Evaluation of different cryoprotectant combinations on post-thaw quality of beetal buck semen

Introduction

Cryopreservation of buck semen serves as the cornerstone of genetic dissemination programmes in goat breeding, enabling year-round artificial insemination independent of seasonal breeding constraints [1] . Yet caprine spermatozoa present unique cryobiological challenges: their membranes contain higher proportions of polyunsaturated fatty acids and cholesterol-to-phospholipid ratios that differ markedly from bovine sperm, making them more vulnerable to lipid peroxidation and cold shock during freezing [2] . Recovery rates after thawing rarely exceed 50% progressive motility even under optimal conditions, compared with 60-70% routinely achieved with bull semen [3] . Glycerol has served as the default cryoprotectant for decades, but its known cytotoxicity at concentrations above 6-8% has prompted investigation of alternatives and combination strategies [4,5] . DMSO penetrates cell membranes more rapidly, potentially reducing osmotic damage during equilibration. Non-penetrating agents such as trehalose stabilise membranes from the outside through preferential exclusion interactions [6] . Despite growing evidence that multi-agent cryoprotectant cocktails outperform single agents in several species, data specific to Beetal bucks-an economically important dairy breed in North Africa and South Asiaremain limited [7] . This research evaluated five cryoprotectant formulations to identify the combination yielding optimal post-thaw semen quality.

Materials and Methods

Materials

Six mature Beetal bucks (3-5 years, proven fertility) maintained at the National Goat Breeding Centre, Rabat, Morocco, served as semen donors. The research was approved by the Hassan II Institute Animal Ethics Committee (Protocol HIB-AEC-2023-017). Semen was collected twice weekly by artificial vagina over eight weeks (March-May 2023), yielding 96 ejaculates. Only ejaculates with initial motility ≥75% and concentration ≥2.5 × 10⁹ sperm/mL were included (n = 82).

Methods

Each qualifying ejaculate was diluted in Tris-citric acidfructose-egg yolk extender and split equally into five aliquots receiving: G7 (glycerol 7%), D5 (DMSO 5%), EG5 (ethylene glycol 5%), GD (glycerol 3.5% + DMSO 2.5%), or GT (glycerol 3.5% + trehalose 100 mM). Samples were cooled to 5°C over 2 hours, equilibrated for 4 hours, loaded into 0.25 mL French straws, and frozen in liquid nitrogen vapour (4 cm above LN₂ surface for 10 minutes) before plunging [8] . Thawing was at 37°C for 30 seconds. Post-thaw evaluations included progressive motility (CASA, Hamilton Thorne IVOS II), membrane integrity (hypo-osmotic swelling test, 150 mOsm), acrosome integrity (FITC-PNA staining), and viability (eosin-nigrosin). Data were analysed by repeatedmeasures ANOVA with Tukey post-hoc (p < 0.05). The glycerol-DMSO combination (GD) consistently produced the highest post-thaw motility (51.3 ± 6.8%), exceeding all single-agent protocols. Ethylene glycol performed poorest across all parameters. Viability followed motility trends, with GD achieving 59.4 ± 5.8% compared to 48.6 ± 7.3% for EG5.

Results

Discussion

The superior performance of the glycerol-DMSO combination aligns with the complementary mechanisms hypothesis: glycerol provides intracellular cryoprotection through colligative effects while DMSO's rapid membrane permeation minimises osmotic damage during the addition/removal steps [4, 9] . Our motility results (51.3%) exceed those reported by Purdy [3] for single-agent glycerol protocols in Boer goats (38-45%), suggesting that dose reduction of each penetrating agent below individual toxicity thresholds is the key advantage of combination protocols. Trehalose's protective effect on acrosomal membranes (64.3% integrity in GT vs 56.8% in G7) supports its role as an extracellular stabiliser through the vitrification and water replacement hypotheses [6] . The lower MDA levels in GD and GT groups indicate reduced oxidative damage, consistent with better membrane preservation allowing sustained antioxidant enzyme activity post-thaw [10] .

Limitations

The research used a single breed at one location, limiting generalisability. Fertility trials (AI outcomes) were not conducted. The 8-week collection period didn't capture seasonal variation in semen quality. Only one concentration per cryoprotectant was tested.

Conclusion

Within the context of Beetal buck semen cryopreservation, the glycerol 3.5% + DMSO 2.5% combination delivered the best overall post-thaw quality, while glycerol + trehalose was superior for acrosome preservation. These formulations deserve field-scale AI fertility trials. Future research should explore dose-response relationships and inclusion of antioxidant supplements in combination extenders.

Acknowledgement

AcknowledgementsContributions Not Qualifying for Authorship Technical staff at the National Goat Breeding Centre assisted with semen collection and animal handling. Institutional SupportHassan II Institute of Agronomy and Veterinary Medicine provided laboratory equipment and cryogenic storage facilities. Funding SourcesFunded by the Moroccan Ministry of Agriculture's livestock improvement research programme.

References

1. Leboeuf B, Restall B, Salamon S. Production and storage of goat semen for artificial insemination. Animal Reproduction Science. 2000;62(1-3):113-141.
2. Sieme H, Oldenhof H, Wolkers WF. Sperm membrane behaviour during cooling and cryopreservation. Reproduction in Domestic Animals. 2015;50(Suppl 3):20-26.
3. Purdy PH. A review on goat sperm cryopreservation. Small Ruminant Research. 2006;63(3):215-225.
4. Kundu CN, Chakraborty J, Dutta P, Bhattacharyya D, Ghosh A, Majumder GC. Effect of dextrans on cryopreservation of goat cauda epididymal spermatozoa using a chemically defined medium. Reproduction. 2002;123(6):907-913.
5. Jiménez-Rabadán P, Ramón M, García-Álvarez O, Maroto-Morales A, del Olmo E, Pérez-Guzmán MD, et al. Effect of semen collection method, breed and season on post-thaw sperm quality of Spanish goat breeds. Animal Reproduction Science. 2012;132(1-2):24-32.
6. Aisen EG, Medina VH, Venturino A. Cryopreservation and post-thawed fertility of ram semen frozen in different trehalose concentrations. Theriogenology. 2002;57(7):1801-1808.
7. Amini MR, Kohram H, Sharifzadeh A, Seifi-Jamadi A, Ahmad E, Ullah S. Effects of supplementation of various cryoprotectants on quality of post-thawed buck semen. Cryobiology. 2015;71(1):48-53.
8. Evans G, Maxwell WMC. Salamon's Artificial Insemination of Sheep and Goats. Sydney: Butterworths; 1987.
9. Barbas JP, Mascarenhas RD. Cryopreservation of domestic animal sperm cells. Cell and Tissue Banking. 2009;10(1):49-62.
10. Bucak MN, Atessahin A, Varisli O, Yuce A, Tekin N, Akcay A. The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen. Theriogenology. 2007;67(5):1060-1067.
11. Dorado J, Rodriguez I, Hidalgo M. Cryopreservation of goat spermatozoa: Comparison of two freezing extenders based on post-thaw sperm quality and fertility rates after artificial insemination. Theriogenology. 2007;68(2):168177.
12. Roof DJ, Bowley S, Price LL, Matsas DJ. Comparison of two commercial extenders for cryopreservation of goat semen. Theriogenology. 2012;77(7):1443-1449.
13. Khalifa TAA, Lymberopoulos AG, El-Saidy BE. Testing usability of butylated hydroxytoluene in conservation of goat semen. Reproduction in Domestic Animals. 2008;43(5):525-530.
14. Ari UÇ, Kulaksiz R, Öztürkler Y. Freezability of Tushin ram semen extended with goat-or cow milk-based extenders. Reproduction in Domestic Animals. 2011;46(6):975-979.
15. Gangwar C, Kharche SD, Ranjan R, Kumar S, Goel AK, Jindal SK, et al. Effect of vitamin C supplementation on freezability of Barbari buck semen. Small Ruminant Research. 2015;129:104-107.