1. Home
  2. Vol 29, No 10 (2025)
  3. Abilash

A Study of the durability of sperm cell motility and deoxyribo-nucleic acid damage during cryopreservation

Doriah Abilash(1), T. B. Sridharan(2),


(1) Gene Cloning Technology Lab, School of Biosciences and Technology, Vellore Institute of Technology, Vellore – 632014, India
(2) Gene Cloning Technology Lab, School of Biosciences and Technology, Vellore Institute of Technology, Vellore – 632014, India
Corresponding Author

Abstract


Semen cryopreservation is an essential technique in artificial insemination (AI) and assisted reproductive technology (ART). The addition of antioxidants to the freezing medium is a promising strategy to reduce cryo-induced damage to sperm cells. While sperm freezing has been extensively studied, this research aimed to improve the post-thaw durability and functionality of sperm. A total of 110 semen samples 55 from fertile and 55 from infertile individuals were collected and processed. After centrifugation, each sample was mixed with a cryoprotectant medium at a 1:1 (v/v) ratio. Three different cryoprotectant formulations (S1, S2, and S3), each containing varying concentrations of protective agents, were evaluated. The samples were then frozen in liquid nitrogen at −196°C. Post-thaw analyses included assessments of sperm count, motility, vitality, morphology, DNA fragmentation, and reactive oxygen species (ROS) levels representing a major strength of the study due to the comprehensive evaluation of sperm quality. The results showed significant differences in DNA fragmentation between fresh and cryopreserved samples. Among the tested formulations, S3 supplemented with citric acid anhydrous and taurine produced the best outcomes. It significantly improved sperm motility and vitality, while effectively reducing oxidative stress and cryodamage. Statistical analysis using one-way ANOVA and Tukey’s HSD test revealed significant differences in sperm morphology between fresh and post-thaw S3-treated samples (p < 0.05), with a 95% confidence level. A limitation of the study was the long interval between the use of fresh control samples and the analysis of cryopreserved samples, which may influence comparative accuracy. Despite this limitation, the findings suggest that the S3 formulation is highly effective in preserving sperm quality. These results support its potential integration into fertility preservation protocols in ART. Furthermore, the outcomes may guide clinical practice and support the development of national policies aimed at improving access to advanced cryopreservation techniques as part of comprehensive reproductive healthcare services.

References


Vickram AS, Rao KA, Pathy MR, Thomas C, Paramswari R

and Sridharan TB. Effects of cryoprotectants on

human sperm motility. Cryoletters. 2015;36(6):405

Ombelet W and Van Robays J. Artificial insemination

history: Hurdles and milestones. Facts Views Vis

Obgyn. 2015;7(2):137–43.

Jones RC. Collection, motility, and storage of spermatozoa

from the African elephant Loxodonta africana.

Nature. 1973;243(5401):38–9.

Bozkurt Y and Yavaş İ. Comparison of different freezing

techniques, cryoprotectants, and cryoprotectants on

quality and fertility of cryopreserved Salmo trutta f.

fario sperm. Acta Sci Technol. 2024;46(1).

Lovelock JE and Polge C. The immobilization of

spermatozoa by freezing and thawing and the

protective action of glycerol. Biochem J.

;58(4):618.

Rota A, Seom B and Linde-Forsberg C. The impact of

cryoprotectants on post-thaw survival of dog semen.

Theriogenology. 1995;44:885–900.

Lingappa HA, Govindashetty AM, Krishnamurthy A,

Puttaveerachary AK, Manchaiah S, Shimoga IC,

Mallaradhya SH and Gowda SB. Quest for an ideal,

simple and cost-effective stain for morphological

assessment of sperms. Journal of clinical and

diagnostic research: JCDR. 2015 Oct 1;9(10):EC01.

Memon AA, Wahid H, Rosnina Y, Goh YM, Ebrahimi M,

Nadia FM and Audrey G. Effect of butylated

hydroxytoluene on cryopreservation of Boer goat

semen in Tris egg yolk extender. Animal

reproduction science. 2011 Nov 1;129(1-2):44-9.

Chakraborty P, Kumar S, Dutta D and Gupta V. Role of

antioxidants in common health diseases. Res J Pharm

Technol. 2009;2(2):238–44.

Dhara S, Gupta HP, Kumar S, Sharma RK and Thakur S.

Effects of heterologous bovine seminal plasma

supplemented egg yolk-based cryoprotectants on

cryosurvivability of Pantja buck semen. Biopreserv

Biobank. 2023;21(4):336–45.

Vickram AS, Rao KA, Archana K, Jayaraman G, Kumar SV

and Sridharan TB. Effects of various semen

cryoprotectants on semen parameters for the purpose

of human male fertility preservation. Cryoletters.

;36(3):182–6

Riva NS, Ruhlmann C, Iaizzo RS, López CAM and

Martínez AG. Comparative analysis between slowfreezing and ultra-rapid freezing for human spermcryopreservation. JBRA Assist Reprod.

;22(4):331–7. doi:10.5935/1518

20180060.

Agarwal A, Majzoub A, Esteves SC, Ko E, Ramasamy R

and Zini A. Clinical utility of sperm DNA

fragmentation testing: Practice recommendations

based on clinical scenarios. Transl Androl Urol.

;5(6):935–50. doi:10.21037/tau.2016.10.03.

Robinson C, Roberts P, Reynolds K and Matson P. The

effect of glycerol and a glycerol-containing

cryoprotective medium upon the motility of human

sperm prior to freezing, and subsequent difficulties

in assessing sperm motility following dilution. J

Reprod Biotechnol Fertil. 2018;7:8–13.

Mohammadi F, Nikzad H, Taherian A, Mahabadi JA and

Salehi M. Effects of herbal medicine on male

infertility. Anat Sci. 2013;10(4).

Kashani HH, Malekzadeh Shiravani S and Hoshmand F.

The effect of aqueous extract of Salep prepared from

root-tubers of Dactylorhiza maculata (Orchidaceae)

on the testes and sexual hormones of immature male

mice. J Med Plants Res. 2012;6(24):4102–6.

World Health Organization. WHO laboratory manual for

the examination and processing of human semen. 6th

ed. Geneva: World Health Organization; 2021.

Shao ZM, Zhu YT, Gu M, Guo SC, Yu H, Li KK, Tang DD,

Xu YP and Lv MR. Novel variants in DNAH6 cause

male infertility associated with multiple

morphological abnormalities of the sperm flagella

(MMAF) and ICSI outcomes. Asian Journal of

Andrology. 2024 Jan 1;26(1):91-8.

Esfandiari N, Sharma RK, Saleh RA, Thomas AJ Jr and

Agarwal A. Utility of the nitroblue tetrazolium

reduction test for assessment of reactive oxygen

species production by seminal leukocytes and

spermatozoa. J Androl. 2003;24(6):862–70.

doi:10.1002/j.1939-4640.2003.tb03137.x

Vahedi V, Hedayat Evrigh N, Behroozlak M and Dirandeh

E. Antioxidant effects of Thyme (Thymus vulgaris)

extract on ram sperm quality during

cryopreservation. Iran J Appl Anim Sci.

;8(2):263–9.

Isachenko V, Todorov P, Seisenbayeva A and Isachenko E.

Cryoprotectant-free vitrification of human

spermatozoa in large (up to 0.5 mL) volume.

Cryobiology. 2020;92:77–83.

doi:10.1016/j.cryobiol.2020.07.002.

Kumaresan A, Sahu SB and Datta TK. Efficacy of fructose

and egg yolk-supplemented semen cryoprotectants

for cryopreservation of Sahiwal bull semen.

Theriogenology. 2022;179:56–62.

doi:10.1016/j.theriogenology.2022.06.021

Mota DA, Martins JAM and Oliveira AA. The role of Tris

buffer in the improvement of post-thaw sperm

quality in rams. Animals. 2022;12(1):148.

doi:10.3390/ani12010148.

Hong SH, Kwon DK, Kim MJ, Cho SJ, Park YS and Park

CK. Effects of citric acid supplementation on the

quality and functional activity of canine sperm

during the cryopreservation process. Animals.

;11(10):2972. doi:10.3390/ani11102972.

Ros-Santaella JL, Nový P, Scaringi M and Pintus E.

Antimicrobial peptides and proteins as alternative

antibiotics for porcine semen preservation. BMC Vet

Res. 2024;20(1):257.

Parameswari R, Rao KA, Manigandan P, Vickram AS,

Archana K and Sridharan TB. (2017). Tea

polyphenol-T. arjuna bark as sperm antioxidant

extender in infertile smokers. Cryoletters, 38(2), 95


Full Text: PDF

Article Metrics

Abstract View : 496 times
PDF Download : 260 times

Refbacks

  • There are currently no refbacks.