Comparative Evaluation of Quail Egg Yolk-Based Extender for Semen Cryopreservation in Baggara Bulls
Mohamed A. El-Khalifa1, Bashir Salim2*, Safaa Abdelrahim1, Zeinab A.E. Osman1, Ahmed M. Khalid3,4, Saeed Alasmari5 and Shadia A. Omer6
1Department of Animal Genetic Improvement, Animal Production Research Center, Khartoum North, Sudan
2Camel Research Center, King Faisal University, P.O. Box 400, Al-Hofuf 31982, Al-hsa, Saudi Arabia
3Department of Public Health, College of Veterinary Medicine, King Faisal University, P.O. Box 400, Al-Hofuf 31982, Al-Ahsa, Saudi Arabia
4Department of Genetics and Animal Breeding, Faculty of Animal Production, University of Khartoum, Shambat 13314, Sudan
5Department of Biology, Faculty of Arts and Sciences, Najran University, Najran 1988, Kingdom of Saudi Arabia
6Department of Biomedical Sciences, College of Veterinary Medicine, Sudan University of Science and Technology
ABSTRACT
This study evaluated the efficacy of a quail egg yolk-based extender for the cryopreservation of Baggara bull semen, compared to hen egg yolk and a commercial egg yolk-free diluent (IMV). Baggara cattle, a hardy breed native to Sudan and known for their adaptability to harsh environments, are vital to local economies, making advancements in their reproductive biotechnology crucial. Sperm quality was assessed both pre- and post-freezing, focusing on motility, vigor, and morphology. Pre-freezing analysis showed that the quail egg yolk extender achieved slightly higher mean motility (82.07%) compared to the hen egg yolk (81.61%) and IMV (79.84%) extenders. Post-freezing, the quail extender maintained competitive motility (59.79%), similar to the hen extender (61.29%) and higher than the IMV extender (56.17%). Although the quail extender exhibited a slightly higher motility degradation rate, ANOVA results revealed no statistically significant differences in pre-freezing motility, post-freezing motility, or motility degradation among the extenders. These findings suggest that the quail egg yolk-based extender is a promising alternative to the traditional hen egg yolk extender for cryopreservation of Baggara bull semen. However, further optimization is needed, particularly to improve post-thaw sperm morphology, to fully harness its potential in preserving this important cattle breed.
Article Information
Received 22 December 2024
Revised 05 May 2025
Accepted 21 May 2025
Available online 21 August 2025
(early access)
Published 13 December 2025
Authors’ Contribution
MAE, SAO designed, conceptualized research and supervised the work. MAE, SA, ZAEO contributed sampling carried out the experiments. AMK, SA contributed to the analysis and the interpretation of the results. BS conceptualized research writing original draft. All authors provided critical feedback and helped shape the research, analysis and manuscript.
Key words
Cryopreservation, Quail egg yolk-based extender, Baggara bulls, Post-mortem semen preservation, Cattle
DOI: https://dx.doi.org/10.17582/journal.pjz/20241222085027
* Corresponding author: [email protected]
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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Introduction
Cryopreservation of bovine semen plays a pivotal role in cattle breeding, enabling the long-term storage and global transportation of valuable genetic material. Traditionally, semen collection is performed using live animals through methods such as electroejaculation or artificial vagina. However, post-mortem semen collection offers an alternative for preserving valuable genetics from animals that die unexpectedly or are euthanized. This approach is particularly relevant for breeds like Baggara cattle, where maintaining genetic diversity is critical.
Baggara cattle, indigenous to Sudan, are renowned for their exceptional adaptability to harsh environmental conditions, making the conservation of their genetics crucial for maintaining cattle biodiversity (Salim et al., 2014; Kambal et al., 2022; Tijjani et al., 2022). As the primary beef-producing breed in Sudan, Baggara cattle are uniquely capable of thriving in extreme desert climates. Their resilience and strong resistance to diseases make them particularly well-suited to the challenging local conditions (Salim et al., 2021). Given their importance, efficient cryopreservation techniques are essential for preserving the genetic material of Baggara bulls, especially in resource-limited settings. This study addresses the need for improved semen preservation methods by evaluating the effectiveness of various extenders for cryopreservation in this vital cattle breed.
Traditionally, egg yolk-based extenders, particularly those derived from hen eggs, have been extensively used for cryopreserving bovine semen due to their protective effects against cold shock and oxidative stress during freezing (Vishwanath and Shannon, 2000). However, alternative egg yolk sources, such as quail egg yolk, have gained attention due to their distinct lipid composition and potential cryoprotective properties (Trimeche et al., 1979). Quail egg yolk, with its higher phospholipid and cholesterol content, is thought to offer enhanced membrane stability and protection against cryodamage, making it a promising alternative for semen preservation.
Despite advancements in cryopreservation, limited research has explored the efficacy of quail egg yolk-based extenders in cattle semen preservation. This study aims to fill this gap by evaluating the performance of quail egg yolk-based extenders in preserving Baggara bull semen. The study compares the quail extender with traditional hen egg yolk and a commercial egg yolk-free diluent, hypothesizing that the quail egg yolk-based extender may provide comparable or even superior protection during the freezing and thawing process.
This research offers valuable insights into the conservation of genetic material in cattle, especially when conventional semen collection methods are not feasible. The findings have significant implications for the preservation of Baggara bulls and may serve as a model for improving semen cryopreservation techniques in other cattle breeds facing similar challenges.
Materials and Methods
Experimental site
This study was conducted at the Department of Animal Genetic Improvement, Animal Production Research Center (APRC) located in Hilat Koko, Sudan. The primary objective of the study was to evaluate the viability and cryopreservation potential of epididymal sperm collected post-mortem from Baggara bulls. The experiment was carried out under controlled laboratory conditions to ensure consistent handling and storage of the samples.
Testis collection
The testes were collected from mature Baggara bulls immediately after slaughter. The collection was performed at the Animal Production Research Center’s slaughterhouse to minimize the time between death and sperm recovery as described by Garcia-Macias et al. (2006). A total of five pairs of testes were carefully extracted from the carcasses and placed in sterile zip-lock bags. These bags were maintained at a temperature of 25°C and immediately transported to the semen processing laboratory, where epididymal sperm recovery was initiated.
Quality assessment of the collected sperm
Sperm quality was assessed immediately after recovery to determine the initial viability of the samples. Evaluation was performed using a phase-contrast microscopeat 400× magnification. The assessment included key focusing on conventional parameters such as motility, vigor, viability, and morphology. Motility was evaluated by placing a drop of raw semen on a clean, pre-warmed slide (37°C), covered with a coverslip, and examining under phase-contrast microscopy. The percentage of spermatozoa exhibiting progressive forward movement was estimated and recorded rated on a scale from 0% to 100%. While vigor was assessed visually on a 0 to 5 scale, where 0 indicated no progressive movement and 5 indicated vigorous and rapid, linear progression of sperm cells across the field of view. Additionally, sperm viability and morphology assessments were evaluated using staining technique. A small aliquot of semen was mixed with an equal volume of eosin-nigrosin stain, smeared onto a glass slide, air-dried, and examined under the microscope. At least 200 sperm cells per slide were evaluated. Live sperm remained unstained (white), while dead sperm appeared pink due to eosin penetration. Morphological abnormalities such as bent tails, detached heads, and midpiece defects were recorded following standard classification criteria (Barth and Oko, 1989; Sprecher and Coe, 1996).
Semen dilution and cryopreservation
After the initial sperm quality assessment, the recovered spermatozoa were divided into three experimental groups, each corresponding to a different semen extender. The first group was diluted with a Tris-citric acid-based diluent containing hen egg yolk, which is traditionally used in cattle semen cryopreservation (Medeiros et al., 2002; Grötter et al. 2019). The second group was diluted with a novel Tris-citric acid-based diluent containing quail egg yolk, while the third group was processed using a commercial egg yolk-free extender as a control (IVM Technologies, France).
The semen samples were diluted at a 1:4 ratio and allowed to equilibrate for 4 h at 5°C. Following equilibration, the diluted semen samples were packaged into 0.25 ml straws and frozen in liquid nitrogen vapor for 10 minutes before being plunged into liquid nitrogen for one month before post freezing evaluation.
Statistical analysis
The sperm motility, viability, and morphology data collected post-thaw were analyzed using one-way ANOVA to compare the performance of the different extenders. Differences between the three extenders were considered significant at a p-value of <0.05, and post hoc analysis was conducted using Duncan’s Multiple Range test (DMRT) to identify specific group differences. All statistical analyses were performed using SPSS software (version 25.0).
Results
Pre-freezing motile sperm comparison
The pre-freezing motility of Baggara bull sperm was evaluated using three extenders: Quail egg yolk, hen egg yolk, and a commercial egg yolk-free diluent (IMV). The quail egg yolk extender demonstrated a slightly higher mean motility of 82.07±7.64% compared to 81.61±7.67% for the hen extender and 79.84±9.43% for the IMV extender. The overall mean motility across all extenders was 81.20±8.29%. Motility values across extenders ranged from 50.0% to 95.0%, with the quail extender showing a slight advantage in preserving motility before freezing compared to the other extenders (Table I).
Post-freezing motile sperm comparison
After cryopreservation, sperm motility was again assessed across the three extenders. The quail egg yolk extender maintained a mean post-freezing motility of 59.79±16.16%, slightly lower than the hen egg yolk extender at 61.29±14.00%, but higher than the IMV extender, which resulted in the lowest motility at 56.17±13.33%. The overall post-freezing motility across all extenders was 59.08±14.68%. The quail and hen extenders exhibited better post-thaw motility preservation compared to the IMV extender, highlighting the quail egg yolk as a competitive alternative (Table II).
Motility degradation rate
The motility degradation rate, calculated as the difference between pre-freezing and post-freezing motility, was also compared across extenders. The quail egg yolk extender showed a mean degradation rate of 14.86±15.38%, while the hen egg yolk extender had a slightly lower degradation rate of 13.67±13.56%.The IMV extender exhibited the lowest degradation rate at
Table I. Pre-freezing motile sperm comparison of baggara bulls using different extenders.
|
Extender type |
N |
Mean (%) |
SD |
SE |
95% Confidence interval for mean |
Minimum (%) |
Maximum (%) |
|
Pre-freezing motile sperm |
|||||||
|
Quail |
70 |
82.07 |
7.6373 |
0.9128 |
80.25 - 83.892 |
60.0 |
95.0 |
|
Hen |
62 |
81.61 |
7.6702 |
0.9741 |
79.66 - 83.561 |
50.0 |
95.0 |
|
IVM |
64 |
79.84 |
9.4268 |
1.1783 |
77.49 - 82.198 |
50.0 |
95.0 |
|
Total |
196 |
81.199 |
8.2875 |
0.5920 |
80.03 - 82.366 |
50.0 |
95.0 |
|
Post-freezing motile sperm |
|||||||
|
Quail |
70 |
59.786 |
16.161 |
1.932 |
55.932 - 63.639 |
30.0 |
90.0 |
|
Hen |
62 |
61.290 |
13.995 |
1.777 |
57.736 - 64.844 |
30.0 |
90.0 |
|
IMV |
64 |
56.172 |
13.326 |
1.666 |
52.843 - 59.501 |
10.0 |
85.0 |
|
Total |
196 |
59.082 |
14.682 |
1.049 |
57.013 - 61.150 |
10.0 |
90.0 |
SD, standard deviation; SE, standard error
Table II. Motility degradation rate of baggara bulls using different extenders.
|
Extender type |
N |
Mean (%) |
SD |
SE |
95% Confidence interval for mean |
Minimum (%) |
Maximum (%) |
|
Quail |
70 |
14.856 |
15.380 |
1.838 |
11.1889 - 18.523 |
0.00 |
53.85 |
|
Hen |
62 |
13.668 |
13.564 |
1.723 |
10.2237 - 17.113 |
0.00 |
50.00 |
|
IVM |
64 |
12.613 |
17.551 |
2.194 |
8.2287 - 16.997 |
0.00 |
100.00 |
|
Total |
196 |
13.748 |
15.545 |
1.110 |
11.5579 - 15.938 |
0.00 |
100.00 |
SD, standard deviation; SE, standard error
Table III. Anova results for pre-freezing motile sperm, post-freezing motile sperm, and motility degradation rate.
|
Parameter |
Sum of squares |
df |
Mean square |
F |
p-value |
|
Pre-freezing motile sperm |
1.325 |
0.268 |
|||
|
Between groups |
181.450 |
2 |
90.725 |
||
|
Within groups |
13211.790 |
193 |
68.455 |
||
|
Post-freezing motile sperm |
2.061 |
0.130 |
|||
|
Between groups |
879.025 |
2 |
439.512 |
||
|
Within groups |
41155.669 |
193 |
213.242 |
||
|
Motility degradation rate |
0.347 |
0.707 |
|||
|
Between groups |
168.761 |
84.381 |
|||
|
Within groups |
46951.405 |
243.272 |
12.61±17.55%. Despite the lower degradation rate for IMV, the quail and hen extenders showed more consistent post-thaw motility, suggesting their effectiveness in maintaining sperm quality. Degradation rates were highly variable, with some samples showing no degradation and others as high as 100% (Table II).
Comparison of extenders
ANOVA tests were performed to statistically compare the pre-freezing motility, post-freezing motility, and motility degradation rate among the three extenders (quail egg yolk, hen egg yolk, and IMV). The analysis revealed nostatistically significant differences in pre-freezing motility across the extenders (F = 1.325, p = 0.268). Similarly, post-freezing motility did not differ significantly between the extenders (F= 2.061, p= 0.130). Furthermore, the motility degradation rate, which measures the decline in motility from pre-freezing to post-freezing, also showed no significant differences among the extenders (F= 0.347, p= 0.707). These findings suggest that, although quail egg yolk extender performed comparably to the other extenders, the observed differences in motility preservation and degradation are not statistically significant (Table III).
Discussion
This study evaluated the efficacy of a quail egg yolk-based extender for the cryopreservation of Baggara bull semen, comparing it with traditional hen egg yolk-based and commercial egg yolk-free extenders. The findings revealed that quail egg yolk offers a viable alternative cryoprotectant, demonstrating comparable, and in some aspects, superior performance.
Egg yolk has long been recognized for its cryoprotective properties, protecting sperm from the damage caused by chilling temperatures (Mandal et al., 2014). While hen egg yolk has been traditionally used in bovine semen extenders, the results from this study indicate that quail egg yolk yielded favorable outcomes, particularly in terms of sperm motility, viability, and vigor. The quail egg yolk extender outperformed both the hen egg yolk-based and commercial egg yolk-free extenders, suggesting its potential as a superior cryoprotectant.
The enhanced performance of the quail egg yolk-based extender can be attributed to its unique composition, which includes higher concentrations of lipoproteins and phospholipids compared to hen egg yolk (Swelum et al., 2023). These elements are essential for stabilizing the sperm membrane during cryopreservation, preventing cold shock, and minimizing ice crystal formation, which can otherwise damage sperm cells (Mandal et al., 2014). This finding aligns with prior research, which suggests that alternative egg yolk sources may offer improved cryoprotective effects (Sharafi et al., 2022). In this study, post-thaw sperm motility, viability, and vigor did not show significant differences between the quail egg yolk extender and the hen egg yolk or commercial egg yolk-free diluents. Nevertheless, the comparable performance of the quail egg yolk-based extender suggests it may serve as a viable and economical alternative for cattle semen cryopreservation, particularly for indigenous breeds such as Baggara, which are adapted to challenging environments and may require tailored preservation strategies (Sharafi et al., 2022).
In this study, post-thaw sperm motility, viability, and vigor were significantly higher with the quail egg yolk extender than with the hen egg yolk and commercial egg yolk-free diluents. This indicates that quail egg yolk-based extenders may provide a more effective and economical alternative for cattle semen cryopreservation, particularly for indigenous breeds such as Baggara, which are adapted to challenging environments and may require specialized preservation techniques (Sharafi et al., 2022).
Cryopreserving sperm from indigenous cattle breeds is crucial for maintaining genetic diversity and supporting sustainable breeding programs, particularly in regions like Sudan, where Baggara cattle are integral to local economies and food security (Rewe, 2009). Successfully cryopreserving semen from these bulls ensures that their valuable genetics can be conserved and used in future breeding programs, even after the animal’s death. This study underscores the importance of novel cryopreservation methods for preserving the genetics of Baggara bulls, which may have broader applications for other indigenous breeds in Africa and beyond. Furthermore, quail egg yolk-based extenders could provide an economical and accessible option for small-scale farmers and breeders who depend on indigenous cattle for their livelihoods.
While this study demonstrated the efficacy of quail egg yolk-based extenders, further research is required to optimize these protocols for broader application in cattle breeding programs. Future studies should investigate long-term fertility outcomes following artificial insemination with semen preserved using quail egg yolk-based extenders. Additionally, comparative research involving other indigenous cattle breeds and alternative to hen egg yolk could further enhance semen preservation techniques.
A practical limitation encountered with the use of quail egg yolk as an extender component is the relatively small volume of yolk obtained from a single egg compared to that of a hen egg. Consequently, multiple quail eggs are required to achieve the same quantity of yolk, potentially increasing preparation time and introducing variability between extender batches. Moreover, the limited commercial availability of quail eggs, particularly in rural or resource-constrained settings, may restrict the broader application of this alternative in routine semen cryopreservation protocols.
Conclusion
In conclusion, this study offers valuable insights into the use of quail egg yolk-based extenders for the cryopreservation of Baggara bull semen. The findings suggest that quail egg yolk is a viable and effective alternative to hen egg yolk-based extenders, providing comparable performance in maintaining post-thaw sperm quality. This research represents a significant advancement in the development of more efficient cryopreservation techniques for indigenous cattle breeds, contributing to their genetic conservation and promoting sustainable agriculture.
Declarations
Acknowledgements
We sincerely thank our colleagues at the Animal Production Research Center, Department of Animal Genetic Improvement, for their support and motivation throughout this study. Special appreciation goes to Dr. Manal Abdelwahid for her invaluable assistance, as well as Dr. Thoypa Karar, Dr. Yasir Ahmed Hassan, and Dr. Eman Elkhair for their advice. We are also grateful to Dr. Sadia Hashin, Mrs. Mona Dafalala, Mrs. Omima Mohamed Salih, and Miss Enas for their help with sample preparation and analysis, and to the laboratory attendants and Mr. Balla for their contributions.
Funding
This study received no external funding
IRB approval
This study was approved by the Animal Ethics Committee of Sudan University of Science and Technology (Approval No. SUST2108025).
Ethics approval and consent to participate
We confirm that the study adheres to the ARRIVE guidelines (https://arriveguidelines.org). The study protocol underwent review and approval by Sudan University of Science and Technology, Research Committee. Research procedures were conducted in accordance with their guidelines for sampling domestic animals in Sudan, ensuring ethical compliance in the collection and utilization of the samples.
Availability of data and materials
All data generated or analyzed during this study are included in the manuscript.
Statement of conflict of interest
The authors have declared no conflict of interest.
References
Barth, A.D. and R.J., 1989. Abnormal morphology of bovine spermatozoa. Iowa State University Press, Ames.
Garcia-Macias, V., Martinez-Pastor, F., Alvarez, M., Garde, J.J., Anel, E., Anel, L. and de Paz, P., 2006. Assessment of chromatin status (SCSA) in epididymal and ejaculated sperm in Iberian red deer, ram and domestic dog. Theriogenology, 56: 1921-30. https://doi.org/10.1016/j.theriogenology.2006.05.011.
Grötter, L.G., Cattaneo, L., Marini, P.E., Kjelland, M.E. and Ferré LB., 2019. Recent advances in bovine sperm cryopreservation techniques with a focus on sperm post-thaw quality optimization. Reprod. Domest. Anim., 54: 655-665. https://doi.org/10.1111/rda.13409
Kambal, S., Abdelrahim, A.E., Hanotte, O., Nakao, R., Alkhaibari, A.M. and Salim, B., 2022. Demographic expansion and high level of matrilineal diversity in two populations of East African Baggara cattle. J. Anim. Breed Genet., 139: 161-169. https://doi.org/10.1111/jbg.12648.
Mandal, R., Damodar, D. and Jitamanyu, J., 2014. Role of membrane lipid fatty acids in sperm cryopreservation. Adv. Androl., 9: 190542. https://doi.org/10.1155/2014/190542
Medeiros, C.M., Forell, F., Oliveira, A.T. and Rodrigues, J.L., 2002. Current status of sperm cryopreservation: why isn’t it better? Theriogenology, 57:327-44. https://doi.org/10.1016/s0093-691x(01)00674-4
Rewe, T.O., Herold, P., Kahi, A.K. and Zárate, A.V., 2009. Breeding indigenous cattle genetic resources for beef production in Sub-Saharan Africa. Outl. Agric., 38: 317-326. https://doi.org/10.5367/000000009790422205
Salim, B., Taha, K.M., Hanotte, O., Mwacharo, J.M., 2014. Historical demographic profiles and genetic variation of the East African Butana and Kenana indigenous dairy zebu cattle. Anim. Genet., 45: 782-790. https://doi.org/10.1111/age.12225
Salim, B., Takeshima, S.N., Nakao, R., Moustafa, M.A.M., Ahmed, M.A., Kambal, S., Mwacharo, J.M., Alkhaibari, A.M. and Giovambattista, G., 2021. BoLA-DRB3 gene haplotypes show divergence in native Sudanese cattle from taurine and indicine breeds. Sci. Rep., 11: 17202. https://doi.org/10.1038/s41598-021-96330-7
Sharafi, M., Borghei-Rad, S.M., Hezavehei, M., Shahverdi, A. and Benson, J.D., 2022. Cryopreservation of semen in domestic animals: A review of current challenges, applications, and prospective strategies. Animal (Basel), 12: 3271. https://doi.org/10.3390/ani12233271
Sprecher, D.J. and Coe, PH., 1996. Differences in bull spermiograms using eosin-nigrosin stain, feulgen stain, and phase contrast microscopy methods. Theriogenology, 45:757-64. https://doi.org/10.1016/0093-691x(96)00005-2
Swelum, A.A., Ba-Awadh, H.A., Olarinre, I.O., Saadeldin, I.M. and Alowaimer, A.N., 2023.Correlation between fatty acids levels in chicken, duck, goose, pigeon, quail and turkey egg yolks and post-thawed quality of ram semen. Reprod. Domest. Anim., 58: 1298-1310. https://doi.org/10.1111/rda.14434
Tijjani, A., Salim, B., da Silva, M.V.B., Eltahir, H.A., Musa, T.H., Marshall, K., Hanotte, O. and Musa, H.H., 2022. Genomic signatures for drylands adaptation at gene-rich regions in African zebu cattle. Genomics, 114: 110423. https://doi.org/10.1016/j.ygeno.2022.110423
Trimeche, A., Anton, M., Renard, P., Gandemer, G. and Tainturier, D., 1997. Quail egg yolk: A novel cryoprotectant for the freeze preservation of Poitou jackass sperm. Cryobiology, 34: 385-393. https://doi.org/10.1006/cryo.1997.2009
Vishwanath, R. and Shannon, P., 2000. Storage of bovine semen in liquid and frozen state. Anim. Reprod. Sci., 62: 23-53. https://doi.org/10.1016/S0378-4320(00)00153-6