Different concentrations of cysteamine, ergothioneine, and serine modulate quality and fertilizing ability of cryopreserved chicken sperm

Abstract

The aim of this study was to evaluate the effects of freezing diluents supplemented in three potential amines/amino acids, namely, antioxidant cysteamine (2-aminoethanethiol [ AET ]), ergothioneine ( ERG ), and serine ( SER ), in optimization of chicken sperm cryopreservation. The semen of 36 Pradu Hang Dum males, selected based on their motility vigor score, was frozen by a simple freezing method using nitrogen vapors and dimethylformamide ( DMF ). In a first experiment, a wide range of AET, ERG, and SER doses were tested. Semen quality was evaluated after incubation at 5°C or after cryopreservation in straws in the Blumberger Hahnen Sperma Verdünner ( BHSV ) diluent + DMF (6% v/v) with or without AET, ERG, or SER. The best targeted doses of AET, ERG, or SER were then selected for experiment 2 that was focused on cryopreserved semen. Frozen-thawed sperm quality was evaluated by different in vitro tests and by evaluation of fertility. Objective motility parameters were evaluated by computer-assisted sperm analysis. Membrane integrity, acrosome integrity, and mitochondria function were evaluated using appropriate dyes and flow cytometry. Lipid peroxide production was assessed by the thiobarbituric acid test (malondialdehyde production). Fertility obtained with frozen-thawed semen supplemented or not in AET, ERG, or SER was evaluated after artificial insemination of laying hens. ERG and AET decreased sperm lipid peroxidation and decreased fertility, even at low doses. The presence of 4 mmol of SER significantly decreased lipid peroxidation, increased the frozen-thawed sperm quality, and increased fertility after sperm cryopreservation (90% vs. control 84%, P < 0.05). In a third experiment, the use of 1 mmol of sucrose (the best result of our previous study) added to 4 mmol of SER-supplemented extender was tested. This addition allowed to the highest levels of fertility (93%). In conclusion, the addition of 4 mmol of SER in semen cryopreservation diluents decreases peroxidation and improves the efficiency of the process.

Poloxamer 188 exerts a cryoprotective effect on rooster sperm and allows decreasing glycerol concentration in the freezing extender

“…The results in other cell types suggest that the composition of the plasma membrane could modulate the interactions of P188. This composition is quite particular in spermatozoa ( Thananurak et al., 2020 ), and it could explain why 0.1% P188 did not show significant improvements.…”

“…Thus, the increase of ATP content and reduction in ROS production could be a consequence of mitochondrial protection by P188, resulting in better motility, increased sperm survival, and lower presence of apoptotic markers. Bird spermatozoa are especially sensitive to oxidative stress because of the composition of their plasma membrane ( Thananurak et al., 2020 ). Therefore, they could benefit from this kind of protection resulting from P188.…”

“…Reactive oxygen species excess due to the imbalance between their production and the antioxidant defense systems results in oxidative stress, causing fertility loss ( Shahin et al., 2020 ). In the case of poultry spermatozoa, sperm membranes are rich in unsaturated fatty acids, thus being prone to produce lipid peroxides in the presence of ROS ( Thananurak et al., 2020 ; Najafi et al., 2020 ).…”

Functional Aspects of Seminal Plasma in Bird Reproduction

“…Negative correlations have been found between sperm DNA fragmentation after freezing and the concentrations of the following amino acids in seminal plasma: valine, alanine, isoleucine, methionine, leucine, tyrosine, phenylalanine, serine, most of which are of hydrophobic nature [63]. Although many of the cryoprotectant mechanisms of free amino acids are not well known, their role on sperm cryoresistance appears to be related to their antioxidant activity as recently shown for valine and serine [63,88], by serving as oxidizable substrates for scavenge free radicals. Furthermore, amino acids may provide protection against the denaturing effects of low water potential during freezing [89]; amino acid might stabilize proteins, thus avoiding denaturation and dissociation that would lead to a greater contact surface between proteins and solutes during freeze-thaw process [90].…”

“…Supplementation of valine to chicken freezing extenders showed a positive effect on DNA fragmentation and fertilizing ability of frozen-thawed sperm, with a better response in a breed considered as the lowest freezer [94]. The addition of appropriate serine levels also showed a positive effect on sperm cryopreservation, partly through the antioxidant action of this amino acid [88].…”