Methionine, Cysteine and Butylated Hydroxytoluene Enhance Cryosurvival of Ram Semen on Post-Thaw and Post-Incubation Time Points

Methionine, Cysteine and Butylated Hydroxytoluene Enhance Cryosurvival of Ram Semen on Post-Thaw and Post-Incubation Time Points | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Methionine, Cysteine and Butylated Hydroxytoluene Enhance Cryosurvival of Ram Semen on Post-Thaw and Post-Incubation Time Points Mehmed Berk Toker, Ibrahim Dogann This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2120718/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 May, 2023 Read the published version in Tropical Animal Health and Production → Version 1 posted 5 You are reading this latest preprint version Abstract Despite there have been many experiments conducted about antioxidants, the best sole or combination use of antioxidants to include as a standard ingredient to freezing extenders yet to be found. This study conducted to investigate the different doses of methionine (2.5 and 5mM), cysteine (1 and 2mM) and butylated hydroxytoluene (BHT) (1 and 2mM) for ram semen cryopreservation on post-thaw and post-incubation (6 h) time points over spermatological parameters. Semen samples collected from Kivircik rams via electro-ejaculator in breeding season. After essential spermatological evaluations, appropriate samples were pooled then split into 7 equal aliquots to create study groups (antioxidant free control, 2.5mM methionine, 5mM methionine, 1mM cysteine, 2mM cysteine, 1mM BHT and 2mM BHT). Semen samples put into French straws (0.25mL) and freezing procedure (two-step) conducted via a programmable gamete freezer. At both time points; motility, HOST, PSA-FITC and TUNEL assays were conducted to discover the impacts of cryopreservation and incubation process over sperm cells. Antioxidant supplemented groups yielded better results comparing to control groups in terms of various spermatological parameters not only at post-thaw time point but after incubation for 6 h of time. The study demonstrated that supplementing sperm freezing extenders with abovesaid antioxidants may create new approaches to cryopreservation procedures and through increasing success rate of freezing, fertility results may increase to better results in near future. Butylated hydroxytoluene Cryopreservation Cysteine Incubation resilience Methionine Ram semen Figures Figure 1 Introduction Ensuring genetic diversity, obtaining high merit traits, hampering infertility and protecting animal health for the future generations have created the necessity of preserving sperm for some time now (Walters, 2009 ). Freezing genetically maximum valued sperm procures the spread of high merit numbers created by male. This advancement, prevents the spreading of venereal diseases, creates new approaches for the fertility programs and secures the future of genetic diversity (Hafez and Hafez, 1987 ). Sperm cells are exposed to physical and biochemical impacts throughout the freezing process. These effects decrease the viability, membrane integrities, movement ability and fertility of the cells (Alcay et al., 2019 ). Various chemical components and freezing methods were evaluated to decrease the effects of external impact factors. Several pH buffers, cryoprotectants, extenders and freezing methods (Gökçe et al., 2017 ; Alcay et al., 2019 ; Toker and Alcay, 2022 ) were examined and successful results were obtained with these trials. Sperm cell membrane consist of 70% phospholipids, 25% of natural lipids and 5% of glycolipid. On the other hand, 70% of the phospholipid layer consists of unsaturated fatty acids (Bucak et al., 2015 ). Due to high amount of unsaturated fatty acids and cholesterol, reactive oxygen species (ROS) are created by lipid peroxidation throughout the process of cooling, freezing and thawing. Lipid peroxidation causes damage in the cells with various ways. In the beginning of this damage process, free oxygen groups transform into very active and effective forms and aim to damage the macromolecules like lipids, proteins and carbohydrates. Furthermore, toxic effects of hydroperoxides cause degeneration on cell membranes and altering cell pH. The effects of reactive oxygen species over lipids have one of the strongest impacts on biological systems (Yarsan, 1998 ). ROS creates lipid peroxidation by effecting very susceptible polyunsaturated fat acids (PUFA) that exists in biological membranes. The sequence of events begins as an initial phase, continues as chain phase and ends as demolition phase. The last molecules that were created in the organelles’ membranes of the very cell expedites the process of dying (Yarsan, 1998 ). Oxidation process between ROS and proteins create a wide diversity of stabile or reactive molecules. By the effect of transition metals, active hydroperoxides may produce additional radicals and the bunch of these radicals can create alteration on the protein functions (Kehrer, 2000 ; Karabulut and Gülay, 2016 ). ROS are very toxic, mutagenic and carcinogenic due to their high activity and tendency to create cellular damage (Nordberg and Arnér, 2001 ). The phenomenon called oxidative stress happens when the ROS increase in the cells and damaging the very cells that located in. Antioxidants which supplemented in sperm extenders are using to restore the balance under the stress conditions (Bucak et al., 2015 ). Methionine is known as the precursor amino acid for glutathione and inhibits oxidative damage in various tissues. Furthermore, it chelates the sulphur and takes an important role on detoxification by moving away these particles (Tuncer et al., 2010 ). Cysteine is a thiol group antioxidant that has low molecular weight. It naturally exits in seminal plasma and nucleic acid of spermatozoa and protects DNA integrity also acts as an antioxidant against ROS originated impacts (Uysal and Bucak, 2007 ). It is also a main element for glutathione and prevents lipid peroxidation that occurs in the cell (Topraggaleh et al., 2014 ). Butylated hydroxytoluene (BHT) is a synthetic analogue of vitamin E and acts as the initiator of the transforming process of peroxy radicals to hydroperoxides [14] . Protecting spermatozoa against cold shock occurs by the BHT through effecting membrane lipids and as a result of reducing the membrane permeability (Khalifa et al., 2008 ). This study designed to discover the effects of various types antioxidants and their doses over sperm cell motility, plasma membrane functional and acrosomal integrity, DNA integrity and the viability of them for 6 h of incubation time. For this purpose, TRIS-citric acid and egg yolk sperm cryopreservation extenders were used. 2.5 and 5 mM of methionine, 1 and 2 mM of cysteine, 1 and 2mM of BHT were supplemented to the extender and a control group was existed to create a comparison amongst the study groups. Kivircik rams were used as sperm donors and these samples were evaluated by hypoosmotic swelling test (HOST), FITC conjugated Pisum sativum agglutinin (PSA-FITC) and TUNEL assay methods throughout the study. Materials And Methods Animals, Collection and Processing of Semen Experiments were done with 5 Kivircik rams (2 to 4 years old) throughout the season of breeding. Animals were at a farm in Bursa (lat. 40°11’N, long. 29°04’E, altitude 155 m asl.), Türkiye with same sheltering conditions. Samples were obtained via electro-ejaculator (Minitüb GmbH, Germany) with at least three days apart (Toker and Alcay, 2022 ). After gathering, the samples were immersed into a warm water bath (32–34°C) within sterile tubes for evaluations. Upon collection, samples went through a series of quality control and were analysed for volume (measured were done with conical tubes), density, mass motion (3–5 on a 0–5 scale), sperm number by haemocytometer (at least 1.5 x 10 9 spermatozoa/mL) and motility (at least 75%) and pooled afterwards (Alcay et al., 2017 ). Preparing Extenders And Semen Dilution Freezing solutions were prepared freshly in the study day with the principal of two-step dilution method. Extender A consisted of 223.7 mmol/L Tris, 66.6 mmol/L citric acid, 55.5 mmol/L fructose, 4 g/L penicillin G, 3 g/L dihydrostreptomycin and 20% egg yolk. Extender B consisted with the same ingredients and also 100.4 mmol/L Trehalose, 4.03 mmol/L EDTA, and 6% of glycerol were added (Onder et al., 2022 ). Whole ingredients were mixed in distilled water after weighing and relevent concentrations of antioxidants were added to the groups. Experiment groups were arranged as; antioxidant free control, 2.5 and 5 mM of methionine, 1 and 2 mM of cysteine, 1 and 2mM of BHT. After pooling the samples, seven equal aliquots were created in every iteration and diluted with the appropriate extender for the final concentration of 150 x 10 6 spermatozoon/mL. Semen Freezing And Evaluations Semen samples that were diluted with extender A were gradually cooled in a water bath with ice cubes in an hour to 4°C. At that temperature, extender B was added by 5 equal aliquots in an hour of time. After that process, diluted sperm cells were held 2 hours at the same temperature for equilibration (Ustuner et al., 2018 ). At the end of time, semen samples were loaded into French straws (0.25 mL). Freezing procedure was conducted by Nicool Plus PC gamete freezing machine (+ 4°C to -8°C with cooling 3°C/min and − 8°C to -120°C with the rate of 15°C/min cooling by the aid of liquid nitrogen) (Air Liquide, Marne-la-Vallée Cedex 3, France). After cooling to -120°C temperature, straws plunged into − 196°C liquid nitrogen for long time storage (Alcay et al., 2017 ). At least ten straws for each experiment group were thawed (37°C, 30 sec) in a water bath. At the same time with the sampling procedure, thawed semen samples were placed in a chamber with 5% CO 2 at 37°C for the examinations of incubation effects over semen characteristics (6 h) (Toker et al., 2016 ). Laboratorial analyses were done by one person at both time points (0 and 6 h). Microscopic evaluations were conducted with a fluorescent attachment specified phase-contrast microscope (Olympus BX51-TF - Olympus Optical Co., Ltd., Japan). Hypo-osmotic swelling test (HOST) was carried out to explore the integrity of sperm membrane function. Curled and swollen tails of sperm cells were examined after incubation of 10 µL of semen sample for 60 min at 37°C with 100 µL HOST solution (9 g fructose and 4.9 g sodium citrate per liter of distilled water). After incubation process, 20µL of mixture over a warm slide that was covered with a slip was examined. At least 200 cells were counted and evaluated for integrity under 1000x magnification and results were recorded as percentiles (Alcay et al., 2021 ). FITC conjugated Pisum sativum agglutinin (PSA-FITC) test was conducted to evaluated the acrosomal integrity of sperm cells. The examination was conducted as the instructions of Toker et al. ( 2016 ). At least 200 cells were examined and the results were presented as percentiles. In Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) used to apply the procedure of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. DNA damage rates were obtained by using with fluorescein microscopy that Toker et al. (2022) indicated. Statistical evaluations were analysed with the aid of SPSS. (SPSS 20.0 for Windows; SPSS, Chicago, IL, USA). Normality of the distribution was tested by using Shapiro Wilk. For parametric and homogenously distributed values One-Way ANOVA was used. Tukey was chosen as Post-Hoc test. Non parametrically distributed parameters were analysed by Kruskal Wallis test then Mann Whitney U test used to determine the significances between study groups. Statistical significance level (P) was chosen as 0.05. Results The effects of various antioxidants (at post-thaw and post-incubation time points) over spermatological parameters throughout the study were analysed. Sperm motility percentages, status of plasma membrane functional integrity, acrosomal integrity and sperm DNA damage results were presented in tables for both time periods (post-thaw and 6 h of incubation). Sperm motility effected and decreased as expected throughout the thawing and incubation procedures (Fig. 1 ). By presenting higher values than control, all of the antioxidant groups differentiate significantly on post-thaw time point (P 0.05). But, 1mM cysteine supplemented group presented the best motility value (56.00%) after thawing. After 6 h of incubation period, status cannot be protected and statistical difference between control and antioxidant groups were vanished. Yet, 2.5mM methionine supplemented group yielded the highest value for motility after incubation (15.67%). Hypo-osmotic swelling test results presented similar statistical effects with motility throughout the study. At post-thaw time point, whole of the antioxidant study groups presented better results comparing to control (P < 0.05). Amongst antioxidant supplemented study groups, 5mM methionine group presented the highest membrane functional integrity value (64.53%). After incubation for 6 h, membrane integriy values presented counterpart results, but 2mM methionine supplemented group yielded the highest integrity by 43.27% and differentiate statistically only against 1mM BHT supplemented group (P 0.05). At post-thawing time point, antioxidant supplemented study groups protected the acrosomal integrity better than comparing to control group (P < 0.05). Amongst antioxidant groups, 1mM cysteine supplemented group yielded 77.60% acrosomal integrity and presented the best performance comparing to others. The interaction regarding the investigations about acrosomal integrity values can be found in Table 1 . Table 1 Sperm resilience parameters on different extender groups at post-thaw (0 h) time point. Group Motility (%) Membrane Integrity (%) Acrosomal Integrity (%) DNA Integrity (%) Control 45.00 ± 1.29 a 51.60 ± 1.40 a 63.00 ± 1.67 a 84.40 ± 1.48 a Methionine 2.5mM 53.67 ± 1.65 b 61.80 ± 1.49 b 77.80 ± 0.75 b 89.87 ± 0.53 bc Methionine 5mM 55.67 ± 1.45 b 64.53 ± 1.69 b 75.60 ± 0.50 cf 89.20 ± 0.95 bce Cysteine 1mM 56.00 ± 1.63 b 62.20 ± 1.33 b 77.60 ± 1.18 bf 90.00 ± 0.61 b Cysteine 2mM 53.33 ± 1.74 b 61.67 ± 2.02 b 74.40 ± 1.18 bdf 89.80 ± 1.16 bc BHT 1mM 53.33 ± 1.93 b 61.87 ± 2.12 b 72.00 ± 1.13 d 86.20 ± 1.19 ade BHT 2mM 52.00 ± 1.75 b 61.87 ± 1.36 b 71.00 ± 1.36 de 87.75 ± 1.14 ac The values are the mean ± standard error of mean (SEM). a,b,c,d,e and f : Values with different superscripts in the same column for each parameter are significantly different (P < 0.05). After 6 h of incubation, the effects of the antioxidant supplementation observed in the results. All antioxidant groups yielded statistically significant results comparing the control group (P < 0.05). 2mM of cysteine group yielded the highest acrosomal integrity after incubation (66.00%). While BHT groups presented better results than control groups, they also differentiated significantly (P < 0.05) from other antioxidant groups (Table 2 ). Table 2 Sperm resilience parameters on different extender groups at post-incubation (6 h) time point. Group Motility (%) Membrane Integrity (%) Acrosomal Integrity (%) DNA Integrity (%) Control 8.33 ± 1.93 39.33 ± 1.68 ab 42.00 ± 1.50 a 81.00 ± 2.60 ad Methionine 2.5mM 15.67 ± 2.53 43.27 ± 2.23 a 62.20 ± 0.80 b 87.33 ± 1.69 bc Methionine 5mM 10.00 ± 2.67 42.00 ± 2.77 ab 61.60 ± 0.94 b 80.25 ± 0.69 d Cysteine 1mM 13.67 ± 2.60 39.73 ± 3.31 ab 63.60 ± 1.20 b 85.75 ± 0.99 ace Cysteine 2mM 10.33 ± 2.60 38.27 ± 1.67 ab 66.00 ± 3.42 b 82.50 ± 1.78 adf BHT 1mM 11.00 ± 2.59 36.27 ± 2.08 bc 54.40 ± 1.37 c 81.33 ± 2.17 ad BHT 2mM 9.33 ± 2.38 38.20 ± 3.80 ac 54.80 ± 0.85 c 89.33 ± 0.33 bef The values are the mean ± standard error of mean (SEM). a,b,c,d,e and f : Values with different superscripts in the same column for each parameter are significantly different (P < 0.05). TUNEL assay results presented significant difference in methionine and cysteine supplemented antioxidant groups in both doses comparing to control after thawing the straws (P 0.05). Amongst all groups, 1mM cysteine group yielded the highest DNA integrity on post-thaw time point (90.00%). After 6 h of incubation, 2.5mM methionine (87.33%) and 2mM BHT (89.33%) supplemented groups yielded statistically difference comparing to control group (P 0.05). Discussion As cryopreservation process has a detrimential effect over sperm cells in various ways, particular methods and approaches have been studied to achieve the best results in terms of spermatological parameters (Toker et al., 2016 ; Alcay et al., 2017 kçe et al., 2017; Ustuner et al., 2018 ; Onder et al., 2022 ). Since there is a balance between ROS and detoxification abilities in normal cellular circumstances, antioxidant matters generally become more important in physical and chemical stress conditions (Sariözkan et al., 2009 ). Cryopreservation itself is a massive stress factor for sperm cells and many molecules are generated due to lipid peroxidation throughout the freezing process (Bucak et al., 2015 ). This study was designed and conducted to investigate and overcome these hampering effects over spermatological parameters by supplementing antioxidant substances for post-thaw and post-incubation (6 h) time points. Toker et al. ( 2016 ) had the approach of same chelating role of methionine for the process of ram semen cryopreservation and supplemented the extenders with various antioxidant agents including 5mM methionine and investigated spermatological parameters for post-thaw and post-incubation (6 h) time points. The results of the study presented very similar results in terms of motility (52.20%), plasma membrane integrity by HOST (67.00%), acrosomal integrity by FITC-PSA (75.60%) and DNA integrity that was obtained with TUNEL method (93.80%) at post-thaw time point. The study investigated the MDA levels of antioxidant supplementation and methionine group yielded significantly better results comparing to control groups (P < 0.05). Also, the results after 6 h incubation were in a mirroring harmony in every aspect with the current study. Omur and Coyan ( 2016 ) aimed the same effects of methionine in their study and used it in sperm extenders with various doses (1mM, 2mM and 4mM). After thawing, the motility values were found to be 64.20%, 57.10% and 58.50% for 1mM, 2mM and 4mM respectively. Despite the differences between methods for sperm plasma membrane integrity (SYBR-14/PI and HOST), 1mM methionine supplemented group yielded similar results with 5mM supplementation of this study. 4mM methionine supplementation yielded relatively low results comparing to our 5mM study group. Since acrosomal status’ of both study investigating with FITC methods, researchers used PNA way and yielded 58.80% integrity in 1mM study group. This result is very low comparing to 75.60% that was obtained in current study. Alcay et al. ( 2016 ) supplemented freeze-dried goat sperm extenders with various antioxidants including 5mM of methionine and investigated the impacts over spermatological parameters. In that study researchers obtained positive statistical differences for methionine against control group in motility and acrosomal integrity at post-thaw time point. After incubation for 6 h, plasma membrane integrity, acrosomal and DNA integity values yielded statistical differences (P < 0.05) comparing to control group. Cysteine has many roles on the antioxidant system of a cell and acts as an inhibitory role for cellular lipid peroxidation metabolism (Naijian et al., 2013 ). Uysal and Bucak ( 2007 ) investigated the effects of some thiol compounds over ram cryopreservation methods and supplemented semen extenders with various doses of cysteine (5mM, 10mM and 20mM). While researchers obtained the best motilty results with 10mM of cysteine supplementation (59.00%), current study yielded same numbers within 1 and 2mM cysteine groups. Also HOST values found to be better in both doses by supplementing 1 and 2mM of cysteine to the ram semen cryopreservation extenders. Bucak et al. ( 2008 ) supplemented extenders with 5mM cysteine and investigated the effects of ram semen cryopreservation of the compound. The results of motility and HOST parameters were in a similar vicinity with Uysal and Bucak ( 2007 )’s research. Çoyan et al. ( 2011 ) studied the effects of cysteine (1mM, 2mM, and 4mM) in a TRIS based extender. Since researchers found high numbers in terms of motility parameters, statistical difference between control and study groups could not obtained in study (P > 0.05). 2mM cysteine supplemented group also yielded statistically significant results in sperm membrane integrity and lipid peroxidation levels comparing to control group (P < 0.05). Sharafi et al. ( 2015 ) performed an investigation for ram semen cryopreservation in lecithin based extender with cysteine (5mM and 10mM). After thawing the straws, 10mM cysteine supplemented study group yielded better results than control in terms of motility parameters (total and progressive) and plasma membrane integrity (P < 0.05). Furthermore, 10mM cysteine supplemented group presented more live and less apoptotic cells comparing to control group of the study (P < 0.05). BHT is a phenolic antioxidant that has a sperm membrane permeability protector role (Farshad et al., 2010 ) and successfully used in cryopreservation process of species’ semen like ram and goat buck (Farshad et al., 2010 ; Naijian et al., 2013 ). Farshad et al. ( 2010 ) investigated the effects of various doses of BHT (0.5, 1, 2 and 3mM) supplementation to the sperm freezing extender. Researchers observed the statistical difference to control group comparing with experimantal groups in terms of motility, live sperm number and acrosomal abnormality status (P < 0.05). Also membrane integrity evaluations of 2mM and 3mM BHT supplemented study groups yielded significant difference comparing to control and other study groups (P < 0.05). Amongst all spermatological parameters 2mM BHT supplemented study group presented the highest values in a positive way comparing to other groups. Palomo et al. ( 2017 ) examined the ram sperm cryopreservation with BHT and effects of either removed and unremoved seminal plasma for the process. Researchers obtained greater results in BHT supplemented study groups comparing to control for motility, sperm kinematics and cell viability parameters. Naijian et al. ( 2013 ), Alcay et al. ( 2016 ) and Khalifa et al. ( 2008 ) are the researchers that investigated the effects of BHT over goat sperm freezing and the impacts over spermatological parameters. All 3 of these studies obtained the positive effects of BHT in terms of freezing ability and the motility of the sperm with different doses (P < 0.05). As a conclusion of the study, methionine, cysteine and BHT supplementation to ram semen cryopreservation extenders offer beneficial contibutions in terms of freezability and the success of the procedure. Furthermore post-thaw and post-incubation spermatological parameters of this study supports the hypothesis and point of origin for this investigation. In addition, as far as our investigation, this is the first report for comparision of this 3 antioxidants with different doses in a comprehensive perspective for the literature. Further studies should aim the success rates of these antioxidants over fertility parameters and field usage. Declarations Additional informations Related the manuscript: Thesis information: This research is originated from PhD thesis of Dr. M. Berk Toker entitled as “In-vitro evaluation of ram sperm, frozen in Tris-Egg yolk extender supplemented with Methionine, Cysteine and BHT”. Project information: This research has not funded by any project. Ethical approval: Experimental designs and studies have been approved by Bursa Uludag University (Bursa, Türkiye) Scientific Ethical Committee. No: 2015-02/01 Acknowledgement This manuscript is originated from PhD thesis of of Dr. M. Berk Toker entitled as “In-vitro evaluation of ram sperm, frozen in Tris-Egg yolk extender supplemented with Methionine, Cysteine and BHT”. Author Contributions MBT and ID designed and performed the experiments. MBT analyzed the data and made the tables. MBT and ID wrote the paper. Both authors reviewed and approved the final manuscript. Data Availability The dataset generated during the current study is available from the corresponding author on reasonable request. Ethical Statement Design and experiments about the study have been approved by the Scientific Ethical Committee of Bursa Uludag University (Bursa, Türkiye) (Approval number: 2015-02/01) Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Competing Interest The authors have no relevant financial or non-financial interests to disclose References Alcay, S., Aktar, A., Koca, D., Kilic, M.A., Akkasoglu, M. and Sagirkaya, H., 2021. Positive effect of autologous platelet rich plasma on Saanen buck semen cryopreservation in non-breeding season Cryobiology, 103, 45–48 (Elsevier Inc.) Alcay, S., Cakmak, S., Cakmak, I., Mulkpinar, E., Gokce, E., Ustuner, B., Sen, H. and Nur, Z., 2019. 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The history of sperm cryopreservation Cambridge University Press, 1, 1–10 Yarsan, E., 1998. Lipid Peroksidasyon Olayı ve Önlenmesine Yönelik Uygulamalar Y.Y.Ü. Vet. Fak. Derg, 9, 89–95 Cite Share Download PDF Status: Published Journal Publication published 22 May, 2023 Read the published version in Tropical Animal Health and Production → Version 1 posted Editorial decision: Major revision with re-assessment 18 Apr, 2023 Reviewers invited by journal 05 Nov, 2022 Reviewers agreed at journal 22 Oct, 2022 Editor assigned by journal 05 Oct, 2022 First submitted to journal 04 Oct, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2120718","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":146287712,"identity":"c554a5b7-b242-4126-bbd7-dce1ea62408d","order_by":0,"name":"Mehmed Berk Toker","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYBACAwYGZiCVwMDA3sB4gIENJkhIywGQFp4DDEhaEojRIpFApBZz9t7Hxh8q0uTMZ74xOMxTZmfXwN68TYLxxz2cWix7jhsnHDiTYyxzOweo5VxycgPPsTIJhoRi3A67kcZ84GBbReIMaaAW3jbmZAaJHDOgFtwuM7j/DKylfobkGZCW+mQG+TcEtNxgY0442JaTICHBA9Jy2I5Bgge/FsueNGaDM2fSDGfwpBUcnHPueAIbT1qxRUIabi3m7MeYJSoqkuUl2A9vfPCmrNqeH8i48cEGtxYMkNgGIknQwMBgT4riUTAKRsEoGBkAAJv/UVUywlMlAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-4033-9749","institution":"Bursa Uludag Universitesi","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mehmed","middleName":"Berk","lastName":"Toker","suffix":""},{"id":146287713,"identity":"aa09c02b-fa0c-4ffd-a532-7153c40a4266","order_by":1,"name":"Ibrahim Dogann","email":"","orcid":"","institution":"Bursa Uludag Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ibrahim","middleName":"","lastName":"Dogann","suffix":""}],"badges":[],"createdAt":"2022-09-30 13:58:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2120718/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2120718/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11250-023-03642-z","type":"published","date":"2023-05-22T20:58:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":28232743,"identity":"b72a1b11-33b6-447a-91f8-c0ec87deead8","added_by":"auto","created_at":"2022-10-25 14:41:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":57343,"visible":true,"origin":"","legend":"\u003cp\u003eChange in motility (%) values between post-thaw and post-incubation time points\u003c/p\u003e","description":"","filename":"Figure.png","url":"https://assets-eu.researchsquare.com/files/rs-2120718/v1/d01b743f71a178b9c23d468b.png"},{"id":44729725,"identity":"5fb893a7-bab1-4ce7-9481-2daf07ab3127","added_by":"auto","created_at":"2023-10-16 21:20:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":353459,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2120718/v1/e04b7bba-a564-4277-85e5-21b8b53b07f2.pdf"}],"financialInterests":"","formattedTitle":"Methionine, Cysteine and Butylated Hydroxytoluene Enhance Cryosurvival of Ram Semen on Post-Thaw and Post-Incubation Time Points","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEnsuring genetic diversity, obtaining high merit traits, hampering infertility and protecting animal health for the future generations have created the necessity of preserving sperm for some time now (Walters, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Freezing genetically maximum valued sperm procures the spread of high merit numbers created by male. This advancement, prevents the spreading of venereal diseases, creates new approaches for the fertility programs and secures the future of genetic diversity (Hafez and Hafez, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Sperm cells are exposed to physical and biochemical impacts throughout the freezing process. These effects decrease the viability, membrane integrities, movement ability and fertility of the cells (Alcay et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Various chemical components and freezing methods were evaluated to decrease the effects of external impact factors. Several pH buffers, cryoprotectants, extenders and freezing methods (G\u0026ouml;k\u0026ccedil;e et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Alcay et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Toker and Alcay, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) were examined and successful results were obtained with these trials.\u003c/p\u003e \u003cp\u003eSperm cell membrane consist of 70% phospholipids, 25% of natural lipids and 5% of glycolipid. On the other hand, 70% of the phospholipid layer consists of unsaturated fatty acids (Bucak et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Due to high amount of unsaturated fatty acids and cholesterol, reactive oxygen species (ROS) are created by lipid peroxidation throughout the process of cooling, freezing and thawing. Lipid peroxidation causes damage in the cells with various ways. In the beginning of this damage process, free oxygen groups transform into very active and effective forms and aim to damage the macromolecules like lipids, proteins and carbohydrates. Furthermore, toxic effects of hydroperoxides cause degeneration on cell membranes and altering cell pH. The effects of reactive oxygen species over lipids have one of the strongest impacts on biological systems (Yarsan, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). ROS creates lipid peroxidation by effecting very susceptible polyunsaturated fat acids (PUFA) that exists in biological membranes. The sequence of events begins as an initial phase, continues as chain phase and ends as demolition phase. The last molecules that were created in the organelles\u0026rsquo; membranes of the very cell expedites the process of dying (Yarsan, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Oxidation process between ROS and proteins create a wide diversity of stabile or reactive molecules. By the effect of transition metals, active hydroperoxides may produce additional radicals and the bunch of these radicals can create alteration on the protein functions (Kehrer, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Karabulut and G\u0026uuml;lay, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). ROS are very toxic, mutagenic and carcinogenic due to their high activity and tendency to create cellular damage (Nordberg and Arn\u0026eacute;r, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The phenomenon called oxidative stress happens when the ROS increase in the cells and damaging the very cells that located in. Antioxidants which supplemented in sperm extenders are using to restore the balance under the stress conditions (Bucak et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMethionine is known as the precursor amino acid for glutathione and inhibits oxidative damage in various tissues. Furthermore, it chelates the sulphur and takes an important role on detoxification by moving away these particles (Tuncer et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Cysteine is a thiol group antioxidant that has low molecular weight. It naturally exits in seminal plasma and nucleic acid of spermatozoa and protects DNA integrity also acts as an antioxidant against ROS originated impacts (Uysal and Bucak, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). It is also a main element for glutathione and prevents lipid peroxidation that occurs in the cell (Topraggaleh et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Butylated hydroxytoluene (BHT) is a synthetic analogue of vitamin E and acts as the initiator of the transforming process of peroxy radicals to hydroperoxides \u003csup\u003e[14]\u003c/sup\u003e. Protecting spermatozoa against cold shock occurs by the BHT through effecting membrane lipids and as a result of reducing the membrane permeability (Khalifa et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study designed to discover the effects of various types antioxidants and their doses over sperm cell motility, plasma membrane functional and acrosomal integrity, DNA integrity and the viability of them for 6 h of incubation time. For this purpose, TRIS-citric acid and egg yolk sperm cryopreservation extenders were used. 2.5 and 5 mM of methionine, 1 and 2 mM of cysteine, 1 and 2mM of BHT were supplemented to the extender and a control group was existed to create a comparison amongst the study groups. Kivircik rams were used as sperm donors and these samples were evaluated by hypoosmotic swelling test (HOST), FITC conjugated Pisum sativum agglutinin (PSA-FITC) and TUNEL assay methods throughout the study.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals, Collection and Processing of Semen\u003c/h2\u003e \u003cp\u003eExperiments were done with 5 Kivircik rams (2 to 4 years old) throughout the season of breeding. Animals were at a farm in Bursa (lat. 40\u0026deg;11\u0026rsquo;N, long. 29\u0026deg;04\u0026rsquo;E, altitude 155 m asl.), T\u0026uuml;rkiye with same sheltering conditions. Samples were obtained via electro-ejaculator (Minit\u0026uuml;b GmbH, Germany) with at least three days apart (Toker and Alcay, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). After gathering, the samples were immersed into a warm water bath (32\u0026ndash;34\u0026deg;C) within sterile tubes for evaluations. Upon collection, samples went through a series of quality control and were analysed for volume (measured were done with conical tubes), density, mass motion (3\u0026ndash;5 on a 0\u0026ndash;5 scale), sperm number by haemocytometer (at least 1.5 x 10\u003csup\u003e9\u003c/sup\u003e spermatozoa/mL) and motility (at least 75%) and pooled afterwards (Alcay et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparing Extenders And Semen Dilution\u003c/h3\u003e\n\u003cp\u003eFreezing solutions were prepared freshly in the study day with the principal of two-step dilution method. Extender A consisted of 223.7 mmol/L Tris, 66.6 mmol/L citric acid, 55.5 mmol/L fructose, 4 g/L penicillin G, 3 g/L dihydrostreptomycin and 20% egg yolk. Extender B consisted with the same ingredients and also 100.4 mmol/L Trehalose, 4.03 mmol/L EDTA, and 6% of glycerol were added (Onder et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Whole ingredients were mixed in distilled water after weighing and relevent concentrations of antioxidants were added to the groups. Experiment groups were arranged as; antioxidant free control, 2.5 and 5 mM of methionine, 1 and 2 mM of cysteine, 1 and 2mM of BHT. After pooling the samples, seven equal aliquots were created in every iteration and diluted with the appropriate extender for the final concentration of 150 x 10\u003csup\u003e6\u003c/sup\u003e spermatozoon/mL.\u003c/p\u003e\n\u003ch3\u003eSemen Freezing And Evaluations\u003c/h3\u003e\n\u003cp\u003eSemen samples that were diluted with extender A were gradually cooled in a water bath with ice cubes in an hour to 4\u0026deg;C. At that temperature, extender B was added by 5 equal aliquots in an hour of time. After that process, diluted sperm cells were held 2 hours at the same temperature for equilibration (Ustuner et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). At the end of time, semen samples were loaded into French straws (0.25 mL). Freezing procedure was conducted by Nicool Plus PC gamete freezing machine (+\u0026thinsp;4\u0026deg;C to -8\u0026deg;C with cooling 3\u0026deg;C/min and \u0026minus;\u0026thinsp;8\u0026deg;C to -120\u0026deg;C with the rate of 15\u0026deg;C/min cooling by the aid of liquid nitrogen) (Air Liquide, Marne-la-Vall\u0026eacute;e Cedex 3, France). After cooling to -120\u0026deg;C temperature, straws plunged into \u0026minus;\u0026thinsp;196\u0026deg;C liquid nitrogen for long time storage (Alcay et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). At least ten straws for each experiment group were thawed (37\u0026deg;C, 30 sec) in a water bath. At the same time with the sampling procedure, thawed semen samples were placed in a chamber with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C for the examinations of incubation effects over semen characteristics (6 h) (Toker et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLaboratorial analyses were done by one person at both time points (0 and 6 h). Microscopic evaluations were conducted with a fluorescent attachment specified phase-contrast microscope (Olympus BX51-TF - Olympus Optical Co., Ltd., Japan).\u003c/p\u003e \u003cp\u003eHypo-osmotic swelling test (HOST) was carried out to explore the integrity of sperm membrane function. Curled and swollen tails of sperm cells were examined after incubation of 10 \u0026micro;L of semen sample for 60 min at 37\u0026deg;C with 100 \u0026micro;L HOST solution (9 g fructose and 4.9 g sodium citrate per liter of distilled water). After incubation process, 20\u0026micro;L of mixture over a warm slide that was covered with a slip was examined. At least 200 cells were counted and evaluated for integrity under 1000x magnification and results were recorded as percentiles (Alcay et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFITC conjugated Pisum sativum agglutinin (PSA-FITC) test was conducted to evaluated the acrosomal integrity of sperm cells. The examination was conducted as the instructions of Toker et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). At least 200 cells were examined and the results were presented as percentiles.\u003c/p\u003e \u003cp\u003eIn Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) used to apply the procedure of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. DNA damage rates were obtained by using with fluorescein microscopy that Toker et al. (2022) indicated.\u003c/p\u003e \u003cp\u003eStatistical evaluations were analysed with the aid of SPSS. (SPSS 20.0 for Windows; SPSS, Chicago, IL, USA). Normality of the distribution was tested by using Shapiro Wilk. For parametric and homogenously distributed values One-Way ANOVA was used. Tukey was chosen as Post-Hoc test. Non parametrically distributed parameters were analysed by Kruskal Wallis test then Mann Whitney U test used to determine the significances between study groups. Statistical significance level (P) was chosen as 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe effects of various antioxidants (at post-thaw and post-incubation time points) over spermatological parameters throughout the study were analysed. Sperm motility percentages, status of plasma membrane functional integrity, acrosomal integrity and sperm DNA damage results were presented in tables for both time periods (post-thaw and 6 h of incubation).\u003c/p\u003e \u003cp\u003eSperm motility effected and decreased as expected throughout the thawing and incubation procedures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). By presenting higher values than control, all of the antioxidant groups differentiate significantly on post-thaw time point (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Nevertheless, neither of the antioxidant supplemented groups yielded statistically significant difference amongst them (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). But, 1mM cysteine supplemented group presented the best motility value (56.00%) after thawing. After 6 h of incubation period, status cannot be protected and statistical difference between control and antioxidant groups were vanished. Yet, 2.5mM methionine supplemented group yielded the highest value for motility after incubation (15.67%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHypo-osmotic swelling test results presented similar statistical effects with motility throughout the study. At post-thaw time point, whole of the antioxidant study groups presented better results comparing to control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Amongst antioxidant supplemented study groups, 5mM methionine group presented the highest membrane functional integrity value (64.53%). After incubation for 6 h, membrane integriy values presented counterpart results, but 2mM methionine supplemented group yielded the highest integrity by 43.27% and differentiate statistically only against 1mM BHT supplemented group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Besides 1mM BHT group, none of the study groups yielded statistical difference amongst them including control (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eAt post-thawing time point, antioxidant supplemented study groups protected the acrosomal integrity better than comparing to control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Amongst antioxidant groups, 1mM cysteine supplemented group yielded 77.60% acrosomal integrity and presented the best performance comparing to others. The interaction regarding the investigations about acrosomal integrity values can be found in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSperm resilience parameters on different extender groups at post-thaw (0 h) time point.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMotility (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMembrane Integrity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcrosomal Integrity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDNA Integrity (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e84.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMethionine 2.5mM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e77.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e89.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMethionine 5mM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003ecf\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e89.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003csup\u003ebce\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCysteine 1mM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e77.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003csup\u003ebf\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e90.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCysteine 2mM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003csup\u003ebdf\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e89.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBHT 1mM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e86.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003csup\u003eade\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBHT 2mM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e87.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003csup\u003eac\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eThe values are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea,b,c,d,e and f\u003c/sup\u003e: Values with different superscripts in the same column for each parameter are significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAfter 6 h of incubation, the effects of the antioxidant supplementation observed in the results. All antioxidant groups yielded statistically significant results comparing the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). 2mM of cysteine group yielded the highest acrosomal integrity after incubation (66.00%). While BHT groups presented better results than control groups, they also differentiated significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from other antioxidant groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSperm resilience parameters on different extender groups at post-incubation (6 h) time point.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMotility (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMembrane Integrity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcrosomal Integrity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDNA Integrity (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e81.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60\u003csup\u003ead\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMethionine 2.5mM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e87.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMethionine 5mM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.77\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCysteine 1mM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.73\u0026thinsp;\u0026plusmn;\u0026thinsp;3.31\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003csup\u003eace\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCysteine 2mM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e82.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003csup\u003eadf\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBHT 1mM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e81.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17\u003csup\u003ead\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBHT 2mM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.80\u003csup\u003eac\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e89.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ebef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eThe values are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea,b,c,d,e and f\u003c/sup\u003e: Values with different superscripts in the same column for each parameter are significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTUNEL assay results presented significant difference in methionine and cysteine supplemented antioxidant groups in both doses comparing to control after thawing the straws (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). BHT supplemented groups yielded better results numerically yet could not present a statistical difference (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Amongst all groups, 1mM cysteine group yielded the highest DNA integrity on post-thaw time point (90.00%). After 6 h of incubation, 2.5mM methionine (87.33%) and 2mM BHT (89.33%) supplemented groups yielded statistically difference comparing to control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Besides these study groups, none of the antioxidant supplemented groups yielded a positive statistical difference to control group (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs cryopreservation process has a detrimential effect over sperm cells in various ways, particular methods and approaches have been studied to achieve the best results in terms of spermatological parameters (Toker et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Alcay et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003ek\u0026ccedil;e et al., 2017; Ustuner et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Onder et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Since there is a balance between ROS and detoxification abilities in normal cellular circumstances, antioxidant matters generally become more important in physical and chemical stress conditions (Sari\u0026ouml;zkan et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Cryopreservation itself is a massive stress factor for sperm cells and many molecules are generated due to lipid peroxidation throughout the freezing process (Bucak et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This study was designed and conducted to investigate and overcome these hampering effects over spermatological parameters by supplementing antioxidant substances for post-thaw and post-incubation (6 h) time points.\u003c/p\u003e \u003cp\u003eToker et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) had the approach of same chelating role of methionine for the process of ram semen cryopreservation and supplemented the extenders with various antioxidant agents including 5mM methionine and investigated spermatological parameters for post-thaw and post-incubation (6 h) time points. The results of the study presented very similar results in terms of motility (52.20%), plasma membrane integrity by HOST (67.00%), acrosomal integrity by FITC-PSA (75.60%) and DNA integrity that was obtained with TUNEL method (93.80%) at post-thaw time point. The study investigated the MDA levels of antioxidant supplementation and methionine group yielded significantly better results comparing to control groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Also, the results after 6 h incubation were in a mirroring harmony in every aspect with the current study. Omur and Coyan (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) aimed the same effects of methionine in their study and used it in sperm extenders with various doses (1mM, 2mM and 4mM). After thawing, the motility values were found to be 64.20%, 57.10% and 58.50% for 1mM, 2mM and 4mM respectively. Despite the differences between methods for sperm plasma membrane integrity (SYBR-14/PI and HOST), 1mM methionine supplemented group yielded similar results with 5mM supplementation of this study. 4mM methionine supplementation yielded relatively low results comparing to our 5mM study group. Since acrosomal status\u0026rsquo; of both study investigating with FITC methods, researchers used PNA way and yielded 58.80% integrity in 1mM study group. This result is very low comparing to 75.60% that was obtained in current study. Alcay et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) supplemented freeze-dried goat sperm extenders with various antioxidants including 5mM of methionine and investigated the impacts over spermatological parameters. In that study researchers obtained positive statistical differences for methionine against control group in motility and acrosomal integrity at post-thaw time point. After incubation for 6 h, plasma membrane integrity, acrosomal and DNA integity values yielded statistical differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) comparing to control group.\u003c/p\u003e \u003cp\u003eCysteine has many roles on the antioxidant system of a cell and acts as an inhibitory role for cellular lipid peroxidation metabolism (Naijian et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Uysal and Bucak (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) investigated the effects of some thiol compounds over ram cryopreservation methods and supplemented semen extenders with various doses of cysteine (5mM, 10mM and 20mM). While researchers obtained the best motilty results with 10mM of cysteine supplementation (59.00%), current study yielded same numbers within 1 and 2mM cysteine groups. Also HOST values found to be better in both doses by supplementing 1 and 2mM of cysteine to the ram semen cryopreservation extenders. Bucak et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) supplemented extenders with 5mM cysteine and investigated the effects of ram semen cryopreservation of the compound. The results of motility and HOST parameters were in a similar vicinity with Uysal and Bucak (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e)\u0026rsquo;s research. \u0026Ccedil;oyan et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) studied the effects of cysteine (1mM, 2mM, and 4mM) in a TRIS based extender. Since researchers found high numbers in terms of motility parameters, statistical difference between control and study groups could not obtained in study (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). 2mM cysteine supplemented group also yielded statistically significant results in sperm membrane integrity and lipid peroxidation levels comparing to control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Sharafi et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) performed an investigation for ram semen cryopreservation in lecithin based extender with cysteine (5mM and 10mM). After thawing the straws, 10mM cysteine supplemented study group yielded better results than control in terms of motility parameters (total and progressive) and plasma membrane integrity (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, 10mM cysteine supplemented group presented more live and less apoptotic cells comparing to control group of the study (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eBHT is a phenolic antioxidant that has a sperm membrane permeability protector role (Farshad et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and successfully used in cryopreservation process of species\u0026rsquo; semen like ram and goat buck (Farshad et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Naijian et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Farshad et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) investigated the effects of various doses of BHT (0.5, 1, 2 and 3mM) supplementation to the sperm freezing extender. Researchers observed the statistical difference to control group comparing with experimantal groups in terms of motility, live sperm number and acrosomal abnormality status (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Also membrane integrity evaluations of 2mM and 3mM BHT supplemented study groups yielded significant difference comparing to control and other study groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Amongst all spermatological parameters 2mM BHT supplemented study group presented the highest values in a positive way comparing to other groups. Palomo et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) examined the ram sperm cryopreservation with BHT and effects of either removed and unremoved seminal plasma for the process. Researchers obtained greater results in BHT supplemented study groups comparing to control for motility, sperm kinematics and cell viability parameters. Naijian et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), Alcay et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and Khalifa et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) are the researchers that investigated the effects of BHT over goat sperm freezing and the impacts over spermatological parameters. All 3 of these studies obtained the positive effects of BHT in terms of freezing ability and the motility of the sperm with different doses (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eAs a conclusion of the study, methionine, cysteine and BHT supplementation to ram semen cryopreservation extenders offer beneficial contibutions in terms of freezability and the success of the procedure. Furthermore post-thaw and post-incubation spermatological parameters of this study supports the hypothesis and point of origin for this investigation. In addition, as far as our investigation, this is the first report for comparision of this 3 antioxidants with different doses in a comprehensive perspective for the literature. Further studies should aim the success rates of these antioxidants over fertility parameters and field usage.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAdditional informations\u003c/p\u003e\n\u003cp\u003eRelated the manuscript:\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003eThesis information: This research is originated from PhD thesis of Dr. M. Berk Toker entitled as \u0026ldquo;In-vitro evaluation of ram sperm, frozen in Tris-Egg yolk extender supplemented with Methionine, Cysteine and BHT\u0026rdquo;.\u003c/li\u003e\n\u003cli\u003eProject information: This research has not funded by any project.\u003c/li\u003e\n\u003cli\u003eEthical approval: Experimental designs and studies have been approved by Bursa Uludag University (Bursa, T\u0026uuml;rkiye) Scientific Ethical Committee. No: 2015-02/01\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript is originated from PhD thesis of of Dr. M. Berk Toker entitled as \u0026ldquo;In-vitro evaluation of ram sperm, frozen in Tris-Egg yolk extender supplemented with Methionine, Cysteine and BHT\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMBT and ID designed and performed the experiments. MBT analyzed the data and made the tables. MBT and ID wrote the paper. Both authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset generated during the current study is available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDesign and experiments about the study have been approved by the Scientific Ethical Committee of Bursa Uludag University (Bursa, T\u0026uuml;rkiye) (Approval number: 2015-02/01)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlcay, S., Aktar, A., Koca, D., Kilic, M.A., Akkasoglu, M. and Sagirkaya, H., 2021. Positive effect of autologous platelet rich plasma on Saanen buck semen cryopreservation in non-breeding season Cryobiology, 103, 45\u0026ndash;48 (Elsevier Inc.)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlcay, S., Cakmak, S., Cakmak, I., Mulkpinar, E., Gokce, E., Ustuner, B., Sen, H. and Nur, Z., 2019. Successful cryopreservation of honey bee drone spermatozoa with royal jelly supplemented extenders Cryobiology, 87, 28\u0026ndash;31 (Elsevier)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlcay, S., Gokce, E., Toker, M.B., Onder, N.T., Ustuner, B., Uzabaci, E., Gul, Z. and Cavus, S., 2016. Freeze-dried egg yolk based extenders containing various antioxidants improve post-thawing quality and incubation resilience of goat spermatozoa Cryobiology, 72\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlcay, S., Toker, M.B., Onder, N.T. and Gokce, E., 2017. Royal jelly supplemented soybean lecithin-based extenders improve post-thaw quality and incubation resilience of goat spermatozoa Cryobiology, 74, 81\u0026ndash;85 (Elsevier Ltd)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBucak, M.N., Ateşşahin, A. and Y\u0026uuml;ce, A., 2008. Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze-thawing process Small Ruminant Research, 75, 128\u0026ndash;134\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBucak, M.N., G\u0026uuml;ng\u0026ouml;r, Ş., Sari\u0026ouml;zkan, S. and Bilgili, A., 2015. Spermanın Dondurulmasında Antioksidanların Etkisi T\u0026uuml;rkiye Klinikleri, 1, 39\u0026ndash;47\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Ccedil;oyan, K., Başpinar, N., Bucak, M.N. and Akalin, P.P., 2011. Effects of cysteine and ergothioneine on post-thawed Merino ram sperm and biochemical parameters Cryobiology, 63, 1\u0026ndash;6\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarshad, A., Khalili, B. and Jafaroghli, M., 2010. Effects of butylated hydroxytoluene on freezability of ram spermatozoa Asian-Australasian Journal of Animal Sciences, 23, 1276\u0026ndash;1281\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG\u0026ouml;k\u0026ccedil;e, E., Al\u0026ccedil;ay, S. and G\u0026uuml;l, Z., 2017. Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5\u0026deg;C Ankara \u0026Uuml;niv Vet Fak Derg, 64, 313\u0026ndash;320\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHafez, E.S.E. and Hafez, B., 1987. Reproduction in Farm Animals,\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarabulut, H. and G\u0026uuml;lay, M.Ş., 2016. Antioksidanlar MAE Vet Fak Derg, 1, 65\u0026ndash;76\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKehrer, J.P., 2000. The Haber-Weiss reaction and mechanisms of toxicity Toxicology, 149, 43\u0026ndash;50\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhalifa, T.A.A., Lymberopoulos, A.G. and El-Saidy, B.E., 2008. Testing usability of butylated hydroxytoluene in conservation of goat semen Reproduction in Domestic Animals, 43, 525\u0026ndash;530\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaijian, H.R., Kohram, H., Shahneh, A.Z. and Sharafi, M., 2013. Effects of various concentrations of BSA on microscopic and oxidative parameters of Mahabadi goat semen following the freeze-thaw process Small Ruminant Research, 113, 371\u0026ndash;375\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNordberg, J. and Arn\u0026eacute;r, E.S.J., 2001. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system1 1This review is based on the licentiate thesis \u0026ldquo;Thioredoxin reductase\u0026mdash;interactions with the redox active compounds 1-chloro-2,4-dinitrobenzene and lipoic acid\u0026rdquo; by Jonas Nordberg, Free Radical Biology and Medicine, 31, 1287\u0026ndash;1312\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOmur, 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 Veterinarni Medicina, 61, 10\u0026ndash;16\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOnder, N.T., Alcay, S. and Nur, Z., 2022. Effects of alpha-lipoic acid on ram semen cryopreservation and post-thaw life span Andrologia, 54, 1\u0026ndash;5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalomo, M.J., Garc\u0026iacute;a, W. and Tabarez, A., 2017. Effect of seminal plasma and butylated hydroxytoluene (BHT) concentration on ram sperm freezability Small Ruminant Research, 153, 66\u0026ndash;70 (Elsevier)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSari\u0026ouml;zkan, S., Bucak, M.N., Tuncer, P.B., Ulutaş, P.A. and Bilgen, A., 2009. The influence of cysteine and taurine on microscopic-oxidative stress parameters and fertilizing ability of bull semen following cryopreservation Cryobiology, 58, 134\u0026ndash;138\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharafi, M., Zhandi, M. and Akbari Sharif, A., 2015. Supplementation of soybean lecithin-based semen extender by antioxidants: complementary flowcytometric study on post-thawed ram spermatozoa Cell and Tissue Banking, 16, 261\u0026ndash;269 (Springer Netherlands)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToker, M.B. and Alcay, S., 2022. Comprehensive Eff ects of Fetal Calf Serum in Soybean-Lecithin Based Goat Semen Cryopreservation Extenders and Impacts on Incubation Resilience Kafkas Universitesi Veteriner Fakultesi Dergisi, 28, 455\u0026ndash;460\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToker, M.B., Alcay, S., Gokce, E. and Ustuner, B., 2016. Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience Cryobiology, 72, 205\u0026ndash;209\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTopraggaleh, T.R., Shahverdi, A., Rastegarnia, A., Ebrahimi, B., Shafiepour, V., Sharbatoghli, M., Esmaeili, V. and Janzamin, E., 2014. Effect of cysteine and glutamine added to extender on post-thaw sperm functional parameters of buffalo bull Andrologia, 46, 777\u0026ndash;783\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuncer, P.B., Bucak, M.N., Sari\u0026ouml;zkan, S., Sakin, F., Yeni, D., \u0026Ccedil;iĝerci, I.H., Ateşşahin, A., Avdatek, F., G\u0026uuml;ndoĝan, M. and B\u0026uuml;y\u0026uuml;kleblebici, O., 2010. The effect of raffinose and methionine on frozen/thawed Angora buck (Capra hircus ancryrensis) semen quality, lipid peroxidation and antioxidant enzyme activities Cryobiology, 61, 89\u0026ndash;93\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUstuner, B., Gokce, E., Toker, M.B., Onder, N.T., Soylu, M.K., Sagirkaya, H. and Nur, Z., 2018. Effect of sperm pooling with seminal plasma collected in breeding or nonbreeding season on Saanen goat sperm cryosurvival Andrologia, 50\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUysal, O. and Bucak, M.N., 2007. Effects of oxidized glutathione, bovine serum albumin, cysteine and lycopene on the quality of frozen-thawed ram semen Acta Veterinaria Brno, 76, 383\u0026ndash;390\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalters, E.M.E.A., 2009. The history of sperm cryopreservation Cambridge University Press, 1, 1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYarsan, E., 1998. Lipid Peroksidasyon Olayı ve \u0026Ouml;nlenmesine Y\u0026ouml;nelik Uygulamalar Y.Y.\u0026Uuml;. Vet. Fak. Derg, 9, 89\u0026ndash;95\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Butylated hydroxytoluene, Cryopreservation, Cysteine, Incubation resilience, Methionine, Ram semen, ","lastPublishedDoi":"10.21203/rs.3.rs-2120718/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2120718/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDespite there have been many experiments conducted about antioxidants, the best sole or combination use of antioxidants to include as a standard ingredient to freezing extenders yet to be found. This study conducted to investigate the different doses of methionine (2.5 and 5mM), cysteine (1 and 2mM) and butylated hydroxytoluene (BHT) (1 and 2mM) for ram semen cryopreservation on post-thaw and post-incubation (6 h) time points over spermatological parameters. Semen samples collected from Kivircik rams via electro-ejaculator in breeding season. After essential spermatological evaluations, appropriate samples were pooled then split into 7 equal aliquots to create study groups (antioxidant free control, 2.5mM methionine, 5mM methionine, 1mM cysteine, 2mM cysteine, 1mM BHT and 2mM BHT). Semen samples put into French straws (0.25mL) and freezing procedure (two-step) conducted via a programmable gamete freezer. At both time points; motility, HOST, PSA-FITC and TUNEL assays were conducted to discover the impacts of cryopreservation and incubation process over sperm cells. Antioxidant supplemented groups yielded better results comparing to control groups in terms of various spermatological parameters not only at post-thaw time point but after incubation for 6 h of time. The study demonstrated that supplementing sperm freezing extenders with abovesaid antioxidants may create new approaches to cryopreservation procedures and through increasing success rate of freezing, fertility results may increase to better results in near future.\u003c/p\u003e","manuscriptTitle":"Methionine, Cysteine and Butylated Hydroxytoluene Enhance Cryosurvival of Ram Semen on Post-Thaw and Post-Incubation Time Points","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-25 14:41:02","doi":"10.21203/rs.3.rs-2120718/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision with re-assessment","date":"2023-04-18T20:20:57+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-11-05T04:25:09+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2022-10-22T15:56:20+00:00","index":0,"fulltext":""},{"type":"editorAssigned","content":"","date":"2022-10-06T02:57:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Animal Health and Production","date":"2022-10-04T07:02:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"56c5eec7-475f-4b70-951b-2727d17c62d9","owner":[],"postedDate":"October 25th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T21:06:51+00:00","versionOfRecord":{"articleIdentity":"rs-2120718","link":"https://doi.org/10.1007/s11250-023-03642-z","journal":{"identity":"tropical-animal-health-and-production","isVorOnly":false,"title":"Tropical Animal Health and Production"},"publishedOn":"2023-05-22 20:58:20","publishedOnDateReadable":"May 22nd, 2023"},"versionCreatedAt":"2022-10-25 14:41:02","video":"","vorDoi":"10.1007/s11250-023-03642-z","vorDoiUrl":"https://doi.org/10.1007/s11250-023-03642-z","workflowStages":[]},"version":"v1","identity":"rs-2120718","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2120718","identity":"rs-2120718","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}