Sperm Plasma Membrane Integrity Test Using Two Different Methods on Frozen Murrah Buffalo Semen

Raden Iis Arifiantini; JOHANA DIAN RAHAYU1 DIAN RAHAYU; BAMBANG PURWANTARA; SYAHRUDDIN SAID

doi:10.14334/jitv.v30i1.3488

Sperm Plasma Membrane Integrity Test Using Two Different Methods on Frozen Murrah Buffalo Semen

Raden Iis Arifiantini, JOHANA DIAN RAHAYU1 DIAN RAHAYU, BAMBANG PURWANTARA, SYAHRUDDIN SAID

DOI

https://doi.org/10.14334/jitv.v30i1.3488

Abstract

The integrity of the sperm plasma membrane in frozen semen is crucial in assessing semen quality and is directly linked to fertility. The plasma membrane integrity of sperm can be tested using several methods, including the Hypo-Osmotic Swelling Test (HOS-Test) and Carboxyfluorescein Diacetate-Propidium Iodide Staining (CFDA-PI). This study compares the integrity of the plasma membrane of frozen Murrah buffalo semen using the HOS-Test and CFDA-PI. The semen was obtained from four buffalo bulls, each representing a different production batch. The straw was thawed individually, and quality was assessed. The plasma membrane integrity of each sample was evaluated using the HOS-Test and CFDA-PI staining methods. A two-sample t-test was used to analyze the data and demonstrate that there is a statistically significant difference in sperm plasma membrane integrity between the HOS-Test (64.83%) and CFDA-PI staining (58.97%) methods (p<0.05). The HOS-Test assessment is optimal for cost-effectiveness, speed, and convenience. It can be implemented in laboratory and field settings with equal ease. In contrast, CFDA-PI staining is a good standard for accuracy and precision. It provides a comprehensive foundation for determining the quality of frozen semen.

Keywords

CFDA-PI, HOS-test, Murrah buffalo, Sperm plasma membrane

References

Agarwal A, Gupta S, Sharma R. 2016. Hypo-osmotic swelling test (HOST). Andrological Evaluation of Male Infertility. Springer. p. 93-96. doi: 10.1007/978-3-319-26797-5_12

Barbagallo F, La Vignera S, Cannarella R, Aversa A, Calogero AE, Condorelli RA. 2020. Evaluation of sperm mitochondrial function: a key organelle for sperm motility. J Clin Med. 9(2):363. doi.org/10.3390/antiox10071131

Bezerra LGP, ALP Souza, HVR Silva, FR Vasconcelos, ADAA Moura, AF Pereira, MFD Oliveira, Silva AR. 2018. Ultrastructural description of fresh and frozen/thawed sperm derived from collared peccaries (Pecari tajacu Linnaeus, 1,758). Microsc Res Tech. 81(11): 1301-1309. Doi: 10.1002/jemt.23138

Bezerra LGP, Silva AM, Dantas MRT, Santos RPD, Moreira SSJ, Pereira AG, Oliveira MFD, Comizzoli P, Silva AR. 2023. Development of assays for the characterization of sperm motility parameters, viability, and membrane integrity in the epididymis and vas deferens of the greater rhea (Rhea americana). Anim Reprod. 20(4):e20230113. doi.org/10.1590/1984-3143-AR2023-0113

Chelucci S, Pasciu V, Succu S, Addis D, Leoni GG, Manca ME, Berlinguer F, Naitana S. 2015. Soybean lecithin–based extender preserves spermatozoa membrane integrity and fertilizing potential during goat semen cryopreservation. Theriogenology. 83(6):1064-1074. doi: 10.1016/j.theriogenology.2014.12.012.

Davey H, Guyot S. 2020. Estimation of microbial viability using flow cytometry. Curr Protoc Cytom. 93(1):72. doi.org/10.1002/cpcy.72

Dolnik M, Mudro?ová D, Pošivák J, Lazar G, Mudro? P. 2019. Flow cytometry in assessment of sperm integrity and functionality–a review. Acta Vet Brno. 88(2):169-175. doi.org/10.2754/avb201988020169

Fischer D, Schneider H, Failing K, Meinecke?Tillmann S, Wehrend A, Lierz M. 2020. Viability assessment of spermatozoa in large falcons (Falco spp.) using various staining protocols. Reprod Domest Anim. 55(10):1383-1392. doi.org/10.1111/rda.13785.

Gaczarzewicz D, Udala J, Piasecka M, Blaszczyk B, Stankiewicz T. 2015. Storage temperature of boar semen and its relationship to changes in sperm plasma membrane integrity, mitochondrial membrane potential, and oxidoreductive capability. Turk J Biol. 39(4):582-594. doi.org/10.3906/biy-1412-76

Garcia-Vazquez FA, Gadea J, Matás C, Holt WV. 2016. Importance of sperm morphology during sperm transport and fertilization in mammals. Asian J Androl. 18(6):844-850. doi: 10.4103/1008-682X.186880

Golher DM, Kumaresan A, Saraf KK, Chhillar S, Nayak S, Tripathi UK, Bhaskar CN, Lathwal SS, Mohanty TK. 2018. Influence of season and climatic variables on testicular cytology, semen quality, and melatonin concentrations in crossbred bucks reared under subtropical climate. Int J Biometeorol. 62:1709-1719. doi.org/10.1007/s00484-018-1571-x

Harrison RAP, Vickers SE. 1990. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. Reproduction. 88(1):343-352. doi.org/10.1530/jrf.0.0880343

Ito C, Toshimori K. 2016. Acrosome markers of human sperm. Anat Sci Int. 91:128-142. doi.org/10.1007/s12565-015-0323-9

Kang SS, Kim UH, Lee MS, Lee SD, Cho SR. 2020. Spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity in 0.25 mL and 0.5 mL straw after frozen-thawing in Hanwoo bull. J Anim Reprod Biotechnol. 35:307–14. doi: 10.12750/JARB.35.4.307.

Kumar P, Srivastava N, Pande M, Prasad JK, Sirohi AS. 2017. Evaluating sperm cell viability and membrane integrity. Protocols in Semen Biology. Springer. p. 57-71. doi: 10.1007/978-981-10-5200-2_6

Matabane MB, Thomas R, Netshirovha TR, Tsatsimpe M, Ng’ambi JW, Nephawe KA, Nedambale TL. 2017. Relationship between sperm plasma membrane integrity and morphology and fertility following artificial insemination. S Afr J Anim Sci. 47(1):102-106 doi.org/10.4314/sajas.v47i1.15

Moore SG, Hasler JF. 2017. A 100-Year Review: Reproductive technologies in dairy science. J of dairy sci. 100(12):10314-10331. doi.org/10.3168/jds.2017-13138

Morrell JM, Valeanu AS, Lundeheim N, Johannisson A. 2018. Sperm quality in frozen beef and dairy bull semen. Acta Vet Scand. 60(1):1-10. doi.org/10.1186/s13028-018-0396-2

Nordhoff V. 2015. How to select immotile but viable spermatozoa on the day of intracytoplasmic sperm injection? An embryologist's view. Andrology. 3(2):156-162. doi.org/10.1111/andr.286

Olexikova L, Miranda M, Kulikova B, Baláži A, Chrenek P. 2019. Cryodamage of the plasma membrane and acrosome region in chicken sperm. Anat Histol Embryol. 48(1):33-39. doi: 10.1111/ahe.12408

Rajoriya JS, Prasad JK, Ramteke SS, Perumal P, Ghosh SK, Singh M, Pande M, Srivastava N. 2016. Enriching membrane cholesterol improves the stability and cryosurvival of buffalo spermatozoa. Anim Reprod Sci.164:72-81. doi.org/10.1016/j.anireprosci.2015.11.014

Ramu S, Jeyendran RS. 2013. The hypo-osmotic swelling test for evaluation of sperm membrane integrity. Spermatogenesis. Springer. p. 21-25. doi: 10.1007/978-1-62703-038-0_3.

Rashedi M, Fazeli MH, Bahreini M. 2016. Polymyxin B changes the plasma membrane integrity of cryopreserved bull semen. Asian Pac. J. Reprod. 5(5):411-415. doi.org/10.1016/j.apjr.2016.07.008

Reis LSLS, Ramos AA, Camargos AS, Oba E. 2016. Integrity of the plasma membrane, the acrosomal membrane, and the mitochondrial membrane potential of sperm in Nelore bulls from puberty to sexual maturity. Arq Bras Med Vet Zootec. 68(03):620-628. doi.org/10.1590/1678-4162-8748

[SNI] National Standardization Agency. 2021. SNI 4869-2: 2021. Frozen semen part 2: Buffalo. National Standardization Agency. Jakarta.

Singh RK, Kumaresan A, Chhillar S, Rajak SK, Tripathi UK, Nayak S, Datta TK, Mohanty TK, Malhotra R. 2016. Identification of suitable combinations of in vitro sperm-function test for the prediction of fertility in buffalo bull. Theriogenology. 86(9):2263-2271. doi.org/10.1016/j.theriogenology.2016.07.022

Sukmawati E, Arifiantini RI, Purwantara B. 2014. Freezing capacity of sperm on various type of superior bulls. JITV. 19(3):168-175. doi:10.14334/jitv.v19i3.1079.

Trimble WS, Grinstein S. 2015. Barriers to the free diffusion of proteins and lipids in the plasma membrane. J Cell Biol 208(3):259-271. doi.org/10.1083/jcb.201410071

Yaghoobi M, Azizi M, Mokhtare A, Javi F, Abbaspourrad A. 2022. Rheotaxis quality index: a new parameter that reveals male mammalian in vivo fertility and low sperm DNA fragmentation. Lab on a Chip. 22(8):1486-1497. doi.org/10.1039/D2LC00150K

Zeidan A, El-Sharabassy A, Mekkawy Y, Amer A, Absy G, Ahmadi ES, Elnady I. 2018. Assessment of the functional integrity of equine spermatozoal membrane using hypo-osmotic swelling test. SCVMJ. 23(2):157-168. doi.org/10.21608/scvmj.2018.60499