L-Proline: An Effective Agent for Frozen and Post-thawed Donkey Semen Storage.
Abstract: The aim of this study was to evaluate the effects of L-proline on the extender quality of frozen and post-thawed jackass semen. Jackass (n = 6) semen samples were collected and cryopreserved in gradient concentrations (0-80 mM) of L-proline in extenders; post-thawed semen samples were cultured in L-proline medium for 10 hours at 37°C. For cryopreservation experiment I, the motile parameters, mitochondrial membrane potential (MMP), and plasma membrane, acrosome, and chromatin structure integrities of post-thawed semen were assessed. For culture experiment II, additional ROS contents were analyzed after incubation. For the fertility trial, jennies (n = 135) were divided into group I (30 mM L-proline in cryopreservation extender), group II (40 mM L-proline in culture medium), and the control. Pregnancy was diagnosed using an ultrasound scanner 30 days after ovulation. The results of experiment I showed that, motile parameters and acrosome and chromatin structure integrities of groups I and 40 mM were significantly higher than the control (P < .05). MMP of group I was significantly higher than the control and 40 mM groups (P < .05). In experiment II, after 4 hours of incubation, motile parameters, MMP, and DNA integrity in group II were significantly higher than the control (P < .05). Additionally, 40 and 80 mM L-proline in culture medium significantly reduced ROS accumulation after 4 and 10 hours of incubation (P < .05). Pregnancy rates of the control and groups I and II were 28.85%, 40%, and 36.84%, respectively. In conclusion, the extenders containing 30 to 40 mM L-proline improved both qualities of frozen and post-thawed semen, and it will be a beneficial agent for donkey frozen spermatozoa or post-thawed semen storage.
Copyright © 2021. Published by Elsevier Inc.
Publication Date: 2021-02-03 PubMed ID: 33993948DOI: 10.1016/j.jevs.2021.103393Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research explores the impact of L-proline, an important amino acid, on the quality of donkey semen during freezing and after thawing. The study found evidence that the use of L-proline helps improve both the quality of the frozen and post-thawed semen samples and the pregnancy rates in the animals used for the study.
Research Methodology
- The study involved the use of semen samples from six male donkeys (jacks). These samples were preserved using various concentrations (0-80 mM) of L-proline as additives.
- Once the samples were thawed, they were placed in an L-proline medium and kept in a controlled environment (37°C) for a duration of 10 hours.
- The quality of the preserved semen was evaluated based on several factors. These include motility parameters, mitochondrial membrane potential (MMP), and the structural integrities of plasma membrane, acrosome, and chromatin in the post-thawed samples.
- In addition, Reactive Oxygen Species (ROS) content was also analyzed after the incubation period.
- For evaluating the efficacy on fertility, 135 female donkeys (jennies) were categorized into three groups: Group I (cryopreservation extender included 30mM L-proline), Group II (culture medium included 40mM L-proline), and a control group. After 30 days of ovulation, ultrasound scanning was used to diagnose pregnancy.
Outcomes of the Research
- The study outcomes pointed towards a positive impact of L-proline on semen quality. The semen samples that had been frozen with L-proline showed significantly better motility parameters, and improved acrosome and chromatin structure integrities than the control group.
- L-proline was found to have a protective effect from Reactive Oxygen Species (ROS), which can damage the structural integrity of sperm, leading to impaired fertility. The L-proline groups showed significantly less ROS accumulation as compared to the control group.
- Pregnancy rates in the animal groups with added L-proline were higher. The control group had a pregnancy rate of 28.85%, while the groups with L-proline additives had rates of 40% and 36.84%.
Conclusion
- The research concludes that L-proline, when added to the extender used in semen cryopreservation, can improve the quality of both frozen and post-thawed donkey semen. These benefits include improved sperm motility parameters, and the structural integrity of acrosome, and chromatin structures even post-thawing.
- The reduction in ROS accumulation further contributes to improving the semen quality and thus, L-proline proves to be a beneficial agent for preserving donkey spermatozoa, either during freezing or after thawing.
- The research also suggests that extenders with 30-40 mM L-proline may be optimal for achieving these benefits.
Cite This Article
APA
Li N, Yu J, Yang F, Shao Y, Wu S, Liu B, Li M, Wang T, Li J, Zeng S.
(2021).
L-Proline: An Effective Agent for Frozen and Post-thawed Donkey Semen Storage.
J Equine Vet Sci, 101, 103393.
https://doi.org/10.1016/j.jevs.2021.103393 Publication
Researcher Affiliations
- National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing, China.
- College of Animal Science and Technology, Northwest A&F University, Yangling, China.
- National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing, China.
- National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing, China.
- National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing, China; College of Animal Science and Technology, Northwest A&F University, Yangling, China.
- College of Animal Science and Technology, Northwest A&F University, Yangling, China.
- College of Animal Science and Technology, Northwest A&F University, Yangling, China.
- College of Animal Science and Technology, Northwest A&F University, Yangling, China.
- Department of Clinical Sciences, College of Veterinary Medicine, China Agricultural University, Beijing, China. Electronic address: jlivet@cau.edu.cn.
- National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing, China. Electronic address: zengsm@cau.edu.cn.
MeSH Terms
- Animals
- Cryopreservation / veterinary
- Cryoprotective Agents
- Equidae
- Female
- Male
- Pregnancy
- Proline / pharmacology
- Semen
- Semen Preservation / veterinary
Citations
This article has been cited 8 times.- Zhang W, Min L, Li Y, Lang Y, Hoque SAM, Adetunji AO, Zhu Z. Beneficial Effect of Proline Supplementation on Goat Spermatozoa Quality during Cryopreservation. Animals (Basel) 2022 Sep 30;12(19).
- Yánez-Ortiz I, Catalán J, Delgado-Bermúdez A, Carluccio A, Miró J, Yeste M. Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm. Vet Sci 2021 Dec 2;8(12).
- Patriarca EJ, Cermola F, D'Aniello C, Fico A, Guardiola O, De Cesare D, Minchiotti G. The Multifaceted Roles of Proline in Cell Behavior. Front Cell Dev Biol 2021;9:728576.
- Dilixiati A, Zhao X, Aihemaiti A, Li W, Fan C, Zhao G, Wusiman A, Wang X. Multi-omics analysis reveals a protective role of endogenous proline in sheep oocyte vitrification and its therapeutic application. Front Cell Dev Biol 2025;13:1714529.
- Moradi M, Jahromi MG, Ghanbari E, Hashemian AH, Faramarzi A. L-Proline supplementation preserves sperm function and chromatin integrity in asthenozoospermic patients during cryopreservation. Basic Clin Androl 2025 Oct 13;35(1):39.
- Feng Q, Yang Y, Zhang B, Shi W, Fang Y, Xu C, Deng Z, Feng W, Shi D. The Effects of Limonin, Myo-Inositol, and L-Proline on the Cryopreservation of Debao Boar Semen. Animals (Basel) 2025 Jul 27;15(15).
- Wu S, Chen G, Zhan S, Wang L, Cao J, Guo J, Li L, Zhang H, Niu L, Zhong T. Liquid chromatograph-mass spectrometry metabolomics uncovers potential biomarkers of semen cryo-injury in goats. Anim Biosci 2025 Apr;38(4):629-640.
- Ramazani N, Mahd Gharebagh F, Soleimanzadeh A, Arslan HO, Keles E, Gradinarska-Yanakieva DG, Arslan-Acaröz D, Zhandi M, Baran A, Ayen E, Dinç DA. The influence of L-proline and fulvic acid on oxidative stress and semen quality of buffalo bull semen following cryopreservation. Vet Med Sci 2023 Jul;9(4):1791-1802.
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