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Methionine, cysteine, and butylated hydroxytoluene enhance cryosurvival of ram semen on post-thaw and post-incubation time points

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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 is yet to be found. This study was designed to investigate the different doses of methionine (2.5 and 5 mM), cysteine (1 and 2 mM), and butylated hydroxytoluene (BHT) (1 and 2 mM) for ram semen cryopreservation on post-thaw and post-incubation (6 h) time points over spermatological parameters. Semen samples were 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, 2.5 mM methionine, 5 mM methionine, 1 mM cysteine, 2 mM cysteine, 1 mM BHT, and 2 mM BHT). Semen samples were put into French straws (0.25 mL), and freezing procedure (two-step) was conducted via a programmable gamete freezer. At both time points, motility, HOST, PSA-FITC, and TUNEL assays were made to discover the impacts of cryopreservation and incubation process over sperm cells. Antioxidant supplemented groups yielded better results compared to the 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 previous 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.

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Data availability

The dataset generated during the current study is available from the corresponding author on reasonable request.

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Acknowledgements

This manuscript 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.”

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  1. Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Bursa Uludag University, 16059, Bursa, Turkey

    Mehmed Berk Toker & Ibrahim Dogan

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  1. Mehmed Berk Toker
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  2. Ibrahim Dogan
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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.

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Correspondence to Mehmed Berk Toker.

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Ethics approval

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).

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The authors declare no competing interests.

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Thesis information: This research is 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.”

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Toker, M.B., Dogan, I. Methionine, cysteine, and butylated hydroxytoluene enhance cryosurvival of ram semen on post-thaw and post-incubation time points. Trop Anim Health Prod 55, 218 (2023). https://doi.org/10.1007/s11250-023-03642-z

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