Effect of Coenzyme Q10 on the Preservation of Porcine Semen at 17℃.

 Artificial insemination (AI) is widely used in swine production and has made a significant contribution to breed improvement and selection [1]. During artificial insemination, semen quality is one of the key factors determining the effectiveness of artificial insemination. During the storage of porcine semen at ambient temperature, reactive oxygen species (ROS) accumulate over time [2], and the large amount of unneutralized ROS reacts with the phospholipids on the cell membranes and destroys their structure [3]. Pig spermatozoa are more sensitive and susceptible to oxidative damage than other species [4]. Therefore, the addition of effective antioxidants during the preservation process is the most efficient and convenient way to reduce the damage of porcine spermatozoa [5-6].

 

Coenzyme Q10 (Co-Q10) is a fat-soluble quinone compound, which is a biologically active antioxidant [7]. This lipophilic antioxidant can diffuse directly into the polyunsaturated lipid chains present in the plasma membrane, generating energy and preventing reactive oxygen species (ROS) from affecting spermatozoa structure and function [8]. Currently, Co-Q10 has been shown to protect spermatozoa in human, dog and broiler tests [9-11], and the results of the test on the addition of CoQ-10 to cockerel semen, which is also sensitive to ROS, showed that CoQ-10 could effectively inhibit lipid peroxidation [12]. At this stage, there are few reports on the effects of Co-Q10 on spermatozoa during ambient storage of porcine semen. The purpose of this experiment was to study the effects of adding different concentrations of Co-Q10 to the diluent on the semen preservation effect, in order to explore the effect and appropriate concentration of adding Co-Q10 to porcine semen preservation, and to provide theoretical references for practical production.

 

1 Materials and Methods

1.1 Main reagents  

1.2 Co-Q10 was purchased from Beijing Solabao Technology Co., Ltd. and the rest of the reagents were purchased from Sigma Corporation (USA) unless otherwise specified. Modena dilution solution: 27.50 g of anhydrous glucose, 6.90 g of sodium citrate, 1.00 g of sodium bicarbonate, 2.60 g of EDTA-Na, 5.65 g of Tris, 2.90 g of citric acid, 0.60 g of penicillin, 1.00 g of streptomycin, 4.00 g of bovine serum albumin, added to 1,000 mL of double-distilled water, and filtered by aseptic operating table. Stored at 4℃.

 

1.2 Test apparatus and equipment

Full-automatic sperm quality analyzer (MD0620 0C, Nanning Songjing Tianlun Biotechnology Co., Ltd.), thermostatic water bath (DK-S12, Shanghai Senxin Co., Ltd.), 17 ℃ thermostatic incubator (FYL-YS-50A, Beijing FUYI Electric Appliance Co., Ltd.), high-speed centrifuge (1-14, Sartorius Co., Ltd.), fluorescence microscope (Ti2-U, Nikon Co., Ltd.) and so on. Ltd.), fluorescence microscope (Ti2-U, Nikon Company), etc.

 

1.3 Porcine semen collection and preservation  

The test semen was provided by the experimental pig farm of Bayi Agricultural and Reclamation University, Heilongjiang, China. A total of 100 mL of semen from 6 healthy long white boars aged 2-3 years and weighing 240-350 kg was collected by hand-holding method and used for mixing. The semen was transported in a way that minimized mechanical damage to the spermatozoa caused by vibration. The semen was centrifuged at 1,800 r/min for 5 min, and the supernatant was removed and the density of porcine spermatozoa was measured with a hemocytometer plate, and then the density was adjusted to 1×108 spermatozoa/mL with Modena diluent. The whole process should be completed quickly within 15 min and mixing should be done every 12 h. The spermatozoa should be mixed with the Modena dilution solution.

 

1.4 Experimental design  

There were five groups in the experiment, and the concentrations of Co-Q10 were 0, 10, 15, 25, and 35 μg/mL, and the data of each group were repeated four times.

 

1.5 Quality testing  

Every 24 hours, 15 μL of the samples were taken out and preheated for 15 min in an incubator at 37℃, and the sperm viability was measured by an automatic sperm analyzer; the pH of the semen was measured by an acidometer; the osmolality of each group of samples was measured by an Osmopro 3250 automatic freezing osmolometer on days 0, 1, 3, and 5; and spermatozoa mitochondrial activity was measured by 500 μL of the samples on days 0, 1, 3, and 5. On days 0, 1, 3 and 5, the mitochondrial activity of spermatozoa was detected by taking 500 μL of the samples, washing them twice with PBS, adding 5 μL of JC-1 staining solution, incubating them for 30 min at 37℃ in a water bath protected from light, and then detecting the proportion of spermatozoa whose tails were orange-red under fluorescence microscope. At the same time to detect the integrity of the sperm plasma membrane, take 100 μL of sample sperm and add 0.1 μL of SYBR-14 working solution, water bath at 37 ℃ for 10 minutes, and then add 0.5 μL of PI working solution to the sample at 37 ℃ and again avoid the light water bath for 10 minutes. The proportion of green spermatozoa was detected by fluorescence microscopy and the data were recorded.

 

1.6 Statistical analysis

The results were analyzed by ANOVA using SPSS 19.0 software, and the results were expressed as mean ± standard deviation, with P＜0.05 indicating significant differences and P＞0.05 indicating non-significant differences.

 

2 Results

2.1 Effect of different concentrations of Co-Q10 on sperm viability As shown in Table 1, the sperm viability of pigs decreased with the increase of storage time. On the 3rd day, the 25 µg/mL group had the best effect, the difference between the 10 and 15 µg/mL groups was not significant; on the 4th day, the 25 µg/mL group had the best sperm viability, which was better than that of the 15 µg/mL group, and the difference between the 10 µg/mL group and the 35 µg/mL group was not significant.

 

Table 1 Effect of different concentrations of Co-Q10 on porcine spermatozoa viability

Save Time	0 µg/mL	10 µg/mL	15 µg/mL	25 µg/mL	35 µg/mL
0 d	89.90±0.43	89.90±0.43	89.90±0.43	89.90±0.43	89.90±0.43
1 d	79.90±0.35d	83.40±0.19bc	84.05±0.35ab	84.70±0.56a	82.70±1.41c
2 d	77.80±1.41	81.80±0.70	82.45±0.49	83.55±1.06	81.10±0.70
3 d	73.60±0.89c	75.80±1.41b	76.30±0.89b	79.25±0.49a	74.85±0.77bc
4 d	66.70±0.70d	71.90±0.70c	73.15±0.63b	76.20±0.70a	71.30±0.49c
5 d	61.10±0.70d	67.70±1.06bc	68.30±0.56b	73.80±0.70a	66.35±0.77c

Note: The same letter in the table indicates that the difference is not significant (P>0.05), while different letters indicate significant difference (P<0.05). The same as below.

2.2 Effects of different concentrations of Co-10 on sperm pH  

As shown in Table 2, the pH of the test group was significantly better than that of the control group on the 2nd and 3rd days of preservation; on the 4th day, the difference between the 15 and 25 µg/mL groups was not significant, and both groups were significantly better than the control group.

 

2.3 Effect of different concentrations of Co-Q10 on the osmotic pressure of spermatozoa  

As shown in Table 3, the differences in sperm osmolality between groups were not significant during 0-1 d of preservation; on the 3rd day, the sperm osmolality of 15 µg/mL group was significantly higher than that of 10 µg/mL group, and the control group was significantly lower than that of 15 and 35 µg/mL groups; the differences between the 10 µg/mL group and the 15 µg/mL group were not significant, and the osmolality of sperm in the 15 µg/mL group was significantly higher than that in the 25 µg/mL group on the 5th day. Sperm osmolality was significantly higher in the 15 µg/mL group than the 25 µg/mL group.

Table 2 Effects of different concentrations of Co-Q10 on pH of porcine spermatozoa

retention time	0 µg/mL	10 µg/mL	15 µg/mL	25 µg/mL	35 µg/mL
0 d	7.36±0.14	7.37±0.10	7.37±0.12	7.30±0.14	7.41±0.13
1 d	7.29±0.12	7.32±0.21	7.31±0.03	7.30±0.21	7.31±0.15
2 d	7.02±0.15b	7.2±0.11a	7.24±0.07a	7.22±0.04a	7.23±0.02a
3 d	6.93±0.11b	7.14±0.12a	7.16±0.03a	7.15±0.12 a	7.14±0.22a
4 d	6.62±0.21b	6.71±0.04ab	6.97±0.06a	6.92±0.12a	6.75±0.25ab
5 d	6.42±0.03	6.57±0.14	6.56±0.18	6.65±0.10	6.49±0.21

 

Table 3 Effect of different concentrations of Co-Q10 on osmolality of porcine spermatozoa mOsm/kg

Save Time	0 µg/mL	10 µg/mL	15 µg/mL	25 µg/mL	35 µg/mL
0 d	316.23±0.61	316.34±0.52	316.21±0.44	316.45±0.31	316.56±0.39
1 d	316.45±0.21	316.57±0.23	316.45±0.35	316.56±0.47	316.73±0.64
3 d	316.42±0.52c	316.32±0.31c	317.57±0.63ab	316.61±0.78bc	317.81±0.53a
5 d	316.87±0.78ab	316.26±0.87ab	317.65±0.54a	316.12±0.79b	316.77±0.67ab

Note: In the table, the same letter on the shoulder of the same peer value indicates that the difference is not significant (P>0.05), and the different letter on the shoulder indicates that the difference is significant (P<0.05).

2.4 Effect of different concentrations of Co-Q10 on mitochondrial activity of spermatozoa  

As shown in Table 4, the percentage of mitochondria-active spermatozoa in each experimental group was significantly higher than that in the control group on the 1st day of preservation; on the 3rd day of preservation, the percentage of mitochondria-active spermatozoa in the 25 µg/mL Co-Q10 group was significantly higher than that in the other groups, and the differences between the 10, 15, and 35 µg/mL groups were not significant, but all of them were significantly higher than the control group. On the 5th day of semen preservation, the percentage of mitochondrionally active spermatozoa was still significantly higher in the 25 µg/mL Co-Q10 group than in the other groups, and the differences between the 10 µg/mL group and the 15 and 35 µg/mL groups were not significant.

 

Table 4 Effect of different concentrations of Co-Q10 on the mitochondrial activity of porcine spermatozoa %

Save Time	0 µg/mL	10 µg/mL	15 µg/mL	25 µg/mL	35 µg/mL
0 d	96.31±0.72	96.31±0.72	96.31±0.72	96.31±0.72	96.31±0.72
1 d	93.82±1.12	94.14±0.78	94.62±0.93	94.76±0.54	94.00±1.03
3 d	87.80±0.87c	90.42±0.54b	89.63±0.76b	92.44±0.43a	90.76±0.78b
5 d	73.36±1.23d	81.53±0.65bc	82.22±0.63b	84.65±0.42a	80.26±0.28c

 

2.5 Effect of different concentrations of Co-Q10 on sperm plasma membrane integrity rate  

As shown in Table 5, on day 1, sperm plasma membrane intactness was better than the control group in all experimental groups, and the difference between the experimental groups was not significant; on day 3, the difference between the 15 µg/mL and 25 µg/mL groups was not significant, and the 25 µg/mL group was significantly better than the 10 µg/mL and 35 µg/mL groups; on day 5, it was still the best result in the 25 µg/mL group, and the difference between the 10 µg/mL and 15 µg/mL groups was not significant, but 2 groups were significantly better than the 35 µg/mL group, and the 35 µg/mL group was significantly better than the control group. At day 5, the 25 µg/mL group was still the most effective group, while the 10 µg/mL and 15 µg/mL groups were not significantly different from each other.

 

Table 5 Effect of different concentrations of Co-Q10 on the plasma membrane intactness of porcine spermatozoa.

 

Save Time	0 µg/mL	10 µg/mL	15 µg/mL	25 µg/mL	35 µg/mL
0 d	91.50±0.30	91.50±0.30	91.50±0.30	91.50±0.30	91.50±0.30
1 d	87.95±0.23c	90.75±0.31ab	90.77±0.37ab	91.15±0.31a	90.20±0.81b
3 d	83.35±0.36c	85.42±0.22b	86.57±0.20a	86.75±0.13a	85.35±0.12b
5 d	60.27±0.91d	66.80±0.42b	67.40±0.12b	71.67±0.52a	65.75±0.59c

3 Discussion

Ambient storage utilizes weak acid conditions to slow down sperm activity [13], but they are still metabolized and consumed during storage [14]. In this study, the addition of Co-Q10 was found to improve sperm motility and spermatozoa could be stored for at least 120 h at 17 °C. The spermatozoa are one of the most important factors in determining fertilization. Sperm viability is one of the most important factors in determining fertilization and plays a key role in the formation of a fertilized egg by sperm binding to the egg after 72 h of storage at 17 ℃. The sperm viability of spermatozoa in the group treated with 25 µg/mL of Co-Q10 was significantly higher than that of the other experimental groups and the control group on the 3rd and 4th days of storage, suggesting that the addition of 25 µg/mL is an effective dose to improve the viability of spermatozoa.

During in vitro preservation of semen, buffers are added to the diluent to maintain the pH of the semen in order to minimize the impact of sperm metabolites on the stability of the internal environment [15]. The pH of fresh porcine spermatozoa is generally 7.2-7.5, and the pH in porcine epididymis is 6.2-6.8, and spermatozoa can survive for 1-2 months in the epididymis [16]. Weak acidic environment is more favorable for sperm preservation. The results of this experiment showed that the addition of appropriate amount of Co-Q10 to the dilution solution could slow down the decrease of pH. Throughout the preservation process, the pH of the 25 µg/mL group decreased significantly slower than that of the other groups, especially on the 5th day of preservation, the 25 µg/mL group had the best pH retention effect among all groups.

 

The osmotic pressure in epididymis is 400 mOsm/kg, the osmotic pressure of pig semen is 290-300 mOsm/kg, and its tolerance pressure is 240-380 mOsm/kg[17] , and sperms can survive in epididymis for two months at most, which is related to the higher osmotic pressure in epididymis, and sperms are slightly dehydrated due to the high pressure, which results in the reduction of the transportation and metabolism ability[18] . However, factors such as the concentration of ions in the diluent should also be taken into account when adding in vitro. In this experiment, the osmolality was controlled in the range of 316-317 mOsm/kg, and there was not much difference between the test groups, which indicated that the addition of Co-Q10 to the diluent did not adversely affect the osmolality of the diluent, and it could be used for the addition of diluent.

 

It has been shown that sperm motility is positively correlated with mitochondrial activity [19]. Spermatozoa are fully differentiated and can only rely on mitochondria to convert sugars into ATP from functional substances in seminal serum or diluent to keep spermatozoa motile. Therefore, the mitochondrial activity can reflect the state of sperm viability. The results of this experiment showed that on the first day of preservation, the mitochondrial activity of each test group was significantly higher than that of the control group, and the percentage of spermatozoa with high mitochondrial activity was the highest in the group treated with 25 µg/mL of Co-Q10 throughout the preservation period.

 

During metabolism, moderate amounts of ROS promote sperm capacitation [20], but large amounts of ROS accumulation can trigger sperm oxidative stress [21]. Lipid peroxidation is the main cause of oxidative damage to spermatozoa during storage [22]. Porcine spermatozoa are more sensitive to ROS, and with prolonged preservation, polyunsaturated fatty acids (PUFAs) in the plasma membrane are more susceptible to lipid peroxidation, which can lead to disruption of the integrity of the plasma membrane [23]. In the present study, the addition of Co-Q10 to semen during the preservation process showed that the 25 µg/mL group had better plasma membrane integrity than the other groups from the 3rd to 5th day of preservation, which is a positive effect on semen preservation.

 

4 CONCLUSIONS

The results of this experiment showed that the addition of Co-Q10 to porcine semen diluent could effectively prolong the storage time of porcine semen at room temperature. On the 5th day of storage, the sperm viability, mitochondrial activity and plasma membrane integrity of the 25 µg/mL group were significantly higher than those of the other groups and the control group, which was the most favorable for semen storage. Meanwhile, the pH of the 25 µg/mL group decreased significantly slower than that of the other groups, and the addition of Co-Q10 did not affect the osmolality of the diluent. Therefore, the appropriate concentration of Co-Q10 for porcine semen preservation at room temperature is 25 µg/mL.

 

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