessment. Current Protocols in Cytometry 13, 7-13.
| Google Scholar |

Evenson D, Wixon R (2006) Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay. Reproductive BioMedicine Online 12(4), 466-472.
| Crossref | Google Scholar | PubMed |

Evenson DP, Wixon R (2008) Data analysis of two in vivo fertility studies using sperm chromatin structure assay–derived DNA fragmentation index vs. pregnancy outcome. Fertility and Sterility 90(4), 1229-1231.
| Crossref | Google Scholar | PubMed |

Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, de Angelis P, Claussen OP (1999) Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Human Reproduction 14(4), 1039-1049.
| Crossref | Google Scholar | PubMed |

Evenson DP, Larson KL, Jost LK (2002) Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. Journal of Andrology 23(1), 25-43.
| Crossref | Google Scholar | PubMed |

Felix MR, Turner RM, Dobbie T, Hinrichs K (2022) Successful in vitro fertilization in the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitation. Biology of Reproduction 107(6), 1551-1564.
| Crossref | Google Scholar | PubMed |

Fernandez-Lopez P, Garriga J, Casas I, Yeste M, Bartumeus F (2022) Predicting fertility from sperm motility landscapes. Communications Biology 5(1), 1027.
| Crossref | Google Scholar | PubMed |

Gacem S, Bompart D, Valverde A, Catalan J, Miro J, Soler C (2020) Optimal frame rate when there were stallion sperm motility evaluations and determinations for kinematic variables using CASA-Mot analysis in different counting chambers. Animal Reproduction Science 223, 106643.
| Crossref | Google Scholar | PubMed |

Gacem S, Castello-Ruiz M, Hidalgo CO, Tamargo C, Santolaria P, Soler C, Yaniz JL, Silvestre MA (2023) Bull sperm SWATH-MS-based proteomics reveals link between high fertility and energy production, motility structures, and sperm–oocyte interaction. Journal of Proteome Research 22, 3607-3624.
| Crossref | Google Scholar |

Gaitskell-Phillips G, Martin-Cano FE, Ortiz-Rodriguez JM, Silva-Rodriguez A, Rodriguez-Martinez H, Gil MC, Ortega-Ferrusola C, Pena FJ (2020) Seminal plasma AnnexinA2 protein is a relevant biomarker for stallions which require removal of seminal plasma for sperm survival upon refrigeration. Biology of Reproduction 103(6), 1275-1288.
| Crossref | Google Scholar | PubMed |

Gaitskell-Phillips G, Martin-Cano FE, Ortiz-Rodriguez JM, Silva-Rodriguez A, da Silva-Alvarez E, Rojo-Dominguez P, Tapia JA, Gil MC, Ortega-Ferrusola C, Pena FJ (2021a) Proteins involved in mitochondrial metabolic functions and fertilization predominate in stallions with better motility. Journal of Proteomics 247, 104335.
| Crossref | Google Scholar | PubMed |

Gaitskell-Phillips G, Martin-Cano FE, Ortiz-Rodriguez JM, Silva-Rodriguez A, Gil MC, Ortega-Ferrusola C, Pena FJ (2021b) In stallion spermatozoa, superoxide dismutase (Cu-Zn) (SOD1) and the aldo-keto-reductase family 1 member b (AKR1B1) are the proteins most significantly reduced by cryopreservation. Journal of Proteome Research 20(5), 2435-2446.
| Crossref | Google Scholar | PubMed |

Gaitskell-Phillips G, Martin-Cano FE, Ortiz-Rodriguez JM, Silva-Rodriguez A, da Silva-Alvarez E, Gil MC, Ortega-Ferrusola C, Pena FJ (2022) The seminal plasma proteins Peptidyl arginine deaminase 2, rRNA adenine N (6)-methyltransferase and KIAA0825 are linked to better motility post thaw in stallions. Theriogenology 177, 94-102.
| Crossref | Google Scholar | PubMed |

Gaitskell-Phillips G, Martin-Cano FE, da Silva-Alvarez E, Tapia JA, Silva A, Gil MC, Ortega-Ferrusola C, Pena FJ (2023) Phosphoproteomics for the identification of new mechanisms of cryodamage: the role of SPATA18 in the control of stallion sperm function. Biology of Reproduction 108(2), 324-337.
| Crossref | Google Scholar | PubMed |

Gallardo Bolanos JM, Balao da Silva C, Martin Munoz P, Plaza Davila M, Ezquerra J, Aparicio IM, Tapia JA, Ortega Ferrusola C, Pena FJ (2014) Caspase activation, hydrogen peroxide production and akt dephosphorylation occur during stallion sperm senescence. Reproduction in Domestic Animals 49(4), 657-664.
| Google Scholar | PubMed |

Garner DL (2001) Sex-sorting mammalian sperm: concept to application in animals. Journal of Andrology 22(4), 519-526.
| Crossref | Google Scholar | PubMed |

Garner DL, Johnson LA (1995) Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biology of Reproduction 53(2), 276-284.
| Crossref | Google Scholar | PubMed |

Garner DL, Thomas CA (1999) Organelle-specific probe JC-1 identifies membrane potential differences in the mitochondrial function of bovine sperm. Molecular Reproduction and Development 53(2), 222-229.
| Crossref | Google Scholar | PubMed |

Garner DL, Johnson LA, Yue ST, Roth BL, Haugland RP (1994) Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide. Journal of Andrology 15(6), 620-629.
| Crossref | Google Scholar | PubMed |

Garner DL, Dobrinsky JR, Welch GR, Johnson LA (1996) Porcine sperm viability, oocyte fertilization and embryo development after staining spermatozoa with SYBR-14. Theriogenology 45(6), 1103-1113.
| Crossref | Google Scholar | PubMed |

Garner DL, Evans KM, Seidel GE (2013) Sex-sorting sperm using flow cytometry/cell sorting. Methods in Molecular Biology 927, 279-295.
| Crossref | Google Scholar | PubMed |

Ghosh S, Carden CF, Juras R, Mendoza MN, Jevit MJ, Castaneda C, Phelps O, Dube J, Kelley DE, Varner DD, Love CC, Raudsepp T (2020) Two novel cases of autosomal translocations in the horse: warmblood family segregating t(4;30) and a cloned Arabian with a de novo t(12;25). Cytogenetic and Genome Research 160(11–12), 688-697.
| Crossref | Google Scholar | PubMed |

Gibb Z, Lambourne SR, Aitken RJ (2014) The paradoxical relationship between stallion fertility and oxidative stress. Biology of Reproduction 91(3), 77.
| Crossref | Google Scholar | PubMed |

Gibb Z, Lambourne SR, Quadrelli J, Smith ND, Aitken RJ (2015) L-carnitine and pyruvate are prosurvival factors during the storage of stallion spermatozoa at room temperature. Biology of Reproduction 93(4), 104.
| Crossref | Google Scholar | PubMed |

Giudice V, Fonseca V, Selleri C, Gadina M (2023) Cell viability multiplexing: quantification of cellular viability by barcode flow cytometry and computational analysis. Methods in Molecular Biology 2644, 99-121.
| Crossref | Google Scholar | PubMed |

Gonzalez-Castro RA, Pena FJ, Herickhoff LA (2022) Validation of a new multiparametric protocol to assess viability, acrosome integrity and mitochondrial activity in cooled and frozen thawed boar spermatozoa. Cytometry Part B: Clinical Cytometry 102(5), 400-408.
| Google Scholar | PubMed |

Gonzalez-Castro RA, Pena FJ, Herickhoff LA (2023) Spermatozoa cooled to 5°C one day after collection from porcine commercial semen doses retain sperm functionality with reduced bacterial load. Andrology 12, 186-197.
| Crossref | Google Scholar |

Goodson SG, Zhang Z, Tsuruta JK, Wang W, O’Brien DA (2011) Classification of mouse sperm motility patterns using an automated multiclass support vector machines model. Biology of Reproduction 84(6), 1207-1215.
| Crossref | Google Scholar | PubMed |

Gravance CG, Garner DL, Baumber J, Ball BA (2000) Assessment of equine sperm mitochondrial function using JC-1. Theriogenology 53(9), 1691-1703.
| Crossref | Google Scholar | PubMed |

Greenbaum S, Averbukh I, Soon E, Rizzuto G, Baranski A, Greenwald NF, Kagel A, Bosse M, Jaswa EG, Khair Z, Kwok S, Warshawsky S, Piyadasa H, Goldston M, Spence A, Miller G, Schwartz M, Graf W, Van Valen D, Winn VD, Hollmann T, Keren L, van de Rijn M, Angelo M (2023) A spatially resolved timeline of the human maternal–fetal interface. Nature 619(7970), 595-605.
| Crossref | Google Scholar | PubMed |

Griffin RA, Baker M, Aitken RJ, Swegen A, Gibb Z (2019) What makes a fertile sperm?. Unique molecular attributes of stallion fertility. Reproduction 158(4), R125-R137.
| Crossref | Google Scholar | PubMed |

Griffin RA, Swegen A, Baker M, Aitken RJ, Skerrett-Byrne DA, Silva Rodriguez A, Martin-Cano FE, Nixon B, Pena FJ, Delehedde M, Sergeant N, Gibb Z (2020) Mass spectrometry reveals distinct proteomic profiles in high- and low-quality stallion spermatozoa. Reproduction 160(5), 695-707.
| Crossref | Google Scholar | PubMed |

Grundler W, Dirscherl P, Beisker W, Weber F, Stolla R, Bollwein H (2004) Quantification of temporary and permanent subpopulations of bull sperm by an optimized SYBR-14/propidium iodide assay. Cytometry Part A 60A(1), 63-72.
| Crossref | Google Scholar | PubMed |

Hicks SA, Andersen JM, Witczak O, Thambawita V, Halvorsen P, Hammer HL, Haugen TB, Riegler MA (2019) Machine learning-based analysis of sperm videos and participant data for male fertility prediction. Scientific Reports 9(1), 16770.
| Crossref | Google Scholar | PubMed |

Hughes JR, McMorrow KJ, Bovin N, Miller DJ (2023) An oviduct glycan increases sperm lifespan by diminishing the production of ubiquinone and reactive oxygen species. Biology of Reproduction 109(3), 356-366.
| Crossref | Google Scholar | PubMed |

Ibanescu I, Siuda M, Bollwein H (2020) Motile sperm subpopulations in bull semen using different clustering approaches – associations with flow cytometric sperm characteristics and fertility. Animal Reproduction Science 215, 106329.
| Crossref | Google Scholar | PubMed |

Janecka JE, Davis BW, Ghosh S, Paria N, Das PJ, Orlando L, Schubert M, Nielsen MK, Stout TAE, Brashear W, Li G, Johnson CD, Metz RP, Zadjali AMA, Love CC, Varner DD, Bellott DW, Murphy WJ, Chowdhary BP, Raudsepp T (2018) Horse Y chromosome assembly displays unique evolutionary features and putative stallion fertility genes. Nature Communications 9(1), 2945.
| Crossref | Google Scholar | PubMed |

Jena SR, Nayak J, Kumar S, Kar S, Dixit A, Samanta L (2021) Paternal contributors in recurrent pregnancy loss: cues from comparative proteome profiling of seminal extracellular vesicles. Molecular Reproduction and Development 88(1), 96-112.
| Crossref | Google Scholar | PubMed |

Ledford H, Callaway E (2020) Pioneers of revolutionary CRISPR gene editing win chemistry Nobel. Nature 586(7829), 346-347.
| Crossref | Google Scholar | PubMed |

Leemans B, Stout TAE, De Schauwer C, Heras S, Nelis H, Hoogewijs M, Van Soom A, Gadella BM (2019) Update on mammalian sperm capacitation: how much does the horse differ from other species? Reproduction 157(5), R181-R197.
| Crossref | Google Scholar | PubMed |

Llavanera M, Delgado-Bermudez A, Ribas-Maynou J, Salas-Huetos A, Yeste M (2022) A systematic review identifying fertility biomarkers in semen: a clinical approach through Omics to diagnose male infertility. Fertility and Sterility 118(2), 291-313.
| Crossref | Google Scholar | PubMed |

Lomakin A, Svedlund J, Strell C, Gataric M, Shmatko A, Rukhovich G, Park JS, Ju YS, Dentro S, Kleshchevnikov V, Vaskivskyi V, Li T, Bayraktar OA, Pinder S, Richardson AL, Santagata S, Campbell PJ, Russnes H, Gerstung M, Nilsson M, Yates LR (2022) Spatial genomics maps the structure, nature and evolution of cancer clones. Nature 611(7936), 594-602.
| Crossref | Google Scholar | PubMed |

Love CC (2005) The sperm chromatin structure assay: a review of clinical applications. Animal Reproduction Science 89(1–4), 39-45.
| Crossref | Google Scholar | PubMed |

Macías Garcia B, Fernandez LG, Ferrusola CO, Salazar-Sandoval C, Rodriguez AM, Martinez HR, Tapia JA, Morcuende D, Pena FJ (2011a) Membrane lipids of the stallion spermatozoon in relation to sperm quality and susceptibility to lipid peroxidation. Reproduction in Domestic Animals 46(1), 141-148.
| Crossref | Google Scholar | PubMed |

Macias Garcia B, Gonzalez Fernandez L, Ortega Ferrusola C, Morillo Rodriguez A, Gallardo Bolanos JM, Rodriguez Martinez H, Tapia JA, Morcuende D, Pena FJ (2011b) Fatty acids and plasmalogens of the phospholipids of the sperm membranes and their relation with the post-thaw quality of stallion spermatozoa. Theriogenology 75(5), 811-818.
| Crossref | Google Scholar | PubMed |

Maitan PP, Bromfield EG, Hoogendijk R, Leung MR, Zeev-Ben-Mordehai T, van de Lest CH, Jansen JWA, Leemans B, Guimaraes JD, Stout TAE, Gadella BM, Henning H (2021) Bicarbonate-stimulated membrane reorganization in stallion spermatozoa. Frontiers in Cell and Developmental Biology 9, 772254.
| Crossref | Google Scholar | PubMed |

Martin-Cano FE, Gaitskell-Phillips G, Ortiz-Rodriguez JM, Silva-Rodriguez A, Roman A, Rojo-Dominguez P, Alonso-Rodriguez E, Tapia JA, Gil MC, Ortega-Ferrusola C, Pena FJ (2020) Proteomic profiling of stallion spermatozoa suggests changes in sperm metabolism and compromised redox regulation after cryopreservation. Journal of Proteomics 221, 103765.
| Crossref | Google Scholar | PubMed |

Martin-Cano FE, Gaitskell-Phillips G, da Silva-Alvarez E, Silva-Rodriguez A, Castillejo-Rufo A, Tapia JA, Gil MC, Ortega-Ferrusola C, Pena FJ (2023) The concentration of glucose in the media influences the susceptibility of stallion spermatozoa to ferroptosis. Reproduction 167, e230067.
| Crossref | Google Scholar |

Martinez IN, Moran JM, Pena FJ (2006) Two-step cluster procedure after principal component analysis identifies sperm subpopulations in canine ejaculates and its relation to cryoresistance. Journal of Andrology 27(4), 596-603.
| Google Scholar | PubMed |

Martinez-Pastor F (2022) What is the importance of sperm subpopulations? Animal Reproduction Science 246, 106844.
| Crossref | Google Scholar | PubMed |

Mateo-Otero Y, Madrid-Gambin F, Llavanera M, Gomez-Gomez A, Haro N, Pozo OJ, Yeste M (2023) Sperm physiology and in vitro fertilising ability rely on basal metabolic activity: insights from the pig model. Communications Biology 6(1), 344.
| Crossref | Google Scholar | PubMed |

Medica AJ, Gibb Z, Sheridan A, Harrison N, Aitken RJ (2022) Causative mechanisms and functional correlates of MTT reduction in stallion spermatozoa. Reproduction 163(6), 341-350.
| Crossref | Google Scholar | PubMed |

Nguyen PC, Nguyen V, Baldwin K, Kankanige Y, Blombery P, Came N, Westerman DA (2023) Computational flow cytometry provides accurate assessment of measurable residual disease in chronic lymphocytic leukaemia. British Journal of Haematology 202(4), 760-770.
| Crossref | Google Scholar | PubMed |

Niu Z-H, Huang X-F, Jia X-F, Zheng J, Yuan Y, Shi T-Y, Diao H, Yu H-G, Sun F, Zhang H-Q, Shi H-J, Feng Y (2011) A sperm viability testusing SYBR-14/propidium iodide flow cytometry as a tool for rapid screening of primary ciliary dyskinesia patients and for choosing sperm sources for intracytoplasmic sperm injection. Fertility and Sterility 95(1), 389-392.
| Crossref | Google Scholar | PubMed |

Nunez-Martinez I, Moran JM, Pena FJ (2005) Do computer-assisted, morphometric-derived sperm characteristics reflect DNA status in canine spermatozoa? Reproduction in Domestic Animals 40(6), 537-543 10.1111/j.1439-0531.2005.00628.x.
| Google Scholar | PubMed |

Odhiambo JF, Sutovsky M, DeJarnette JM, Marshall C, Sutovsky P (2011) Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis. Theriogenology 76(6), 1168-1176.
| Crossref | Google Scholar | PubMed |

Ortega-Ferrusola C, Sotillo-Galan Y, Varela-Fernandez E, Gallardo-Bolanos JM, Muriel A, Gonzalez-Fernandez L, Tapia JA, Pena FJ (2008) Detection of “apoptosis-like” changes during the cryopreservation process in equine sperm. Journal of Andrology 29(2), 213-221.
| Crossref | Google Scholar | PubMed |

Ortega-Ferrusola C, Macias Garcia B, Suarez Rama V, Gallardo-Bolanos JM, Gonzalez-Fernandez L, Tapia JA, Rodriguez-Martinez H, Pena FJ (2009) Identification of sperm subpopulations in stallion ejaculates: changes after cryopreservation and comparison with traditional statistics. Reproduction in Domestic Animals 44(3), 419-423.
| Google Scholar | PubMed |

Ortega-Ferrusola C, Anel-Lopez L, Martin-Munoz P, Ortiz-Rodriguez JM, Gil MC, Alvarez M, de Paz P, Ezquerra LJ, Masot AJ, Redondo E, Anel L, Pena FJ (2017) Computational flow cytometry reveals that cryopreservation induces spermptosis but subpopulations of spermatozoa may experience capacitation-like changes. Reproduction 153(3), 293-304.
| Crossref | Google Scholar | PubMed |

Ortiz-Rodriguez JM, Martin-Cano FE, Gaitskell-Phillips G, Silva A, Tapia JA, Gil MC, Redondo E, Masot J, Ortega-Ferrusola C, Pena FJ (2020) The SLC7A11: sperm mitochondrial function and non-canonical glutamate metabolism. Reproduction 160(6), 803-818.
| Crossref | Google Scholar | PubMed |

Ortiz-Rodriguez JM, Martin-Cano FE, Gaitskell-Phillips GL, Silva A, Ortega-Ferrusola C, Gil MC, Pena FJ (2021) Low glucose and high pyruvate reduce the production of 2-oxoaldehydes, improving mitochondrial efficiency, redox regulation, and stallion sperm function. Biology of Reproduction 105(2), 519-532.
| Crossref | Google Scholar | PubMed |

Pena FJ (2023) Expanding the use of flow cytometry in semen analysis: the rise of flow spermetry. Cytometry Part A 103(6), 465-469.
| Crossref | Google Scholar | PubMed |

Pena FJ, Gibb Z (2022) Oxidative stress and reproductive function: oxidative stress and the long-term storage of horse spermatozoa. Reproduction 164(6), F135-F144.
| Crossref | Google Scholar | PubMed |

Pena FJ, Ball BA, Squires EL (2018) A new method for evaluating stallion sperm viability and mitochondrial membrane potential in fixed semen samples. Cytometry Part B: Clinical Cytometry 94(2), 302-311.
| Crossref | Google Scholar | PubMed |

Pena FJ, O’Flaherty C, Ortiz Rodriguez JM, Martin Cano FE, Gaitskell-Phillips G, Gil MC, Ortega Ferrusola C (2022) The stallion spermatozoa: a valuable model to help understand the interplay between metabolism and redox (de)regulation in sperm cells. Antioxidants & Redox Signaling 37(7–9), 521-537.
| Crossref | Google Scholar | PubMed |

Ramal-Sanchez M, Bernabo N, Tsikis G, Blache M-C, Labas V, Druart X, Mermillod P, Saint-Dizier M (2020) Progesterone induces sperm release from oviductal epithelial cells by modifying sperm proteomics, lipidomics and membrane fluidity. Molecular and Cellular Endocrinology 504, 110723.
| Crossref | Google Scholar | PubMed |

Ramirez-Agamez L, Hernandez-Aviles C, Ortiz I, Love CC, Varner DD, Hinrichs K (2024) Lactate as the sole energy substrate induces spontaneous acrosome reaction in viable stallion spermatozoa. Andrology 12, 459-471.
| Crossref | Google Scholar |

Rubbens P, Props R (2021) Computational analysis of microbial flow cytometry data. mSystems 6(1), 10.1128/msystems.00895-20.
| Crossref | Google Scholar |

Saeys Y, Van Gassen S, Lambrecht BN (2016) Computational flow cytometry: helping to make sense of high-dimensional immunology data. Nature Reviews Immunology 16(7), 449-462.
| Crossref | Google Scholar | PubMed |

Santorsola M, Lescai F (2023) The promise of explainable deep learning for omics data analysis: adding new discovery tools to AI. New Biotechnology 77, 1-11.
| Crossref | Google Scholar | PubMed |

Schaum N, Lehallier B, Hahn O, Palovics R, Hosseinzadeh S, Lee SE, Sit R, Lee DP, Losada PM, Zardeneta ME, Fehlmann T, Webber JT, McGeever A, Calcuttawala K, Zhang H, Berdnik D, Mathur V, Tan W, Zee A, Tan M, The Tabula Muris Consortium, Pisco AO, Karkanias J, Neff NF, Keller A, Darmanis S, Quake SR, Wyss-Coray T (2020) Ageing hallmarks exhibit organ-specific temporal signatures. Nature 583(7817), 596-602.
| Crossref | Google Scholar | PubMed |

Spinelli JB, Haigis MC (2018) The multifaceted contributions of mitochondria to cellular metabolism. Nature Cell Biology 20(7), 745-754.
| Crossref | Google Scholar | PubMed |

Swegen A, Curry BJ, Gibb Z, Lambourne SR, Smith ND, Aitken RJ (2015) Investigation of the stallion sperm proteome by mass spectrometry. Reproduction 149(3), 235-244.
| Crossref | Google Scholar | PubMed |

Thakur B, Hasooni LP, Gera R, Mitra S, Bjorndahl L, Darreh-Shori T (2023) Presence of key cholinergic enzymes in human spermatozoa and seminal fluid. Biology of Reproduction 110, 63-77.
| Crossref | Google Scholar |

Tollefson J (2023) This pioneering nuclear-fusion lab is gearing up to break more records. Nature 617(7959), 13-14.
| Crossref | Google Scholar | PubMed |

Torra-Massana M, Jodar M, Barragan M, Soler-Ventura A, Delgado-Duenas D, Rodriguez A, Oliva R, Vassena R (2021) Altered mitochondrial function in spermatozoa from patients with repetitive fertilization failure after ICSI revealed by proteomics. Andrology 9(4), 1192-1204.
| Crossref | Google Scholar | PubMed |

Tsai VFS, Zhuang B, Pong Y-H, Hsieh J-T, Chang H-C (2020) Web- and artificial intelligence–based image recognition for sperm motility analysis: verification study. JMIR Medical Informatics 8(11), e20031.
| Crossref | Google Scholar | PubMed |

Umair M, Claes A, Buijtendorp M, Cuervo-Arango J, Stout TAE, Henning H (2023) In vitro aging of stallion spermatozoa during prolonged storage at 5°C. Cytometry Part A 103(6), 479-491.
| Crossref | Google Scholar | PubMed |

Wagner AO, Turk A, Kunej T (2023) Towards a multi-omics of male infertility. The World Journal of Men’s Health 41(2), 272-288.
| Crossref | Google Scholar | PubMed |

Wang H, Fu T, Du Y, Gao W, Huang K, Liu Z, Chandak P, Liu S, Van Katwyk P, Deac A, Anandkumar A, Bergen K, Gomes CP, Ho S, Kohli P, Lasenby J, Leskovec J, Liu T-Y, Manrai A, Marks D, Ramsundar B, Song L, Sun J, Tang J, Velickovic P, Welling M, Zhang L, Coley CW, Bengio Y, Zitnik M (2023) Scientific discovery in the age of artificial intelligence. Nature 620(7972), 47-60.
| Crossref | Google Scholar | PubMed |

WHO (2021) ‘WHO laboratory manual for the examination and processing of human semen.’ 6th edn. (World Health Organization) Available at https://iris.who.int/bitstream/handle/10665/343208/9789240030787-eng.pdf?sequence=1

Xu Y, Ritchie SC, Liang Y, Timmers PRHJ, Pietzner M, Lannelongue L, Lambert SA, Tahir UA, May-Wilson S, Foguet C, Johansson A, Surendran P, Nath AP, Persyn E, Peters JE, Oliver-Williams C, Deng S, Prins B, Luan J, Bomba L, Soranzo N, Di Angelantonio E, Pirastu N, Tai ES, van Dam RM, Parkinson H, Davenport EE, Paul DS, Yau C, Gerszten RE, Malarstig A, Danesh J, Sim X, Langenberg C, Wilson JF, Butterworth AS, Inouye M (2023) An atlas of genetic scores to predict multi-omic traits. Nature 616(7955), 123-131.
| Crossref | Google Scholar | PubMed |

Yaniz JL, Silvestre MA, Santolaria P, Soler C (2018) CASA-Mot in mammals: an update. Reproduction, Fertility and Development 30(6), 799-809.
| Crossref | Google Scholar | PubMed |

Yao Z, Liu H, Xie F, Fischer S, Adkins RS, Aldridge AI, Ament SA, Bartlett A, Behrens MM, Van den Berge K, Bertagnolli D, de Bezieux HR, Biancalani T, Booeshaghi AS, Bravo HC, Casper T, Colantuoni C, Crabtree J, Creasy H, Crichton K, Crow M, Dee N, Dougherty EL, Doyle WI, Dudoit S, Fang R, Felix V, Fong O, Giglio M, Goldy J, Hawrylycz M, Herb BR, Hertzano R, Hou X, Hu Q, Kancherla J, Kroll M, Lathia K, Li YE, Lucero JD, Luo C, Mahurkar A, McMillen D, Nadaf NM, Nery JR, Nguyen TN, Niu S-Y, Ntranos V, Orvis J, Osteen JK, Pham T, Pinto-Duarte A, Poirion O, Preissl S, Purdom E, Rimorin C, Risso D, Rivkin AC, Smith K, Street K, Sulc J, Svensson V, Tieu M, Torkelson A, Tung H, Vaishnav ED, Vanderburg CR, van Velthoven C, Wang X, White OR, Huang ZJ, Kharchenko PV, Pachter L, Ngai J, Regev A, Tasic B, Welch JD, Gillis J, Macosko EZ, Ren B, Ecker JR, Zeng H, Mukamel EA (2021) A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex. Nature 598(7879), 103-110.
| Crossref | Google Scholar | PubMed |