Results of the “test-freeze” approach in a commercial program of stallion sperm cryopreservation and the relationship between pre-freeze sperm quality and “freezability.”.
Abstract: In the current study, we report the effect of different commercially available semen freezing extenders utilized for the "test-freeze" procedure for 13 years (2010-2023) as part of a commercial program of stallion sperm cryopreservation. Ejaculates obtained from sexually active and healthy stallions (n = 124) were cryopreserved using up to five commercially available semen freezing extenders (Lactose-EDTA [LE], MFR5, CryoMax LE [CMLE], CryoMax MFR5 [CMMFR5] or BotuCrio [BC]). Post-thaw sperm motility (total motility - TM [%]; progressive motility - PM [%]; and curvilinear velocity - VCL [μm/s]), sperm viability (VIAB [%]), and sperm DNA damage (COMP [%]) were compared among semen freezing extenders. Overall, TM, PM, and VIAB were similar for extenders LE, CMLE, and BC (P > 0.05) while higher than for extenders MFR5 or CMMFR5 (P < 0.05). Mean VCL was lower for extender LE than for extender BC (P < 0.05). An effect of semen extenders on mean COMP was not observed (P > 0.05). The proportion of ejaculates that yielded four different post-thaw sperm "freezability" indices, based on the average value between post-thaw TM and VIAB (MV; MV ≤ 41 % [LOW]; MV 42-54 % [AVERAGE]; or MV ≥ 55 % [HIGH]), or post-thaw PM: ≥30 %, was calculated. Extenders MFR5 and CMMFR5 yielded a higher proportion of stallions in the LOW category (36 and 37 %, respectively) compared to extenders LE (22 %), CMLE (20 %), or BC (21 %; P < 0.05). For the HIGH and PM ≥ 30 % categories, extenders LE (16 and 65 %), CMLE (15 and 66 %), and BC (15 and 61 %) yielded a higher proportion of stallions than extenders MFR5 (4 and 40 %) or CMMFR5 (2 and 45 %), respectively (P < 0.05). For the AVERAGE category, an effect of semen extenders was not observed (60-63 %; P > 0.05). The relationship between pre-freeze sperm quality parameters (i.e., sperm motility, morphology [Normal], viability, and COMP) and the indices of post-thaw "freezability" (LOW, AVERAGE, HIGH, PM ≥ 30 %) was also determined. The percentages of TMOT and Normal in fresh semen yielded the highest ROC values related to "freezability," irrespective of the index used: 1) LOW = % Normal - AUC 0.8328 (P < 0.0001); 2) AVERAGE = TMOT - AUC 0.5875 (P = 0.055); 3) HIGH = % TMOT - AUC: 0.8015 (P < 0.0001); 4) PM ≥ 30 % = % Normal - AUC 0.8392 (P < 0.0001). This study provides clinically relevant data regarding the effect of different semen freezing extenders on post-thaw stallion sperm quality and the potential relationship between some sperm quality parameters commonly assessed in practice and post-thaw sperm "freezability" in stallions.
Published by Elsevier Inc.
Publication Date: 2025-03-03 PubMed ID: 40049076DOI: 10.1016/j.theriogenology.2025.117373Google Scholar: Lookup
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Summary
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Research Overview
Ultraprocessing sperm from stallions using commercially available freezing extenders was analyzed over 13 years to evaluate post-thaw sperm quality and to identify pre-freeze sperm quality markers that predict the ability of sperm to survive freezing (“freezability”).
Background and Purpose
- The study focused on cryopreservation, a process of freezing and thawing stallion sperm for commercial use.
- Different commercially available semen freezing extenders were tested to determine which best preserved sperm quality after freezing.
- Another goal was to understand how pre-freeze sperm quality parameters relate to the success of sperm preservation (“freezability”).
Materials and Methods
- Subjects: 124 sexually active, healthy stallions were involved over a 13-year period (2010-2023).
- Freeze Extenders Tested: Five extenders — Lactose-EDTA (LE), MFR5, CryoMax LE (CMLE), CryoMax MFR5 (CMMFR5), and BotuCrio (BC).
- Measured Parameters Post-Thaw:
- Total motility (TM): Percentage of sperm moving.
- Progressive motility (PM): Percentage of sperm moving forward actively.
- Curvilinear velocity (VCL): Speed of sperm movement in μm/s.
- Sperm viability (VIAB): Percentage of live sperm.
- Sperm DNA damage (COMP): Percentage of sperm with DNA damage.
- Freezability Categories: Grouped based on averages of TM and VIAB or PM into LOW, AVERAGE, and HIGH freezability.
Key Findings
- Post-thaw Sperm Quality:
- Extenders LE, CMLE, and BC showed similar and better outcomes for TM, PM, and VIAB than MFR5 and CMMFR5.
- VCL was lower with LE compared to BC, suggesting sperm moved slower with LE.
- No significant differences in DNA damage (COMP) were observed among extenders.
- Freezability Indices Distribution:
- MFR5 and CMMFR5 extenders resulted in a higher proportion of sperm samples falling into the LOW freezability category.
- LE, CMLE, and BC extenders produced more samples classified as HIGH freezability and with PM ≥ 30%.
- AVERAGE category did not significantly differ among extenders.
- Relationship Between Pre-Freeze Sperm Quality and Freezability:
- Percentage of normal morphology (% Normal) and total motility (TMOT) in fresh semen were strong predictors of post-thaw freezability.
- Receiver Operating Characteristic (ROC) analysis showed high Area Under the Curve (AUC) values for these parameters, indicating good predictive value:
- LOW freezability best predicted by % Normal morphology (AUC = 0.83).
- HIGH freezability best predicted by % TMOT (AUC = 0.80).
- Progressive motility ≥30% also strongly associated with % Normal morphology (AUC = 0.84).
Implications and Conclusions
- The choice of sperm extender significantly affects post-thaw sperm quality, with LE, CMLE, and BC providing better preservation than MFR5 and CMMFR5.
- Evaluating sperm motility and morphology before freezing can help predict sperm’s ability to withstand freezing and thawing.
- These findings are valuable for optimizing commercial stallion sperm cryopreservation programs for better fertility outcomes.
- Continued monitoring and selection of extenders and pre-freeze sperm quality assessments can enhance success rates in horse breeding.
Cite This Article
APA
Hernández-Avilés C, Varner DD, Ramírez-Agámez L, Samper JC, Love CC.
(2025).
Results of the “test-freeze” approach in a commercial program of stallion sperm cryopreservation and the relationship between pre-freeze sperm quality and “freezability.”.
Theriogenology, 238, 117373.
https://doi.org/10.1016/j.theriogenology.2025.117373 Publication
Researcher Affiliations
- Equine Fertility Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA. Electronic address: chernandez@cvm.tamu.edu.
- Equine Fertility Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
- Equine Fertility Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
- Equine Fertility Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
- Equine Fertility Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
MeSH Terms
- Animals
- Male
- Cryopreservation / veterinary
- Cryopreservation / methods
- Horses / physiology
- Semen Preservation / veterinary
- Semen Preservation / methods
- Spermatozoa / physiology
- Semen Analysis / veterinary
- Cryoprotective Agents / pharmacology
- Sperm Motility
Conflict of Interest Statement
Conflicts of interest None.
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