Iranian Journal of animal Science

Iranian Journal of animal Science

Effect of different levels of soy lecithin nanoliposomes in extender on the quality of canine sperm after freeze-thawing

Document Type : Research Paper

Author
Touba Nadri
Department of Animal Science, Urmia University
10.22059/ijas.2026.408382.654114
Abstract
Cryopreservation imposes physical and chemical stress on sperm, resulting in decreased motility, viability, and overall quality. This study aimed to investigate the effect of different concentrations of soy lecithin nanoliposomes (1, 2, and 3%) on the quality of sperm from Sarabi dogs after freezing-thawing. Sperm collection was performed manually from three Sarabi dogs using hand stimulation and a suitable collection device in the presence of a female dog in heat to stimulate sexual desire. The evaluation parameters included sperm motility, abnormalities (Hancock), sperm membrane integrity (eosin-nigrosin), acrosome membrane integrity (PSA), and membrane activity (HAST) after the freezing-thawing process. The results of this study showed that the extender containing 2% lecithin nanoliposomes exhibited higher total motility (65.5±1.8) compared to other nanoliposomal treatments, although this difference was not statistically significant compared to the control group (66.7±1.8) (P<0.05). The progressive motility results showed that the L2 group (55.5±1.5) was significantly higher than the other groups, although it did not differ significantly from the control treatment (52.3±1.5) (P<0.05). Similarly, the highest percentage of integrity, membrane activity, and acrosome integrity was related to the 2% lecithin nanoliposomes treatment, which did not differ significantly from the control treatment (P<0.05). Sperm abnormalities in this experiment were not significantly affected by the type of extender. Therefore, it seems that the use of the extender containing 2% lecithin Soy lecithin nanoliposomes can improve the properties of dog sperm after freeze-thaw.
Keywords
Dog
Sperm
Egg yolk
Lecithin
Freezing
Nanoparticle
Liposome
Subjects

Extended Abstract

Introduction

Sperm cryopreservation has significantly improved assisted reproductive technologies (ART) and has enabled the conservation of endangered dog breeds (Dalmazzo et al., 2018; Nguyen et al., 2019). However, a major challenge in cryopreservation is the risk of contamination from animal-derived components such as egg yolk or milk, which are commonly used in extenders (Bousseau et al., 1998). Egg yolk is widely known as a cryoprotectant that lowers the freezing point of the medium and reduces the formation of ice crystals (Farstad, 2000). Based on this, we hypothesized that a soy lecithin nanoliposome-based diluent would demonstrate better efficacy for canine sperm cryopreservation compared to a traditional egg yolk-based diluent. The development of such an optimal extender could improve canine sperm cryopreservation protocols for artificial insemination. However, the underlying cellular mechanisms through which egg yolk or soy lecithin affects in vivo fertility potential in dogs have not yet been fully elucidated. Therefore, this study aimed to evaluate a comprehensive set of parameters, including motility, morphological abnormalities and plasma membrane integrity, acrosome membrane integrity, and membrane activity to evaluate the optimal cryopreservation extender. Therefore, this study aimed to evaluate the effect of different concentrations of soy lecithin nanoliposome on the quality of frozen-thawed Sarabi dog sperm.

 

Materials and methods

Semen samples were collected from three healthy Sarabi dogs aged 3 to 4 years. Semen collection was performed manually by an experienced technician. To stimulate sexual desire, the samples were collected in the presence of a female dog in heat. Immediately after collection, each ejaculate was evaluated for color, volume, concentration, sperm motility, viability (plasma membrane integrity), morphology, and motility. Evaluation parameters included sperm motility, abnormalities (Hancock), sperm membrane integrity (eosin-nigrosin), acrosome membrane integrity (PSA), and membrane activity (HASt) after freeze-thaw.

 

Results and Discussion

The results of this study showed that the L2 and EY groups had the highest total mobility (65.5 ± 1.8 and 66.7 ± 1.8), which were significantly different from the L1 group (58.2 ± 1.8). Also, the results of progressive mobility showed that the L2 group (55.5 ± 1.5) was significantly higher than the other groups, although there was no significant difference with the control treatment (52.3 ± 1.5) (P<0.05).

The results of eosin-nigrosin staining are given in Table 2. The results of this evaluation showed that the L2 treatments (69.5 ± 1.3) and the control group (62.1 ± 0.3) showed the highest cell membrane integrity, and no significant difference was observed between these two treatments (P < 0.05). The lowest percentage of cell membrane integrity was in the L3 group (53.1 ± 0.3), which showed a significant difference with the L2 and control groups (P < 0.05). The sperm acrosome integrity results are given in Table 2. This study showed that the treatment based on 2% soy lecithin nanoliposomes had the highest percentage of acrosome integrity (1.78 ± 2.2), which was significantly different from other nanoliposome-based treatments.

 

Conclusion

This study showed that an extender containing 2% lecithin nanoliposomes was highly effective for canine sperm cryopreservation. The positive results observed establish it as an effective plant-based alternative for canine sperm cryopreservation and cryopreservation protocols. Therefore, it appears that soy lecithin could be successfully used as a plant-based alternative to egg yolk-based extender in canine cryopreservation environments.

 

Author Contributions

Touba Nadri: Conceptualization, Validation, Methodology, Software, Formal analysis,  Investigation, Writing - Original Draft, Supervision, Formal analysis, Investigation.

 

Data Availability Statement

The data supporting the reported results are collected and used from published articles, to which we have referred in the manuscript.

 

Ethical considerations

The authors avoided data fabrication, falsification, plagiarism, and misconduct.

 

Conflict of interest

The author declares no conflict of interest.

REFERENCES
Agarwal, A., Virk, G., Ong, C., & Du Plessis, S. S. (2014). Effect of oxidative stress on male reproduction. The world journal of men's health, 32(1), 1-17. DOI: 10.5534/wjmh.2014.32.1.1
Beccaglia, M., Anastasi, P., & Luvoni, G. (2009). Freezing of canine semen in an animal-free protein extender. Veterinary Research Communications, 33, 77-80.  DOI: 10.1007/s11259-009-9249-9
Bousseau, S., Brillard, J., Marquant-Le Guienne, B., Guerin, B., Camus, A., & Lechat, M. (1998). Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents. Theriogenology, 50(5), 699-706. DOI: 10.1016/s0093-691x(98)00175-7
Bucak, M. N., Ateşşahin, A., Varışlı, Ö., Yüce, A., Tekin, N., & Akçay, A. (2007). The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen: microscopic and oxidative stress parameters after freeze–thawing process. Theriogenology, 67(5), 1060-1067.  DOI: 10.1016/j.theriogenology.2006.12.004
Dalmazzo, A., Losano, J. D. A., Rocha, C. C., Tsunoda, R. H., Angrimani, D. d. S. R., Mendes, C. M., Assumpção, M. E. O. D. Á., Nichi, M., & Barnabe, V. H. (2018). Effects of soy lecithin extender on dog sperm cryopreservation. Animal biotechnology, 29(3), 174-182.  DOI: 10.1080/10495398.2017.1334662
Dix, T. A., & Aikens, J. (1993). Mechanisms and biological relevance of lipid peroxidation initiation. Chemical research in toxicology, 6(1), 2-18.  https://doi.org/10.1021/tx00031a001
Farstad, W. (2000). Assisted reproductive technology in canid species. Theriogenology, 53(1), 175-186. Doi.org/10.1016/S0093-691X(99)00250-2
Forouzanfar, M., Sharafi, M., Hosseini, S., Ostadhosseini, S., Hajian, M., Hosseini, L., Abedi, P., Nili, N., Rahmani, H., & Nasr-Esfahani, M. (2010). In vitro comparison of egg yolk–based and soybean lecithin–based extenders for cryopreservation of ram semen. Theriogenology, 73(4), 480-487.  DOI: 10.1016/j.theriogenology.2009.10.005
Helmerich, G., & Koehler, P. (2003). Comparison of methods for the quantitative determination of phospholipids in lecithins and flour improvers. Journal of agricultural and food chemistry, 51(23), 6645-6651.  https://doi.org/10.1021/jf0345088
Hermansson, U., Johannisson, A., & Axnér, E. (2021). Cryopreservation of dog semen in a Tris extender with two different 1% soybean preparations compared with a Tris egg yolk extender. Veterinary Medicine and Science, 7(3), 812-819.  DOI: 10.1002/vms3.445
Judde, A., Villeneuve, P., Rossignol‐Castera, A., & Le Guillou, A. (2003). Antioxidant effect of soy lecithins on vegetable oil stability and their synergism with tocopherols. Journal of the American Oil Chemists' Society, 80(12), 1209-1215.  https://doi.org/10.1007/s11746-003-0844-4
Kmenta, I., Strohmayer, C., Müller-Schlösser, F., & Schäfer-Somi, S. (2011). Effects of a lecithin and catalase containing semen extender and a second dilution with different enhancing buffers on the quality of cold-stored canine spermatozoa. Theriogenology, 75(6), 1095-1103.  DOI: 10.1016/j.theriogenology.2010.11.018
Nadri, T., Towhidi, A., Zeinoaldini, S., Riazi, G., Sharafi, M., Zhandi, M., Kastelic, J., & Gholami, D. (2022). Supplementation of freezing medium with encapsulated or free glutathione during cryopreservation of bull sperm. Reproduction in Domestic Animals, 57(5), 515-523.  DOI: 10.1111/rda.14088
Nadri, T., Towhidi, A., Zeinoaldini, S., Riazi, G., Sharafi, M., Gholami, D., Riazi, GH., & Martínez-Pastor, F. (2025). Impact of particle size of nanoliposomes on the biological response of frozen-thawed sperm in Holstein bulls. Cryobiology, Volume 118,105208. DOI.org/10.1016/j.cryobiol.2025.105208.
Nasir, M. I., Bernards, M. A., & Charpentier, P. A. (2007). Acetylation of soybean lecithin and identification of components for solubility in supercritical carbon dioxide. Journal of agricultural and food chemistry, 55(5), 1961-1969.  DOI: 10.1021/jf0618832
Nguyen, V. V., Ponchunchoovong, S., Kupittayanant, S., & Kupittayanant, P. (2019). Effects of egg yolk and soybean lecithin on sperm quality determined by computer‐assisted sperm analysis and confocal laser scanning microscope in chilled canine sperm. Veterinary Medicine and Science, 5(3), 345-360.  DOI: 10.1002/vms3.158
Parks, J. E., & Hammerstedt, R. H. (1985). Development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane. Biol Reprod, 32(3), 653-668.  DOI: 10.1095/biolreprod32.3.653
Parks, J. E., & Lynch, D. V. (1992). Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes. Cryobiology, 29(2), 255-266.  DOI: 10.1016/0011-2240(92)90024-v
Pearodwong, P., Suwimonteerabutr, J., Rungruangsak, J., & Tummaruk, P. (2019). Comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of boar semen.  DOI: orcid.org/0000-0001-7000-4371 
Reed, M. L., Ezeh, P. C., Hamic, A., Thompson, D. J., & Caperton, C. L. (2009). Soy lecithin replaces egg yolk for cryopreservation of human sperm without adversely affecting postthaw motility, morphology, sperm DNA integrity, or sperm binding to hyaluronate. Fertility and sterility, 92(5), 1787-1790.  DOI: 10.1016/j.fertnstert.2009.05.026
Shah, S. A. H., Andrabi, S. M. H., Ahmed, H., & Qureshi, I. Z. (2017). Chicken egg yolk plasma in tris-citric acid extender improves the quality and fertility of cryopreserved water buffalo (Bubalus bubalis) spermatozoa. Theriogenology, 89, 32-40.  DOI: 10.1016/j.theriogenology.2016.10.009
Sun, L., Fan, W., Wu, C., Zhang, S., Dai, J., & Zhang, D. (2020). Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation. Cryobiology, 92, 146-150.  DOI: 10.1016/j.cryobiol.2019.12.004
Tavilani, H., Doosti, M., Nourmohammadi, I., Mahjub, H., Vaisiraygani, A., Salimi, S., & Hosseinipanah, S. M. (2007). Lipid composition of spermatozoa in normozoospermic and asthenozoospermic males. Prostaglandins, leukotrienes and essential fatty acids, 77(1), 45-50.  DOI: 10.1016/j.plefa.2007.07.001
Zhao, F., Li, R., Liu, Y., & Chen, H. (2023). Perspectives on lecithin from egg yolk: Extraction, physicochemical properties, modification, and applications. Frontiers in nutrition, 9, 1082671.  https://doi.org/10.3389/fnut.2022.1082671
Iranian Journal of animal Science
Volume 57, Issue 3
Summer 2026
Pages 517-527

  • Receive Date 22 December 2025
  • Revise Date 21 April 2026
  • Accept Date 27 April 2026