Influence of Egg Yolk Substitution with Nano-L-α phosphatidylcholine on Sperm Kinetics, Lipid Peroxidation, and Antioxidant Activity of Cryopreserved Buffalo Bull Spermatozoa

Ghania, Asmaa; Abdel-Ghaffar, Alaa Elsayed; Sosa, Gamal Abdel-raheem Mohamed; Badr, Magdy Ramadan; Agag, Mohsen Abd Elhafeez; Kandiel, Mohamed Mahmoud M.; Sakr, Abdelaziz M.; Kortam, Laila E.

doi:10.21608/javs.2024.323359.1427

Influence of Egg Yolk Substitution with Nano-L-α phosphatidylcholine on Sperm Kinetics, Lipid Peroxidation, and Antioxidant Activity of Cryopreserved Buffalo Bull Spermatozoa

Document Type : Original Article

Authors

¹ Theriogenology Department, Faculty of Veterinary Medicine, Benha University, Benha, Egypt

² Theriogenology Department, Faculty of Veterinary Medicine, Benha University, Benha, Egypt. Department of Clinical Veterinary Sciences, Faculty of Veterinary Medicine, Delta University for Science and Technology, Gamasa, Dakahlia, Egypt.

³ Department of Artificial Insemination and Embryo Transfer, Animal Reproduction Research Institute (ARRI), Agriculture Research Center (ARC), 12556 Haram, Giza, Egypt.

⁴ Theriogenology Department, Faculty of Veterinary Medicine, Benha University, Benha, Egypt.

⁵ Animal Production Research Institute, Agricultural Research Center (A RC), Dokki, Giza, Egypt

⁶ Immunity Unit, Animal Reproduction Research Institute (ARRI), Agriculture Research Center (ARC), 12556 Haram, Giza, Egypt

10.21608/javs.2024.323359.1427

Abstract

This study aimed to examine how varying concentrations (0.5%, 1.0%, 2.0%, 3.0%, and 4.0%) of nano-L-α phosphatidylcholine (nano-L-α-PC) affected the quality of post-thawed buffalo bull semen compared to the same concentrations of L-α-phosphatidylcholine (L-α-PC), while 20% egg yolk (EY) was used as a control. Ejaculates (n = 72) were collected from fertile buffalo bulls (n = 12) for 6 weeks. The ejaculates were pooled and divided into 11 groups (3 aliquots per group) extended with 20% EY (Group 1 without supplement), 0.5%, 1.0%, 2.0%, 3.0%, and 4.0% nano-L-α-PC (Groups 2–6), and 0.5%, 1.0%, 2.0%, 3.0%, and 4.0% L-α-PC (Groups 7–11) in tris buffer. After cryopreservation procedures, semen samples were thawed at 37 °C and then evaluated for sperm kinematics, acrosomes, plasma membranes, and DNA integrities. The seminal plasma was analyzed for malondialdehyde (MDA), superoxide dismutase (SOD), glutathione reduced (GSH), and catalase (CAT). Results demonstrated that extended semen samples containing 1.0-3.0% nano-L-α-PC and 1.0-4.0% L-α-PC showed high total motility compared to control, whereas at 0.5–4.0% of nano-L-α-PC and L-α-PC showed high progressive motility. Significant (P< 0.05) wobble levels were seen at 0.5, 1.0, and 3.0% nano-L-α-PC, and distance average path and velocity average path at 2.0% L-α-PC. Acrosome and plasma membrane integrities were markedly elevated (P< 0.0001) at 2.0% nano-L-α-PC and 4.0% L-α-PC. High DNA integrity metrics were noticed at 0.5–1.0% nano-L-α-PC, as well as 3.0% and 4% L-α-PC. Low MDA and high (SOD, GSH, and CAT) levels were observed at 2.0% nano-L-α-PC and 4.0% L-α-PC. In conclusion, the optimum concentration of nano-L-α-PC that improved semen quality was 2.0%, which is roughly equivalent to the effect of 4.0% L-α-PC.

Keywords

Main Subjects

Theriogenelogy

References

ABDEL-KHALEK, A. E., DOWIDAR, Y. A., EL-NAGAR, H. A., WAFA, W. M., EL-RATEL, I. T., and MOUSBAH, A. M., 2022. A Review on various antioxidants utilized in bovine semen extenders. Journal of Applied Veterinary Sciences.7(2): 13–24. https://doi.org/10.21608/JAVS.2022.115377.1120

ABOELMAATY, A., EL-SEADAWY, I.E.S., EL-DEBAKY, H.A., HOZYEN, H.F., EL-BADRY, D.A., ANWER, A.M., TSVETKOV, T., and DANEVA, T., 2023. The optimum concentration of soya-lecithin for replacing egg yolk in INRA-82-modified semen extenders improved the cryopreserved semen quality and DNA integrity of Arabian stallions. Applied Veterinary Research. 2(4): 2023019-2023019..https://doi.org/10.31893/avr.2023019

ADAMI, L.N.G., DE LIMA, B.T., ANDRETTA, R.R., BERTOLLA, R.P., and NICHI, M., 2020. Carnosine treatment during human semen processing by discontinuous density gradient. Andrologia. 5(2): e13497. https://doi.org/10.1111/and.13497.

AKHTER, S., ANSARI, M.S., RAKHA, B.A., ANDRABI, S.M.H., IQBAL, S., and ULLAH, N., 2010. Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcell® extender. Theriogenology. 74(6): 951–955. https://doi.org/10.1016/j.theriogenology.2010.04.024.

AKHTER, S., ANSARI, M.S., RAKHA, B.A., ULLAH, N., ANDRABI, S.M.H., and KHALID, M., 2011. In vitro evaluation of liquid‐stored buffalo semen at 5 C diluted in soya lecithin based extender (Bioxcell®), tris‐Citric egg yolk, skim milk and egg yolk‐citrate extenders. Reproduction in Domestic Animals. 46(1): 45–49. https://doi.org/10.1111/j.1439-0531.2009.01561.x.

ALMADALY, E.A., TAWFIK, F.S., EL-KON, I.I., HELEIL, B.A., and FATTOUH, E.S., 2019. Effect of different cryoprotectants on the post-thaw sperm characteristics and in vivo fertility of buffalo (Bubalus bubalis) bull semen. Slovenian Veterinary Research / Slovenski Veterinarski Zbornik. 56: 541-551.https://doi.org/10.26873/SVR-792-2019.

ALTHOUSE, G.C. 2008. Sanitary procedures for the production of extended semen. Reproduction in Domestic Animals. 43(2): 374–378. https://doi.org/10.1111/j.1439-0531.2008.01187.

ANSARI, M.S., RAKHA, B.A., ANDRABI, S.M.H., and AKHTER, S., 2010. Usefulness of powdered and fresh egg yolk for cryopreservation of Zebu bull spermatozoa. Reproductive Biology. 10(3): 235–240. https://doi.org/10.1016/S1642-431X(12)60043-6.

BECCAGLIA, M., ANASTASI, P., CHIGIONI, S., and LUVONI, G.C., 2009. Tris‐lecithin extender supplemented with antioxidant catalase for chilling of canine semen. Reproduction in Domestic Animals. 44: 345–349. https://doi.org/10.1111/j.1439-0531.2009.01410.x

BENCHARIF, D., AMIRAT, L., ANTON, M., SCHMITT, E., DESHERCES, S., DELHOMME, G., LANGLOIS, M.-L., BARRIÈRE, P., LARRAT, M., and TAINTURIER, D., 2008. The advantages of LDL (low density lipoproteins) in the cryopreservation of canine semen. Theriogenology.70(9):1478–1488. https://doi.org/10.1016/j.theriogenology.2008.06.095

BENDOU, O., GUTIÉRREZ-FERNÁNDEZ, I., MARCOS-BARBERO, E.L., BUENO-RAMOS, N., GONZÁLEZ-HERNÁNDEZ, A.I., MORCUENDE, R., and ARELLANO, J.B., 2021. Theoretical and experimental considerations for a rapid and high throughput measurement of catalase in vitro. Antioxidants. 11(1): 21. https://doi.org/10.3390/antiox11010021.

BOE-HANSEN, G.B., MORRIS, I.D., ERSBØLL, A.K., GREVE, T., and CHRISTENSEN, P., 2005. DNA integrity in sexed bull sperm assessed by neutral Comet assay and sperm chromatin structure assay. Theriogenology. 63(3):1789–1802. https://doi.org/10.1016/j.theriogenology.2004.08.004.

CAMPOS-SHIMADA, L.B., HIDEO GILGLIONI, E., FERNANDES GARCIA, R., RIZATO MARTINS-MACIEL, E., LUIZA ISHII-IWAMOTO, E., and LUZIA SALGUEIRO-PAGADIGORRIA, C., 2020. Superoxide dismutase: a review and a modified protocol for activities measurements in rat livers. Archives of Physiology and Biochemistry. 126(4): 292–299. https://doi.org/10.1080/13813455.2018.1520891.

CASTIGLIONE MORELLI, M.A., OSTUNI, A., GIANGASPERO, B., CECCHINI, S., CARLUCCIO, A., and BONI, R., 2021. Relationships between seminal plasma metabolites, semen characteristics and sperm kinetics in donkey (Equus asinus). Animals 11(1): 201.https://doi.org/10.3390/ani11010201

CELEGHINI, E.C.C., DE ARRUDA, R.P., DE ANDRADE, A.F.C., NASCIMENTO, J., RAPHAEL, C.F., and RODRIGUES, P.H.M., 2008. Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin. Animal Reproduction Science. 104, 119–131. https://doi.org/10.1016/j.anireprosci.2007.02.001

CHELUCCI, S., PASCIU, V., SUCCU, S., ADDIS, D., LEONI, G.G., MANCA, M.E., NAITANA, S., and BERLINGUER, F., 2015. Soybean lecithin–based extender preserves spermatozoa membrane integrity and fertilizing potential during goat semen cryopreservation. Theriogenology. 83(8): 1064–1074. https://doi.org/10.1016/j.theriogenology.2014.12.012.

CHINOY, N.J., RANGA, G.M., HIGHLAND, H.N., D’SOUZA, K.J., and SEQUEIRA, E., 1992. A modified method for the differential staining of spermatozoa using alcoholic acidic silver nitrate. International Journal of Fertility. 37(4): 232–236.

FEUGANG, J.M., RHOADS, C.E., MUSTAPHA, P.A., TARDIF, S., PARRISH, J.J., WILLARD, S.T., and RYAN, P.L., 2019. Treatment of boar sperm with nanoparticles for improved fertility. Theriogenology. 137: 75–81. https://doi.org/10.1016/j.theriogenology.2019.05.040.

FOROUZANFAR, M., SHARAFI, M., HOSSEINI, S.M., OSTADHOSSEINI, S., HAJIAN, M., HOSSEINI, L., ABEDI, P., NILI, N., RAHMANI, H.R., and NASR-ESFAHANI, M.H., 2010. In vitro comparison of egg yolk–based and soybean lecithin–based extenders for cryopreservation of ram semen. Theriogenology. 73(4): 480–487. https://doi.org/10.1016/j.theriogenology.2009.10.005.

FRASER, L. 2004. Structural damage to nuclear DNA in mammalian spermatozoa: its evaluation techniques and relationship with male infertility. Polish Journal of Veterinary Science. 7(4): 311–321.

GAMAL, A., EL-NATTAT, W.S., EL-SHESHTAWY, R.I., and EL-MAATY, A.M.A., 2016. Substitution of egg yolk with different concentrations of soybean lecithin in tris-based extender during bulls’ semen preservability. Asian Pacific Journal of Reproduction. 5(6): 514–518. https://doi.org/10.1016/j.apjr.2016.10.011.

GONZALES, R., ROSALES, M., PEREA, F., VELARDE, J., SOTO, E., PALOMARES, R., HERNANDEZ, H., and PALASZ, A.T., 2003. 98 conception rates using Brahman bull semn frozen in milk based extender containing egg yolk or soybean lipids; A filed study in a tropical enviroment. Reproduction, Fertility and Development. 16(2): 170–171. https://doi.org/10.1071/RDv16n1Ab98

HASHEM, N.M., and GONZALEZ-BULNES, A., 2020. State-of-the-art and prospective of nanotechnologies for smart reproductive management of farm animals. Animals, 10(5): 840. https://doi.org/10.3390/ani10050840.

HO, R. 2013. Handbook of univariate and multivariate data analysis with IBM SPSS. 2^ndEdition. CRC press. Taylor and Francis Group, pp. 84-96.

HOLT, W. V. 2000. Basic aspects of frozen storage of semen. Animal Reproduction Science. 62(1-3): 3–22. https://doi.org/10.1016/S0378-4320(00)00152-4.

IVANOVA, M.G., GRADINARSKA, D.G., GENOV, M.S., TSVETKOV, T.S., and GEORGIEV, B.A., 2019. Changes in the phospholipid asymmetry of buffalo sperm plasma membrane in relation to cryopreservation technology. Pakistan Veterinay Journal. 39(2): 289-229. 10.29261/pakvetj/2019.020

KADIRVEL, G., KUMAR, S., and KUMARESAN, A., 2009. Lipid peroxidation, mitochondrial membrane potential and DNA integrity of spermatozoa in relation to intracellular reactive oxygen species in liquid and frozen-thawed buffalo semen. Animal Reproduction Science. 114(1-3): 125–134. https://doi.org/10.1016/j.anireprosci.2008.10.002.

KHALIFA, T.A.A. 2001. Effect of some antioxidants on viability of preserved buffalo and ram semen. Ph. D. Thesis, Theriogenology Dept., Faculty of Veterinary Medicine. Cairo University, Egypt.

KHALIL, W.A., EL-HARAIRY, M.A., ZEIDAN, A.E.B., and HASSAN, M.A.E., 2019. Impact of selenium nano-particles in semen extender on bull sperm quality after cryopreservation. Theriogenology. 126: 121–127. https://doi.org/10.1016/j.theriogenology.2018.12.017.

KMENTA, I., STROHMAYER, C., MÜLLER-SCHLÖSSER, F., and 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. https://doi.org/10.1016/j.theriogenology.2010.11.018

KUMARESAN, A., ANSARI, M.R., GARG, A., and KATARIA, M., 2006. Effect of oviductal proteins on sperm functions and lipid peroxidation levels during cryopreservation in buffaloes. Animal Reproduction Science. 93(3-4): 246–257. https://doi.org/10.1016/j.anireprosci.2005.06.030.

LIPAR, J.L., KETTERSON, E.D., NOLAN JR, V., and CASTO, J.M., 1999. Egg yolk layers vary in the concentration of steroid hormones in two avian species. General and Comparative Endocrinology. 115(2): 220–227. https://doi.org/10.1006/gcen.1999.7296.

MAFOLO, K.S., PILANE, C.M., CHITURA, T., and NEDAMBALE, T.L., 2020. Use of phosphatidylcholine in tris-based extender with or without egg yolk to freeze Bapedi ram semen. South African Journal of Animal Science. 50(3): 389–396. 10.4314/sajas.v50i3.5.

MANJUNATH, P., NAUC, V., BERGERON, A., and MÉNARD, M., 2002. Major proteins of bovine seminal plasma bind to the low-density lipoprotein fraction of hen’s egg yolk. Biology of Reproduction. 67(4): 1250–1258. https://doi.org/10.1095/biolreprod67.4.1250.

MASOUDI, R., SHARAFI, M., ZAREH SHAHNEH, A., TOWHIDI, A., KOHRAM, H., ESMAEILI, V., SHAHVERDI, A., and DAVACHI, N.D., 2016. Fertility and flow cytometry study of frozen-thawed sperm in cryopreservation medium supplemented with soybean lecithin. Cryobiology. 73(1): 69–72. https://doi.org/10.1016/j.cryobiol.2016.05.010.

MOTLAGH, M.K., SHARAFI, M., ZHANDI, M., MOHAMMADI-SANGCHESHMEH, A., SHAKERI, M., SOLEIMANI, M., and ZEINOALDINI, S., 2014. Antioxidant effect of rosemary (Rosmarinus officinalis L.) extract in soybean lecithin-based semen extender following freeze–thawing process of ram sperm. Cryobiology. 69(2): 217–222. https://doi.org/10.1016/j.cryobiol.2014.07.007.

MOUSAVI, S.M., TOWHIDI, A., ZHANDI, M., AMOABEDINY, G., MOHAMMADI-SANGCHESHMEH, A., SHARAFI, M., and HUSSAINI, S.M.H., 2019. Comparison of two different antioxidants in a nano lecithin-based extender for bull sperm cryopreservation. Animal Reproduction Science. 209: 106171. https://doi.org/10.1016/j.anireprosci.2019.106171.

NADRI, T., TOWHIDI, A., ZEINOALDINI, S., MARTÍNEZ-PASTOR, F., MOUSAVI, M., NOEI, R., TAR, M., MOHAMMADI and SANGCHESHMEH, A., 2019. Lecithin nanoparticles enhance the cryosurvival of caprine sperm. Theriogenology. 133: 38–44. https://doi.org/10.1016/j.theriogenology.2019.04.024.

NAJAFI, A., DAGHIGH-KIA, H., DODARAN, H.V., MEHDIPOUR, M., and ALVAREZ-RODRIGUEZ, M., 2017. Ethylene glycol, but not DMSO, could replace glycerol inclusion in soybean lecithin-based extenders in ram sperm cryopreservation. Animal Reproduction Science. 177: 35–41. https://doi.org/10.1016/j.anireprosci.2016.12.004.

NAZ, S., UMAIR, M., and IQBAL, S., 2018. Comparison of Tris egg yolk‐based, Triladyl® and Optixell® extender on post‐thaw quality, kinematics and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Andrologia. 50(8): e13063. https://doi.org/10.1111/and.13063

NOWIER, A., and SAAD, M.F., 2022. Effect of substitution egg yolk with different levels of soybean lecithin in Tris-based extender on freezing and fertilizing capacity of Holstein bull semen. Egyptian Journal of Animal Production. 59(1): 1–7. 10.21608/ejap.2022.109751.1030.

OGNJANOVIĆ, B.I., MARKOVIĆ, S.D., PAVLOVIĆ, S.Z., ŽIKIĆ, R. V., and ŠTAJN, A.Š., 2008. Effect of chronic cadmium exposure on antioxidant defense system in some tissues of rats: protective effect of selenium. Physiological Research. 57(3): 403-411.

OKE, M., JACOB, J.K., and PALIYATH, G., 2010. Effect of soy lecithin in enhancing fruit juice/sauce quality. Food Research International. 43(1): 232–240. https://doi.org/10.1016/j.foodres.2009.09.021.

PARTYKA, A., ŁUKASZEWICZ, E., and NIŻAŃSKI, W., 2012. Lipid peroxidation and antioxidant enzymes activity in avian semen. Animal Reproduction Science. 134(3-4): 184–190. https://doi.org/10.1016/j.anireprosci.2012.07.007.

PERIS-FRAU, P., SOLER, A.J., INIESTA-CUERDA, M., MARTÍN-MAESTRO, A., SÁNCHEZ-AJOFRÍN, I., MEDINA-CHÁVEZ, D.A., FERNÁNDEZ-SANTOS, M.R., GARCÍA-ÁLVAREZ, O., MAROTO-MORALES, A., MONTORO, V., and GARDE, J.J., 2020. Sperm cryodamage in ruminants: Understanding the molecular changes induced by the cryopreservation process to optimize sperm quality. International Journal of Molecular Science. 21(8): 2781. https://doi.org/10.3390/ijms21082781

RAWASH, Z.M., LEIL, A.Z.A., ANWER, A.M., and EL-BADRY, D.A., 2020. Influence of combinations of some permeable cryoprotectants with chicken and duck egg yolks on freezability and DNA integrity of buffalo spermatozoa. Journal of Applied Veterinary Science. 5(3): 49–56. https://doi.org/10.21608/JAVS.2020.98371

REVELL, S.G., and MRODE, R.A., 1994. An osmotic resistance test for bovine semen. Animal Reproduction Science. 36(1-2): 77–86. https://doi.org/10.1016/0378-4320(94)90055-8 Get rights and content.

ROOF, D.J., BOWLEY, S., PRICE, L.L., and MATSAS, D.J., 2012. Comparison of two commercial extenders for cryopreservation of goat semen without sperm washing. Theriogenology. 77(2): 412–420. https://doi.org/10.1016/j.theriogenology.2011.08.015

ROS-SANTAELLA, J.L., and PINTUS, E., N., 2021. Plant Extracts as Alternative Additives for Sperm Preservation. Antioxidants. 10(5): 772. https://doi.org/10.3390/antiox10050772.

ŞAHIN, D., BAŞTAN, İ., ÇIL, B., TEKIN, K., AKÇAY, E., DAŞKIN, A., and STELLETTA, C., 2020. The number of false mounting affects the quality of semen in bulls. Lalahan Hayvancılık Araştırma Enstitüsü Derg. 60(1): 9–14. https://doi.org/10.46897/lahaed.701388.

SHOKRY, D.M., BADR, M.R., SAKR, A.-A.M., ELMESIRY, A.M., ASSY, M.M., RAWASH, Z., and Abd ELDAIM, M.A., 2024. Enhancement potential of Moringa oleifera leaves extract on buffalo bull cryopreserved semen quality and fertilization capacity. Animal Reproduction Science. 262, 107414. https://doi.org/10.1016/j.anireprosci.2024.107414.

SIMPSON, A.M., SWAN, M.A., and WHITE, I.G., 1986. Action of phosphatidylcholine in protecting ram sperm from cold shock. Gamete Research. 15(1): 43–56. https://doi.org/10.1002/mrd.1120150106

SINGH, A.K., KUMAR, A., HONPARKHE, M., KAUR, S., KAUR, H., GHUMAN, S.P.S., and BRAR, P.S., 2018. Comparison of in vitro and in vivo fertilizing potential of buffalo bull semen frozen in egg yolk‐, soya bean lecithin‐and liposome‐based extenders. Reproduction in Domestic Animals. 53(1): 195–202. https://doi.org/10.1111/rda.13092

SUN, L., HE, M., WU, C., ZHANG, S., DAI, J., and ZHANG, D., 2021. Beneficial influence of soybean lecithin nanoparticles on rooster frozen–thawed semen quality and fertility. Animals. 11(6): 1769. https://doi.org/10.3390/ani11061769.

TAŞDEMIR, U., BÜYÜKLEBLEBICI, S., TUNCER, P.B., COŞKUN, E., ÖZGÜRTAŞ, T., AYDIN, F.N., BÜYÜKLEBLEBICI, O., and GÜRCAN, İ.S., 2013. Effects of various cryoprotectants on bull sperm quality, DNA integrity and oxidative stress parameters. Cryobiology. 66(1): 38–42. https://doi.org/10.1016/j.cryobiol.2012.10.006.

TOKER, M.B., ALCAY, S., GOKCE, E., and USTUNER, B., 2016. Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience. Cryobiology. 72(3): 205–209. https://doi.org/10.1016/j.cryobiol.2016.05.001.

UGUR, M.R., SABER ABDELRAHMAN, A., EVANS, H.C., GILMORE, A.A., HITIT, M., ARIFIANTINI, R.I., PURWANTARA, B., KAYA, A., and MEMILI, E., 2019. Advances in cryopreservation of bull sperm. Frontiers in Veterinary Science. 6: 268. https://doi.org/10.3389/fvets.2019.00268.

WANG, D.-H., LIU, Y.-L., CAI, Z.-G., AN, J.-H., LAN, J.-C., CHEN, J.-S., LI, Y., HE, L., ZHANG, Y., and HE, P., 2020. Effects of extender type on the quality of post-thaw giant panda (Ailuropoda melanoleuca) semen. Cryobiology. 94, 95–99. https://doi.org/10.1016/j.cryobiol.2020.04.003.

WATERHOUSE, K.E., GJELDNES, A., TVERDAL, A., DE ANGELIS, P.M., FARSTAD, W., HÅÅRD, M., and KOMMISRUD, E., 2010. Alterations of sperm DNA integrity during cryopreservation procedure and in vitro incubation of bull semen. Animal Reproduction Science. 117(1-2): 34–42. https://doi.org/10.1016/j.anireprosci.2009.04.011.

WOJTUSIK, J., STOOPS, M.A., and ROTH, T.L., 2018. Comparison of soy lecithin, coconut water, and coconut milk as substitutes for egg-yolk in semen cryodiluent for black rhinoceros (Diceros bicornis) and Indian rhinoceros (Rhinoceros unicornis). Theriogenology. 121: 72–77. https://doi.org/10.1016/j.theriogenology.2018.07.042

ZAKOŠEK PIPAN, M., CASAL, M.L., ŠTERBENC, N., VIRANT KLUN, I., and MRKUN, J., 2020. Vitrification using soy lecithin and sucrose: A new way to store the sperm for the preservation of canine reproductive function. Animals. 10(4): 653. https://doi.org/10.3390/ani10040653.