Factors affecting motion characteristics of frozen-thawed stallion spermatozoa.
Abstract: Five experiments were conducted to evaluate damage incurred in each processing step for cryopreservation of stallion spermatozoa. In Experiment 1, semen was centrifuged for 9 centrifugation times and the percentage of spermatozoa recovered after each treatment was calculated and spermatozoal motion characteristics analysed. Recovery of spermatozoa was > or = 80% when spermatozoa were centrifuged for > or = 10 min. Experiment 2 evaluated spermatozoa cryopreserved at 5 different concentrations in each of 2 extenders (skim milk-egg yolk-glycerol, SM-EYG; and lactose-EDTA, LAC). In SM-EYG, TMOT and PMOT were higher at spermatozoal concentrations of 20, 200 and 400 x 10(6)/ml (51%/41%, 52%/44%, 50%/43%, respectively) than for samples frozen at > or = 800 x 10(6) spermatozoa/ml (41%/35%, 32%/27%; P < 0.05). Spermatozoa frozen in LAC at a concentration of 20 x 10(6)/ml resulted in the highest TMOT and PMOT (43% and 30%, respectively, P < 0.05). The effect of freezing rate on motion characteristics of spermatozoa was evaluated in Experiment 3. The VCL of spermatozoa frozen in SM-EYG was the only parameter affected by freezing rate (P < 0.05). Experiment 4 evaluated motion characteristics after cryopreservation of spermatozoa in different sized straws (0.5 or 2.5 ml) in each of 2 extenders (SM-EYG and LAC). In SM-EYG, PMOT (38%) and VCL (109 microns/s) were highest when spermatozoa were frozen in 0.5 ml straws (P < 0.05). In Experiment 5, spermatozoa thawed immediately after cryopreservation or thawed after storage in liquid nitrogen for 24-48 h were evaluated. There was no effect of length of storage in liquid nitrogen on spermatozoal motion characteristics (P < 0.05). Experiment 6 evaluated the effects of cooling time to 5 degrees C (0, 2.5 and 5 h) on motion characteristics of spermatozoa cryopreserved in 2 extenders (SM-EYG and LAC). TMOT and PMOT were effected by cooling time, and there was a cooling-time-by-extender interaction (P < 0.05). In SM-EYG, TMOT and PMOT were higher if spermatozoa were cooled to 5 degrees C prior to initiation of freezing than if freezing was initiated at 20 degrees C (P < 0.05). A suggested protocol for cryopreservation of stallion spermatozoa would include: 1) centrifugation at 400 g for 14 to 16 min; 2) extension at 23 degrees C with SM-EYG to 400 x 10(6) spermatozoa/ml; 3) cool to 5 degrees C for 2.5 h; 4) package in 0.5 ml straws at 5 degrees C; 5) freeze in liquid nitrogen vapour at -160 degrees C; and 6) thaw for 30 s in 37 degrees C water.
Publication Date: 1996-01-01 PubMed ID: 8565953DOI: 10.1111/j.2042-3306.1996.tb01589.xGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research studied the processing steps of cryopreserving (freezing then reviving) stallion spermatozoa in order to minimize damage to the sperm. They found the most effective steps involved centrifuging at a particular speed for a specific time, preserving the spermatozoa in certain extenders at a specific concentration, and following a precise freezing and thawing protocol.
Objective of the Study
- The goal was to determine the optimum procedure for cryopreserving stallion spermatozoa. This was broken down into several separate experiments, each analyzing a different factor such as centrifugation times, spermatozoa concentration in different preservatives, freezing rate, straw size, and storage in liquid nitrogen.
Methods and Procedures
- The researchers conducted five separate experiments, each one looking at a different factor of the cryopreservation process.
- In Experiment 1, they examined different centrifugation times to see how they affected spermatozoa recovery rates.
- In Experiment 2, the effect of different sperm concentrations on their movement characteristics after freezing was studied.
- Experiment 3 explored the impact of the freezing rate on the motion characteristics of the spermatozoa.
- In Experiment 4, they evaluated the difference in motion characteristics of spermatozoa after cryopreservation in different-sized straws.
- Experiment 5 assessed whether storage duration in liquid nitrogen affected the motion characteristics of spermatozoa upon revival.
- Lastly, Experiment 6 delved into the effects of cooling time to 5 degrees Celsius on motion characteristics of spermatozoa in two preservatives.
Key Findings
- The study found that centrifuging sperm for more than 10 minutes led to a sperm recovery rate of more than 80%.
- It was found that the skim milk-egg yolk-glycerol (SM-EYG) extension maintained the highest motion characteristics when frozen at certain concentrations.
- Spermatozoa frozen in lactose-EDTA (LAC) at a concentration of 20×10(6)/ml resulted in the highest total motility (TMOT) and progressive motility (PMOT).
- Freezing rate affected only one factor, the curvilinear velocity (VCL), when preserved in SM-EYG.
- The optimum straw size for freezing sperm was found to be 0.5 ml, especially with the SM-EYG extender.
- The storage duration in liquid nitrogen showed no discernable impact on the motion characteristics of thawed spermatozoa.
- The study also discovered an interplay between cooling time and extender type. Cooling the spermatozoa before freezing boosted their TMOT and PMOT if done correctly.
Suggested Protocol for Cryopreservation
- The researchers provided a list of recommended steps for optimal cryopreservation. These incorporate their findings from the experiments and suggest the best practices for each parameter studied, from initial centrifuging stages to eventual thawing of the spermatozoa.
Cite This Article
APA
Heitland AV, Jasko DJ, Squires EL, Graham JK, Pickett BW, Hamilton C.
(1996).
Factors affecting motion characteristics of frozen-thawed stallion spermatozoa.
Equine Vet J, 28(1), 47-53.
https://doi.org/10.1111/j.2042-3306.1996.tb01589.x Publication
Researcher Affiliations
- Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins 80523, USA.
MeSH Terms
- Animals
- Cryopreservation / methods
- Cryopreservation / standards
- Cryopreservation / veterinary
- Edetic Acid / standards
- Egg Yolk
- Glycerol / standards
- Horses / physiology
- Lactose / standards
- Male
- Milk / standards
- Semen / physiology
- Semen Preservation / methods
- Semen Preservation / standards
- Semen Preservation / veterinary
- Sperm Motility / physiology
- Spermatozoa / physiology
- Time Factors
Citations
This article has been cited 3 times.- Ortiz-Rodriguez JM, Balao da Silva C, Masot J, Redondo E, Gazquez A, Tapia JA, Gil C, Ortega-Ferrusola C, Peña FJ. Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through regulating Akt phosphorylation and reduction of caspase 3. PLoS One 2019;14(7):e0211994.
- Di Iorio M, Rusco G, Lauriola F, Antenucci E, Roncarati A, Cerolini S, Schiavitto M, Iaffaldano N. Validating Sperm Concentration in Rabbit Cryopreservation Protocol: Implications for Fertility, Litter Size, and Offspring Growth. Vet Sci 2025 Jul 18;12(7).
- Di Iorio M, Lauriola F, Rusco G, Antenucci E, Schiavitto M, Iaffaldano N. Cryopreserving Rabbit Semen: Impact of Varying Sperm Concentrations on Quality and the Standardization of Protocol. Vet Sci 2023 Dec 22;11(1).
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