Effect of addition of l‐carnitine to cryopreservation extender on rabbit post‐thaw semen parameters, antioxidant capacity, mitochondrial function, apoptosis and ultrastructure changes

Abstract

l‐Carnitine (LC) is considered to be a natural antioxidant agent that could be used to improve the efficiency of reproduction. However, the precise machinery of the effect of LC supplementation on frozen–thawed rabbit sperm has not been evaluated. Thus, the aim of this study was to evaluate the effect of the addition of LC to a freezing medium on parameters and ultrastructure changes in frozen–thawed rabbit sperm. Rabbit bucks (7 months of age) were involved, and semen was collected using the artificial vagina method. Pooled rabbit semen was cryopreserved in a tris yolk fructose extender without any supplement (LC0, control group) or with LC at levels of 1, 2 or 4 mM (LC1, LC2 and LC4, respectively). The samples were then loaded into 0.25‐ml straws and frozen over liquid nitrogen vapours before being plunged into the liquid nitrogen and stored at −196°C until evaluation. Data showed that the addition of LC significantly increased sperm motility, viability and membrane function, while sperm abnormalities decreased (p < .001). Sperm‐like apoptosis (early, late and necrosis spermatozoa) was lower in the LC4 group compared with the other groups. l‐Carnitine addition significantly enhanced the total antioxidant capacity and superoxide dismutase and glutathione peroxide activities and significantly reduced the protein carbonyl and malondialdehyde levels compared with the control group. Moreover, electron microscopy images demonstrated that LC addition (2 or 4 mM) preserved the acrosome and plasma membrane and protected the ultrastructure integrity of the cryopreserved spermatozoa in relation to the control group. Spermatozoa treated with LC exhibited higher mitochondria membrane potential (MMP) values compared with the control group. We conclude that the addition of LC (4 mM) to the freezing extender enhanced the quality, increased the antioxidant capabilities, preserved the ultrastructure integrity and reduced lipid and protein peroxidation as well as increased MMP activity of frozen–thawed rabbit sperm.

Supplementation of Alpha-lipoic acid-loaded nanoliposomes in semen extender improves freezability of buffalo spermatozoa

“…The ratio of progressive sperm motility in individually semen sample was assessed via phase contrast microscope (Leica DM 500, Schweiz) provided with a hot stage set at 37 °C. Concisely, aliquots of diluted sperm (10 µl diluted semen) were placed on pre-warmed glass slides and covered with a glass cover slip 4 , 23 . Examination progressive motility was detected in 4–5 various fields of microscope for individually semen sample.…”

“…Although cryopreservation system offers many benefits as previously described, it causes impairment to various sperm cell parts including protein, DNA, and membrane lipids which may decrease sperm functionality and ultimately ability to fertilization 2 , 3 . In our previous works, sperm structural and functionality was changed by cryopreservation method in different animals 4 – 6 . Furthermore, structural fluctuations of sperm plasma membranes, principally lipid–protein composition ratio, kind of extender, nature of cryoprotectants, cooling–thawing rates, and periodic differences, restrict the prosperity of cryopreservation prevalence among all species 7 , 8 .…”

“…Naturally, spermatozoa have an enzymatic antioxidant defense system 11 . The imbalance between the antioxidant defense and oxidative stress generations (including ROS) in spermatozoa during the cryopreservation leading to damages sperm membrane, and DNA injury as well as induce reduction in sperm quality and subsequently constrain the ability to fertilize 4 , 5 , 12 . However, the natural antioxidant defense system in spermatozoa is ineligible to resist the ROS triggered impairments.…”

Responses of sperm mitochondria functionality in animals to thermal stress: The mitigating effects of dietary natural antioxidants

“…ALC may promote sperm mitochondrial functionality recovery after HS exposure by increasing antioxidant capacity, decreasing oxidative stress and increasing autophagy and apoptosis (Qiao et al, 2021). In our previous work on frozen-thawed rabbit sperm, , Abdelnour, Hassan, et al, 2022 suggested that supplementation of ALC (4 mM) to the freezing extender enhanced the ultrastructure integrity, MMP activity, antioxidant capabilities as well as decreased lipid and protein peroxidation.…”

“…Heat stress (HS) undesirably impacts the reproductive capacity and could reduce fertility in animals (Abdelnour, El‐Ratel, et al, 2022, Abdelnour, Hassan, et al, 2022; Bradford et al, 2003; Hansen, 2009). For many decades, the adversative effects of HS on animal reproduction have been documented widely (Hansen, 2009; Kim et al, 2012).…”