Cryopreservation of equine sperm: optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry.
Abstract: Optimization of equine sperm cryopreservation protocols requires an understanding of the water permeability characteristics and volumetric shrinkage response during freezing. A cell-shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine sperm suspensions at cooling rates of 5 degrees C/min and 20 degrees C/min in the presence and absence of cryoprotective agents (CPAs), i.e., in the Kenney extender and in the lactose-EDTA extender, respectively. The equine sperm was modeled as a cylinder of length 36.5 microm and a radius of 0.66 microm with an osmotically inactive cell volume (V(b)) of 0.6V(o), where V(o) is the isotonic cell volume. Sperm samples were collected using water-insoluble Vaseline in the artificial vagina and slow cooled at < or = 0.3 degrees C/min in an Equitainer-I from 37 degrees C to 4 degrees C. By fitting a model of water transport to the experimentally obtained DSC volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The combined best-fit parameters of water transport (at both 5 degrees C/min and 20 degrees C/min) in Kenney extender (absence of CPAs) are L(pg) = 0.02 microm min(-1) atm(-1) and E(Lp) = 32.7 kcal/mol with a goodness-of-fit parameter R(2) = 0.96, and the best-fit parameters in the lactose-EDTA extender (the CPA medium) are L(pg)[cpa] = 0.008 microm min(-1) atm(-1) and E(Lp)[cpa] = 12.1 kcal/mol with R(2) = 0.97. These parameters suggest that the optimal cooling rate for equine sperm is approximately 29 degrees C/min and is approximately 60 degrees C/min in the Kenney extender and in the lactose-EDTA extender. These rates are predicted assuming no intracellular ice formation occurs and that the approximately 5% of initial osmotically active water volume trapped inside the cells at -30 degrees C will form innocuous ice on further cooling. Numerical simulations also showed that in the lactose-EDTA extender, equine sperm trap approximately 3.4% and approximately 7.1% of the intracellular water when cooled at 20 degrees C/min and 100 degrees C/min, respectively. As an independent test of this prediction, the percentage of viable equine sperm was obtained after freezing at 6 different cooling rates (2 degrees C/min, 20 degrees C/min, 50 degrees C/min, 70 degrees C/min, 130 degrees C/min, and 200 degrees C/min) to -80 degrees C in the CPA medium. Sperm viability was essentially constant between 20 degrees C/min and 130 degrees C/min.
Publication Date: 2001-12-26 PubMed ID: 11751286DOI: 10.1095/biolreprod66.1.222Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research article presents a study on equine sperm cryopreservation where the optimal cooling rate in different conditions was determined using a measure called differential scanning calorimetry (DSC).
Objective and Methodology
- The study sought to optimize the preservation of equine sperm by understanding its water permeability characteristics and its response to changes in volume during the freezing process. This was done using a method called differential scanning calorimetry (DSC).
- The researchers modeled the sperm cells for their experiments. The cells were considered as cylinders with specific dimensions and a degree of osmotically inactive cell volume. This was done to better study the water transport properties of the sperm.
- Two kinds of extenders were used, Kenney extender (absence of CPAs or cryoprotective agents) and lactose-EDTA extender (presence of CPAs), and the cooling rates were systematically changed to three different rates to examine the effects on the sperm cells.
Results and Findings
- The research found that sperm cells preserved in the Kenney extender had best-fit membrane permeability parameters under certain conditions. The same was true for the sperm in the lactose-EDTA extender.
- The optimal cooling rate for the sperm was discovered to be approximately 29 degrees C/min in the Kenney extender and approximately 60 degrees C/min in the lactose-EDTA extender. These rates were found by assuming that no intracellular ice formation occurs during the freezing process.
- The study also provided that there is a limited amount of osmotically active water volume trapped inside the cells when frozen at -30 degrees C, which would form innocuous ice upon further cooling.
- The research showed that in the lactose-EDTA extender, sperm trapped only a small percentage of the intracellular water when cooled at different rates. This suggests the importance of cooling rate in managing the volume of trapped water.
- Finally, the research also showed that the percentage of viable sperm after freezing was basically constant between cooling rates of 20 degrees C/min and 130 degrees C/min in the CPA medium.
Conclusions
- This study gives valuable insights into the optimal conditions for sperm cryopreservation, specifically concerning the cooling rates and the presence/absence of CPAs.
- The findings could potentially lead to improved methods for equine sperm preservation and higher viability rates, important considerations in veterinary science, artificial insemination, and equine breeding practices.
Cite This Article
APA
Devireddy RV, Swanlund DJ, Olin T, Vincente W, Troedsson MH, Bischof JC, Roberts KP.
(2001).
Cryopreservation of equine sperm: optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry.
Biol Reprod, 66(1), 222-231.
https://doi.org/10.1095/biolreprod66.1.222 Publication
Researcher Affiliations
- Department of Mechanical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA.
MeSH Terms
- Algorithms
- Animals
- Calorimetry, Differential Scanning
- Cell Membrane / physiology
- Cell Membrane / ultrastructure
- Cell Survival / physiology
- Computer Simulation
- Cryopreservation
- Cryoprotective Agents / pharmacology
- Freezing
- Horses / physiology
- In Vitro Techniques
- Male
- Semen Preservation
- Spermatozoa / drug effects
- Spermatozoa / physiology
- Temperature
- Water / metabolism
Citations
This article has been cited 12 times.- Bitton A, Frishling A, Kalo D, Roth Z, Arav A. The Impact of Precisely Controlled Pre-Freeze Cooling Rates on Post-Thaw Stallion Sperm. Animals (Basel) 2025 Dec 21;16(1).
- 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).
- Sharafi M, Borghei-Rad SM, Hezavehei M, Shahverdi A, Benson JD. Cryopreservation of Semen in Domestic Animals: A Review of Current Challenges, Applications, and Prospective Strategies. Animals (Basel) 2022 Nov 24;12(23).
- Johnson S, Hall C, Das S, Devireddy R. Freezing of Solute-Laden Aqueous Solutions: Kinetics of Crystallization and Heat- and Mass-Transfer-Limited Model. Bioengineering (Basel) 2022 Oct 10;9(10).
- Kilbride P, Meneghel J, Fonseca F, Morris J. The transfer temperature from slow cooling to cryogenic storage is critical for optimal recovery of cryopreserved mammalian cells. PLoS One 2021;16(11):e0259571.
- Meneghel J, Kilbride P, Morris GJ. Cryopreservation as a Key Element in the Successful Delivery of Cell-Based Therapies-A Review. Front Med (Lausanne) 2020;7:592242.
- Wolkers WF, Oldenhof H. Principles Underlying Cryopreservation and Freeze-Drying of Cells and Tissues. Methods Mol Biol 2021;2180:3-25.
- Shu Z, Hughes SM, Fang C, Hou Z, Zhao G, Fialkow M, Lentz G, Hladik F, Gao D. Determination of the Membrane Permeability to Water of Human Vaginal Mucosal Immune Cells at Subzero Temperatures Using Differential Scanning Calorimetry. Biopreserv Biobank 2016 Aug;14(4):307-13.
- Hagiwara M, Choi JH, Devireddy RV, Roberts KP, Wolkers WF, Makhlouf A, Bischof JC. Cellular biophysics during freezing of rat and mouse sperm predicts post-thaw motility. Biol Reprod 2009 Oct;81(4):700-6.
- Kleinhans FW, Mazur P. Determination of the water permeability (Lp) of mouse oocytes at -25 degrees C and its activation energy at subzero temperatures. Cryobiology 2009 Apr;58(2):215-24.
- Pinisetty D, Huang C, Dong Q, Tiersch TR, Devireddy RV. Subzero water permeability parameters and optimal freezing rates for sperm cells of the southern platyfish, Xiphophorus maculatus. Cryobiology 2005 Jun;50(3):250-63.
- Thirumala S, Huang C, Dong Q, Tiersch TR, Devireddy RV. A theoretically estimated optimal cooling rate for the cryopreservation of sperm cells from a live-bearing fish, the green swordtail Xiphophorus helleri. Theriogenology 2005 Jun;63(9):2395-415.
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