Animal Reproduction (AR)
Animal Reproduction (AR)

A preliminary study on the effects of E-Z Mixin® and EquiPlus® extenders supplemented with Edible Bird’s Nest on the quality of chilled Arabian stallion semen

Khalid Al-Khaldi; Nurhusien Yimer; Samir Al-Bulushi; Abd Wahid Haron; Mark Hiew; Abdul Salam Babji

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The aim of this study was to evaluate the effects of adding different concentrations of edible bird’s nest (EBN) which is secreted by swiftlet birds (Aerodramus fuciphagus), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using computer assisted sperm analysis (CASA) and viability were assessed using Hoechst33342/PI stain. In both extenders, total motility (TM) and progressive motility (PM) were significantly higher at 0.12% and 0.24% compared to 0.24% + SP at 24 and 48 h. At 0.12%, E-Z mixin® treated semen had significantly higher TM and PM than EquiPlus® at 24 and 48 h. At 0.12% and 0.24%, average path velocity (VAP), straight-line velocity (VSL) and curvilinear velocity (VCL) were significantly higher in E-Z mixin® treated semen compared to EquiPlus® at 24 and 48 h. Comparisons between the two extender types at different concentrations of EBN showed no significant difference in lateral head amplitude (ALH), linearity (LIN), straightness (STR), beat cross frequency (BCF) and viability, irrespective of the storage time. The percentage of viable was significantly higher in E-Z mixin® than EquiPlus® at 0 and 48 h in control and 0.12%. Supplementation of the E-Z mixin® extender with 0.12% and 0.24% EBN concentrations in the absence of SP provided better CASA parameters such as TM, PM, VAP, VSL, and VCL at 24 and 48 h storage time. In conclusion, the results of this study indicated that chilled semen from Arabian stallion that was extended using E-Z mixin® and supplemented with 0.12% and 0.24% EBN concentrations performed better and yielded superior results in sperm kinetic parameters and % viable compared to EquiPlus® at 24 and 48 h storage time.


Arabian stallion, EBN, chilled semen, EquiPlus®, E-Z Mixin®


Ahmad H, Abdul Khalid SK, Rashed AA, Aziz SA. Effects of sialic acid from edible bird nest on cell viability associated with brain cognitive performance in mice. World J Traditional China Med. 2019;5(4):214-19.

Aitken RJ, Bronson R, Smith TB, De Iuliis GN. The source and significance of DNA damage in human spermatozoa; a commentary on diagnostic strategies and straw man fallacies. Mol Hum Reprod. 2013;19(8):475-85.

Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction. 2001;122(4):497-506. PMid:11570956.

Alamaary MS, Haron AW, Ali M, Hiew MWH, Adamu L, Peter ID. Effects of four extenders on the quality of frozen semen in Arabian stallions. Vet World. 2019;12(1):34-40. PMid:30936651.

Albishtue AA, Yimer N, Zakaria MZA, Haron AW, Babji AS, Abubakar AA, Almhanawi BH. Effects of EBN on embryo implantation, plasma concentrations of reproductive hormones, and uterine expressions of genes of PCNA, steroids, growth factors and their receptors in rats. Theriogenology. 2019;126:310-19. PMid:30605790.

Al-Bulushi S, Manjunatha BM, Bathgate R, Rickard JP, Graaf SP. Liquid Storage of dromedary camel semen in different extenders’. Anim Reprod Sci. 2019;207:95-106. PMid:31208848.

Al-Essawe EM, Johannisson A, Wulf M, Aurich C, Morrell JM. Improved cryosurvival of stallion spermatozoa after colloid centrifugation is independent of the addition of seminal plasma. Cryobiology. 2018a;81:145-52. PMid:29397923.

Al-Essawe EM, Wallgren M, Wulf M, Aurich C, Macías-García B, Sjunnesson Y, Morrell JM. Seminal plasma influences the fertilizing potential of cryopreserved stallion sperm. Theriogenology. 2018b;115:99-107. PMid:29747159.

Al-Kass Z, Spergser J, Aurich C, Kuhl J, Schmidt K, Morrell JM. Effect of presence or absence of antibiotics and use of modified single layer centrifugation on bacteria in pony stallion semen. Reprod Domest Anim. 2019;54(2):342-9. PMid:30351456.

Alvarenga MA, Le˜ao KM, Papa FO, Landim-Alvarenga FC, Medeiros ASL, Gomes GM. The use of alternative cryoprotectors for freezing stallion semen. In: Proceedings of Workshop on Transporting Gametes and Embryos, Havemeyer Foundation; 2003; Brewster, Massachusetts, USA. Newmarket, UK: R&W Publications; 2003. p. 74-6.

Amidi F, Pazhohan A, Shabani Nashtaei M, Khodarahmian M, Nekoonam S. The role of antioxidants in sperm freezing: a review. Cell Tissue Bank. 2016;17(4):745-56.

Baumber J, Ball BA, Linfor JJ. Assessment of the cryopreservation of equine spermatozoa in the presence of enzyme scavengers and antioxidants. Am J Vet Res. 2005;66(5):772-9. PMid:15934604.

Blanchard TL, Varner DD, Miller C, Roser J. Recommendations for clinical GnRH challenge testing of stallions. J Equine Vet Sci. 2010;20:682-6.

Consuegra C, Crespo F, Bottrel M, Ortiz I, Dorado J, Diaz Jimenez M, Pereira B, Hidalgo M. Stallion sperm freezing with sucrose extenders: a strategy to avoid permeable cryoprotectants. Anim Reprod Sci. 2018;191:85-91. PMid:29482878.

Dowsett KF, Knott LM. The influence of age and breed on stallion semen. Theriogenology. 1996;46(3):397-412.

Ghassem M, Arihara K, Mohammadi S, Sani NA, Babji AS. Identification of two novel antioxidant peptides from edible bird’s nest (Aerodramus fuciphagus) protein hydrolysates. Food Funct. 2017;8(5):2046-52. PMid:28497137.

Hernández-Avilés C, Love CC, Serafini R, Ramírez-Agámez L, Friedrich M, Ghosh S, Teague SR, LaCaze KA, Brinsko SP, Varner DD. Effects of glucose concentration in semen extender and storage temperature on stallion sperm quality following long-term cooled storage. Theriogenology. 2020;147:1-9. PMid:32070880.

Hou ZM, Imam U, Ismail M, Azmi NH, Ismail N, Ideris A, Mahmud R. Lactoferrin and ovotransferrin contribute toward antioxidative effects of Edible Bird’s Nest against hydrogen peroxide-induced oxidative stress in human SH-SY5Y cells. Biosci Biotechnol Biochem. 2015;79(10):1570-8. PMid:26057702.

Hu Q, Li G, Yao H, He S, Li H, Liu S, Wu Y, Lai X. Edible bird’s nest enhances antioxidant capacity and increases lifespan in Drosophila Melanogaster. Cell Mol Biol. 2016;62(4):116-22. PMid:27188745.

Ismail M, Al-Naqeep G, Chan KW. Nigella sativa thymoquinone-rich fraction greatly improves plasma antioxidant capacity and expression of antioxidant genes in hypercholesterolemic rats. Free Radic Biol Med. 2010;48(5):664-72. PMid:20005291.

Kathiravan P, Kalatharan J, Edwin MJ, Veerapandian C. Computer automated motion analysis of crossbred bull spermatozoa and its relationship with in vitro fertility in zona-free hamster oocytes. Anim Reprod Sci. 2008;104(1):9-17. PMid:17254723.

LeFrapper L, Walston L, Whisnant C. Comparison of various extenders for storage of cooled stallion spermatozoa for 72 hours. J Equine Vet Sci. 2010;30(4):200-4.

Ma F, Liu D. Sketch of the edible bird’s nest and its important bioactivities. Food Res Int. 2012;48(2):559-67.

Marcone MF. Characterization of the edible bird’s nests the “Caviar of the East”. Food Res Int. 2005;38(10):1125-34.

Morton K, Billah M, Skidmore J. Effect of sperm diluent and dose on the pregnancy rate in dromedary camels after artificial insemination with fresh and liquid-stored semen. J Camelid Sci. 2013;6:49-62.

Neuhauser S, Bollwein H, Siuda M, Handler J. Comparison of the effects of five semen extenders on the quality of frozen-thawed equine epididymal sperm. J Equine Vet Sci. 2019;79:1-8. PMid:31405486.

Oliveira RA, Wolf CA, De Oliveira VMAO, Gambarini ML. Addition of glutathione to an extender for frozen equine semen. J Equine Vet Sci. 2013;33(12):1148-52.

Ortega-Ferrusola C, Sotillo-Galan Y, Varela-Fernandez E, Gallardo-Bolanos JM, Muriel A, Gonzalez-Fernandez L, Tapia JA, Pena FJ. Detection of “apoptosis-like” changes 485 during the cryopreservation process in equine sperm. J Androl. 2007;29(2):213-21. PMid:17978341.

Peris SI, Bilodeau JF, Dufour M, Bailey JL. Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm. Mol Reprod Dev. 2007;74(7):878-92. PMid:17186553.

Pojprasath T, Lohachit C, Techakumphu M, Stout T, Tharasanit T. Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender. Theriogenology. 2011;75(9):1742-9. PMid:21458054.

Squires EL, Keith SL, Graham JK. Evaluation of alternative cryoprotectants for preserving stallion spermatozoa. Theriogenology. 2004;62(6):1056-65. PMid:15289047.

Usuga A, Rojano B, Restrepo G. Effect of seminal plasma components on the quality of fresh and cryopreserved stallion semen. J Equine Vet Sci. 2017;58:103-11.

Vidament M, Yvon JM, Couty I, Arnaud G, Nguekam-Feugang J, Noue P, Cottron S, Le Tellier A, Noel F, Palmer E, Magistrini M. Advances in cryopreservation of stallion semen in modified INRA82. Anim Reprod Sci. 2001;68(3-4):201-18. PMid:11744265.

Waheed MM, Al-Khaldi KO, Pratap N. Effect of extenders and insemination protocol on the fertilizing capacity of cryopreserved Arabian horse semen. SOJ Veterinary Sciences. 2016;2(2):1-8.

Whittington K, Ford WCL. Relative contribution of leukocytes and of spermatozoa to reactive oxygen species production in human sperm suspensions. Int J Androl. 1999;22(4): 229-235.

Wong RSY. Edible bird’s nest: food or medicine. Chin J Integr Med. 2013;19(9):643-9.

Yagi H, Yasukawa N, Yu SY, Guo CT, Takahashi N, Takahashi T, Bukawa W, Suzuki T, Khoo KH, Suzuki Y, Kato K. The expression of sialylated high-antennary N-glycans in edible bird’s nest. Carbohydr Res. 2008;343(8):1373-7. PMid:18439991.

Yida Z, Imam MU, Ismail M, Der Jiun O, Sarega N, Azmi NH, Ismail N, Chan KW, Hou Z, Yusuf NB. Edible bird’s nest prevents high fat diet-induced insulin resistance in rats. J Diabetes Res. 2015;2015:760535. PMid:26273674.

Yusop DR, Yimer N, Yusoff R, Haron AW, Ibrahim Khushairay ES. Edible Birds’ Nest (EBN) hydrolysate for bovine sperm cryopreservation. JSM Invitro Fertil. 2016;1(1):1004.

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