Cryopreservation Strategies for Poultry Semen: A Comprehensive Review of Techniques and Applications
Simple Summary
Abstract
1. Introduction
2. Semen Collection and Evaluation
2.1. Pre-Collection Preparations
2.2. Collection Methods
2.2.1. Manual Collection (Massage Method)
2.2.2. Modern Collection
- Electroejaculation: Electroejaculation involves the use of mild electrical pulses to stimulate ejaculation in male birds. This technique is particularly useful when manual collection methods are ineffective or when a male bird is unresponsive to traditional collection methods. Studies, such as the one conducted by Santiago-Moreno et al. [28], have demonstrated the successful application of electroejaculation in galliform birds, including quail and pheasants. Electroejaculation has become an essential tool in both research and conservation settings, allowing for the collection and preservation of semen from valuable males without the need for natural mating. It is also a key technique in breeding programs where traditional methods may fail.
- Hormonal stimulation: Hormonal treatments can be used to stimulate semen production in males that are not responsive to conventional methods. This method typically involves the use of hormones like gonadotropins to enhance sperm production and induce ejaculation. Hormonal stimulation has been explored in poultry to improve the consistency and volume of semen collection, particularly in males with low sperm production [29].
2.3. Frequency of Semen Collection
- Variation by species: The optimal frequency for semen collection differs across poultry species. Chickens may require collection every 2–3 days, whereas turkeys maybe collected once a week [31].
- Reproductive phase: Semen collection often coincides with the bird’s reproductive phases especially during peak breeding times, when collection may be more frequent [31].
- Purpose-driven collection: The intended use of semen, whether for AI or research purposes, influences the frequency of collection. Adjustments are often made to meet specific breeding goals [32].
- Health and welfare issues: it is essential to ensure that the collection frequency does not adversely affect the bird’s health, as frequent collection may impact this [32].
- Optimal practices: adhering to established protocols [31] during semen collection is vital for preserving semen quality and optimize fertility rates in breeding initiatives.
2.4. Evaluation of Semen Quality
2.4.1. Semen Appearance (Macroscopic Evaluation)
2.4.2. Sperm Motility Assessment
- Light microscopy: Light microscopy is a widely used method where sperm cells are directly observed, providing a qualitative measure of motility. However, this approach is subjective and lacks a detailed analysis of sperm movement characteristics [40].
- Manual sperm motility scoring: manual sperm motility scoring classifies sperm as rapid, slow, or non-motile based on visual assessment, a technique that, while inexpensive, relies heavily on the technician’s experience.
- Viscosity test: the viscosity test evaluates the sperm’s ability to move through fluids of varying viscosities, simulating the challenges sperm face in the reproductive tract.
- Computer-assisted semen analysis (CASA): CASA utilizes advanced imaging and analytical software (Sperm Class Analyzer (SCA 6.5)) to automate the assessment of sperm parameters such as motility, concentration, and morphology [41]. It quantifies sperm movement using metrics like straight-line velocity (VSL), curvilinear velocity (VCL), and average path velocity (VAP). Sperm are categorized as slow (<10 μm/s), medium (10–50 μm/s), or rapid (>50 μm/s), providing a precise evaluation of their fertilization potential. The method’s high accuracy and reproducibility make it invaluable for research and breeding programs [42].
- Flow cytometry: This method assesses sperm motility, viability, concentration, and DNA integrity by analyzing light scatter and fluorescence. For example, light scatter indicates cell size and structure, while fluorescence reveals membrane integrity or DNA fragmentation. Flow cytometry (FlowJo (10.8)) enables rapid high-throughput analysis of sperm quality [43].
- Digital image analysis (DIA): DIA captures high-resolution images of sperm and uses specialized software (ImageJ (1.53) for quantitative motility assessments. Unlike manual scoring, it minimizes human error and provides detailed morphological and motility data, making it ideal for large-scale evaluations.
- High-resolution video microscopy: This technique records sperm movement in high definition at elevated frame rates, enabling the detection of subtle motility defects and abnormal behaviors. For instance, it can identify irregular flagellar motion or hyperactivated motility patterns, offering deeper insights into reproductive performance. Combining traditional and advanced techniques enhances the reliability of semen evaluation. For instance, integrating CASA with manual motility scoring ensures a comprehensive assessment: CASA provides precise quantitative data, while manual scoring offers practical insights in resource-limited settings. Studies have shown significant correlations between sperm concentration, motility, and morphology, further emphasizing the value of combining these methods [42].
2.4.3. Sperm Morphology
2.4.4. Biochemical Tests
3. Semen Preservation Techniques
3.1. Short-Term Preservation
3.1.1. Dilution with Extenders and Cryoprotectants
- Glycerol is used in extenders like the Lake PC extender to prevent ice formation and stabilize sperm membranes.
- Dimethylformamide (DMF) is utilized in the BHSV extender to lower the freezing point and protect against osmotic stress.
- Dimethylacetamide (DMA) is found in the FEB extender to reduce ice formation and osmotic damage during freezing.
- Ethylene glycol (EG) is added to several extenders to penetrate sperm membranes and prevent intracellular ice formation.
- Propylene glycol (PG) stabilizes sperm membranes and reduces damage during freezing.
- Dimethyl sulfoxide (DMSO) protects sperm by penetrating the cell membrane and preventing ice formation.
- Trehalose stabilizes proteins and lipids during freezing, enhancing sperm membrane protection.
- Egg yolk provides protection due to its high lipid content, stabilizing sperm membranes during freezing and thawing.
- Polyvinylpyrrolidone (PVP) enhances sperm motility and protects membranes during freezing.
- Myo-inositol is an antioxidant that reduces oxidative damage during freezing and thawing.
- Citrate maintains sperm pH and reduces oxidative stress, enhancing sperm quality.
- Vitamins (e.g., Vitamin E and C) are antioxidants that neutralize free radicals, protecting sperm from oxidative damage.
- Formamide is an external CPA that has proven effective in some species for protecting sperm during cryopreservation.
3.1.2. Importance of Dilution
3.1.3. Limitations of Short-Term Preservation
3.1.4. Extenders in Current Use
3.1.5. Recommended Dilution Rates
3.1.6. Research Gaps and Future Directions
3.1.7. Procedure Overview
- Gently mixing the semen with the extender.
- Cooling the mixture to 4 °C for 15 min.
- The extender containing the internal cryoprotective agents (11% glycerol, 6% DMA, or 6% DMF) was also equilibrated at 4 °C.
- The CPA-enriched extender was added to the semen samples and allowed to equilibrate at 4 °C.
- Semen was subsequently transferred into sealed 0.5 mL plastic freezing straws and then placed in a biological freezer unit and frozen using the controlled cooling rates detailed for each experiment and treatment in Table 1 before being plunged into liquid nitrogen. All of the following experiments on dilution rate, MET assay, insemination dose, storage duration, and R+ family restoration relied on glycerol-based freezing protocol.
3.1.8. Pricing Information
Internal Cryoprotectant | GLY | EG | DMF | DMA |
---|---|---|---|---|
Extender | Lake PC | BHSV | BHSV | FEB |
External cryoprotectant | PVP | Myo-inositol | Myo-inositol | PVP |
Semen collection | 1 mL semen in 1 mL extender | 1 mL semen in 1 mL extender | 1 mL semen in 1 mL extender | 1 mL semen in 1 mL extender |
Cooling | 4 °C, 15 min | 4 °C, 15 min | 4 °C, 15 min | 4 °C, 15 min |
% Internal CPA | 11% | 10% | 6% | 6% |
CPA addition | In 1 mL extender | In 1 mL extender | In 1 mL extender | In 1 mL extender |
Equilibriation time | 10 min | 10 min | 4 min | 2 min |
Freezing rate | −7 °C/min | −1/min | −15 °C/min | −60 °C/min |
Thawing (°C -min) | 4 °C, 3 min | 4 °C, 3 min | 4 °C, 3 min | 40 °C, 5 s |
Glycerol removal | Dilution 1/20, 550 g 4 °C—15 min | No glycerol removal | No glycerol removal | No glycerol removal |
No. of female/treatment | 20 | 20 | 20 | |
IA dose | 400 × 106 spz | 400 × 106 spz | 400 × 106 spz | 400 × 106 spz |
No. of AI | 5 | 5 | 5 | 5 |
Frequency of AI | Every day 4 d | Every day 4 d | Every day 4 d | Every day 4 d |
Egg collection | J2 to J5 | J2 to J5 | J2 to J5 | J2 to J5 |
Cost per 100 mL Cryoprotectant | USD 50–USD 150 | USD 50–USD 150 | USD 50–USD 150 | USD 50–USD 150 |
Cost per freezing straw | USD 0.20–USD 0.50 | USD 0.20–USD 0.50 | USD 0.20–USD 0.50 | USD 0.20–USD 0.50 |
Cost per liter of liquid nitrogen | USD 1.00–USD 3.00 | USD 1.00–USD 3.00 | USD 1.00–USD 3.00 | USD 1.00–USD 3.00 |
Labor and equipment costs per experiment | USD 20–USD 50 | USD 20–USD 50 | USD 20–USD 50 | USD 20–USD 50 |
Estimated cost per sample | USD 20–USD 50 | USD 20–USD 50 | USD 20–USD 50 | USD 20–USD 50 |
3.1.9. Specific Breeds of Hens
- White Leghorn: Renowned for its high fertility rates, this breed is commonly used in commercial egg production. Its reproductive efficiency makes it a prime candidate for cryopreservation studies, where maintaining high post-thaw fertility is essential.
- Rhode Island Red: Valued for its hardiness and adaptability, this breed is capable of thriving in various environmental conditions. Research indicates that its sperm may exhibit different viability characteristics during cryopreservation, making it an important breed to consider in the optimization of freezing protocols [44].
- Understanding the specific traits of these breeds is crucial for enhancing the effectiveness of cryopreservation techniques and ensuring successful fertility outcomes in poultry breeding programs.
3.2. Mid-Term Preservation
Conventional and Directional Freezing Techniques
3.3. Long-Term Preservation
3.3.1. CPAs Preservation Technique
3.3.2. Sperm Vitrification Techniques
3.3.3. Primordial Germ Cell Isolation and Cryopreservation
- Optimization of Cryopreservation Techniques
- Mechanism of Cryoinjury
- In Vitro Regeneration of Sperm
- Transgenic Applications
- Long-Term Viability Studies
3.3.4. Gonad Cryopreservation and Transplantation
4. Factors Affecting Semen Preservation
5. Evaluation of Preserved Semen
5.1. Post-Thaw Motility and Viability
5.1.1. Motility Parameters
5.1.2. Plasma Membrane Functionality
5.1.3. Lipid Peroxidation Malondialdehyde (MDA)
5.1.4. Mitochondrial Activity
- To evaluate mitochondrial activity, various assays are used to measure mitochondrial membrane potential, ATP production, and mitochondrial DNA integrity. Common techniques include the following:
- Mitochondrial membrane potential (Δψm): The mitochondrial membrane potential is a key indicator of mitochondrial health. The JC-1 assay is widely used, where healthy mitochondria accumulate the JC-1 dye and emit red fluorescence, while depolarized mitochondria emit green fluorescence.
- ATP measurement: ATP levels can be measured using bioluminescence-based assays, like the luciferase reaction. Decreased ATP levels are indicative of mitochondrial dysfunction.
- Mitochondrial DNA integrity: the integrity of mitochondrial DNA can be assessed using PCR-based techniques or by measuring oxidative damage markers.
- Recent Studies on Mitochondrial Activity and Cryopreservation:
- Mitochondria-Targeted Antioxidants:Astudy investigated the use of mitochondria-targeted antioxidants, such as MitoQ, to mitigate mitochondrial dysfunction and oxidative stress during sperm cryopreservation. The addition of MitoQ at a concentration of 0.02 μM improved post-thaw sperm motility, plasma membrane integrity, and sustained sperm motility for a longer duration. MitoQ supplementation prevented the significant reduction in mitochondrial membrane potential and reduced superoxide production, resulting in lower lipid peroxidation of the sperm plasma membrane after cryopreservation [122].
- Mitochondrial Bioenergetics:Another study evaluated the bioenergetic map of mitochondrial metabolism in cryopreserved bovine sperm. The research highlighted the impact of cryopreservation on mitochondrial function and its subsequent effect on sperm motility and viability [123].
- Antioxidant Supplementation:Research has also explored the effects of antioxidant supplementation, such as MitoTEMPO, before cryopreservation. MitoTEMPO has been reported to maintain mitochondrial function and viability during the freezing–thawing process through the inhibition of mitochondrial ROS production [124].
6. Applications of Poultry Semen Preservation
6.1. Conservation of Rare Breeds
6.2. Artificial Insemination
7. Future Directions and Research Needs
8. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Item |
L&R-84 pH = 7.08 343 mOsm/kg |
Lake 7.1 pH = 7.1 370 mOsm/kg |
EK media pH = 7.8 390 mOsm/kg |
BPSE pH = 7.5 333 mOsm/kg |
ASG pH = 7.1 325 mOsm/kg |
---|---|---|---|---|---|
Sodium-L-glutamate | 19.2 g | 15.2 g | 14 g | 8.76 g | 12.11 g |
Glucose | 8 g | 6.0 g | 7 g | 5.26 g | |
Magnesium acetate 4 H2O | 0.8 g | 0.8 g | 0.64 g | ||
Potassium acetate | 5.0 g | ||||
Polyvinyl pyrrolidone | 3.0 g | 1 g | |||
Potassium citrate tribasic 1 H2O | 1.28 g | 1.4 g | 0.6 g | 1.02 g | |
D-fructose | 2 g | 5.0 g | |||
TES | 1.95 g | ||||
Sodium acetate 3 H₂O | 4.3 g | ||||
Sodium hydroxide 1 N | 58 mL | 1.85 mL | |||
Sodium dihydrogen phosphate | 2.1 g | ||||
Disodium hydrogen phosphate | 9.8 g | ||||
BES | 30.5 g | 24.3 g | |||
Potassium diphosphate 3 H2O | 12.7 g | ||||
Potassium monophosphate | 0.65 g | ||||
Magnesium chloride | 0.34 g | ||||
Inositol | 7 g | ||||
Protamine sulfate | 0.2 g | ||||
H2O | 1000 mL | 1000 mL | 1000 mL | 1000 mL | 100 mL |
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Arif, A.; Zahoor, N.; Tang, J.; Tang, M.; Dong, L.; Khan, S.Z.; Dai, G. Cryopreservation Strategies for Poultry Semen: A Comprehensive Review of Techniques and Applications. Vet. Sci. 2025, 12, 145. https://doi.org/10.3390/vetsci12020145
Arif A, Zahoor N, Tang J, Tang M, Dong L, Khan SZ, Dai G. Cryopreservation Strategies for Poultry Semen: A Comprehensive Review of Techniques and Applications. Veterinary Sciences. 2025; 12(2):145. https://doi.org/10.3390/vetsci12020145
Chicago/Turabian StyleArif, Areej, Nousheen Zahoor, Jianqiang Tang, Meihui Tang, Liyue Dong, Sardar Zarq Khan, and Guojun Dai. 2025. "Cryopreservation Strategies for Poultry Semen: A Comprehensive Review of Techniques and Applications" Veterinary Sciences 12, no. 2: 145. https://doi.org/10.3390/vetsci12020145
APA StyleArif, A., Zahoor, N., Tang, J., Tang, M., Dong, L., Khan, S. Z., & Dai, G. (2025). Cryopreservation Strategies for Poultry Semen: A Comprehensive Review of Techniques and Applications. Veterinary Sciences, 12(2), 145. https://doi.org/10.3390/vetsci12020145