Effects of Vitamin C and Vitamin E on Cryopreservation of Guanzhong Donkey Semen

Xiaohui Yu, Shuai He, Liqiang Wang, Mengyang Kang, Yanjiao Zhu, Shuhui Wang and Xiuzhu Sun*

College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China

ABSTRACT

Sperm cryopreservation offers an effective method to protect Guanzhong donkey, a precious species. The objective of this study was to reveal the effect of different antioxidants on the semen cryopreservation in Guangzhong donkey by testing indices of sperm motility, mitochondrial activity, membrane integrity and arosome integrity. The results illustrated that compared with controls, above-mentioned indices of sperm were significantly improved under concentrations of Vitamin C (400 mg/L) and Vitamin E (600 mg/L) (P < 0.05). Moreover, physiological indices of donkey semen were significantly higher than the control groups (P < 0.05) when Vitamin C (200 mg/L) and Vitamin E (200 mg/L) were present simultaneitily in the medium. Overall, the results suggested both antioxidants (Vitamin C and Vitamin E) could increase frozen semen quality and the ability of antioxidant. Nevertheless, the effect on compatibility of the two antioxidants was better than single addition.

Article Information

Received 25 May 2017

Revised 22 May 2019

Accepted 19 September 2018

Available online 24 June 2019

Authors’ Contribution

XY and XS conceived and designed the research. MK conducted the experiments. LW provided experimental materials. XY wrote the manuscript and YZ and SW edited it. SH performed the statistical analysis.

Key words

Guanzhong donkey, Semen, Cryopreservation, Vitamin C and E.

DOI: http://dx.doi.org/10.17582/journal.pjz/2019.51.5.1777.1781

* Corresponding author: [email protected]

0030-9923/2019/0005-1777 $ 9.00/0

Copyright 2019 Zoological Society of Pakistan

Introduction

Guanzhong donkey lives in Guanzhong Weihe River basin of China at the earliest. During late 1980, traditional function of donkey was replaced by agricultural mechanization, resulting in donkey’s population decreasing year by year. The potential value of Guanzhong donkey in meat puts importance to protecting, developing and utilizing Guanzhong donkey. Studies of donkey artificial insemination began with 1960s-1970s in China, but some causes such as low reproduction rate, long pregnancy, sensitivity of sperm to low temperature blow, easy damage of sperm during the process of freezing-thawing, and hydroperoxidation restricted the development of frozen semen in donkey, which provides a new method finding appropriate cryoprotectants to improve semen quality. Under chemical, osmotic, thermal and mechanical stress, sublethal sperm injury has been generated, which may result in destruction of acrosomal plasma membrane (Johnston et al., 2012). Adding Vitamin C to semen extender improved sperm motility and viability after cryopreservation of bull semen (Beconi et al., 1993; Mittal et al., 2014). The addition of Vitamin C to an extender can reduce sperm cell damage through its continuous radical-scavenging action (Reza et al., 2011). Vitamin E can be used to reduce the damage caused by high concentrations of free radicals, providing protection to the semen DNA and cellular membranes against peroxidation (Nordberg and Arner, 2001). However, reports about the use of Vitamin C and Vitamin E in donkey’s pcryoprotectant solution are scarce. This study aims at selecting an optimum semen extender and demonstrate the best type and concentration of antioxidants for donkey by semen.

 

Materials and methods

Animals and semen collection

Semen was collected by artificial vagina from four mature Guanzhong donkeys that had gross sexual appetites and strong build in Shaanxi Agriculture And Animal Husbandry Seed Multiplication Farm. Donkeys were conducted according to international ethical standard and we took our efforts to minimize donkeys’ discomfort. Semen quality was assessed by observing the morphology of sperm under the microscope at 400X and evaluating its motility subjectively. The density of sperm was determined by counting number of sperms in cell counting chamber after diluting the semen. Ultimately, only the semen free from extraneous odour, milk-white in colour, with abnormal morphology of less than 15% and sperm vitality more than 0.75 was used in the study.

For semen dilution, five formulae which are usually used in Equus were adopted in this study (Cui et al., 2010; Zhou et al., 2006; Zhao et al., 2010). Refrigerating fluid which contained 75% of basic liquid (Table I), 25% of egg yolk, 5% of glycerine was used in all experiments.

Semen processing and subpackaging

Semen samples were centrifuged at 900 RPM for 10 min at 20 to 25 °C. A part of the super-natant was removed. The remaining super-natant alongwith the pellet was mixed with diluent at a ratio of 1:1 and then placed at 4 °C for 60 min. Next 5% glycerinum was added to diluent and kept at 4 °C for 60 min. Later, semen was suctioned into 0.25 mL precooled thin tube for the final semen subpackage. In the process of packing, 1 cm interval of the thin tube was reserved to accommodate the expansion of the frozen semen. Finally the tubes were sealed by heated tweezers and properly marked and labelled.

Semen freezing

For freezing semen, liquid nitrogen was used. For this purpose, a certain volume of liquid nitrogen was placed in the incubator and then self-made floating plate with cryogenic thermometer probe was placed on the surface of liquid nitrogen. Then thin tubes were placed on the hot plate at 4 °C. When the temperature was about 130 °C, the hob was put on the floating plate immediately. Foam insulation box was capped tightly and then fumigated for 10-15 min with the thermometer changing between -100 °C to -130 °C during freezing. The change of freezing temperature and freezing time should be recorded and controlled. After the fumigation, thin tube was quickly plunged into liquid nitrogen for storage.

For thawing semen, frozen tubes were immersed in water bath at 37 °C for about 20 sec.

Effect of vitamins C and E on cryopreservation of semen

Different concentrations of vitamin C (200, 400, 600, 400 mg/L) and Vitamin E (200, 400, 600, 400 mg/L) were added in the selected diluent and their protective effect was determinded on sperms.

For sperm quality evaluation sperm motility, sperm acrosome integrity (FITC-PNA staining method), mitochondrial membrane integrity (Rh123-PI combination dyeing) and plasma membrane integrity (HOST) to determine thoroughly and comprehensively the sperm quality after freezing-thawing.

Statistical analysis

All analysis was performed using SPSS18.0 version for Windows Software. Data of semen analysis were expressed as means ± SEM. Comparison of means values among treatments was performed using One-way ANOVA. The level of significance was set at P value < 0.05.

 

Results

Effect of different diluents on cryopreservation of spermatozoa

Table II shows that among five frozen diluents, the sperm living rate, the acrosome intact rate, the rate of plasma membrane integrity and mitochondrial activity were significantly higher in group II than in other groups (P < 0.05). Group II worked best in all groups, therefore the formula of group II was used as based fluid in the subsequent experiments.

Effect of vitamin C on cryopreservation of spermatozoa

Table III showed that after adding different concentrations of Vitamin C in the diluent, the sperm viability, the acrosome integrity, plasma membrane integrity and mitochondrial activity were all significantly improved after thawing compared with the control group (P < 0.05). Vitamin C concentration of 400 mg/L and 600 mg/L gave the best results.

Effect of vitamin E on cryopreservation of spermatozoa

Table III shows the effect of different concentrations of vitamin E in diluent on sperm activity indicators. Vitamin E concentration of 600 mg/L gave best results.

 

Table I.- Components of base liquids.

Components	I	II	III	IV	V
Distilled-water (mL)	100	100	100	100	100
Sucrose (g)	11	7.3	7.3	-	-
Glucose (g)	-	2.0	2.0	3.0	6.0
Fructose (g)	-	-	-	-	2.0
Sodium citrate (g)	-	0.12	-	1.4	-
NaHCO3 (g)	-	0.04	0.04	-	-
EDTA (g)	-	0.13	0.13	-	1.0
Penicillin sodium (IU)	25000	25000	25000	10000	10000
Streptomycin sulfate (g)	-	-	0.04	0.04	0.04

 

Table II.- Effect of different diluents on cryopreservation of Guanzhong donkey spermatozoa.

Dilutents	Sperm viability (%)	Acrosome integrity (%)	Membrane integrity (%)	Mitochondrial activity (%)
I	31.00±1.57d	47.22±0.88d	48.20±1.50c	38.13±1.14d
II	44.32±0.76a	62.75±0.88a	63.49±1.26a	50.17±1.78a
III	37.91±1.05b	56.19±1.07b	54.75±0.97b	46.88±1.15b
IV	25.64±1.46e	41.14±1.17e	39.00±1.21d	36.88±0.92d
V	35.58±0.65c	51.38±1.41c	50.87±0.77c	42.13±0.98c

The different letters of each column in the table are on behalf significant difference (P>0.05), the same letter indicate the difference was not significant (P>0.05).

 

Table III.- Effect of vitamins C and E, alone and in concentration on cryopreservation of Guanzhong donkey spermatozoa.

Concentration (mg/L)	Sperm viability(%)	Acrosome integrity (%)	Membrane integrity (%)	Mitochondrial activity (%)
Vitamin C
Control	44.32±0.76 b	62.75±0.88 c	63.49±1.26 b	50.17±1.78 b
200	44.33±0.84 b	62.90±0.46 c	63.45±0.70 b	50.22±0.79 b
400	46.13±1.18 a	65.75±0.49 a	66.17±0.71 a	54.25±0.97 a
600	45.05±0.89 ab	64.53±0.72 b	65.91±0.77 a	53.73±0.45 a
800	44.33±1.05 b	62.92±0.68 c	62.92±0.85 b	50.28±1.32 b
Vitamin E
Control	44.32±0.76 b	62.75±0.88 c	63.49±1.26 b	50.17±1.78 b
200	44.37±0.84 b	62.78±0.61 c	63.51±0.77 b	50.22±0.65 b
400	45.01±1.15 ab	65.53±0.72 b	64.90±1.24 a	53.36±0.90 a
600	46.21±1.11 a	65.72±0.41 a	66.10±0.60 a	54.17±0.86 a
800	44.31±1.18 b	62.92±0.68 c	62.92±0.85 b	50.28±1.32 b
Vit. C+ Vit. E
0+400	45.01±1.15 b	64.51±0.68 c	64.90±1.24 cd	53.36±0.90 ab
600+0	45.05±0.89 b	64.53±0.72 c	65.91±0.77 abc	53.73±0.45 ab
200+200	46.97±1.02 a	66.22±1.06 a	66.59±0.42 a	54.95±1.69 a
200+400	45.87±0.94 ab	65.66±0.63 ab	66.14±0.71 ab	53.74±0.87 ab
600+200	45.39±0.94 b	64.71±0.90 ba	65.37±0.85 bcd	53.80±1.58 ab
600+400	44.51±1.04 b	64.65±0.65 ba	64.73±0.71 d	52.49±1.05 b

The different letters of each column in the table are on behalf significant difference (P>0.05), the same letter indicate the difference was not significant (P>0.05).

 

Combined effect of vitamins C and E on cryopreservation of spermatozoa

Table III shows that the combined effect of vitamins C and E improved the indicators of frozen sperm to a certain extent. In addition, the groups Vit. C + Vit. E (200+200) and Vit. C + Vit. E (200+400) proved to be best preservatives with living sperm rate, acrosome intact rate, mitochondria intact rate higher than other groups. However, the former is more superior to the latter in spite of some indicators having no significant difference (P> 0.05).

Discussion

Vitamin C that plays a regulatory role in the oxidation reduction and metabolic reactions is a very common water-soluble vitamin. It has already been widely used in many laboratories for animal sperm cryopreservation. The experiments proved that vitamin C improved the sperm activity of cow after thawing (Foote et al., 2002; Singh and Sharma, 2018). Cheede’s (2011) study showed that vitamin C also had good effect on the preservation of semen in buffalo, and it could improve the conception rate of buffalo after artificial insemination. Moreover, the vitamin C also had the ability of reducing the cohesion of thawed sperm, which was very helpful to sperm liquefaction after thawing (Mustafa and Esref, 2004). The effect of vitamin C on semen cryopreservation in Guanzhong donkey has not been reported so far. This experiment aims to detect all indices by adding different concentrations of vitamin C into donkey semen to suggest its antioxidant effect in Guanzhong donkey semen cryopreservation and reveal the optimal concentration. The reactive oxygen species composition would be stimulated during semen cryopreservation. Jones and Mann (1997) reported that vitamin C could not only neutralize the reactive oxygen species composition that did not reach the dynamic balance but also could effectively remove free radicals. Besides, it can prevent the peroxidatic reaction of sperm phospholipid, and can effectively maintain the integrity of sperm DNA (Mangoli et al., 2014). Certain concentration of vitamin C could significantly improve sperm living rate, acrosome integrity rate and mitochondrial activity in this experiment, suggesting that vitamin C in sperm cryopreservation plays a protective role mainly by reducing the oxidative damage. Vitamin C is also easy to be catalyzed to generate hydrogen peroxide and hydroxyl radicals in the presence of metal ions and increasing peroxide stress of sperm, which is likely to explain why the effect of group with high concentration was inferior to the one of 400 mg/L.

Vitamin E known as alpha tocopherol is a kind of fat-soluble vitamins, and it is also one of the most common type of antioxidants. Its antioxidant capacity is derived from its own structure that there is a lively hydroxyl on the benzene ring. In addition, the saturated carbon chain on one side of five carbon ring brings the fat-soluble feature to vitamin E. When oxygen free radicals (ROS) entered into fat, Vitamin E could neutralize ROS immediately to conduct the antioxidant effect. Vitamin E as an antioxidant was applied widely in frozen semen (Ollero, 1998), and its role adjusting sexual function and extending the life of sperm in terms of reproductive function has been recognized by academic circles. It can be concluded from this experiment that the addition of 600 mg/L of vitamin E could have very good protection effect on sperm, consistant with many results from researchers before (Cui, 2010; Zhou, 2006; Zhao, 2010). But when the concentration of Vitamin E in semen diluent is in excess, the sperm may die.

In previous experiments, vitamin C and vitamin E were applied to bull semen diluent to study in vitro fertilization rate. The results showed that the combination of the two increased the fertilization rate to 74%. The use of vitamin C or vitamin E separately resulted in 50% and 47% fertilization rates, respectively which showed that the two vitamins had a synergistic effect (Dalvit, 1998). The mechanism of synergy is not clear, hence further investigations are required in this directions. In future study, L-tryptophan , which can be used in activation medium for trouts, could be considered potentially positive effect on cryopreservation of Guanzhong donkey semen (Filiz, 2018).

 

Conclusion

Addition of antioxidants vitamin C and vitamin E can improve the quality of frozen thawed semen, and the optimum amount is 400 mg/L and 600 mg/L, respectively. The effect of combination of two antibiotics is apparently better, which can significantly improve the indicators of sperm, superior to adding alone. The best compatibility concentration of vitamin C and vitamin E are 200 mg/L for vitamin C and 200 mg/L for vitamin E.

 

Acknowledgements

The authors are indebted to the manager in Shaanxi agriculture and Animal Husbandry Seed Multiplication Farm for his help with sample collection and experiment instruction.

 

Statement of conflict of interest

The authors declare no conflict of interest.

 

References

Bucak, M.N., Ates, S., Ahin, A., Variali, O., Yuce, A., Tekin, N. and Akcay, E., 2007. The influence of trehalose, taurine, cysteamine and hyaluronanon ram semen, microscopic and oxidative stress parameters after freeze-Thawing process. Theriogenology, 67: 1060-1067. https://doi.org/10.1016/j.theriogenology.2006.12.004

Beconi, M.T., Francia, C.R. and Mora, N.G., 1993. Effect of natural antioxidants in frozen bovine semen preservation. Theriogenology, 40: 841-851. https://doi.org/10.1016/0093-691X(93)90219-U

Cheede, G.S., Pangaonkar, G.R. and Singh, J., 2011. Effect of ascorbic acid and chloroquine diphosphate on the quality and fertility of buffalo semen. Indian J. Anim. Sci., 79: 858-860.

Cui, X., Luo, J.H., Li, W.D., Li, F.H., Meng, D.N., Lu, J.W., Mu, D. and Ni, H.M., 2010. Effect of different concentrations of caffeine, VE on the freezing semen of small-tailed Han sheep. Chinese agric. Sci. Bull., 26: 1-6.

Dalvit, G.C., Cetica, P.D. and Beconi, M.T., 1988. Effect of alpha-tocopherol and ascorbic acid on bovinein bitrofertilization. Theriogenology, 49: 619-627. https://doi.org/10.1016/S0093-691X(98)00012-0

Filiz, K., 2018. In vitro effect of L-tryptophan on the quality and fertilizing capacity of sperms of endangered species of trouts. Pakistan J. Zool., 50: 903-910.

Foote, R.H., Brockett, C.C. and Kaproth, M.T., 2002. Motility and fertility of bull sperm in whole milk extender containing antioxidants. Anim. Reprod. Sci., 71: 13-23. https://doi.org/10.1016/S0378-4320(02)00018-0

Jeyendran, R.S., Vander, H.H., Perez-Pelaez, M., Crabo, B.G. and Zaneveld, L.J., 1984. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Fertil., 70: 219-228. https://doi.org/10.1530/jrf.0.0700219

Johnston, S.D., Satake, N., Zee, Y., Lópezfernández, C., Holt, W.V. and Gosálvez, J., 2012. Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function. Reproduction, 143: 787-797. https://doi.org/10.1530/REP-11-0436

Jones, R. and Mann, T., 1997. Damage to ram spermatozoa by peroxidantion endogenous phospholipids. Reprod. Fertil., 50: 261-268.

Li, J.D., 2009. Test report of application of two kinds of diluent frozen to donkey semen. Contemporary Anim. Husband., 2009: 36-38.

Mangoli, E., Talebi, A., Halvaei, I. and Taheri, F., 2014. Vitamin C can protect spermatozoa from detrimental effects of diabetes mellitus; effects on sperm parameters and DNA integrity using different cytochemical tests. Iranian J. Reprod. Med., 12: 26.

Mittal, P. K., Anand, M., Madan, A.K., Yadav, S. and Kumar, J., 2014. Antioxidative capacity of vitamin E, vitamin C and their combination in cryopreserved bhadavari bull semen. Vet. World, 7: 1108-1112. https://doi.org/10.14202/vetworld.2014.1127-1131

Mustafa, S. and Eserlf, D., 2004. The effect of ascorbic acid on the freez-ability of ram semen diluted with extenders containing different proportion’s of glycerol. Turk. J. Vet. Anim. Sci., 28: 893-910.

Nordberg, J. and Arner, E.S., 2001. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med., 31: 1287-1312. https://doi.org/10.1016/S0891-5849(01)00724-9

Ollero, M., Perezpe, R., Muiñoblanco, T. and Cebrianperez, J.A., 1998. Improvement of ram sperm cryopreserbation protocols assessed by sperm quality parameters and heterogeneity analysis. Cryobiology, 37: 1-12. https://doi.org/10.1006/cryo.1998.2092

Reza, A., Razi, J. and Hossein, T.N., 2011. Influence of added vitamin C and vitamin E on frozen-thawed bovine sperm cryopreserved in citrate and tris-based extenders. Vet. Res. Forum, 2: 37-44.

Singh, M. and Sharma, A., 2018. Improvement in quality of frozen jersey bull semen following fortification with chloroquine diphosphate and ascorbic acid. Indian J. Anim. Sci., 88: 1-5.

Wang, X.P., Wang, L.B., Zhang, Y.M. and Zhang, L.B., 1997. Horse and donkey semen stored in low temperature. Heilongjiang J. Anim. Reprod., 1997: 29-30.

Zeng, W.G. and Wang, Y.P., 2009. Advance in stallion and donkey semen preservation. China Herbiv., 29: 59-62.

Zhao, X.L., Zan, L.S., Hu, J.H., Gui, L.H. and Hai, R.J., 2010. The antioxidation protective effects of vitamin E on frozen-thawed semen in Qinchuan cattle. Chinese J. Anim. Vet. Sci., 2010: 117-122.

Zhou, D.X., Qiu, S.D., Zhang, J., Tian, H. and Wang, H.X., 2006. The protective effect of vitamin E against oxidative damage caused by formaldehyde in the testes of adult rats. Asian J. Androl., 8: 584-588. https://doi.org/10.1111/j.1745-7262.2006.00198.