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Animals
Special Issues
Current Status and Advances in Semen Preservation—Second Edition
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Current Status and Advances in Semen Preservation—Second Edition

A special issue of Animals (ISSN 2076-2615). This special issue belongs to the section "Animal Reproduction".

Deadline for manuscript submissions: 31 October 2025 | Viewed by 3864

Special Issue Editors

Dr. Anna Dziekońska

E-Mail Website
Guest Editor
Department of Animal Biochemistry and Biotechnology, University of Warmia and Mazury, Oczapowskiego 5, 10-718 Olsztyn, Poland
Interests: animal reproduction; sperm quality; liquid storage; cryopreservation; membrane integrity; antioxidants; energy metabolism; proteomic
Special Issues, Collections and Topics in MDPI journals
Dr. Agnieszka Partyka

E-Mail Website
Guest Editor
Department of Reproduction and Clinic of Farm Animals, Wroclaw University of Environmental and Life Sciences, pl. Grunwaldzki 49, 50-366 Wroclaw, Poland
Interests: animal reproduction; andrology; sperm quality assessment; flow cytometry; CASA system; semen cryopreservation; antioxidants
Special Issues, Collections and Topics in MDPI journals
Dr. Magdalena Koziorowska-Gilun

E-Mail Website
Guest Editor
Department of Animal Biochemistry and Biotechnology, University of Warmia and Mazury, Oczapowskiego 5, 10-718 Olsztyn, Poland
Interests: animal reproduction; sperm quality; oxidative stress; antioxidant activity, semen analysis; seasonality; environmental science; lipid peroxidation; animal science; reactive oxygen species

Special Issue Information

Dear Colleagues,

We invite you to submit original research papers and review articles to the “Current Status and Advances in Semen Preservation—Second Edition” Special Issue.

One of the most dynamically developing assisted reproductive techniques (ARTs) used in animals is the preservation of gametes, especially sperm. Improving sperm preservation methods, both liquid and frozen, requires the consideration of various factors, including temperature and storage time, the composition of the diluent used, and species-specific as well as individual differences in the structure and functioning of an animal's reproductive system (e.g., tissues of the reproductive tract). Storage in a liquid state allows for the short-term preservation of the biological properties of sperm, often providing better fertilizing abilities of sperm compared to cryopreservation. Sperm cryopreservation (both ejaculated and epididymal) enables the long-term preservation of valuable genetic material for reproductive purposes. The use of an appropriate preservation procedure allows sperm to retain their fertilizing ability, as predicted by laboratory analyses and ultimately confirmed by the results of their use in in vitro and in vivo fertilization procedures.

We particularly welcome manuscripts presenting new technological solutions for improving sperm preservation methods and demonstrating new possibilities of using various laboratory techniques (including computer, enzymatic, fluorescence, and proteomic analyses) to predict the fertilization potential of preserved sperm in different animal species.

Dr. Anna Dziekońska
Dr. Agnieszka Partyka
Dr. Magdalena Koziorowska-Gilun
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Animals is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2400 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • semen preservation
  • liquid state
  • cryopreservation
  • semen quality
  • fertility
  • assisted reproductive technologies
  • energy metabolism
  • antioxidants
  • oxidative stress
  • proteomic

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Published Papers (5 papers)

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Research

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18 pages, 5002 KiB  
Article
Differential Metabolomic Signatures in Boar Sperm with Varying Liquid Preservation Capacities at 17 °C
by Serge L. Kameni, Notsile H. Dlamini and Jean M. Feugang
Animals 2025, 15(15), 2163; https://doi.org/10.3390/ani15152163 - 22 Jul 2025
Viewed by 452
Abstract
In the swine industry, artificial insemination (AI) primarily uses chill-stored semen, making sperm preservation crucial for reproductive success. However, sperm quality declines at varying rates during chilled storage at 17 °C, distinguishing high-survival semen from low-survival semen. This study investigates the metabolomic profiles [...] Read more.
In the swine industry, artificial insemination (AI) primarily uses chill-stored semen, making sperm preservation crucial for reproductive success. However, sperm quality declines at varying rates during chilled storage at 17 °C, distinguishing high-survival semen from low-survival semen. This study investigates the metabolomic profiles of boar sperm with different abilities to survive liquid storage. We analyzed sperm motility, kinematics, and morphology in freshly extended (Day 0) and 7-day stored AI semen doses. The AI semen doses were classified as high-motile (HM) or low-motile (LM) based on sperm motility after 7 days of storage (Day 7). Metabolomic data were collected in positive (ESI+) and negative (ESI−) ion modes using a Vanquish Flex UPLC coupled with a Q Extractive Plus. We consistently detected 442 metabolites (251 in ESI+, 167 in ESI−, and 24 in both) across samples and storage durations. In freshly extended and 7-day stored AI doses, we identified 42 and 56 differentially expressed metabolites (DEMs), respectively. A clustering analysis showed significant changes in DEMs between the HM and LM samples. These DEMs were mainly enriched in amino acid metabolism, the pentose phosphate pathway, glycerolipid metabolism, glyoxylate and dicarboxylate metabolism, terpenoid backbone biosynthesis, etc. In summary, this study highlights the metabolomic differences between semen doses with varying abilities to survive liquid storage. Glyceric acid and lysoPC(20:3) emerged as potential markers for sperm preservation. Full article
(This article belongs to the Special Issue Current Status and Advances in Semen Preservation—Second Edition)
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13 pages, 1055 KiB  
Article
Effect of Centrifugation of Stallion Semen Through a Low Density Colloid Prior to Freezing on Sperm Cryosurvival
by Ziyad Al-Kass, Jane M. Morrell and Theodoros Ntallaris
Animals 2025, 15(13), 1881; https://doi.org/10.3390/ani15131881 - 25 Jun 2025
Viewed by 414
Abstract
Sperm quality is adversely affected by cryopreservation due to the increased production of reactive oxygen species, which affects the integrity of sperm membranes, motility, and DNA fragmentation. Three methods for removing seminal plasma, washing (centrifuging extended semen at 800× g for 10 min) [...] Read more.
Sperm quality is adversely affected by cryopreservation due to the increased production of reactive oxygen species, which affects the integrity of sperm membranes, motility, and DNA fragmentation. Three methods for removing seminal plasma, washing (centrifuging extended semen at 800× g for 10 min) and Single Layer Centrifugation with high or low density Equicoll, were used to prepare 29 ejaculates from ten stallions for freezing. Sperm quality parameters (kinematics, plasma membrane integrity, superoxide and hydrogen peroxide production, mitochondrial membrane potential, and DNA fragmentation) were evaluated before and after freezing using kinematic and flow cytometric analysis. The parameters for fresh samples were within the normal range for stallion semen but were lower after thawing. There were few differences between the three preparation methods. Interestingly, DNA fragmentation was affected most by the sperm preparation method, being lowest for SLC through high density Equicoll, although SLC through low density Equicoll was effective for some stallions. Some differences were observed in the proportions of live or dead spermatozoa positive for hydrogen peroxide. In conclusion, all of these methods would be suitable for the preparation of semen prior to cryopreservation, but Single Layer Centrifugation through high density Equicoll was the most effective in removing spermatozoa with damaged DNA. Full article
(This article belongs to the Special Issue Current Status and Advances in Semen Preservation—Second Edition)
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15 pages, 642 KiB  
Article
Innovative Approaches to Avoid Antibiotic Use in Equine Semen Cryopreservation: Advancing Sustainable Reproductive Technologies
by Sonsoles Mercedes Zabala, Consuelo Serres, Natalia Montero, Francisco Crespo, Pedro Luis Lorenzo, Verónica Pérez-Aguilera, Agustín Oliet, Virginia Hijón, Santiago Moreno, Bruno González-Zorn and Luna Gutiérrez-Cepeda
Animals 2025, 15(10), 1368; https://doi.org/10.3390/ani15101368 - 9 May 2025
Viewed by 631
Abstract
This study evaluated the impact of different processing techniques on microbial load and sperm quality in frozen–thawed equine semen to identify alternatives to reduce the preventive use of antibiotics. Semen was obtained and processed under rigorous hygiene measures from ten stallions, using four [...] Read more.
This study evaluated the impact of different processing techniques on microbial load and sperm quality in frozen–thawed equine semen to identify alternatives to reduce the preventive use of antibiotics. Semen was obtained and processed under rigorous hygiene measures from ten stallions, using four protocols: Simple Centrifugation with antibiotics (S+) and Simple Centrifugation (S−), Filtration (F−) and Single-Layer Colloidal Centrifugation (C−) in an antibiotic-free extender. Microbial load in different culture media, sperm viability and motility were assessed. Microbial load results were consistent across protocols, except in Columbia 5% Sheep Blood Agar media, where S− exhibited higher microbial load than S+ (p < 0.05). However, F− and C− showed similar microbial loads to S+. No significant differences were observed in progressive motility, average path velocity, straight-line velocity or wobble parameters between protocols. Total motility and viability were significantly higher in S+ compared to other treatments (p < 0.05). Thus, regardless of antibiotics, the proposed methods achieved results similar to the traditional antibiotic-inclusive protocol in terms of microbial load and the most relevant semen quality parameters. These findings suggest that the use of F− and C−, combined with optimized hygiene measures, offers an effective alternative to reduce the prophylactic use of antibiotics in semen extenders. Full article
(This article belongs to the Special Issue Current Status and Advances in Semen Preservation—Second Edition)
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12 pages, 1424 KiB  
Article
Sustainable Alternatives to the Reduction of Plastic Straws Used with Chilled Equine Semen
by Noelia González, Aroa Peñalosa, Ignacio de Blas and Lydia Gil
Animals 2024, 14(23), 3388; https://doi.org/10.3390/ani14233388 - 25 Nov 2024
Viewed by 1024
Abstract
Currently, plastic is used being without any limitations. The livestock sector is no stranger to its use. For example, artificial insemination involves the use of different plastic materials, including semen straws and insemination catheters, which increase the levels of waste in our environment. [...] Read more.
Currently, plastic is used being without any limitations. The livestock sector is no stranger to its use. For example, artificial insemination involves the use of different plastic materials, including semen straws and insemination catheters, which increase the levels of waste in our environment. Finding a sustainable solution to avoid plastic materials is a challenge. The aim of this study was to examine different biodegradable materials as alternatives to conventional syringes used for refrigerated equine semen. The materials tested were bamboo, avocado, grass, paper, Kraft paper, wheat, and rice. Bamboo and avocado were selected to continue testing with the refrigerated semen with a lifespan extended by INRA 96®; the rest of the materials did not meet the requirements necessary for preservation. Motility and movement kinetics tests were performed at 24 and 96 h of refrigeration to evaluate semen quality; relative to the control, significantly better results were displayed in regard to the total and progressive motility avocado straws. The kinetic parameters were comparable to the control for both materials and within normal ranges. After performing a microbiological control process for both straws, it was confirmed that there was no bacterial growth in either straw. We can confirm that avocado straws can be used to preserve chilled equine semen, guaranteeing seminal quality while being respectful to the environment. Full article
(This article belongs to the Special Issue Current Status and Advances in Semen Preservation—Second Edition)
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Review

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18 pages, 931 KiB  
Review
Sperm Membrane: Molecular Implications and Strategies for Cryopreservation in Productive Species
by Macarena Castro, Karla Leal, Felipe Pezo and María José Contreras
Animals 2025, 15(12), 1808; https://doi.org/10.3390/ani15121808 - 19 Jun 2025
Viewed by 586
Abstract
Sperm cryopreservation is a fundamental reproductive biotechnology, enabling the long-term storage of genetic material and supporting assisted reproduction programs. Despite its widespread application, the process is associated with significant limitations due to the cryo-induced cellular damage that occurs during freezing and thawing. These [...] Read more.
Sperm cryopreservation is a fundamental reproductive biotechnology, enabling the long-term storage of genetic material and supporting assisted reproduction programs. Despite its widespread application, the process is associated with significant limitations due to the cryo-induced cellular damage that occurs during freezing and thawing. These injuries primarily affect the plasma membrane, nuclear DNA, and motility, thereby compromising the fertilizing potential of spermatozoa. Furthermore, interspecies variability in terms of cryo-sensitivity poses a major challenge to the development of standardized cryopreservation protocols. Recent advances have focused on mitigating cryodamage through the use of various strategies. The inclusion of antioxidants in cryopreservation media has proven effective in reducing oxidative stress, thereby enhancing cellular protection. Similarly, the addition of lipid-based supplements contributes to membrane stabilization, improving post-thaw sperm viability and functionality. Moreover, the application of omics technologies, such as transcriptomics and proteomics, has facilitated a deeper understanding of molecular damage and protective responses, paving the way for the development of tailored, species-specific protocols. These integrated approaches optimize cryopreservation conditions, maximizing post-thaw survival and the fertilizing capacity of sperm. Enhancing cryopreservation techniques not only improves the outcomes of assisted reproductive technologies, but also plays a crucial role in the conservation of genetically valuable livestock species. In conclusion, the integration of biotechnological and molecular tools holds significant promise for overcoming the current limitations and advancing the efficacy of sperm cryopreservation. Full article
(This article belongs to the Special Issue Current Status and Advances in Semen Preservation—Second Edition)
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