Caspase 3 Activity and Lipoperoxidative Status in Raw Semen Predict the Outcome of Cryopreservation of Stallion Spermatozoa.
Abstract: Stallion-to-stallion variability in the quality of cryopreserved ejaculates postthaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry. We studied three markers of oxidative stress in sperm, including 8-iso-PGF2alpha, 8-OH guanosine, and 4-hydroxynonenal (4-HNE); the presence of active caspase 3; and their changes after sperm cryopreservation. Although 4-HNE levels increased after cryopreservation (from 7% to 33%, P < 0.001), 8OH-guanosine and 8-ISO-PGF2alpha levels decreased after cryopreservation (from 130 to 35 arbitrary fluorescence units, P < 0.01, and from 1280 to 1233, P < 0.01, respectively). Postthaw sperm quality was classified as poor, average, or good using the 25th and 75th percentiles of all assays of sperm quality studied (motility, velocity, membrane functionality, and thiol content) as thresholds. Using these values, a sperm postthaw quality index was proposed. Receiver operating characteristic curves and the Youden J statistic were used to investigate the value of the measured parameters in fresh sperm as predictors of potential freezability. Using these techniques, we identified markers of bad freezers (percentages of caspase 3-positive dead sperm [area under the curve (AUC) = 0.820, P < 0.05] and percentages of caspase 3- and 4-HNE-positive sperm [AUC = 0.872, P < 0.05]) and good freezers (percentages of caspase 3-negative live sperm [AUC = 0.815, P < 0.05], percentages of live sperm with high thiol content [AUC = 0.907, P < 0.01], and percentages of 8-ISO-PGF2alpha-positive sperm [AUC = 0.900, P < 0.01]. Moreover, we described for the first time the presence of 8-ISO-PGF2alpha in stallion spermatozoa and revealed the importance of considering different markers of oxidative stress.
© 2016 by the Society for the Study of Reproduction, Inc.
Publication Date: 2016-07-14 PubMed ID: 27417910DOI: 10.1095/biolreprod.116.139444Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research article explores how variability in the quality of stallion semen post-cryopreservation impacts its commercial value, with a focus on identifying indicators of good and bad freezing outcomes. Using various statistical techniques, the researchers identified markers related to caspase 3 activity and peroxidative status, which could predict the outcome of sperm cryopreservation.
Objective and Methodology
- The researchers aim to understand the variability in the quality of cryopreserved stallion semen, which affects its commercial utilization in the equine industry.
- They explored molecular mechanisms related to sperm damage during cryopreservation.
- The study also aimed to identify markers that could predict the viability of the semen even before the freezing process begins. These markers include 8-iso-PGF2alpha, 8-OH guanosine, 4-hydroxynonenal (4-HNE), and the presence of active caspase 3.
Oxidative Stress Markers and Caspase 3
- The researchers observed an increase in 4-HNE levels after cryopreservation. However, the levels of 8OH-guanosine and 8-ISO-PGF2alpha decreased post-cryopreservation.
- They focused on the activity of caspase 3, a protein involved in apoptosis or programmed cell death, to check its viability after sperm cryopreservation.
Establishing Post-thaw Quality Index
- The team classified post-thaw sperm into three categories (poor, average, good) based on the 25th and 75th percentiles of all sperm quality assays, including motility, velocity, membrane functionality, and thiol content.
- They further proposed a sperm post-thaw quality index basis these observations.
Predicting Freezability of Sperm
- Receiver operating characteristic curves and the Youden J statistic were employed to study the predictive value of fresh sperm parameters in indicating potential freezability.
- The results highlighted ‘bad freezers’ represented by higher percentages of caspase 3-positive dead sperm and caspase 3/4-HNE-positive sperm.
- ‘Good freezers’ were identified by higher percentages of caspase 3-negative live sperm, live sperm with high thiol content, and 8-ISO-PGF2alpha-positive sperm.
- Researchers reported, for the first time, the presence of 8-ISO-PGF2alpha in stallion sperm and emphasized the importance of different oxidative stress markers.
Cite This Article
APA
Muñoz PM, Ferrusola CO, Lopez LA, Del Petre C, Garcia MA, de Paz Cabello P, Anel L, Peña FJ.
(2016).
Caspase 3 Activity and Lipoperoxidative Status in Raw Semen Predict the Outcome of Cryopreservation of Stallion Spermatozoa.
Biol Reprod, 95(3), 53.
https://doi.org/10.1095/biolreprod.116.139444 Publication
Researcher Affiliations
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, León, Spain.
- Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, León, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, León, Spain.
- Department of Molecular Biology, University of León, León, Spain.
- Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, León, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain fjuanpvega@unex.es.
MeSH Terms
- Aldehydes / metabolism
- Animals
- Caspase 3 / metabolism
- Cryopreservation / methods
- Dinoprost / analogs & derivatives
- Dinoprost / metabolism
- Guanosine / analogs & derivatives
- Guanosine / metabolism
- Horses
- Male
- Oxidative Stress / physiology
- Semen / metabolism
- Semen Preservation / methods
- Sperm Motility / physiology
- Spermatozoa / cytology
- Spermatozoa / metabolism
Citations
This article has been cited 11 times.- Montes-Garrido R, Riesco MF, Anel-Lopez L, Neila-Montero M, Palacin-Martinez C, Boixo JC, de Paz P, Ortega-Ferrusola C, Hassan MAA, Anel L, Alvarez M. Application of ultrasound technique to evaluate the testicular function and its correlation to the sperm quality after different collection frequency in rams. Front Vet Sci 2022;9:1035036.
- Schäfer-Somi S, Colombo M, Luvoni GC. Canine Spermatozoa-Predictability of Cryotolerance. Animals (Basel) 2022 Mar 15;12(6).
- Neila-Montero M, Riesco MF, Alvarez M, Montes-Garrido R, Boixo JC, de Paz P, Anel-Lopez L, Anel L. Centrifugal force assessment in ram sperm: identifying species-specific impact. Acta Vet Scand 2021 Nov 4;63(1):42.
- Bazzano M, Laus F, Spaterna A, Marchegiani A. Use of nutraceuticals in the stallion: Effects on semen quality and preservation. Reprod Domest Anim 2021 Jul;56(7):951-957.
- Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, Silva-Rodríguez A, Gil MC, Ortega-Ferrusola C, Peña FJ. 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. J Proteome Res 2021 May 7;20(5):2435-2446.
- Peña FJ, O'Flaherty C, Ortiz Rodríguez JM, Martín Cano FE, Gaitskell-Phillips GL, Gil MC, Ortega Ferrusola C. Redox Regulation and Oxidative Stress: The Particular Case of the Stallion Spermatozoa. Antioxidants (Basel) 2019 Nov 19;8(11).
- Ortiz-Rodriguez JM, Balao da Silva C, Masot J, Redondo E, Gazquez A, Tapia JA, Gil C, Ortega-Ferrusola C, Peña FJ. Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through regulating Akt phosphorylation and reduction of caspase 3. PLoS One 2019;14(7):e0211994.
- Ortiz-Rodriguez JM, Ortega-Ferrusola C, Gil MC, Martín-Cano FE, Gaitskell-Phillips G, Rodríguez-Martínez H, Hinrichs K, Álvarez-Barrientos A, Román Á, Peña FJ. Transcriptome analysis reveals that fertilization with cryopreserved sperm downregulates genes relevant for early embryo development in the horse. PLoS One 2019;14(6):e0213420.
- Anel-Lopez L, Ortega-Ferrusola C, Álvarez M, Borragán S, Chamorro C, Peña FJ, Morrell J, Anel L, de Paz P. Improving sperm banking efficiency in endangered species through the use of a sperm selection method in brown bear (Ursus arctos) thawed sperm. BMC Vet Res 2017 Jun 26;13(1):200.
- Ullah A, Chen W, Shi L, Wang M, Geng M, Na J, Akhtar MF, Khan MZ, Wang C. Challenges and Enhancing Strategies of Equine Semen Preservation: Nutritional and Genetic Perspectives. Vet Sci 2025 Aug 25;12(9).
- Khalil WA, El-Rais MS, Hegazy MM, Hassan MAE, El-Raghi AA, El-Moghazy MM. The Effect of Metallic Nanoparticles Supplementation in Semen Extender on Post-thaw Quality and Fertilizing Ability of Egyptian Buffalo (Bubalus bubalis) Spermatozoa. Biol Trace Elem Res 2025 May;203(5):2636-2653.
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