Abstract
Little information is available regarding the effect of melatonin on the quality and fertilization capability of sex-sorted bull sperm, and even less about the associated mechanism. Sex-sorted sperm from three individual bulls were washed twice in wash medium and incubated in a fertilization medium for 1.5 h, and each was supplemented with melatonin (0, 10−3 M, 10−5 M, 10−7 M, and 10−9 M). The reactive oxygen species (ROS) and endogenous antioxidant activity (glutathione peroxidase (GPx); superoxide dismutase (SOD); catalase (CAT)), apoptosis (phosphatidylserine [PS] externalization; mitochondrial membrane potential (Δψm)), acrosomal integrity events (malondialdehyde (MDA) level; acrosomal integrity), capacitation (calcium ion [Ca2+]i level; cyclic adenosine monophosphate (cAMP); capacitation level), and fertilization ability of the sperm were assessed. Melatonin receptor 1 (MT1) and 2 (MT2) expression were examined to investigate the involvement of melatonin receptors on sex-sorted bull sperm capacitation. Our results show that treatment with 10−5 M melatonin significantly decreased the ROS level and increased the GPx, SOD, and CAT activities of sex-sorted bull sperm, which inhibited PS externalization and MDA levels, and improved Δψm, acrosomal integrity, and fertilization ability. Further experiments showed that melatonin regulates sperm capacitation via MT1. These findings contribute to improving the fertilization capacity of sex-sorted bull sperm and exploring the associated mechanism.
Highlights
Flow cytometry has been widely used to sort sperm based on the difference in DNA content of X and Y spermatozoa
Our results showed that the level of reactive oxygen species (ROS) was significantly lower in all of the melatonin-treated groups compared to the controls (Figure 1B), indicating that ROS was removed efficiently from these groups
Our results showed that melatonin treatment significantly increased the activities of CAT, GPx, and superoxide dismutase (SOD). This evidence indicates that melatonin significantly enhances the activities of endogenous antioxidants, which helps to explain the lower level of ROS in the melatonin-treated group (Figure 1B)
Summary
Flow cytometry has been widely used to sort sperm based on the difference in DNA content of X and Y spermatozoa. High quality frozen sex-sorted sperm plays an important role in the prevention and control of sex-linked genetic diseases, factory production of sexed embryos, and shortening the animal breeding cycle [1]. The preparation of sex-sorted frozen sperm involves the processes of attenuation, dying, laser light, freezing and thawing, all of which produce a large number of reactive oxygen species (ROS). Several studies have shown that the low quality and fertilization capability of sex-sorted sperm are closely associated with an excessive level of ROS [2,3,4]. Cells use a variety of antioxidant enzymes to remove excess ROS to reduce them to a steady state. Disrupts the interior balance of the sperm’s antioxidant defense system, and induces oxidative injury to sperm [7]
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