Submit or Track your Manuscript LOG-IN

Effect of Different Sonication Protocols on Merino Ram Sperm Parameters


Effect of Different Sonication Protocols on Merino Ram Sperm Parameters

Caner Öztürk1*, Mücahid Onay1 and Neşe Hayat Aksoy2

1Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Aksaray University, Aksaray, Turkey

2Department of Biochemistry, Faculty of Veterinary Medicine, Aksaray University,

Aksaray, Turkey


The biochemical analysis and the determination of the intracellular-extracellular contents of the sperm cells are important in the spermatological studies. Since homogenization processes reveal the intracellular contents of the sperm cells, we aimed to examine the optimal sonication procedure and the protective effect of the antioxidants against sonication. Four merino rams (2–3 years old) were used for semen collection, and the ejaculates were pooled and divided into five equal aliquots. The samples were diluted with a solution having different additives at 37 °C. The first two groups contained L-Cystine as an additive (2 and 4 mM), the next two groups had methionine as an additive (2 and 4 mM), and the last group had no additives (control). After dilution, the semen samples were cooled at 5 °C for 2 h and then stored in liquid nitrogen. For further evaluation, the samples were thawed at 38 °C for 30 s. A phase-contrast microscope (400x) was used to determine the sperm motility. PNA-FITC staining was used to examine the acrosome integrity under a fluorescence microscope. Total oxidant status (TOS) and total antioxidant status (TAS) were measured using ELISA. The methionine group showed an increase in the post-thaw motility (53.08 ±3.6%) and viability (47.5 ±2.9) percentages compared to the control group (46.68 ±2.9; 40 ±4.08%, p<0.05). All the groups with antioxidants showed decreased levels of TOS in all sonication replicates compared to the control group (p<0.05). The methionine group showed statistical differences in the TAS level measurements between the 4 and 8-repeat sonication compared to the control group (p<0.05). Upon the examination of the damaged sperm rates, no difference was found between 8 and 12-repetitions (p>0.05). The result of our study showed that methionine had a cryoprotective effect on motility, acrosome integrity, and TOS levels and was also found to have beneficial effects at TAS levels. Considering the percentage of the damaged sperms, applying 8-repeat (4 seconds) was found to be the most suitable measurement.

Article Information

Received 04 November 2020

Revised 05 December 2020

Accepted 15 December 2020

Available online 14 May 2021

Authors’ Contribution

CÖ and MO designed the study and collected the samples. CÖ, MO and NHA did the analysis. CÖ and MO wrote the manuscript.

Key words

Sonication, Sperm viability, Methionine additive, Spermatozoa, TOS, TAS level, Ram serum, PNA-FITC staining


* Corresponding author:

0030-9923/2021/0004-1259 $ 9.00/0

Copyright 2021 Zoological Society of Pakistan


Sperm freezing is an innovative assisted reproductive technology that enables the usage of valuable genetic resources over a long period, which can increase the cross-country exchange of perfect fathers (Awda et al., 2009). While studying sperm cryopreservation, it is important to evaluate the oxidative stress parameters to determine the sperm’s quality (Akalın et al., 2016; Bucak et al., 2020). Ultrasonication under optimum conditions is necessary to break down the sperm cells and organelle membranes to obtain the biochemical contents (Tateno et al., 2000). It was determined that mouse and human sperm cells subjected to 5 s of sonication could lead to separation of the head and tail of more than 95% of the cells.

Sonication involves the propagation of acoustic waves of a specific frequency. This method is used in the laboratory to separate microbial cells before culture and for further analysis. It is also extremely fast, with the splitting of the cells occurring in a short time (Amalfitano and Fazi, 2008). Separation occurs as the effect of external forces acting on a biofilm surface when the biofilm matrix exceeds the internal cohesion (Hunt et al., 2004).

During the homogenization process using sonication, the intensity and duration of ultrasonic waves can release the free oxygen radicals and disrupt the physical and chemical integrity of the sperm cells. The radicals that are released as an effect of high heat and pressure formed during the process can disrupt the integrity of the sperm membrane (Karabiga et al., 2007). Due to the free radical damage of purine and pyrimidine bonds, DNA is damaged, which causes low blastocyst formation (Agarwal et al., 2014). Sound waves propagate as waves based on the properties of the environment, such as solid, liquid, and gas (Akalın et al., 2016). The oxidative status of sperm samples is determined by the total oxidant status (TOS), while the antioxidative status is evaluated by measuring the levels of total antioxidant status (TAS).

Plasma membrane and acrosome are the main structures in the sperm cell that are affected by a cold shock (Hammerstedt et al., 1990). Ram sperm cells are highly sensitive to oxidative stress due to a higher number of polyunsaturated fatty acids in their plasma membranes (Allai et al., 2016). Exogenous antioxidants are required to maintain the balance between ROS production and scavenging. Many antioxidants, coenzyme Q10, trehalose, Gallic, and carnosic acids have been extensively added to semen extenders for minimizing the deleterious effect of oxidative stress (Gungor et al., 2019; Öztürk et al., 2020; de Albuquerque et al., 2020).

Methionine is a methyl and thiol provider, and its depletion in the presence of repeated ROS formation causes the pro-oxidant state (Bhardwaj and Yadav, 2013). In the antioxidant defense mechanism, methionine plays an important role because it easily reacts with the oxidants to form methionine sulfoxide (Levine et al., 1999). Glutathione synthesis occurs due to the presence of cysteine in the cell (De Matos and Furnus, 2000), and Cysteamine is a thiol compound that reacts with cystine to form a mixed disulfide, which is taken up by the mammalian cell and then cleaved to cysteine in the cytoplasm (Swami et al., 2017).

This study aims to determine the effects of antioxidants in semen extender on the oxidative stress parameters and investigate the effects of both additives and different sonication protocols on oxidative stress. Also, our study aimed to reveal the optimal sonication method for other studies as well.


Animals and semen collection

Four adult merino rams were used for this study, and semen samples were collected via an artificial vagina. Ethics Committee permission was obtained (No 2018/441).

Experimental design

The ejaculates were collected once a week for four weeks, and the ones with more than 80% motility and concentrations higher than 2x109 spermatozoa/mL were used. Tris base extender (TB) was used to dilute the semen (15% egg yolk, 6% glycerol), and the following additives were added based on which the groups were determined: TB + L-Cystine (2 mM), TB + L-Cystine (4 mM), TB + methionine (2mM), TB + methionine (4mM).

Diluted semen samples were loaded into 0.25 mL straws and equilibrated at 5°C for 2 h. Straws were frozen by placing them 5 cm above liquid nitrogen vapor (-110 to -120 °C) for 15 min, which was then stored in liquid nitrogen (-196 °C). The straws were later thawed in a water bath at 37 °C for 30 s for further evaluation.

Sperm motility

Spermatozoa motility was determined subjectively using a phase-contrast microscope (400x) and was estimated in five different microscopic areas. The mean of the five successive estimations was recorded as the final motility score.

Plasma membrane integrity

A modified staining protocol of Garner and Johnson (1995) was used. The semen samples were diluted at a ratio of 1: 3 with the tris stock solution, and then 30 µL of semen was mixed with 6 µL of SYBR-14 and 2.5 µL of propidium iodide (PI). Samples were incubated at 37 °C for 20 min in the dark and then 10 µL of Hancock solution was added to terminate the process.

Acrosome integrity

The acrosome integrity was determined as described by Nagy et al. (2003). The thawed straws were diluted with tris buffer at a ratio of 1: 3, then 60 µL semen, 10 µL FITC-PNA, and 2.5 µL of PI were mixed. The samples were incubated at 37 °C for 20 min in the dark, and 10 µL of Hancock solution (Schafer and Holzmann, 2000) was added for fixation.

Semen preparation for the enzymatic assays

To separate the spermatozoa, the diluted ejaculate was centrifuged three times at 4 oC for 15 min at 800 g. The cells were then washed with PBS and finally resuspended in 0.5 mL of PBS. For homogenization, the sperm suspension was placed in a 2 mL chamber of ice water. Each antioxidant group was further divided into four groups; 4- repeat (4 seconds), 8- repeat (4 seconds), 12- repeat (4 seconds) sonication, and the 4th group was the control group, which was sonicated for 4-seconds (no repeat).

Measurement of total antioxidant capacity

Total antioxidant status (Relassay, Turkey) was measured using the method of Erel (2004), which is based on bleaching of the characteristic color of a more stable ABTS (2,2 ′- Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) radical cation by antioxidants. This test has excellent sensitivity values of less than 3%, and the results are expressed as mmol Trolox equivalent/L.

Measurement of total oxidant status

TOS levels were measured using commercially available kits (Relassay, Turkey). The oxidants present in the sample oxidizes the ferric ion-o-dianisidine complex to the ferric ion, and this ferric ion produces a colored complex with xylenol orange in an acidic environment. The intensity of the color is measured spectrophotometrically, which is related to the total amount of oxidant molecules in the sample. The assay was calibrated with hydrogen peroxide, and the results were expressed in terms of micromolar hydrogen peroxide equivalent per liter (µmol H2O2 equivalent/L) (Erel, 2005).

Statistical analysis

The normality and homogeneity of the variances were confirmed by the Shapiro-Wilk test. The results of post-thaw sperm samples were expressed as mean±standard deviation with a One-way analysis of variance (ANOVA) used for evaluation. Analysis of variance was followed by Duncan’s post hoc test to determine the differences between the groups. Differences with values of P<0.05 were considered to be statistically significant. Analyses were performed using the SPSS 21 package program.


As shown in Table I, the freezing extender having methionine led to higher percentages of sperm viability in comparison to the control group (p<0.05). Sperm acrosome integrity did not show any significant differences compared to the control group (p<0.05). The number of repetitions increased the level of the total oxidant status (TOS) with statistical differences between the groups with additives and the control group in all the repetitions (p<0.05) (Table II). The highest TAS levels were achieved in the group having 4 mM methionine with a statistically significant difference observed between the control, 4- repeat (4 seconds), and 8- repeat (4 seconds) groups whereas, no significant differences were observed in the 12- repeat (4 seconds) group (Table II). As a result of sonication, the sperm damage percentage rates were found to be statistically different between the groups and the control (p<0.05), but there was no difference between the 8 and 12- repetition groups (p>0.05) (Table II).


This study was conducted to define an optimal sonication time for sperm cells and to evaluate the protective effects of antioxidants on both thawed and sonicated cells (Akalın et al., 2016). Sperm cells are sensitive to the damage caused by high levels of ROS. Seminal plasma is an important source of antioxidants in semen (Agarwal et al., 2014). However, the existing antioxidant defense system is disrupted by the freeze-thawing procedure, which causes oxidative damage, imparting the toxic effects of free radicals (Bucak et al., 2015).


Table I. Flourescent staining parameters(Mean±SEM) in frozen–thawed Merino ram semen.


Sperm viability (%)

Acrosome integrity (%)



Cystine 2mM




Cystine 4mM

50.13±4 ab



Methionine 2mM




Methionine 4mM













(*p < 0.05).


Table II. TOS levels, TAS levels and % damaged sperm rate (Mean±SEM) in thawed ram semen.



4 replicates

8 replicates

12 replicates


Cystine 2mM





Cystine 4mM





Methionine 2mM





Metiyonin 4mM















TAS (mmol/L)

Cystine 2mM





Cystine 4mM





Methionine 2mM





Methionine 4mM















Damaged sperm rates

Sonication 4 second 4 Repetitions

66.0 ±2.2b

Sonication 4 second 8 Repetitions


Sonication 4 second 12 Repetitions


Control 4 second





(*p < 0.05).


In this study, a repeated sonications lead to an increase in the AOP levels of all the groups as the number of repeats increased. The application of antioxidants showed a protective effect when compared to the control group. Sonication is an easy method used to separate the head and tail of the sperm cells. However, due to the high temperature during the sonication, undesirable free radical molecules are released from the cells that cause lipid peroxidation (Akalın et al., 2016). Researchers have observed pronuclear formation in their injection using sperm heads separated by sonication but have not shown the developmental competence of the zygotes (Yanagida et al., 1991; Katayose et al., 1992). Baker et al. (2002) reported that the membrane proteins of mouse sperm were damaged and detached by sonication of 15-seconds, followed by 1-minute cooling with three repetitions. Considering the TOS values and sperm damage percentage, our results determined that 3-repetitive application for 8s was the most suitable method.

Antioxidants added to the extender in the cryopreservation of semen have beneficial effects on the preservation of sperm motility and acrosome integrity (Salamon and Maxwell, 2000). Methionine and thiol-containing antioxidants are used to protect spermatozoa from oxidative damage, thus, providing detoxification (Caylak et al., 2008). The claim that methionine has the property of clearing ROS makes it an important antioxidant (Levine et al., 1999). Methionine added to sperm extender showed an increase in the motility of sperm cells along with statistically non-significant differences in the LPO levels between the groups (Çoyan et al., 2010). In the present study, significant differences were observed in the TOS levels between the methionine and the control group. Similar to our study Omur and Çoyan (2016) reported that semen extender supplemented with methionine preserved the integrity of plasma membrane and acrosome.

It is reported that thiol compounds such as cysteamine and cystine stimulate oocyte GSH synthesis until the highest GSH levels are reached in the oocytes. Low molecular weight thiol compounds such as cysteamine reduce cystine to cysteine, promoting cysteine uptake in the cells while increasing the synthesis of GSH (Gasparrini et al., 2006). Pradieé et al. (2016) had reported that cysteine did not affect the post-thaw viability and oxidative activity of ram sperm, which was similar to our study, where the spermatological parameters showed a differently positive effect on the TOS level.


We observed that the additives added to the semen extender had positive effects on the spermatological parameters and as a result, the antioxidant substances that resulted from sonication decreased the TOS values. Also, TAS values showed a decrease with an increase in the number of repetitions of sonication, but the antioxidants provided a protective effect. There was no difference observed in the percentage of damaged semen in the number of repetitions of sonication between the 8 and 12 repetition. Thus, we concluded that 8 repetition was sufficient.


This research was funded by The Scientific and Technological Research Council of Turkey (TÜBİTAK) with number 1919B011802699.

Statement of conflict of interest

The authors have declared no conflict of interest.


Agarwal, A., Durairajanayagam, D., Halabi, J., Peng, J. and Vazquez-Levin, M., 2014. Proteomics, oxidative stress and male infertility. Reprod. Biomed. Online, 29: 32-58.

Akalin, P.P., Bucak, M.N., Güngör, Ş., Başpinar, N., Coyan, K., Dursun, Ş., İli, P., Aksoy, A., Karaşör, Ö.F., Bilgili, A., Sariözkan S. and Yeni, D., 2016. Influence of lycopene and cysteamine on sperm and oxidative stress parameters during liquid storage of ram semen at 5C. Small Rum. Res., 137: 117-123.

Allai, L., Druart, X., Ozturk, M., BenMoula, A., Nasser, B. and El Amiri, B., 2016. Protective effects of Opuntia ficus-indica extract on ram sperm quality, lipid peroxidation and DNA fragmentation during liquid storage. Anim. Reprod. Sci., 175: 1–9.

Amalfitano, S., and Fazi, S., 2008. Recovery and quantification of bacterial cells associated with streambed sediments. J. Microbiol. Methods, 75: 237–243.

Awda, B.J., Mackenzie-Bell, M. and Buhr, M.M. 2009. Reactive oxygen species and boar sperm function. Biol. Reprod., 81: 553-561.

Baker, S.S., Cardullo, R.A. and Thaler, C.D., 2002. Sonication of mouse sperm membranes reveals distinct protein domains. Biol. Reprod., 66: 57–64.

Bhardwaj, P. and Yadav, R.K., 2013. Chronic pancreatitis: Role of oxidative stress and antioxidants. Free Radic. Res., 47: 941-949.

Bucak, M.N., Ataman, M.B., Başpınar, N., Uysal, O., Taşpınar, M., Bilgili, A., Ozturk, C., Gungor, Ş., İnanç, M.E. and Akal, E., 2015. Lycopene and resveratrol improve post-thaw bull sperm parameters: Sperm motility, mitochondrial activity and DNA integrity. Andrologia, 47: 545–552.

Bucak, M.N., Keskin, N., Ili, P., Bodu, M., Akalın, P.P., Öztürk, A.E., Özkan, H., Topraggaleh, T.R., Sari, F., Başpınar, N. and Dursun, Ş., 2020. Decreasing glycerol content by co-supplementation of trehalose and taxifolin hydrate in ram semen extender: Microscopic, oxidative stress, and gene expression analyses. Cryobiology, 96: 19-29.

Caylak, E., Aytekin, M., Halifeoğlu, I., 2008. Antioxidant effects of methionine, a-lipoic acid, N-acetylcysteine and homocysteine on lead-induced oxidative stress to erythrocytes in rats. Exp. Toxicol. Pathol., 60: 289–294.

Çoyan, K., Başpınar, N., Bucak, M.N., Akalın, P.P., Ataman, M.B., Ömür, A.D., Güngör, Ş., Küçükgünay, S., Ozkalp, B. and Sarıözkan, S., 2010. Influence of methionine and dithioerythritol on sperm motility, lipid peroxidation and antioxidant capacities during liquid storage of ram semen. Res. Vet. Sci., 89: 426-431.

De Albuquerque, L.M., da Silva, G.C., Cortes, S.F., Luz, S.B., de Resende, A.C., de Castro Alves, N., Wenceslau, R. and Stahlberg, R., 2020. Does coenzyme Q10 exert antioxidant effect on frozen equine sperm? J. Equine Vet. Sci., pp. 102964.

De Matos, D.G. and Furnus, C.C., 2000. The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development: effect of β-mercaptoethanol, cysteine and cystine. Theriogenology, 53: 761-771.

Edmonds, P.D. and Sancier K.M., 1983. Evidence for free radical production by ultrasonic cavitation in biological media. Ultras. med. Biol., 9: 635–639.

Erel, O., 2004. A novel automated method to measure total antioxidant response against potent free radical reactions. Clin. Biochem., 37: 112-119.

Erel, O., 2005. A new automated colorimetric method for measuring total oxidant status. Clin. Biochem., 38: 1103-1111.

Garner, D.L. and Johnson, L.A., 1995. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol. Reprod., 53: 276–284.

Gasparrini, B., Boccia, L., Marchandise, J., Di Palo, R., George, F., Donnay, I. and Zicarelli, L., 2006. Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development. Theriogenology, 65: 275-287.

Gungor, S., Inanc, M.E., Ozturk, C., Korkmaz, F., Bastan, I., Cil, B. and Kastelic, J.P., 2019. Gallic and carnosic acids improve quality of frozen-thawed ram spermatozoa. Andrologia, 51: 13393.

Hammerstedt, R.H., Graham, J.K. and Nolan, J.P., 1990. Cryopreservation of mammalian sperm: What we ask them to survive. J. Androl., 11: 73-88.

Hunt, S.M., Werner, E.M., Huang, B.C., Hamilton, M.A. and Stewart, P.S., 2004. Hypothesis for the role of nutrient starvation in biofilm detachment. Appl. environ. Microbiol., 70: 7418–7425.

Karabiga, M., Kiriş, İ., Yılmaz, N., Altuntaş, İ., Karahan, N. and Okutan, H., 2007. The effect of aprotinin on renal injury in an experimental model of aortic ischemia-reperfusion. Turk. J. Vasc. Surg., 165: 9–18.

Katayose, H., Matsuda, J., Yanagimachi, R., 1992. The ability of dehydrated hamster and human sperm nuclei to develop into pronuclei. Biol. Reprod., 47: 277-284.

Levine, R.L., Berlett, B.S., Moskovitz, J., Mosoni, L. and Stadtman, E.R. 1999. Methionine residues may protect proteins from critical oxidative damage. Mech. Ageing Dev., 107: 323-332.

Nagy, S., Jansen, J., Topper, E.K. and Gadella, B.M. 2003. A triple-stain flow cytometric method to assess plasma and acrosomemembrane integrity of cryopreserved bovine sperm immediately after thawing in presence of egg-yolk particles. Biol. Reprod., 68: 1828-1835.

Omur, A.D. and Coyan, K., 2016. Protective effects of the antioxidants curcumin, ellagic acid and methionine on motility, mitochondrial transmembrane potential, plasma membrane and acrosome integrity in freeze-thawed Merino ram sperm. Vet. Med., 61: 10-16.

Öztürk, A.E., Bodu, M., Bucak, M.N., Ağir, V., Özcan, A., Keskin, N., İli., P., Topraggaleh, T.R., Sidal, H., Başpınar, N. and Dursun, Ş., 2020. The synergistic effect of trehalose and low concentrations of cryoprotectants can improve post-thaw ram sperm parameters. Cryobiology, 95: 157-163.

Pradieé, J., Cardoso, T.F., Silva, E.F., Gonçalves, A.O., Gastal, G.D.A., Rosa, C.E., Mondadori, R.G., Pegoraro, L.M.C., Vieira, A.D. and Lucia, Jr, T., 2016. Effect of β-mercaptoetanol and cysteine on post-thawing quality and oxidative activity of ram sperm and on the viability of vitrified sheep embryos. Arq. Bras. Med. Vet. Zootec., 68: 1309-1315.

Salamon, S. and Maxwell, W.M.C., 2000. Storage of ram semen. Anim. Reprod. Sci., 62: 77-111.

Schafer, S. and Holzmann, A., 2000. The use of transmigration and spermac stain to evaluate epididymal cat spermatozoa. Anim. Reprod. Sci., 59: 201–211.

Swami, D.S., Kumar, P., Malik, R.K., Saini, M., Kumar, D. and Jan, M.H., 2017. Cysteamine supplementation revealed detrimental effect on cryosurvival of buffalo sperm based on computer-assisted semen analysis and oxidative parameters. Anim. Reprod. Sci., 177: 56-64.

Tateno, H., Kimura, Y. and Yanagimachi, R., 2000. Sonication per se is not as deleterious to sperm chromosomes as previously inferred. Biol. Reprod., 63: 341–346.

Yanagida, K., Yanagimachi, R., Perreault, S.D. and Kleinfeld, R.G., 1991. Thermostability of sperm nuclei assessed by microinjection into hamster oocytes. Biol. Reprod., 44: 440-447.

To share on other social networks, click on any share button. What are these?

Pakistan Journal of Zoology


Vol. 53, Iss. 3, Pages 801-1200


Click here for more

Subscribe Today

Receive free updates on new articles, opportunities and benefits

Subscribe Unsubscribe