The Effect of the Addition of Co-Q10 on the Preservation of Boar Semen at 17°C

Artificial insemination technology has been widely used in boar production and breeding; it has contributed significantly to breed improvement and selection (Knox, 2016). Semen quality is one of the key factors that determine the process of artificial insemination. ROS accumulate over time during the preservation, causing a large amount of ROS that cannot be neutralized to undergo lipid peroxidation reactions with phospholipids on cell membranes, destroying their structures (Allai, 2018). Boar sperms are more sensitive and susceptible to oxidative damage than other species (Dong et al., 2021). Therefore, adding effective antioxidants during the preservation process is the most effective and convenient method to reduce the degree of damage to boar sperm.
 
Co-Q10 is a lipid-soluble quinone compound that is an antioxidant with strong biological activity (Alahmar et al., 2021). When this lipophilic antioxidant is added to the semen, it can spread directly to the polyunsaturated lipid chain present in the plasma membrane to generate energy and prevent ROS from affecting the sperm’s structure and function (Appiah et al., 2020). At present, Co-Q10 has been proved to have an antioxidant protective effect on sperm in humans (Alahma et al., 2021), dogs (Kobayashi et al., 2021), broiler (Sharideh et al., 2020)  and other experiments. It was also confirmed that CoQ-10 could effectively inhibit lipid peroxidation in rooster semen (Masoudi et al., 2019) supplementation experiments that are also sensitive to ROS.
 
The quality of spermatozoa is seriously affected due to the inevitable oxidative damage during storage at 17°C. Co-Q10 can effectively reduce the damage during semen preservation as a strong and effective antioxidant. In the present study, different concentrations of Co-Q10 were added to study the effect of Co-Q10 on semen. A control group and 4 concentration groups were set up in this experiment. The sperm motility, mitochondrial activity, T-AOC, SOD activity and MDA content were tested and analyzed to explore the effect and appropriate concentration of boar semen.

The indicators of sperm motility, mitochondrial activity, T-AOC, SOD and MDA were measured at 0, 1, 3 and 5 days of semen preservation. The sperm motility can directly reflect sperm quality and reproductive status of the boar and determine the fertilization ability. ROS generated by sperm metabolism can cause lipid peroxidation of sperm membranes and T-AOC, SOD and MDA are antioxidant indicators, which can reflect the antioxidant capacity of semen The experimental records were taken and each experimental group was repeated 4 or 5 times.

Except otherwise stated, all reagents used for this experiment were purchased from Sigma. The boar semen was collected from 6 boars on the porcine farm of Heilongjiang Bayi Agricultural University.
 
Preparation of spermatozoa
 
The boar semen used in the experiment was collected from 6 healthy Landrace boars aged 12-16 months in the experimental pig farm of Bayi Agricultural Reclamation University in Heilongjiang. The fresh semen retrieved from the experimental boar farm was centrifuged at 1800 RPM for 5 min in a centrifuge. The supernatant was removed, diluted and mixed in proportion and a haemocytometer detected the sperm concentration in the semen. Then, the concentration of the original semen was adjusted to 1×10⁸/mL with Modena dilution and then divided into 10 mL centrifuge tubes with different concentrations of Co-Q10, placed in a 17°C incubator mixed every 12 hours.
 
Determination index
 
Sperm motility was detected by a MAILANG automatic sperm analyzer every 24 h during semen preservation. The diluted semen was shaken before detection with 15 μL pipetted from the dilution mix and preheated in a 37°C carbon dioxide incubator for 15 min. This was dropped on a glass slide and covered with a coverslip. After testing, records of the experiment were taken.
 
Semen (500 μL) was taken, washed twice with PBS, resuspended and the concentration was adjusted to 1×10⁶/mL. We added 5 μL of JC-1 staining working solution to the suspension and incubated it in a 37°C water bath for 30 min in the dark. Then, the suspension was washed once with PBS, resuspended and observed under a fluorescence microscope. The tail of sperm with high mitochondrial activity was orange-red.
 
The determination of T-AOC, SOD and MDA of sperm was carried out according to the instruction of the kits.
 
Statistical analysis
 
Data from four replicated trials were analyzed by one-way ANOVA using the STATVIEW 5.0 software (Abacus Concepts, Inc., Berkeley, CA, USA). If the P-value was smaller than 0.05 in the ANOVA, Bonferroni/Dunn test was carried out using the same program. All data were expressed as means±SD. Findings were considered significantly different at P<0.05.

The effects of adding different concentrations of Co-Q10 to diluted semen on sperm motility are shown in Table 1. It can be seen from Table 1 that the motility of boar semen decreased with the increase in storage time. On the 1^st day of storage, the viability of the 4 groups supplemented with Co-Q10 was significantly (P>0.05) better than the control group, but there was no significant difference between the 10 µg/mL, 15 µg/mL and 25 µg/mL groups. On the 3^rd to 5^th day of storage, the addition amount of 25 µg/mL Co-Q10 group was significantly (P<0.05) higher than the other experimental groups. The effects of adding different concentrations of Co-Q10 to the semen dilution on sperm mitochondrial activity are shown in Table 2. On the 1^st day, the percentage of spermatozoa with mitochondrial activity in each experimental group was significantly (P<0.05) higher than that in the control group (Table 2). On the 3^rd day, the percentage of spermatozoa with mitochondrial activity in the 25 µg/mL Co-Q10 group was significantly (P<0.05) higher than the other groups but all of the experimental groups were significantly (P<0.05) higher than the control group. When the semen was stored on the 5^th day, the percentage of spermatozoa with mitochondrial activity was still significantly (P<0.05) higher in the 25 µg/mL Co-Q10 group than in the other groups but the control group was the lowest.


 

 
Although the frequency of sperm motility can be slowed down during storage at 17°C, it will still be metabolized and consumed during the storage process (Torres et al., 2018). During the metabolic process, the excessive accumulation of ROS produced by the sperm tail and the ROS produced by the plasma membrane can trigger sperm oxidative stress (Aitken, 2017; Vongpralub et al., 2016). This experimental study found that adding Co-Q10 can improve sperm motility and mitochondrial activity and that it can be stored at 17°C for at least 120 h. Similar results were reported by Kobayashi Masanori (2021) in the dog sperm treatment experiment. Lin and Liu (2021) reported that sperm motility was significantly improved in the group with Co-Q10 supplementation. Masoudi (2018) also reported that supplementation with Co-Q10 resulted in greater sperm motility and mitochondrial activity. Sperm motility is a key factor in determining fertilization and plays a key role in whether sperm can combine with an egg to fertilize it. The spermatozoa were more easily oxidized after being stored at 17°C for 72 h than the sperm motility of the group treated with 25 µg/mL. Co-Q10 in the dilution was significantly higher than the other experimental and control groups on the 3^rd and 4^th days of storage, indicating that this concentration is an effective dose for enhancing motility. Studies have shown that sperm motility is positively correlated with mitochondrial activity (Malo et al., 2012). The results of this experiment showed that the mitochondrial activity of each experimental group was significantly higher than that of the control group during the five days and the percentage of sperm with high mitochondrial activity in the 25 µg/mL Co-Q10 treatment group was the highest throughout the storage period.
 
The effects of different concentrations of Co-Q10 in semen dilutions on T-AOC activity in semen are shown in Fig 1. On the first day of storage, the T-AOC activity of semen increased with the increase of Co-Q10 concentration and the test group was significantly(P<0.05) higher than the control group (Fig 1). On the 3^rd and 5^th day of storage, the T-AOC activity of all the experimental groups was significantly(P<0.05) higher than the control group and the 25 µg/mL Co-Q10 group had the strongest T-AOC activity and was significantly higher than 10 µg/mL, 15 µg/mL and 30 µg/mL Co-Q10 group.
 

 
This experiment proved that on the 5^th day of storage, the T-AOC in the semen when the concentration of Co-Q10 used was 25 μg/mL were significantly (P<0.05) higher than those of other experimental groups and control groups. Wu et al., (2020) proved this with similar results and the T-AOC was improved markedly in supplemented groups with Co-Q10. Moreover, the sperm motility and mitochondrial activity of the 25 μg/mL Co-Q10 group on the 5^th day of storage were also excellent, which could be related to be the effect of the antioxidant capacity of Co-Q10.
 
The effects of different concentrations of Co-Q10 on the SOD activity in semen are shown in Fig 2. In the 1^st day, the SOD activity of the 25 µg/mL Co-Q10 group was significantly higher (P<0.05) than the other groups (Fig 2). On the third day, the SOD activity of the 25 µg/mL Co-Q10 group was significantly higher than that of the other groups and the other experimental groups were also significantly higher than the control group. On the 5^th day of storage, the SOD activity of the 25 µg/mL Co-Q10 group was also significantly higher (P<0.05) than the other groups. Overall, adding 25 µg/mL Co-Q10 group could improve the SOD activity in semen.
 

 
SOD can highly specifically remove superoxide anion groups in semen and plays an important antioxidant role in organisms (Torres et al., 2021). Fouad et al., (2011) revealed that Co-Q10 could suppress oxidative stress in the testis by inhibiting lipid peroxidation and enhancing antioxidant enzyme activity. Therefore, reducing SOD activity will make scavenging superoxide anion free radicals worse and the lipid peroxidation products caused will destroy the proteins and enzymes of sperm cells and reduce sperm quality.

The effects of different concentrations of Co-Q10 on the MDA content in semen are shown in Fig 3. On the 1^st, 3^rd and 5^th days of storage, the content of MDA in the experimental group added with Co-Q10 was significantly lower (P<0.05) than the control group. And the higher the concentration of Co-Q10, the lower the MDA content and the groups with 25 µg/mL and 35 µg/mL had the best effect.
 

 
During the preservation of semen in vitro, due to the limited antioxidant capacity of the antioxidative enzyme system of the semen itself (Anzar et al., 2011), oxidative stress is one of the main reasons for the decrease in the success rate of artificial insemination. An important lipid peroxidation product is MDA and its content will directly affect the quality of semen preservation (Aitken, 2017). The results of this experiment showed that with the increase of the concentration of Co-Q10, the MDA content in semen showed a downward trend and the groups with 25 µg/mL and 35 µg/mL Co-Q10 had the best effect. In addition, as a marker of lipid peroxidation, the MDA levels were significantly reduced after Co-Q10 treatment, as published by Bakhshayeshkaram (2018). This indicates that the addition of Co-Q10 reduces sperm lipid peroxidation and protects sperm.

Adding Co-Q10 to boar semen dilution can effectively prolong the storage time of boar semen at 17°C. On the 5^th day, the sperm motility and mitochondrial activity of the 25 µg/mL group were significantly higher than the other groups, which was the most favorable for semen preservation. Meanwhile, adding the Co-Q10 can also improve the antioxidant level and preservation quality of boar semen. On the 5^th day, the SOD and T-AOC ability were the strongest in the 25 μg/mL group. In terms of MDA content, the 25 μg/mL and 35 μg/mL Co-Q10 groups were significantly lower than the control group on the 5^th day of storage. Therefore, the appropriate concentration of Co-Q10 in boar semen stored at 17°C is 25 μg/mL.

This project was supported by the Postdoctoral scientific research developmental fund of Heilongjiang Province (No.LBH-Q18100); The Doctoral Starting up Foundation of Heilongjiang Bayi Agricultural University (No.XYB-2016-04;No.XYB201910); Heilongjiang Bayi Agricultural University Support Program for San Zong (No. ZRCPY202107). We would like to thank Dr Samson O. ADENIRAN (Department of Biological Sciences, College of Basic and Applied Sciences, Mountain Top University Ibafo, Ogun State Nigeria) for English language editing.

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