Comparative analysis of various step-dilution techniques on the quality of frozen Limousin bull semen

Abstract

Background and Aim: Indonesia has two National Artificial Insemination centers and 17 Regional Artificial Insemination Centers. The frozen semen production techniques differed between the centers, including the type of diluent and semen dilution technique. The aim of the research was to compare the quality of frozen Limousin bull semen diluted using different techniques. Materials and Methods: Semen was collected from three sexually mature Limousin bulls using an artificial vagina. Immediately after collection, the semen was evaluated macroscopically and microscopically. Semen that had >70% motile sperm and <20% sperm abnormality was divided into three tubes and diluted with skim milk-egg yolk (SMEY) using three different dilution techniques: One-step dilution (100% SMEY with 8% glycerol) at room temperature ([RT] 20°C until 25°C) two-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol after 1 h stored at 5°C); and three-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol added twice at 1 h and 1.5 h after being stored at 5°C). The diluted semen was loaded into 0.25 mL mini straws, equilibrated, and frozen using a freezing machine. Sperm motility, viability, membranes, DNA integrity, and concentrations of malondialdehyde (MDA) and aspartate aminotransferase (AST) enzymes were evaluated after thawing. Results: The results showed that there were no significant differences in sperm motility and DNA integrity between dilutions (p>0.05). However, sperm viability and membrane intactness of one-step dilutions were higher than those of three-step dilutions. The concentrations of MDA and AST enzymes of sperm in one-step dilutions were lower than those of three-step dilutions (p<0.05). Conclusion: It was concluded that the one-step-dilution technique was better than three-step dilution for cryopreservation of Limousin bull semen.

Evaluation of the effects of hesperidin on fresh and frozen-thawed semen quality using two different cryopreservation methods in Simmental bull

“…The current study showed the effects of different doses of Hesp and two different semen freezing methods on Simmental bulls' sperm characteristics and oxidative stress status. Different cryopreservation methods have been designed to achieve a higher quality of frozenthawed semen (Arif et al, 2020;Jha et al, 2019). The comparison of the one and two-step dilution protocols showed less toxicity for sperm in the two-step dilution because of the slower exposure of sperm to glycerol (Brito et al, 2017); On the other hand, less contamination and easy handling of the one-step dilution method increased this protocol usage in AI stations (Schulze et al, 2018).…”

“…Based on the results, there were no significant alterations between the oneand the two-step dilution methods, but higher MDA and AST levels were detected in the threestep compared to the one-step dilution and higher MDA and AST levels in the one-and threestep compared to the two-step dilution. The sperm plasma membrane integrity could not be evaluated perfectly, but there was a significant reduction between the one-and three-step dilutions (Arif et al, 2020). Moreover, fresh boar semen was collected from three European AI centers to assess the one-and two-step dilution methods.…”

“…However, the two-step dilution presented less glycerol toxicity in the cooling and equilibration stage of sperm freezing, thereby minimizing its harmful influence (Brito et al, 2017;Peña and Linde-Forsberg, 2000). In two-step dilution, glycerol addition was performed in the second stage of dilution, which showed better cryoprotective potential than one and three-step dilution methods in dog, bulls, and rams (Peña and Linde-Forsberg, 2000;Arif et al, 2020;Colas, 1975). But, one-step dilution showed better cryoprotective effect on rams' semen compared to two-step dilution, which could indicate that glycerol concentration was not the only issue for semen protection (Jha et al, 2019).…”

The Quality of Frozen Semen of Limousin Bull in Various Semen Diluents

“…Sperm viability was assessed by mixing 50 µL of semen with 100 µL of eosin nigrosine stain, smeared, and dried at 37 ℃. Examination of the integrity of the sperm plasma membrane was performed by mixing 50 µL of semen with 950 µL of HOS solution (Arif et al, 2020).…”

Effect of glycerol addition time on the cryopreserved Korean native brindle cattle (Chikso) sperm quality

“…The most obvious difference between most of protocols is the addition of glycerol before and after cooling during the Chikso cryopreserved semen process. It has been reported that post-thaw motility of bison sperm was not observed significant effects by glycerol addition time (Vilela et al, 2017;Arif et al, 2020). However, other colleage have been showed that sperm motility was altered by glycerol addition time in freezing-thawing Mediterranean buffalo (B.bubalis) sperm (Fabbrocini et al, 2000).…”