Influence of different centrifugation protocols on equine semen preservation.
Abstract: Three experiments were conducted to evaluate the impact of centrifugation on cooled and frozen preservation of equine semen. A standard centrifugation protocol (600 x g for 10 min=CP1) was compared to four protocols with increasing g-force and decreased time period (600 x g, 1200 x g, 1800 x g and 2400 x g for 5 min for CP2, 3, 4, and 5, respectively) and to an uncentrifuged negative control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on semen quality following centrifugation was assessed by monitoring several sperm parameters (membrane integrity using SYBR14-PI, acrosomal status using PSA-FITC, percentage total motility (TM), percentage progressive motility (PM) and beat cross frequency (BCF) obtained with computer assisted sperm analysis (CASA)) immediately after centrifugation and daily during chilled storage for 3 d. The use of CP1 resulted in a sperm loss of 22%. Increasing the centrifugation force to 1800 x g and 2400 x g for 5 min led to significantly lower sperm losses (7.4% and 2.1%, respectively; P<0.05). Compared to the uncentrifuged samples, centrifugation of semen resulted in a better sperm quality after chilled storage. There were minimal differences between the CPs although total motility was lower for CP2 than for the other treatments (P<0.005). In experiment 2, the centrifuged samples were cryopreserved using a standard freezing protocol and analyzed immediately upon thawing. Samples centrifuged according to CP2 resulted in a higher BCF (P<0.005), whereas CP3 and CP5 yielded a lower BCF (P<0.05) when compared to CP1. There were no post thaw differences between CP1 and CP4. In experiment 3, DNA integrity of the different samples was analyzed using TUNEL. Although DNA integrity decreased over time, CP had no impact. In conclusion, the loss of sperm cells in the supernatant after centrifugation can be substantially reduced by increasing the g-force up to 1800 x g or 2400 x g for a shorter period of time (5 min) compared to the standard protocol without apparent changes in semen quality, resulting in a considerable increase in the number of insemination doses per ejaculate.
Publication Date: 2010-03-06 PubMed ID: 20207406DOI: 10.1016/j.theriogenology.2010.01.022Google Scholar: Lookup
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- Comparative Study
- Journal Article
Summary
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This is a study on the impact of different centrifugation protocols on the preservation of equine semen. The results suggest that higher g-force centrifugation for shorter periods reduces sperm loss compared to standard centrifugation, without notably affecting the quality of the semen.
Overview of the research
The research focused on how different centrifugation processes can affect the preservation of horse semen, either cooled or cryopreserved (frozen). By changing the g-force (gravitational force) and the duration of the centrifugation, the scientists evaluated if these variations could reduce sperm cell loss and maintain sperm quality.
Structure of the Experiments
- The researchers compared a standard centrifugation procedure (termed CP1) to four alternatives (CP2-CP5) that used increased g-force and decreased time. The goal was to determine how much lowering the time and raising the g-force could decrease sperm loss. An uncentrifuged control group was also included.
- The effects on sperm cells after centrifugation were compared using various sperm indicators, such as membrane integrity, acrosomal status, total motility, progressive motility, and beat cross frequency (BCF). The post-centrifugation parameters were examined both immediately after the procedure and after three days of chilled storage.
- The centrifuged samples were also frozen using a standard method, after which the samples were defrosted and evaluated.
- In the final experiment, the researchers tested DNA integrity for each protocol variant. This was an important aspect to establish if the g-force had any effect on the DNA structure.
Results of the Study
- The standard procedure (CP1) led to a 22% sperm loss. By increasing the centrifugation force, the loss dropped significantly, reaching the lowest at 2.1% for a protocol that increased the g-force to 2400 x g for 5 minutes.
- The centrifuged semen had higher quality parameters after being stored chilled, compared to the uncentrifuged control batch. Among the centrifugation protocols, there were minor differences, but generally the variance of g-force and time period did not lead to drastic changes.
- In the freezing experiment, different CPs yielded different BCF results. For instance, the CP2 protocol had better results than CP3 and CP5, but there were no significant differences among CP1 and CP4.
- The DNA integrity analysis revealed that while DNA integrity decreased over time overall, the centrifugation process did not have a significant effect on this decrease.
Conclusions from the Research
The main finding of the study is that increasing the g-force during centrifugation, while reducing the duration, can decrease sperm loss without impacting the quality of sperm. This could lead to a higher quantity of insemination doses per ejaculation and be a considerable improvement for practices involving artificial insemination of horses.
Cite This Article
APA
Hoogewijs M, Rijsselaere T, De Vliegher S, Vanhaesebrouck E, De Schauwer C, Govaere J, Thys M, Hoflack G, Van Soom A, de Kruif A.
(2010).
Influence of different centrifugation protocols on equine semen preservation.
Theriogenology, 74(1), 118-126.
https://doi.org/10.1016/j.theriogenology.2010.01.022 Publication
Researcher Affiliations
- Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke, Belgium. maarten.hoogewijs@UGent.be
MeSH Terms
- Acrosome / ultrastructure
- Animals
- Cell Membrane / ultrastructure
- Centrifugation / adverse effects
- Centrifugation / methods
- Centrifugation / veterinary
- Cryopreservation / methods
- Cryopreservation / veterinary
- DNA Damage
- Fluorescent Dyes
- Horses
- Hot Temperature
- In Situ Nick-End Labeling
- Male
- Semen Preservation / methods
- Semen Preservation / veterinary
- Sperm Count
- Sperm Motility
- Spermatozoa / chemistry
- Spermatozoa / physiology
- Spermatozoa / ultrastructure
- Time Factors
Citations
This article has been cited 11 times.- 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).
- Sinagra L, Polisca A, Donato G, Caspanello T, Pettina G, Pastore S, De Majo M, Cristarella S, Quartuccio M, Zappone V. Enhancing canine semen quality through a second centrifugation after 48 hours of storage: a comparative study. Acta Vet Scand 2024 Sep 11;66(1):47.
- Sugai N, Werre S, Cecere JT, Balogh O. Comparing different sperm concentrations for optimizing cooled semen use in the dog. Front Vet Sci 2023;10:1339840.
- Sugai N, Werre S, Cecere J, Balogh O. Defining an Optimal Range of Centrifugation Parameters for Canine Semen Processing. Animals (Basel) 2023 Apr 21;13(8).
- 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.
- Sadeghi S, Del Gallego R, García-Colomer B, Gómez EA, Yániz JL, Gosálvez J, López-Fernández C, Silvestre MA. Effect of Sperm Concentration and Storage Temperature on Goat Spermatozoa during Liquid Storage. Biology (Basel) 2020 Sep 19;9(9).
- Marzano G, Moscatelli N, Di Giacomo M, Martino NA, Lacalandra GM, Dell'Aquila ME, Maruccio G, Primiceri E, Chiriacò MS, Zara V, Ferramosca A. Centrifugation Force and Time Alter CASA Parameters and Oxidative Status of Cryopreserved Stallion Sperm. Biology (Basel) 2020 Jan 27;9(2).
- Leemans B, Stout TAE, Soom AV, Gadella BM. pH-dependent effects of procaine on equine gamete activation†. Biol Reprod 2019 Nov 21;101(5):1056-1074.
- El-Bahrawy KA. The influence of caffeine supplementation and concerted utilization of enzymatic and mechanical semen liquefaction on freezability of dromedary camel spermatozoa. Int J Vet Sci Med 2017 Dec;5(2):121-127.
- Ahmed T, Islam R, Lone FA, Malik AA. Effect of washing on the post-thaw quality of cryopreserved ram epididymal spermatozoa. Vet World 2016 May;9(5):519-23.
- Gloria A, Carluccio A, Wegher L, Robbe D, Befacchia G, Contri A. Single and double layer centrifugation improve the quality of cryopreserved bovine sperm from poor quality ejaculates. J Anim Sci Biotechnol 2016;7:30.
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