Improving the Cryostorage Parameters of Ram Germ Stem Cells by Supplementing with Soybean Lecithin as a Cryoprotectant

Subject Areas : Camel

S. Ghahremani ¹ , M. Sharafi ² , B. Ghasemi Panahi ³ , A. Javanmard ⁴

1 -
2 -
3 -
4 -

Received: 2024-03-01 Accepted : 2024-11-15 Published : 2024-12-01

Keywords: cryopreservation, cryoprotectants, Ghezel sheep, soybean lecithin, sper-matogonial stem cells,

Abstract :

To date, the challenging problem of reducing cryodamage caused by the freezing and thawing process of spermatogonial stem cells (SSCs) is still at the experimental stage, and understanding the complexity of this puzzle is crucial. More recently, soybean lecithin (SL) has been claimed to be a suitable molecule to protect SSCs from cold shock throughout the cryostorage process. Based on this motivation, the main aim of the present research is to evaluate the cryostorage parameters of ram germinal stem cells by SL supplementa-tion as a cryoprotectant agent. For this purpose, SSCs were isolated from the testicular tissue of a 2-month-old ram and we then added different concentrations of SL (control, 0.5, 1 and 1.5 percent) and then added them to the basic media described. Next, cryostorage parameters (cell viability, number and area of colo-nies) were assessed per treatment. To examine this statistically, we calculated a one-way ANOVA and HSD test with SAS and a P < 0.05 was considered significant. Interestingly, our results showed that cell viability and colony number and area were significantly higher during supplementation with 0.5% SL than the other treatments (P<0.05). However, SSCs preserved in 1.5% SL have adverse effects due to their toxicity rate. On this basis, our current findings demonstrate an improvement in cryostorage parameters of ram germ cells by supplementation with SL. This assumption could be addressed in future studies.

References:

Aliakbari F., Gilani M.A.S., Amidi F., Baazm M., Korouji M., Izadyar F., Yazdekhasti H. and Abbasi M. (2016). Improving the efficacy of cryopreservation of spermatogonia stem cells by antioxidant supplements. Cell. Reprogram. 18(2), 87-95.
Aliakbari F., Sedighi Gilani M.A., Yazdekhasti H., Koruji M., Asgari H.R., Baazm M., Izadyar F., Kharrazi Nejad E., Khanezad M. and Abbasi M. (2017). Effects of antioxidants, catalase and α-tocopherol on cell viability and oxidative stress variables in frozen-thawed mice spermatogonial stem cells. Artif. Cells. Nanomed. Biotechnol. 45(1), 63-68.
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(6), 1064-1074.
Dalmazzo A., Losano J.D.A., Rocha C.C., Tsunoda R.H., Angri-mani D.R., Mendes C.M., Assumpção M.E.O.D.Á., Nichi M. and Barnabe V.H. (2018). Effects of soy lecithin extender on dog sperm cryopreservation. Anim. Biotechnol. 29(3), 174-182.
El-Shahat K. and Hammam A. (2018). Effect of different types of cryoprotectants on developmental capacity of vitrified-thawed immature buffalo oocytes. Anim. Reprod. 11(4), 543-548.
Fathi M., Zaher R., Ragab D., Gamal I., Mohamed A., Abu-El Naga E. and Badr M. (2019). Soybean lecithin–based extender improves Damascus goat sperm cryopreservation and fertiliz-ing potential following artificial insemination. Asian. Pac. J. Reprod. 8(4), 174-184.
Feng T.Y., Li Q., Ren F., Xi H.M., Lv D.L., Li Y. and Hu J.H. (2020). Melatonin protects goat spermatogonial stem cells against oxidative damage during cryopreservation by improv-ing antioxidant capacity and inhibiting mitochondrial apop-tosis pathway. Oxid. Med. Cell. Longev. 5, 1-10.
Forouzanfar M., Sharafi M., Hosseini S., Ostadhosseini S., Hajian M., Hosseini L., Abedi P., Nili N., Rahmani H. and 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.
Ibtisham F. and Honaramooz A. (2020). Spermatogonial stem cells for in vitro spermatogenesis and in vivo restoration of fer-tility. Cells. 9(3), 745-753.
Ibtisham F., Awang-Junaidi A.H. and Honaramooz A. (2020). The study and manipulation of spermatogonial stem cells using animal models. Cell. Tissue. Res. 380(2), 393-414.
Ibtisham F., Zhao Y., Nawab A., Wu J., Mei X., Honaramooz A. and An L. (2021). In vitro production of haploid germ cells from murine spermatogonial stem cells using a two-dimensional cell culture system. Theriogenology. 162, 84-94.
Izadyar F., Matthijs-Rijsenbilt J., Den Ouden K., Creemers L.B., Woelders H. and de Rooij D.G. (2002). Development of a cryopreservation protocol for type A spermatogonia. J. An-drol. 23(4), 537-545.
Jeong D., McLean D.J. and Griswold M.D. (2003). Long‐term culture and transplantation of murine testicular germ cells. J. Androl. 24(5), 661-669.
Kazemzadeh S., Mohammadpour S., Madadi S., Babakhani A., Shabani M. and Khanehzad M. (2022). Melatonin in cryopre-servation media improves transplantation efficiency of frozen–thawed spermatogonial stem cells into testes of azoospermic mice. Stem Cell Res. Ther. 13(1), 346-356.
Kim K.J., Lee Y.A., Kim B.J., Kim Y.H., Kim B.G., Kang H.G., Jung S.E., Choi S.H., Schmidt J.A. and Ryu B.Y. (2015). Cryopreservation of putative pre-pubertal bovine spermatogo-nial stem cells by slow freezing. Cryobiology. 70(2), 175-183.
Konyak P., Mandal A., Mondal M., Bhakat C., Das S., Rai S., Ghosh M. and Karunakaran M. (2018). Preservation of black Bengal buck semen in soybean lecithin based chemically de-fined extender. Indian J. Anim. Res. 52(8), 1151-1154.
Kubota H., Avarbock M.R. and Brinster R.L. (2004). Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc. Natl. Acad. Sci. 101(47), 16489-16494.
Mehdipour M., Kia H.D., Moghaddam G. and Hamishehkar H. (2018). Effect of egg yolk plasma and soybean lecithin on rooster frozen-thawed sperm quality and fertility. Therio-genology. 116, 89-94.
Miguel-Jimenez S., Del Alamo M.M.R., Álvarez-Rodríguez M., Hidalgo C.O., Peña A.I., Muiño R., Rodríguez-Gil J.E. and Mogas T. (2020). In vitro assessment of egg yolk-, soya bean lecithin-and liposome-based extenders for cryopreservation of dairy bull semen. Anim. Reprod. Sci. 215, 1-11.
Mirzapour T., Movahedin M., Tengku Ibrahim T.A., Haron A.W. and Nowroozi M.R. (2013). Evaluation of the effects of cryo-preservation on viability, proliferation and colony formation of human spermatogonial stem cells in vitro culture. Andrologia. 45(1), 26-34.
Mohebbi M., Moghaddam G., Qasemi-Panahi B., Daghigh Kia H., Rafat S. and Hamidian G. (2021). Cryopreservation of Sper-matogonial Stem Cells of Native Goat of Iran. Iranian J. Appl. Anim. Sci. 11(2), 351-356.
Motta J.P.R., Paraguassú-Braga F.H., Bouzas L.F. and Porto L.C. (2014). Evaluation of intracellular and extracellular trehalose as a cryoprotectant of stem cells obtained from umbilical cord blood. Cryobiology. 68(3), 343-348.
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. Anim. Reprod. Sci. 209, 106171-106181.
Pirnia A., Assadollahi V., Alasvand M., Moghadam A. and Gholami M.R. (2020). A comparison of the effects of fetal bo-vine serum and newborn calf serum on cell growth and main-tenance of cryopreserved mouse spermatogonial stem cells. Mol. Biol. Rep. 47(12), 9609-9614.
Qasemi-Panahi B., Movahedin M., Moghaddam G., Tajik P., Koruji M., Ashrafi-Helan J. and Rafat S.A. (2018). Isolation and proliferation of spermatogonial cells from ghezel sheep. Avicenna J. Med. Biotechnol. 10(2), 93-97.
Salmani H., Towhidi A., Zhandi M., Bahreini M. and Sharafi M. (2014). In vitro assessment of soybean lecithin and egg yolk based diluents for cryopreservation of goat semen. Cryobiol-ogy. 68(2), 276-280.
SAS Institute. (2013). SAS®/STAT Software, Release 9.4. SAS Institute, Inc., Cary, NC. USA.
Shamsollahi M., Daghigh Kia H., Moghaddam G. and Taghizade A. (2018). Effects of centrifugation and different levels of soybean lecithin-based extender on post-thaw sperm quality of Ghezel ram semen. Iranian J. Appl. Anim. Sci. 8(2), 257-262.
Sharafi M., Zhandi M. and Akbari Sharif A. (2015). Supplementa-tion of soybean lecithin-based semen extender by antioxidants: Complementary flowcytometric study on post-thawed ram spermatozoa. Cell. Tissue Bank. 16(2), 261-269.
Shi Y.Q., Wang Q.Z., Liao S.Y., Zhang Y., Liu Y.X. and Han C.S. (2006). In vitro propagation of spermatogonial stem cells from KM mice. Front. Biosci. 11(3), 2614-2622.
Strober W. (1997). Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol. 21(1), 1-9.
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), 1769-1779.
Ustuner B., Alçay S., Nur Z., Sagirkaya H. and Soylu M.K. (2014). Effect of egg yolk and soybean lecithin on tris-based extender in post-thaw ram semen quality and in vitro fertility. Kafkas Univ. Vet. Fak. Derg. 20(3), 393-398.
Wang P., Li Y., Hu X.C., Cai X.L., Hou L.P., Wang Y.F., Hu J.H., Li Q.W., Suo L.J., Fan Z.G. and Zhang B. (2014). Cryo-protective effects of low-density lipoproteins, trehalose and soybean lecithin on murine spermatogonial stem cells. Zygote. 22(2), 158-163.
Zargarzadeh M., Tajik P., Movahedin M., Akbari G. and Qasemi-Panahi B. (2019). Immediate cryopreservation of isolated Ghezel ram spermatogonial stem cells and subsequent co-culture with Sertoli cells preserve their survival and functional capacity. Eurasian J. Biosci. 13(2), 2614-2622.
Zhao X., Wan W., Zhang X., Wu Z. and Yang H. (2021). Sper-matogonial stem cell transplantation in large animals. Animals. 11(4), 257-262.