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Home / Equine Research Bank / Reprod Domest Anim / Niacin Improves Cryopreserved Equine Sperm Quality and Gene ...
Reproduction in domestic animals = Zuchthygiene2026; 61(1); e70173; doi: 10.1111/rda.70173

Niacin Improves Cryopreserved Equine Sperm Quality and Gene Expression: An Artificial Intelligence Assisted Evaluation.

Abstract: Niacin acts as an antioxidant that protects cells from oxidative damage. This study evaluated the effects of adding niacin to the equine semen freezing extender on sperm quality and gene expression after cryopreservation. Ejaculates from ten stallions were frozen using the INRA 96 extender (control) or extenders supplemented with 10- and 20-mM niacin. After thawing, sperm were analysed for motility, kinematics, viability, membrane integrity, mitochondrial potential, lipid peroxidation, nitrite, hydrogen peroxide, malondialdehyde and reactive oxygen species (ROS) concentrations, DNA integrity, sperm binding to bovine oviduct explants, and expression of apoptosis related (BCL2, BAX), mitochondrial (ROMO1), sperm binding (SPACA3) and DNA repair (OGG1) genes. Data were tested for normality (Shapiro-Wilk) and analysed by randomised block ANOVA followed by Tukey's test (p < 0.05). Machine learning algorithms (Logistic Regression, MLP, XGBoost, KNN and SVM) with SHAP analysis ranked the most influential parameters associated with sperm quality. The addition of 10 mM niacin improved sperm motility, mitochondrial activity, and the number of sperm bound to bovine oviduct explants, while reducing ROS levels and expression of BAX and ROMO1, and increasing BCL2 and SPACA3 genes. The 20 mM treatment also enhanced sperm binding and upregulated SPACA3 expression compared to the control. However, 20 mM niacin showed lower binding activity than 10 mM. Machine learning identified sperm binding to oviduct explants and SPACA3 expression as the most influential variables for classifying samples. In conclusion, both niacin concentrations improved equine cryopreserved sperm quality, although 10 mM showed superior antioxidant, anti-apoptotic, and functional effects, representing optimal supplementation level.
© 2026 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.
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Publication Date: 2026-01-10 PubMed ID: 41518220DOI: 10.1111/rda.70173Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Research Overview

  • This study investigated the effects of niacin supplementation in the freezing extender used for cryopreserving equine sperm, focusing on sperm quality and gene expression after thawing.
  • Using artificial intelligence techniques, the researchers identified key parameters that influence sperm quality and found that 10 mM niacin was the optimal concentration for improving sperm function and protecting against oxidative stress.

Background

  • Niacin (vitamin B3) acts as an antioxidant, potentially protecting cells from oxidative damage during cryopreservation.
  • Cryopreservation of equine sperm can cause oxidative stress, impair sperm quality, functionality, and DNA integrity.
  • Improving cryopreservation protocols with antioxidant additives like niacin might enhance post-thaw sperm viability and fertilization ability.

Experimental Design and Methods

  • Samples: Ejaculates collected from ten stallions.
  • Treatments:
    • Control group: Sperm frozen using INRA 96 extender without niacin.
    • 10 mM niacin group: Extender supplemented with 10 mM niacin.
    • 20 mM niacin group: Extender supplemented with 20 mM niacin.
  • Post-thaw analyses included:
    • Sperm motility and kinematics (movement characteristics).
    • Viability and membrane integrity.
    • Mitochondrial potential (energy status).
    • Lipid peroxidation and various markers of oxidative stress (nitrite, hydrogen peroxide, malondialdehyde, reactive oxygen species).
    • DNA integrity.
    • Sperm binding to bovine oviduct explants (functional assay indicating sperm-oviduct interaction).
    • Gene expression related to:
      • Apoptosis (BCL2, BAX genes).
      • Mitochondrial function (ROMO1 gene).
      • Sperm binding function (SPACA3 gene).
      • DNA repair (OGG1 gene).
  • Statistical analysis:
    • Data normality checked with Shapiro-Wilk test.
    • Randomised block ANOVA followed by Tukey’s post-hoc test used for comparisons between groups (significance at p < 0.05).
  • Machine learning and AI techniques:
    • Algorithms applied: Logistic Regression, Multi-layer Perceptron (MLP), XGBoost, K-Nearest Neighbours (KNN), and Support Vector Machine (SVM).
    • SHAP (SHapley Additive exPlanations) analysis used to rank the most influential parameters affecting sperm quality post-thaw.

Key Findings

  • 10 mM Niacin supplementation effects:
    • Significant improvement in sperm motility and mitochondrial activity.
    • Increased number of sperm bound to bovine oviduct explants, indicating enhanced functional capacity.
    • Reduction in oxidative stress markers (lower reactive oxygen species levels).
    • Altered gene expression:
      • Downregulation of pro-apoptotic BAX and mitochondrial oxidative stress-related ROMO1 genes, indicating reduced cell death signals.
      • Upregulation of anti-apoptotic BCL2 gene and sperm binding-related SPACA3 gene, suggesting improved cell survival and function.
  • 20 mM Niacin supplementation effects:
    • Improvement in sperm binding to oviduct explants compared to the control, but less than the 10 mM group.
    • Upregulation of SPACA3 gene expression.
    • Less pronounced antioxidant and anti-apoptotic effects compared to 10 mM treatment.

Machine Learning Insights

  • Sperm binding to oviduct explants and expression of the SPACA3 gene were identified as the strongest predictors for classifying sperm quality post-cryopreservation.
  • AI-assisted analysis confirmed the biological importance of functional sperm-oviduct interactions and related gene expression in determining sperm viability and quality after freezing and thawing.

Conclusions and Implications

  • Both 10 mM and 20 mM niacin supplementation improve aspects of equine sperm quality after cryopreservation, but 10 mM is superior in terms of antioxidant protection, anti-apoptotic gene regulation, and functional parameters.
  • Incorporating 10 mM niacin into equine semen extenders may optimize sperm preservation outcomes, likely leading to better fertility rates following artificial insemination.
  • The use of AI and machine learning provided valuable insights into the key biological markers and functions that correlate with sperm quality, suggesting these tools can enhance evaluation strategies in reproductive biotechnology.

Cite This Article

APA
Alves NC, Freitas MM, Faria JRD, Horta CL, Martins-Filho OA, Araújo MSS, Costa GMJ, Costa EA, de Almeida FRL, Amaral PHR, Pérez JCG, Lana ÂQ, Coelho IMP, da Costa AJA, Lagares MA. (2026). Niacin Improves Cryopreserved Equine Sperm Quality and Gene Expression: An Artificial Intelligence Assisted Evaluation. Reprod Domest Anim, 61(1), e70173. https://doi.org/10.1111/rda.70173

Publication

Reproduction in domestic animals = Zuchthygiene
ISSN: 1439-0531
NlmUniqueID: 9015668
Country: Germany
Language: English
Volume: 61
Issue: 1
Pages: e70173

Researcher Affiliations

Alves, Natália de Castro
  • Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil.
Freitas, Marina Morra
  • Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil.
Faria, Jade Raquel Dias
  • Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil.
Horta, Cesar Lopes
  • Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil.
Martins-Filho, Olindo Assis
  • Centro de Pesquisa René Rachou, Fiocruz, Laboratório de diagnóstico e monitoramento de biomarcadores, Belo Horizonte, Brazil.
Araújo, Márcio Sobreira Silva
  • Centro de Pesquisa René Rachou, Fiocruz, Laboratório de diagnóstico e monitoramento de biomarcadores, Belo Horizonte, Brazil.
Costa, Guilherme Mattos Jardim
  • Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
Costa, Erica Azevedo
  • Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil.
de Almeida, Fernanda Radicchi Lobato
  • Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
Amaral, Paulo Henrique Ribeiro
  • Departamento de Física, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
Pérez, Juan Carlos González
  • Departamento de Física, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
Lana, Ângela Quintão
  • Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil.
Coelho, Isadora Martins Pinto
  • Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil.
da Costa, Ailton Junior Antunes
  • Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil.
Lagares, Monique de Albuquerque
  • Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil.

MeSH Terms

  • Animals
  • Cryopreservation / veterinary
  • Male
  • Semen Preservation / veterinary
  • Semen Preservation / methods
  • Horses
  • Semen Analysis / veterinary
  • Spermatozoa / drug effects
  • Spermatozoa / physiology
  • Niacin / pharmacology
  • Sperm Motility / drug effects
  • Cryoprotective Agents / pharmacology
  • Female
  • Gene Expression / drug effects
  • Reactive Oxygen Species
  • Cattle

Grant Funding

  • 140683/2019-4 / Conselho Nacional de Desenvolvimento Científico e Tecnológico

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