Sperm cryopreservation has been widely used in men needing an assisted reproductive technology. In addition to sperm cryopreservation for infertile male patients, fertility preservation and banking for male patients with a certain type of cancer are commonly practiced in reproductive medicine [11]. In 1953, Bunge and Sherman reported a protocol for the preservation of human spermatozoa using 10% glycerol solution and dry ice. With this method, a 67%-sperm survival rate was obtained after cryopreservation. These authors also reported three cases of pregnancies following fertilization of frozen spermatozoa [12]. Later, Sherman [13] et al. successfully preserved semen samples in liquid nitrogen. Despite decades of development and improvement, the quality of sperm cryopreservation remains unsatisfactory. In particular, when non-liquefied semen is preserved using cryogenic method, the quality of spermatozoa decreases. This is due to the non-liquefaction of the seminal plasma as well as to the lack of related substance conducing to a reduced sperm viability. Normal semen seminal plasma contains endogenous antioxidants and non-enzymatic antioxidants that play a protective role in sperm cryopreservation [14]. Therefore, normal seminal plasma was employed to replace non-liquefied seminal plasma in the current study, when non-liquefied semen was preserved at low temperature.
We found that normal seminal plasma was able to maintain the viability of spermatozoa derived from non-liquefied semen. When the semen was left for a longer period of time, normal seminal plasma is better able to maintain spermatozoon viability than non-liquefied seminal plasma. It has been reported that spermatozoa protein Ⅰ (Semenogelin Ⅰ, Sg-I) covered the surface of spermatozoa by binding with Epididymis protease inhibitor (Eppin) to form Sg-I-Eppin complex. Thereby inhibiting the forward motion of sperm. [15]. Normal seminal plasma provides nutrients for spermatozoon activity, and participates in the de-capacitation and capacitation of sperm. In this way, spermatozoa can maintain a good vitality. Zinc in the seminal plasma slows down the lipid oxidation of the cell membrane, thereby maintaining the permeability and stability of the cell membrane, which is necessary for the good viability of spermatozoa. Fructose is able to provide energy for spermatozoa. The citrate in seminal plasma maintains the osmotic pressure, appropriate pH and good buffering capacity of the semen. The above substances are conducive to the survival of spermatozoa, and meet well their acticity needs [16, 17]. Normal seminal plasma employed for cryopreservation can play a cryoprotective role in sperm. In the study conducted by Montoya Páez et al. [18], it is reported that 10% homologous seminal plasma provides protection for sperm freezing, improves the sperm viability, and maintains the membrane integrity and morphology of sperm. Zoca et al. [19] also reported that seminal plasma provides an increased acrosomal integrity and a reduced remodeling of the F-actin cytoskeleton. Similar to these studies, our results show that during cryopreservation of non-liquefied semen-derived sperm, the addition of normal seminal plasma reduces the sperm cryoinjury, increases the viability and survival of sperm, and maintains the sperm morphology and plasma membrane integrity..
The oxidative stress is one of the most important factors conducing to an impaired sperm function during cryopreservation. Spermatozoa produce appropriate amounts of Reactive Oxygen Species (ROS), a byproduct of redox, which regulates the physiological functions of spermatozoa at physiological concentration, including energy acquisition, hyper-activation, acrosome reaction, and binding to zona pellucida [20]. During cryopreservation, antioxidant-rich seminal plasma is diluted and the antioxidant activity of sperm is reduced. Sperm damage, dead spermatozoa and leukocytes caused by cryopreservation also produce peroxides in excess [21. The balance between ROS production and the antioxidant capacity of the biological milieu is then disrupted and the excessive ROS produces oxidative stress damages. The ROS in excess, at first, results in the lipid peroxidation (LPO) of the sperm plasma membrane. After then, this peroxidation conduces to the decarboxylation of unsaturated fatty acids, which gives rise to malondialdehyde (MDA), a compound capable of impairing the permeability and fluidity of the sperm plasma membrane [22]. The above processes also cause damages to the proteins, mitochondria, and DNA of sperm [23]. For sperm proteins, the oxidation of the thiol groups makes the sperm cells susceptible to leukocyte attack. An excessive ROS production affects the phosphorylation and glycosylation of protein, and production of mitochondrial ATP, which subsequently affects the sperm fertilization [24]. The present study also confirms that sperm freezing leads to an oxidative stress with an increased expression of MDA and a decreased expression of SOD and GSH-Px in seminal plasma. The addition of normal seminal plasma not only enhances the anti-oxidative stress ability of sperm, but also exerts a certain cryoprotective effect, which reduces the expression of MDA and increases the expression of SOD and GSH-Px. This is consistent with the fact that normal semen seminal plasma contains superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, as well as non-enzymatic antioxidants such as glutathione, ascorbic acid, vitamin E, albumin, and taurine [6–7]. These compounds are capable of counteracting the oxidative stress damages caused by ROS. Under normal conditions, endogenous antioxidants such as CAT, SOD and GSH-Px in seminal plasma can protect sperm from oxidative stress injury [25]. However, during the freezing process of oligo-zoosperm and non-liquefied semen, the dynamic balance of the antioxidant system is disrupted. In such a situation, the endogenous antioxidants themselves cannot fully protect sperm cells from the oxidative stress damage. The resort to exogenous antioxidants is then necessary to resist the oxidative stress damage [26]. In this way, the addition of the exogenous antioxidant melatonin into the semen was able to improve sperm motility after thawing. It also protects the acrosome and plasma membrane integrities, and increases the activities of T-AOC, T-SOD, GSH-Px and CAT, whereas reduces the MDA and 4-HNE activities [27].
At the same time, the excessive ROS causes DNA damage in spermatozoa, with a high frequency of single- and double-stranded DNA breaks [28]. Disruption of DNA integrity in the sperm nucleus is the underlying cause of irreversible sperm damage. Human sperm DNA fragmentation suggests sperm damage and is a factor of male infertility. The percentage of sperm DNA damages correlates with the fertilization rate and the unfavorable outcomes in embryo development [29–30]. Sperm DNA fragment Index (DFI) is the main indicator of sperm DNA integrity. In the current study, we also found that the DFI increased after sperm freezing, and neither non-liquefied seminal plasma nor no-seminal plasma exerted a good protection of DNA integrity. The normal semen plasma may contain antioxidants that exert a more convenient DNA integrity protection. This suggests that normal seminal plasma plays an important role in antioxidant protection, which protects spermatozoa from freezing damages.
In conclusion, our results demonstrate that the seminal plasma of non-liquefied semen could not resist to the damage of cryopreservation, which may lead to the occurrence of abnormal frozen sperm. The normal seminal plasma, however, containing antioxidants, fatty acids and nutrients, can exert a protective effect against oxidative species and resist cryopreservation risks. The addition of normal seminal plasma into the sperm extracted from non-liquefied semen improves the viability, survival rate and plasma membrane integrity of sperm, maintains DNA integrity and normal morphology of cryopreserved sperm, and reduces oxidative stress damage.