Reproduction in domestic animals = Zuchthygiene2026; 61(8); e70307; doi: 10.1111/rda.70307

The Effect of Different Extenders and Testicular Anaesthesia During Orchiectomy on Post-Thawed Spermatological Parameters of Stallion Epididymal Spermatozoa.

Cinar, H
·
Herdogan, M
·
Mart, F
·
Cay, HA
·
Danisman, IT
·
Inanc, ME
·
Balkan, BM
·
Gungor, S
·
Ata, A

Abstract: Cryopreservation of epididymal stallion spermatozoa is a valuable tool for fertility preservation, particularly after orchiectomy. However, the potential interactions between local anaesthesia and extender composition on post-thaw spermatozoal quality remain poorly understood. This study investigated the effects of intratesticular lidocaine injection and the efficacy of various extender (egg yolk [EY] vs. lecithin [LC]) and cryoprotectant (glycerol [GLY] vs. dimethyl sulfoxide [DMSO]) combinations on stallion epididymal spermatological parameters. Using a paired factorial design, epididymal spermatozoa were collected from both lidocaine-injected (right) and non-injected (left) testes of eight stallions. Samples were cryopreserved in Tris-based extenders supplemented with EY + GLY, EY + DMSO, LC + GLY or LC + DMSO. Spermatozoal motility (fresh, post-equilibration and post-thaw), viability, plasma membrane and acrosome integrity (PMAI), mitochondrial reactive oxygen species (MROS), spermatozoa malondialdehyde (MDA) and glutathione (GSH) levels were evaluated. Intratesticular lidocaine administration had no significant effect on spermatozoal motility at any evaluation stage, and no differences were detected in any biochemical or spermatologic parameters assessed post-thaw (p > 0.05). Regarding cryopreservation media, a significant main effect of extender type was observed for spermatozoal viability (p < 0.05) and MROS levels (p < 0.001), with EY-based extenders providing superior protection compared to LC-based formulations. A significant Extender × Cryoprotectant interaction was detected for GSH levels (p < 0.05), with the EY + GLY combination yielding the highest antioxidant capacity. No significant differences were observed in MDA levels among treatment groups (p > 0.05). Intratesticular lidocaine administration during orchiectomy does not compromise epididymal spermatozoal quality after cryopreservation. Among the evaluated cryopreservation strategies, the combination of egg yolk and glycerol in a Tris-based extender provides the most effective protection against oxidative stress and membrane damage in stallion epididymal spermatozoa.

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Publication Date: 2026-08-13 PubMed ID: 42593151DOI: 10.1111/rda.70307Google Scholar: Lookup

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Affiliations
MeSH
References
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Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

This study examined how local anaesthesia (lidocaine) and different sperm freezing solutions (extenders and cryoprotectants) affect the quality of frozen-thawed sperm collected from stallion testes after castration.
The researchers found lidocaine injection into the testes did not affect sperm quality, while using an egg yolk-based extender combined with glycerol provided the best protection to sperm cells during freezing and thawing.

Research Context and Objectives

Cryopreservation of epididymal spermatozoa is important for preserving male fertility, especially when testicular tissue is surgically removed (orchiectomy).
Local anaesthesia like intratesticular lidocaine is often used during orchiectomy to reduce pain, but its effects on sperm quality after freezing are not well-studied.
Choosing the right extender (a fluid that supports sperm during freezing) and cryoprotectant (a chemical that protects cells from ice damage) is critical for maintaining sperm viability after thawing.
This study aimed to:
- Evaluate whether intratesticular lidocaine affects sperm quality post-thaw.
- Compare different extender and cryoprotectant combinations for their ability to protect stallion epididymal spermatozoa during cryopreservation.

Methods

Sperm samples were collected from both testes of eight stallions; the right testis received a lidocaine injection while the left did not, enabling paired comparison.
Sperm were cryopreserved using Tris-based extenders formulated with two types of protectants:
- Extenders: Egg yolk (EY) or lecithin (LC)
- Cryoprotectants: Glycerol (GLY) or dimethyl sulfoxide (DMSO)
This resulted in four treatment combinations: EY+GLY, EY+DMSO, LC+GLY, LC+DMSO.
Sperm parameters assessed included:
- Motility at different stages: fresh, after equilibration, and post-thaw
- Viability and plasma membrane & acrosome integrity (PMAI)
- Biochemical markers: mitochondrial reactive oxygen species (MROS), malondialdehyde (MDA, indicating lipid peroxidation), and glutathione (GSH, an antioxidant)

Key Findings

Effect of Intratesticular Lidocaine:
- No significant differences in sperm motility or any assessed biochemical parameters between lidocaine-injected and non-injected testes.
- Indicates lidocaine use during orchiectomy does not compromise post-thaw sperm quality.
Effect of Extender and Cryoprotectant Types:
- Egg yolk-based extenders significantly improved sperm viability compared to lecithin-based extenders.
- Mitochondrial reactive oxygen species (MROS) levels were significantly lower with EY extenders, indicating better protection from oxidative stress.
- Combination of EY+GLY yielded the highest glutathione (GSH) levels, pointing to greater antioxidant capacity in this group.
- No significant differences in malondialdehyde (MDA) levels among the groups, suggesting similar levels of lipid peroxidation across treatments.

Interpretation and Implications

Using local lidocaine for anaesthesia during orchiectomy appears safe with respect to preserving sperm quality during subsequent cryopreservation.
Choosing the right extender and cryoprotectant is crucial:
- Egg yolk effectively enhances sperm membrane protection and viability.
- Glycerol as a cryoprotectant with egg yolk improves antioxidant defenses.
These findings support the use of EY+GLY in Tris-based extenders as an optimal method to maintain post-thawed stallion epididymal sperm quality and reduce oxidative damage.
This can advance fertility preservation strategies for stallions undergoing orchiectomy or other situations requiring sperm cryopreservation.

Summary

The study systematically evaluated the effects of testicular lidocaine injection and extender/cryoprotectant combinations on the quality of frozen-thawed epididymal sperm from stallions.
Lidocaine administration is safe during orchiectomy without detrimental effects on sperm quality post-thaw.
Egg yolk extenders combined with glycerol provide superior cryoprotection by enhancing sperm viability and antioxidant capacity.
These insights guide improved cryopreservation protocols for maintaining stallion fertility potential after testicular removal.

Cite This Article

APA

Cinar H, Herdogan M, Mart F, Cay HA, Danisman IT, Inanc ME, Balkan BM, Gungor S, Ata A. (2026). The Effect of Different Extenders and Testicular Anaesthesia During Orchiectomy on Post-Thawed Spermatological Parameters of Stallion Epididymal Spermatozoa. Reprod Domest Anim, 61(8), e70307. https://doi.org/10.1111/rda.70307

Publication

Reproduction in domestic animals = Zuchthygiene

ISSN: 1439-0531

NlmUniqueID: 9015668

Country: Germany

Language: English

Volume: 61

Issue: 8

Pages: e70307

Researcher Affiliations

Cinar, Harun

Department of Surgery, Faculty of Veterinary Medicine, Burdur Mehmet Akif Ersoy University, Burdur, Turkey.

Herdogan, Mine

Department of Reproduction and Artificial Insemination, Health Science Institute, Burdur Mehmet Akif Ersoy University, Burdur, Turkey.

Mart, Feyzanur

Department of Reproduction and Artificial Insemination, Health Science Institute, Burdur Mehmet Akif Ersoy University, Burdur, Turkey.

Cay, Hasan Ali

Department of Reproduction and Artificial Insemination, Health Science Institute, Burdur Mehmet Akif Ersoy University, Burdur, Turkey.
Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Burdur Mehmet Akif University, Burdur, Turkey.

Danisman, Ibrahim Taha

Department of Surgery, Faculty of Veterinary Medicine, Burdur Mehmet Akif Ersoy University, Burdur, Turkey.

Inanc, Muhammed Enes

Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Burdur Mehmet Akif University, Burdur, Turkey.

Balkan, Burcu Menekse

Department of Biochemistry, Faculty of Veterinary Medicine, Burdur Mehmet Akif University, Burdur, Turkey.

Gungor, Sukru

Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Burdur Mehmet Akif University, Burdur, Turkey.

Ata, Ayhan

Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Burdur Mehmet Akif University, Burdur, Turkey.

MeSH Terms

Animals
Male
Horses / physiology
Cryopreservation / veterinary
Cryopreservation / methods
Semen Preservation / veterinary
Semen Preservation / methods
Cryoprotective Agents / pharmacology
Spermatozoa / drug effects
Lidocaine / pharmacology
Lidocaine / administration & dosage
Orchiectomy / veterinary
Testis / drug effects
Egg Yolk
Sperm Motility / drug effects
Epididymis / cytology
Anesthetics, Local / pharmacology
Anesthetics, Local / administration & dosage
Lecithins / pharmacology
Glycerol / pharmacology
Malondialdehyde
Dimethyl Sulfoxide / pharmacology
Glutathione
Reactive Oxygen Species

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