test Effects of Melatonin and Coenzyme Q 10 Added to Extenders on Spermatological Properties in Cryopreservation of Epididymal Rat Sperm|Journal of the Hellenic Veterinary Medical Society

Effects of Melatonin and Coenzyme Q 10 Added to Extenders on Spermatological Properties in Cryopreservation of Epididymal Rat Sperm


Published: Nov 10, 2025
Keywords:
Rat Sperm Analysis Apoptosis Cryopreservation Antioxidant Melatonin Coenzyme Q10
F Yılmaz Dayanç
https://orcid.org/0000-0003-0526-389X
C Yıldız
Abstract

With the use of advanced genome editing technology, the production of genetically modified mutant rats worldwide, and the successful freezing and storage of their gametes, have become important in biotechnology. In the present study, the effects of different doses of melatonin (0.5mM, 1mM, 2mM) and coenzyme Q10 (25µM, 50µM, 75µM) added to the spermatozoon freezing medium containing 8% lactose monohydrate, 23% egg yolk (EY), and 10% trisaminomethane on the freezability of rat spermatozoa were investigated. After freezing and thawing, motility, dead spermatozoa rate, plasma membrane integrity, abnormal acrosome rate, motility longevity, apoptotic and necrotic cell rates were evaluated. Compared to the control group (12.90±1.34%), the highest motility value was found in the 1mM Melatonin group (24.58±1.68%) and 25µM Coenzyme Q10 group (24.23±1.95%) (p<0.01). The lowest dead spermatozoa rate was determined in the 1mM Melatonin group (63.08±4.89%) compared to the control group (75.47±2.27%) (p<0.01). Plasma membrane integrity rates were higher in the 1mM Melatonin (35.50±1.82%) and 25µM Coenzyme Q10 (31.31±1.93%) groups compared to the control group (18.21±1.36%) (p<0.01). The best motility longevity was observed in the 1mM melatonin and 25µM coenzyme Q10 groups compared to the control group (p<0.01). There was no difference between the groups in terms of abnormal acrosome rates (p>0.01). Apoptotic and necrotic cell rates were found to be lower in the 1mM Melatonin or 25µM Coenzyme Q10 groups compared to the control group (p<0.01). However, it was found that the use of 1mM melatonin had a better protective effect than 25µM coenzyme Q10 (p<0.01). In conclusion, the addition of 1mM Melatonin or 25µM Coenzyme Q10 to the rat sperm diluent effectively protected spermatozoa against cryo-damage during cryopreservation process.

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References
AbdelHafez F, Bedaiwy M, El-Nashar SA, Sabanegh E, Desai N
(2009)T echniques for cryopreservation of individual or small
numbers of human spermatozoa: a systematic review. Human reproduction
update, 15(2), 153-164.
Agarwal A, Gupta S, Sharma R (2016) Eosin-Nigrosin staining procedure.
In Andrological Evaluation of Male Infertility, Springer,
Cham, 73-77.
Aitken RJ ve Baker MA (2006) Oxidative stress, sperm survival and
fertility control. Molecular and Cellular Endocrinology, 250(1-
, 66-69.
Alipour-Jenaghard P, Daghigh-Kia H, Masoudi R (2023) Preservation
of the quality and fertility potential of post-thawed rooster sperm
using MitoQ. Theriogenology, 208, 165-170.
Anzar M, He L, Buhr M M, Kroetsch TG, Pauls KP (2002) Sperm
apoptosis in fresh and cryopreserved bull semen detected by flow
cytometry and its relationship with fertility. Biology of Reproduction,
(2), 354-360.
Appiah MO, Asante-Badu B, Zhao J, Liu H, Wang J, Lu W (2020)
Possible protective mechanisms of coenzyme Q10 action on spermatozoa
during cryopreservation or cooled-stored condition. Cryoletters,
(5), 246-256.
Asadzadeh N, Abdollahi Z, Esmaeilkhanian S, Masoudi R (2021)
Fertility and flow cytometry evaluations of ram frozen semen in
plant-based extender supplemented with Mito-TEMPO. Animal
Reproduction Science, 233, 106-836.
Awad H, Halawa F, Mostafa T, Atta H (2006) Melatonin hormone
profile in infertile males. İnternational journal of andrology, 29(3),
-413.
Ball BA, Medina V, Gravance CG, Baumber J (2001b) Effect of
antioxidants on preservation of motility, viability and acrosomal
integrity of equine spermatozoa during storage at 5 C. Theriogenology,
(4), 577-589.
Bhattacharya S (2018) Cryoprotectants and their usage in cryopreservation
process. Cryopreservation Biotechnology in Biomedical
and Biological Sciences, s: 7-19.
Bucak MN, Karaşör ÖF, Sarı A, Bodu M, Ili P, Narlıçay S, Sari F
(2024) Lipid mixtures (from a liposome kit) and melatonin improve
post-thawed Angora goat sperm parameters. Cryobiology, 115,
ChaithraShree AR, Ingole SD, Dighe VD, Nagvekar AS, Bharucha
SV, Dagli N. R, Kharde SD (2020) Effect of melatonin on
bovine sperm characteristics and ultrastructure changes following
cryopreservation. Veterinary Medicine and Science, 6(2), 177-186.
Charreau B, Tesson L, Soulillou JP, Pourcel C, Anegon I (1996)
Transgenesis in rats: technical aspects and models. Transgenic
Research, 5, 223-234.
Chaudhary SC, Aeksiri N, Wanangkarn A, Liao YJ, Inyawilert W
(2021) Effects of melatonin on cryopreserved semen parameters and
apoptosis of Thai swamp buffalo bull (Bubalus bubalis) in different
thawing conditions. Adv. Anim. Vet. Sci, 9, 238-245
Chen XJ, Zhang Y, Jia GX, Meng QG, Bunch T D, Liu GS, Zhou
GB (2016) Effect of melatonin supplementation on cryopreserved
sperm quality in mouse. Cryoletters, 37(2), 115-122.
Daghigh Kia H, Bolooki Z, Vaseghi Dodran H, Mahdipour M (2017)
Effect of adding coenzyme Q10 and Ellagic acid during cryopreservation
on post-thaw quality of ram semen. Iranian Journal of
Applied Animal Science, 7(3), 445-451.
Divar MR, Azari M, Mogheiseh A, Ghahramani S (2022) Supplementation
of melatonin to cooling and freezing extenders improves
canine spermatozoa quality measures. BMC Veterinary
Research, 18(1), 86.
Doidar YA, El-Nagar HA, Elrefy A, Mousbah AM (2018) Cryopreservation
and quality assessment of buffalo bull (Bubalus bubalis)
semen using new Moringa extender and antioxidant CoQ10. Journal
of Animal and Poultry Production, 9(9), 375-381.
Elkhawagah AR, Ricci A, Bertero A, Poletto ML, Nervo T, Donato
GG, Martino NA (2024 ) Supplementation with MitoTEMPO before
cryopreservation improves sperm quality and fertility potential
of Piedmontese beef bull semen. Frontiers in Veterinary Science,
, 1376057.
El-Tohamy MM, Kotp MS, El Nattat WS, Mohamed AH (2012)
Semen characteristics and oxidative/antioxidative status in semen
and serum of male rabbits supplemented with antioxidants during
heat stress. 175-183.
Eraslan Sakar A, Yalcin OK, Mazi A, Yildiz C (2025) The effects of
supplemented coenzyme Q10 to extender on the endoplasmic reticulum
stress-related genes and sperm quality parameters in cryopreservation
of mouse spermatozoa. Biopreservation and Biobanking.
Fadl AM, Ghallab ARM, Abou‐Ahmed MM, Moawad AR (2021)
Melatonin can improve viability and functional integrity of cooled
and frozen/thawed rabbit spermatozoa. Reproduction in Domestic
Animals, 56(1), 103-111.
Fayez E, Samir H, El Sayed MA, Rawash ZM, Salama A (2023) Melatonin
supplementation to the freezing extender improves the quality
of canine epididymal spermatozoa. Andrologia, 2023(1), 7873399.
Festing MFW, Greenhouse DD (1992) Abbreviated list of inbred strains
of rats. Rat News Letter, 26, 10-22
Gardela J, Ruiz-Conca M, Palomares A, Olvera-Maneu S, Garcia-
Calvo L, Lopez-Bejar M, Alvarez-Rodriguez M (2023)
Effect of Honey, Coenzyme Q10, and β-Carotene/α-Tocopherol as
Novel Additives in Rabbit-Sperm Cryopreservation Extender. Animals,
(14), 2392.
Hancock JL (1952) The morphology of bull spermatozoa. Journal of
Experimental Biology, 29(3): 445-453.
Hezavehei M, Sharafi M, Kouchesfahani HM, Henkel R, Agarwal
A, Esmaeili V, Shahverdi A (2018) Sperm cryopreservation: A
review on current molecular cryobiology and advanced approaches.
Reproductive Biomedicine Online, 37(3), 327-339.
Izadpanah G, Zare-Shahneh A, Zhandi M, Yousefian I, Emamverdi
M (2015 ) Melatonin has a beneficial effect on stallion sperm
quality in cool condition. Journal of Equine Veterinary Science,
(7), 555-559.
Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Crabo BG (1984)
Development of an assay to assess the functional integrity of the
human sperm membrane and its relationship to other semen characteristics.
Reproduction, 70(1), 219-228.
Kapoor P, Kapoor A (2013) Coenzyme Q10-a novel molecule. J Indian
Acad Clin Med, 14(1), 37-45.
Karimfar MH, Niazvand F, Haghani K, Ghafourian S, Shirazi R,
Bakhtiyari S (2015) The protective effects of melatonin against
cryopreservation-induced oxidative stress in human sperm. International
journal of immunopathology and pharmacology, 28(1), 69-76.
Khazravi B, Khodaei-Motlagh M, Masoudi R, Yahyaei M (2024)
Coenzyme Q10 preserves buck’s sperm quality during cryopreservation
process in plant-based extender. Animal Reproduction
Science, 265, 107487.
Lobo V, Patil A, Phatak A, Chandra N (2010) Free radicals, antioxidants
and functional foods: Impact on human health. Pharmacogn
Rev. 4(8),118-126
Luo X, Wu D, Liang M, Huang S, Shi D, Li X (2023) The effects of
melatonin, glutathione and vitamin E on semen cryopreservation
of Mediterranean buffalo. Theriogenology, 197, 94-100.
Mancini A, Milard, D, Conte G, Festa R, De Marinis L, Littarru GP
(2005) Seminal antioxidants in humans: preoperative and postoperative
evaluation of coenzyme Q10 in varicocele patients. Hormone
and metabolic research, 37(07), 428-432.
Masoudi R, Sharafi M, Pourazadi L (2019a) Improvement of rooster
semen quality using coenzyme Q10 during cooling storage in the
Lake extender. Cryobiology, 88, 87-91.
Masoudi R, Sharafi M, Shahneh AZ, Kohram H, Nejati-Amiri E,
Karimi H, Shahverdi A (2018) Supplementation of extender with
coenzyme Q10 improves the function and fertility potential of rooster
spermatozoa after cryopreservation. Animal reproduction science,
, 193-201.
Masoudi R, Sharafi M, Shahneh AZ (2019b) Effects of Co Q10 on the
quality of ram sperm during cryopreservation in plant and animal
based extenders. Animal Reproduction Science, 208, 106103.
Mehaisen GM, Partyka A, Ligocka Z, Nizanski W (2020) Cryoprotective
effect of melatonin supplementation on post-thawed rooster
sperm quality. Animal reproduction science, 212, 106238.
Monteiro MM, Silva RAJA, Arruda LCP, de Oliveira AS, da Cunha
Mergulhao FC, Júnior PLJM, Guerra MMP (2022) Effect of
melatonin in different extenders on the quality of frozen semen of
goats. Emerging Animal Species, 5, 100015.
Morris GJ, Acton E, Murray BJ, Fonseca F (2012) Freezing injury:
the special case of the sperm cell. Cryobiology, 64(2), 71-80.
Morris GJ (2006) Rapidly cooled human sperm: no evidence of intracellular
ice formation. Human Reproduction, 21(8), 2075-2083.
Nakagata N, Mikoda N, Nakao S, Nakatsukasa E, Takeo T (2020)
Establishment of sperm cryopreservation and in vitro fertilisation
protocols for rats. Scientific reports, 10(1), 93.
Nakatsukasa E, Inomata T, Ikeda T, Shino M, Kashiwazaki N (2001)
Generation of live rat offspring by intrauterine insemination with epididymal
spermatozoa cryopreserved at-196 0C. Reproduction-Cambridge,
(3), 463-467.
Nakatsukasa E, Kashiwazaki N, Takizawa A, Shino M, Kitada K,
Serikawa T, Nakagata N (2003) Cryopreservation of spermatozoa
from closed colonies, and inbred, spontaneous mutant, and
transgenic strains of rats. Comparative medicine, 53(6), 639-641.
Nogueira BG, Pereira RR, Bitencourt JL, Milan B, Reis WV, Junior
MV, Souza MI (2022) Coenzyme Q10 and melatonin protect
cryopreserved equine sperm against lipoperoxidation. Animal Reproduction
Science, 243, 107027.
O’connell MN. Mcclure S, Lewis SEM (2002) The effects of cryopreservation
on sperm morphology, motility and mitochondrial
function. Human Reproduction, 17(3), 704-709
Ofosu J, Qazi IH, Fang Y, Zhou G (2021) Use of melatonin in sperm
cryopreservation of farm animals: A brief review. Animal reproduction
science, 233, 106850.
Perumal P, Khan MH, Chang S, Ezung E, Vupru K, Khate K (2018)
Anti-apoptotic effect of melatonin in sperm of mithun. Indian J.
Anim. Sci, 88(4), 412-414.
Pindaru LP, Cenariu M, Pall E, Groza IS (2015) Effects of coenzyme
Q10 on sperm viability during storage of boar semen at 17°
C. Veterinary Medicine, 32-36
Putri F, Karja NWK, Setiadi MA, Kaiin EM (2023) Influence of
Sperm Number and Antioxidant Melatonin in Extender on the Quality
of Post-Thawing Sheep Spermatozoa. Indonesian Journal of Animal
& Veterinary Sciences/Jurnal Ilmu Ternak dan Veteriner, 28(1).
Quinn P, Kerin JF, Warnes GM (1985) Improved pregnancy rate in
human in vitro fertilization with the use of a medium based on the
composition of human tubal fluid. Fertility and sterility, 44(4),
-498.
Raouf SM, Taha AT (2021) Effects of CO Q10 with vitamin E supplementation
on semen quality and seminal plasma parameters of
broiler breeder males. Iraqi Journal of Veterinary Sciences, 35(1),
-70.)
Ruan Q, Yang S, Hua S, Zhang W, Li D, Yang Y, Meng Z (2024)
Supplementation of Extender with Melatonin Improves the Motility,
Mitochondrial Membrane Potential, and Fertilization Ability of
Cryopreserved Brown-Marbled Grouper Sperm. Animals, 14(7),
Rudolph AS, Crowe JH, Crowe LM (1986) Effects of three stabilizing
agents proline, betaine, and trehalose on membrane phospholipids.
Archives of Biochemistry and Biophysics, 245(1), 134-143.
Saeed AM, El-Nagar HA, Wafa WM, Hussein YS (2016) Effect of
coenzyme Q10 as an antioxidant added to semen extender during
cryopreservation of buffalo and cattle semen. Journal of Animal
and Poultry Production, 7(11), 403-408.
Said TM, Gaglani A, Agarwal A (2010) Implication of apoptosis in
sperm cryoinjury. Reproductive Biomedicine Online, 21(4), 456-
Santos-Ocaña C, Do TQ, Clarke CF, Padilla S, Navas P (2002) Uptake
of exogenous coenzyme Q and transport to mitochondria is
required for bc1 complex stability in yeast coq mutants. Journal
of Biological Chemistry, 277(13), 10973-10981.
Shahat AM, Thundathil JC, Kastelic JP (2022) Melatonin or L-arginine
in semen extender mitigate reductions in quality of frozen-
thawed sperm from heat-stressed rams. Animal Reproduction
Science, 238, 106934.
Sharideh H, Zeinoaldini S, Zhandi M, Zaghari M, Sadeghi M, Akhlaghi
A, Peebles ED (2020) Use of supplemental dietary coenzyme
Q10 to improve testicular function and fertilization capacity in aged
broiler breeder roosters. Theriogenology, 142, 355-362.
Sharideh H, Zhandi M, Zenioaldini S, Zaghari M, Sadeghi M (2019)
The effect of coenzyme Q10 on rooster semen preservation in cooling
condition. Theriogenology, 129, 103-109.
Sieme H, Oldenhof H, Wolkers WF (2016) Mode of action of cryoprotectants
for sperm preservation. Animal Reproduction Science,
, 2-5.
Silvestre MA, Yaniz JL, Pena FJ, Santolaria P, Castello-Ruiz M
(2021) Role of antioxidants in cooled liquid storage of mammal
spermatozoa. Antioxidants, 10(7), 1096.
Somfai T, Bodo SZ, Nagy SZ Gocza E, Ivancsics J (2002) Simultaneous
Evaluation of Viability and Acrosome Integrity of Mouse
Spermatozoa Using light Microscopy. Biotechnic & Histochemistry,
(3), 117-120.
Sun TC, Li HY, Li XY, Yu K, Deng SL, Tian L (2020) Protective
effects of melatonin on male fertility preservation and reproductive
system. Cryobiology, 95, 1-8.
Tamura H, Takasaki A, Taketani T, Tanabe M, Kizuka F, Lee L,
Sugino N (2012) The role of melatonin as an antioxidant in the
follicle. Journal of Ovarian Research, s: 5, 1-9.
Tas DO, Ozkavukcu S, Inanc I, Kose SK, Erdemli E (2023) The
effects of coenzyme Q10 and curcumin supplementation in freezing
medium for human sperm cryopreservation. European Journal of
Obstetrics & Gynecology and Reproductive Biology, s: 287, 36-45.
Triyaningrum T, Fahrudin M, Karja NWK, Prasetyaningtyas WE
(2024) The quality of chilled ram semen after storage in extender
with addition of melatonin at 4°C. Jurnal Kedokteran Hewan-Indonesian
Journal of Veterinary Sciences, s: 18(1).
Varisli O, Uguz C, Agca C, Agca Y (2009) Effect of chilling on the
motility and acrosomal integrity of rat sperm in the presence of
various extenders. Journal of the American Association for Laboratory
Animal Science, 48(5), 499-505.
World Health Organization, WHO laboratory manual for the examination
of human semen and sperm‐cervical mucus interactions. 4th
edn. Cambridge University Press, Cambridge, UK, 1999.
Yamashiro H, Han YJ, Sugawara A, Tomioka I, Hoshino Y, Sato E
(2007) Freezability of rat epididymal sperm induced by raffinose
in modified Krebs–Ringer bicarbonate (mKRB) based extender
solution. Cryobiology, 55(3), 285-294.
Yamashiro H, Toyomizu M, Kikusato M, Toyama N, Sugimura S,
Hoshino Y, Sato E (2010a) Lactate and adenosine triphosphate
in the extender enhance the cryosurvival of rat epididymal sperm.
Journal of the American Association for Laboratory Animal Science,
(2), 160-166.
Yamashiro H, Toyomizu M, Toyama N, Aono N, Sakurai M, Hiradate
Y, Sato E (2010b) Extracellular ATP and dibutyryl cAMP enhance
the freezability of rat epididymal sperm. Journal of the American
Association for Laboratory Animal Science, 49(2), 167-172.
Yang S, Fan B, Chen X, Meng Z (2021) Supplementation of the freezing
medium with Coenzyme Q10 attenuates oxidative stress and
improves function of frozen-thawed giant grouper (Epinephelus
lanceolatus) spermatozoa. Theriogenology, 175, 77-82.
Yang S, Zeng Z, Chen X, Fan B, Meng Z (2023) Melatonin supplementation
to the freezing medium enhances post-thaw sperm quality
and fertility of giant grouper (Epinephelus lanceolatus). Aquaculture
Reports, 31, 101662.
Yi X, Qiu Y, Tang X, Lei Y, Pan Y, Raza SHA, Sun X (2024) Effect
of Five Different Antioxidants on the Effectiveness of Goat Semen
Cryopreservation. Reproductive Sciences, s: 1-15.
Yousefian I, Emamverdi M, Karamzadeh-Dehaghani A, Sabzian-
Melei R, Zhandi M, Zare-Shahneh A (2018) Attenuation
of cryopreservation-induced oxidative stress by antioxidant: Impact
of Coenzyme Q10 on the quality of post-thawed buck spermatozoa.
Cryobiology, 81, 88-93.
Yousefian I, Zare-Shahneh A, Zhandi M (2014) The effect of coenzyme
Q10 and α-tocopherol in skim milk–based extender for preservation
of caspian stallion semen in cool condition. Journal of
Equine Veterinary Science, 34(8), 949-954.
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