The influence of L‐proline and fulvic acid on oxidative stress and semen quality of buffalo bull semen following cryopreservation

Abstract Background This study investigates the effects of cryopreservation and supplementation of Azeri water buffalo's semen with proline (Lp) and fulvic acid (FA). Objectives Therefore, this study aimed to assess motility parameters, sperm viability, oxidative stress parameters, and DNA damage to detect the optimum concentrations of Lp and FA for buffalo semen cryopreservation. Methods Thirty semen samples of three buffalo bulls were diluted in Tris‐egg yolk extender and divided into 12 equal groups including control (C), Lp‐10, Lp‐20, Lp‐40, Lp‐60, Lp‐80 (containing 10, 20, 40, 60, 80 mM L‐proline, respectively), FA‐0.2, FA‐0.5, FA‐0.8, FA‐1.1, FA‐1.4 and FA‐1.7 (containing 0.2%, 0.5%, 0.8%, 1.1%, 1.4% and 1.7% fulvic acid, respectively). Results The velocity parameters, TM and PM were improved by FA‐1.7, FA‐1.4, Lp‐40 and Lp‐60 groups compared to the C group but no significant difference was found regarding the amplitude of lateral head displacement and straightness compared to the control groups. The percentage of sperm viability and PMF were increased by FA‐1.7, FA‐1.4, FA‐1.1, Lp‐40 and Lp‐60 groups compared to C group, while in terms of sperm DNA damage FA‐1.7, FA‐1.4, FA‐1.1, Lp‐10, Lp‐20, Lp‐40 and Lp‐60 groups showed better results compared to C group. The results also showed that FA‐1.7, FA‐1.4, FA‐1.1, Lp‐20, Lp‐40 and Lp‐60 groups could improve TAC, SOD, GSH and decrease MDA levels. Also, FA‐1.7, FA‐1.4, Lp‐20 and Lp‐40 groups could improve GPx levels but just FA‐1.7, and Lp‐40 groups could improve CAT levels compared to C group. Conclusions Thus, it can be concluded that L‐proline and fulvic acid supplementations can improve the quality parameters of post‐thawed buffalo bull semen.


INTRODUCTION
Genetic diversity and characteristics of native breeds are recognised as valuable resources that cannot be recovered if extinct (Antkowiak et al., 2012). In addition to the physical protection of native species and ecotypes, the protection of their genetic pool should also be considered (Aminafshar et al., 2008). Azari ecotype is small in size with black skin and high-quality milk production rate as well as high fat and a long milking period per year (Borghese & Mazzi, 2005;Pour Azary et al., 2004).
This native endangered ecotype may further proceed to extinction in the future due to climate change. Therefore, it necessitates preserving genetic reserves of the buffalo that is considered as a vital genetic source in the production of milk and other products. Therefore, sperm freezing of this valuable ecotype is very important (Tavakolian, 2000).
Moreover, cryopreservation and thawing of semen produces reactive oxygen species (ROS), thus exposing the sperm to oxidative stress.
The physiological levels of ROS affect the fertilisation potency and capacitation and acrosomal reaction of spermatozoa; at extremely high levels, ROS lead to apoptosis, decreased motility, chromatin damage, and impaired fertilisation ability (Elsayed et al., 2019). According to previous evidence (Izanloo et al., 2022;Partyka et al., 2011), it also increases sperm susceptibility to lipid peroxidation (LPO). Glutathione peroxidase (GPx), glutathione reductase, superoxide dismutase (SOD), and catalase (CAT) as enzymatic or natural antioxidants, as well as glutathione (GSH), ascorbic acid, carotenoids, vitamin E, selenium, coenzyme Q, and zinc as nonenzymatic or synthetic antioxidants have a defence mechanism against free radicals and LPO (Kasimanickam et al., 2006;Tuncer et al., 2010;Partyka et al., 2015;Papas et al., 2019Papas et al., , 2020. Buffalo semen has an anti-oxidative stress system that includes enzymatic and nonenzymatic antioxidants (Turaja et al., 2019), but during cryopreservation, due to the low concentration of these antioxidants in semen, the endogenous defence system is insufficient for countering this stress (Ansari et al., 2012). Low temperatures during the buffalo semen freezing and thawing cycle cause excessive adenosine triphosphate consumption, cell membrane lesions, DNA apoptosis, increased osmotic pressure, early acrosome reactions, and oxidative stress, reducing sperm motility and fertilisation capacity (De Lamirande et al., 1997;Tariq et al., 2015;Ezz et al., 2017). Therefore, an improvement in sperm cryopreservation leads to increased productivity in the breeding program of this animal species (Topraggaleh et al., 2014). According to some studies, antioxidants protect cells against oxidative damage and are also essential for sperm integrity and motility. For this reason, adding antioxidants to buffalo semen is necessary to improve fertilisation capacity and semen quality (Beheshti et al., 2011;Ansari et al., 2012;Saeed et al., 2016;Soleimanzadeh et al., 2020).
L-proline is one of the essential amino acids that has proteinogenic properties (Moradi et al., 2022); it also has various functions such as being an osmoprotectant, stabilising cellular structures and enzymes, scavenging ROS and keeping up redox balance in adverse situations (Meena et al., 2019). Some researchers have reported that L-proline has antioxidant and natural osmoprotective properties in relation to the destructive effects of freezing on various species, including human, donkey and ram sperm as well as mice oocytes (Sangeeta et al., 2015;Zhang et al., 2016;Li et al., 2021;Moradi et al., 2022).
Fulvic acids, a type of humic acids, are organic substances obtained from peat. Fulvic acids are often found in soil and natural water systems and have greater biological activities and are soluble in both acid and alkali solutions (Islam et al., 2005;Bai et al., 2013). On the other hand, Fulvic acids are rich in many reactive functional groups (phenols, quinones and hydroxyl), and these groups cause anti-oxidative activity Jayasooriya et al., 2016). It has been showed that humic and fulvic acid could improve sexual desire and semen quality under heat-stress conditions (Abdel-Khalek et al., 2019). On the other hand, Xiao and coworkers observed that fulvic acid have a cryoprotective effect on freezing goat buck semen (Xiao et al., 2018).
Today, although the technology of freezing and preserving semen for artificial insemination, especially in bulls and buffalo, has advanced beyond other species, there are still prominent gaps in the foundation of this knowledge and technology. Still, sperm survival rates after thawing are low and vary considerably among breeding bulls. These weaknesses are significant because they prevent progress in the fundamental science of reproductive biotechnology. However, the protective effects of L-proline and fulvic acids on buffalo semen are still unknown.
Hence, this study aims to determine the effects of L-proline and fulvic acid supplementations to the extender following the freezing of Azeri water buffalo, on sperm characteristics and oxidative stress parameters.

Chemicals
All required chemical compounds were obtained from Sigma (St. Louis, MO, USA) and Merck (Darmstadt, Germany).

Collection and processing of the semen
All animals were healthy and under the same management conditions.
The artificial vagina method was used for semen collection twice a week. Overall, 30 ejaculations from three Iranian water buffalo bulls (Bubalus bubalis, Azari ecotype), the fertility of which had already been confirmed, were obtained during five weeks. The samples were examined for sperm quality; only those with a concentration greater than 500 × 10 6 spermatozoa/mL, volume between 2-6 mL, total motility greater than 65% and abnormal morphology less than 20% per ejaculation were considered normal and investigated.
A standard Tris-based extender (Tris: 2.66 g per 100 mL, citric acid: 1.47 g per 100 mL, glucose: 0.63 g per 100 mL, egg yolk: 20% (v/v), glycerol: 7% v/v, penicillin: 1 mg per 100 mL, streptomycin: 1 mg per 100 mL and pH: 6.8), according to previous research (Topraggaleh et al., 2014), was used as base extender in this study. In each replicate, semen samples (n = 3) were pooled and equally divided to 12 parts and each part was diluted to a final concentration of 15 × 10 6 spermatozoa/mL (Tahmasbian et al., 2022) with one of the following extenders including: Group 1: Control (Tris-based extender without antioxidant); Lp-10: Tris-based extender + L-proline (10 mM); Lp-20: Tris-based extender   (Xiao et al., 2018). The straws were put in a refrigerator set at 4˚C for 2 h and then semen was frozen according to the following protocol: from + 4 • C to −12˚C at a rate of -4˚C /min, from -12˚C to -40˚C at a rate of -40˚C/min and from -40˚C up to -140˚C at a rate of -50˚C/min. Then they were transferred into goblets floating in liquid nitrogen. After thawing at 38˚C for 30 s, three samples from each frozen pie were analysed for the quality parameters of semen (Soleimanzadeh et al., 2020).

Assessment of enzymatic antioxidants activity
Semen biochemical analyses were performed using six straws (one for each extender). After thawing, 120 μL of the semen was centrifuged at 1600 × g at 25 • C for 5 min. The supernatant was removed, and 360 μL of 1% Triton X-100 was precipitated for 20 min for extraction. The resulting mixture was centrifuged at 4000 × g at 25 • C for 30 min. The suspending of the precipitate was repeated once more, and the supernatant was obtained as the crude extract of the enzymes present in the sperm.

Catalase evaluation
A 2-step method using a commercial CAT kit (Nactaz™ Catalase Activity Assay Kit; Navand Salamat Company, Urmia, Iran) was considered to evaluate the activity of CAT. An absorbance rate of 550 nm was taken into consideration for determining CAT after incubation for 10 min at room temperature. The CAT activity in the plasma was expressed as U/mL.

Glutathione evaluation
A GSH commercial kit (NarGul™ -Glutathione Assay Kit-GSH; Navand Salamat Company, Urmia, Iran) was utilised for GSH activity, and its value was recorded as U/L.

Statistical analysis
The obtained data were analysed by SPSS (Version 26.0; IBM CO., Chicago, USA) using one-way ANOVA and Tukey's post hoc test and p ≤ 0.05 was considered statistically significant.

DISCUSSION
The present study demonstrated that using Lp and FA supplementation could improve some sperm quality parameters (including sperm motility and velocity parameters, sperm viability and PMF, DNA integrity and antioxidant profile). Similar to our study, it was demonstrated that using various antioxidants also improved post-thawed semen quality parameters (Longobardi et al., 2017;Lone et al., 2018;Ahmed et al., 2020b;Soleimanzadeh et al., 2020).
The cryopreservation process leads to the overproduction of ROS, and to cope with this issue, and maintain the balance of ROS, antioxidants are necessary. Some studies showed that the fertility rate of cryopreserved semen is lower than that of the fresh sperm due to reduced nucleus and mitochondrial membrane integrity, plasma loss, protein denaturation, DNA damage, and oxidative stress (Ejaz et al., 2014;Ahmed et al., 2016). Reactive oxygen species are produced during the cryopreservation process, which reduces motility, viability, intracellular enzyme activity and sperm fertility potential (Selvaraju et al., 2008). In addition, the antioxidant system of the buffalo semen is insufficient for protecting the sperm from the harmful effects of oxidative stress during the cryopreservation process (Bansal & Bilaspuri, 2011). The production of free radicals and ROS is directly related to protein dephosphorylation. Therefore, it is assumed that the compounds used in this study maybe inhibiting protein dephosphorylation through the cAMP-PKA pathway in semen during storage (Fu et al., 2018). Different enzymes such as CAT, GPx and reductase, and SOD are the main defenders of the sperm plasma membrane against ROS and LPO (Selvaraju et al., 2008). Xiao et al. (2018) showed that FA addi- to the C group. Therefore, it is assumed that the Lp and FA used in this study maybe inhibiting protein dephosphorylation during cryopreservation (Fu et al., 2018). In another study on buffalo spermatozoa, Iqbal et al. (2016) found that the activity of these enzymes was significantly improved after thawing by adding 30 mM trehalose to the extender.
Based on the reports of Soleimanzadeh et al. (2020) the caffeic acid (100 μM) supplemented extender improved the antioxidant profile of buffalo semen after thawing. Reddy et al. (2010) also demonstrated that adding 50 mM taurine to the semen extender could significantly improve the antioxidant status.
Sperm motility and velocity parameters have positive correlation with sperm fertility and pregnancy in buffalo and cattle (Kasai et al., 2002;Kefer et al., 2009;Soleimanzadeh et al., 2020). Also, Shahzad et al. (2016) suggested that in order to check the fertility rate in buffalo, sperm motility rate can be evaluated after thawing, and the reason for that can be the damage to the sperm membrane that occurs during the cryopreservation process. The results of the current study showed that the addition of Lp and FA to extender could improve TM, PM and velocity parameters, except for ALH and STR, especially at FA-1.7, FA-1.4 and Lp-40, which had the highest yield. Similarly, Sangeeta et al. (2015) reported that Lp at a concentration of 25 to 50 mM improved TM and PM in ram semen. Additionally, other study showed that the proline can increased sperm motility after thawing in buck semen (Farshad & Hosseini, 2013). Also, Li et al. (2021)  protein dephosphorylation and cause them to improve sperm motility after freeze-thawing process (Fu et al., 2018).
The main cause of sperm cell damage during freezing is the impairment in plasma membrane (Ugur et al., 2019). Sperm cryo-injury occurs during cryopreservation due to changes in ice crystal damage. These injuries of sperm lead to decreased sperm viability and functionality (Khan et al., 2021). Also, in the course of the cooling process, limitations of phospholipid lateral motion induce a liquid-to-gel phase shift, making the plasma membrane rigid and fragile. These changes lead to lipid phase separation, and thus, the proteins are irreversibly clus-  (Topraggaleh et al., 2014), Royal jelly (Shahzad et al., 2016) or green tea extract (Ahmed et al., 2020a).
According to previous evidence, infertile males have more DNA damage than fertile males, and this damage may have a negative effect

F I G U R E 4
Continued on male fertility (Narayana et al., 2002;Talebi et al., 2011). Sperm freezing, as an adverse process, has been shown to destabilise chromatin and lead to sperm DNA fragmentation in boars and birds (Fraser & Strzeżek, 2007;Gliozzi et al., 2011). Double-stranded DNA breaks are due to high levels of ROS production, disruption of DNA repair enzymes and mechanical stress of DNA molecular genomic regions where chromatin density increases due to cell contraction (Bogle et al., 2017). Some studies have shown that DNA damage can be a cause of infertility in males (Narayana et al., 2002;Talebi et al., 2011). The results of current study showed that FA-1.7, FA-1.4, FA-1.1, FA-0.8, Lp-60, Lp-40, Lp-20 and Lp-10 groups could reduce DNA damage in buffalo semen after cryopreservation. In the present study, the concentration of 40 mM Lp had the greatest effect in minimising the amount of DNA damage. The high level of sperm DNA damage at higher doses (80 mM) of LP may be due to the fact that increasing the amount of L-proline creates a toxic state for sperm. This is confirmed by the MDA values shown in this study, which increase the amount of malondialdehyde in the cells as the dose of L-proline increases. In this respect, Topraggaleh et al. (2014) found that the concentration of 7.5 mM cysteine supplementation in the extender reduced amount of DNA damage in buffalo semen. In the study by Ejaz et al. (2014) adding 20 ng/mL arachidic acid to the extender also improved sperm chromatin integrity in buffalo semen. Dorostkar et al. (2012) reported that the addition of 2 mg/mL sodium selenite to extender reduced damage to buffalo sperm DNA, which is in line with our findings.

CONCLUSION
In conclusion, the addition of Lp and FA to semen extender could improve sperm motility and had antioxidant properties against ROS and free radicals (especially at the Lp-40 and FA-1.7 groups). It also improved sperm antioxidant capacity, reduced damage to sperm viability, and improved sperm PMF. Therefore, the addition of Lp and FA can improve the quality of frozen-thawed semen in buffaloes. Further studies are warranted to determine the effects of Lp and FA supplementations into the Tris-based extender on fertility.