Introduction
The use of cryopreserved semen in artificial insemination has many advantages in animal husbandry, especially in breeding programmes. These advantages include protecting animals from stress caused by transportation for mating, the risk of disease transmission during copulation, and favouring the preservation of good genetic material (Vidal et al., Reference Vidal, Batista, da Silva, Gomes, Pelinca, Silva and Guerra2013). Nevertheless, cryopreservation severely damages sperm, leading to a decreased success rate compared with natural breeding (Qian et al., Reference Qian, Yu and Zhou2016). Several cryoprotectants such as glutathione, ascorbic acid, trehalose, glycerol, 1,2 propanediol, sucrose, and Laminaria japonica polysaccharide are added to freezing extenders to minimise sperm cryodamage (Hu et al., Reference Hu, Li, Li, Jiang, Bu, Yang and Wang2009, Reference Hu, Sun, Li, Zhang, Hu, Hu and Wan2013; Nur et al., Reference Nur, Zik, Ustuner, Sagirkaya and Ozguden2010; Yeste et al., Reference Yeste, Estrada, Pinart, Bonet, Miró and Rodríguez-Gil2014; Giaretta et al., Reference Giaretta, Estrada, Bucci, Spinaci, Rodríguez-Gil and Yeste2015).
Humic acids (HAs) are formed via decomposition and transformation of microbial materials and plant and animal residues (Janos, Reference Janos2003). Fulvic acids (FAs), a type of HAs, are organic substances obtained from peat and different types of coal such as weathered coal and lignite. FAs are often found in soil and natural water systems and are compatible with all aquatic life forms (Islam et al., Reference Islam, Schumacher and Groop2005). Compared with other HAs, FAs are soluble in both acid and alkali solutions, are lower in molecular weight, and have greater biological activities (Bai et al., Reference Bai, Chang, Shi and Shan2013). They are rich in many reactive functional groups such as carboxyls, hydroxyls, quinones and phenols (Aiken et al., Reference Aiken, McKnight, Wershaw and MacCarthy1985). These reactive groups endow FAs with many beneficial activities such as metal chelation (Plaza et al., Reference Plaza, García-Gil, Polo, Senesi and Brunetti2005), antioxidant activity immunostimulation (Chang et al., Reference Chang, Bai, Shi, Shan, Wei, Yu and Tong2013), anti-inflammatory activities and antiviral properties (Huck et al., Reference Huck, Porter and Bushed1991; Yang et al., Reference Yang, Chiu and Lu1996) and anti-cancer activity (Jayasooriya et al., Reference Jayasooriya, Dilshara, Kang, Lee, Choi, Jeong and Kim2016). However, reports on the cryoprotective ability of FAs on goat buck sperm are few. The present study aimed to investigate the protective effect of FAs on cryopreserved goat buck sperm.
Materials and methods
Ethics statement
This study was approved by the ethics committee of the International Peace Maternity and Child Health Hospital of China Welfare Institute, China.
Materials
Fulvic acids were purchased from Shanxi Jintai Biological Co., Ltd (Shanxi, China). All other agents used were of analytical reagent grade.
Semen collection
Semen was collected once at 3-day intervals from six mature Lubei White male goats and maintained under uniform nutritional conditions. Male goats were kept in sheltered pens and away from females, fed with hay and concentrate, and given water ad libitum. Male goats (aged 2–4 years) were trained to ejaculate into an artificial vagina at a doe mount, and the ejaculate was collected into a pre-warmed empty tube (Liu et al., Reference Liu, Dong, Ma, Li, Han, Luo, Chang and Tan2016).
Cryopreservation
The Tris-based egg yolk extender used consisted of Tris (24.2 g/l), citric acid (14.8 g/l), glucose (11 g/l), egg yolk [20% (v/v)], glycerol [6.4% (v/v)], benzyl penicillin (1 × 106 IU/l), streptomycin (1 g/l), and distilled water. Each pooled ejaculate was divided into six equal aliquots and diluted with the freezing extender containing FAs (0.2, 0.4 or 0.6%, w/w). Diluted semen samples were then loaded into 2 ml plastic straws and sealed with polyvinyl. After equilibration, the straws were frozen in liquid nitrogen vapour for 10 min and plunged into liquid nitrogen for 2 weeks of storage.
Thawing and semen evaluation
To evaluate sperm quality, the frozen straws were thawed by immersing in a water bath at 37°C for 1 min. The thawed semen was slowly diluted with 4.75 ml of Beltsville thawing solution at room temperature (23–25°C) to reduce sperm sticking to the glassware used during motility analysis.
Sperm motility was assayed according to the methods of Qian et al. (Reference Qian, Yu and Zhou2016). The percentage of sperm with intact acrosome was assayed according to Ahmad et al. (Reference Ahmad, Anzar, Shahab, Ahmad and Andrabi2003). Sperm-membrane integrity was determined using the combination of propidium iodide (PI) and carboxyfluorescein diacetate (CFDA), as described by Vidal et al. (Reference Vidal, Batista, da Silva, Gomes, Pelinca, Silva and Guerra2013). Sperm abnormality was determined according to the eosin–nigrosin staining method of Bearden and Fuquay (Bearden & Fuquay, Reference Bearden, Fuquay, Bearden and Fuquay1997).
Malondialdehyde (MDA)
MDA content in the sperm membrane was determined by the thiobarbituric acid (TBA) method. One millilitre of diluted semen solution (~3 × 106) was added to 1 ml of cold trichloroacetic acid (20%, w/v) and mixed well. The mixture was centrifuged at 1600 g for 10 min, and ~1 ml of the supernatant was mixed with 1 ml of TBA (0.67%, w/v), incubated at 100°C for 10 min, and then cooled. MDA content was assayed based on the absorbance at 534 nm recorded on a spectrophotometer (Shanghai Spectrophotometer Co., Ltd, China).
Biochemical parameters assay
In total, 120 ml of thawed semen sample were centrifuged for 5 min at 1600 g and 25°C. The resulting precipitate was extracted using 360 µl of Triton X-100 (1%) for 20 min and then centrifuged again for 30 min at 5000 g and 25°C. The supernatant obtained was used as the crude enzyme extract in the semen. SOD activity was assayed according to the method described by Flohe & Otting (Reference Flohe and Otting1984). One SOD activity unit was defined by the enzyme amount required to inhibit the nitroblue tetrazolium and expressed as nanomole per millilitre. Catalase (CAT) activity was assayed by the method of Goth (Reference Goth1991).
Statistical analysis
Data are presented as mean ± standard deviation (SD), and analysis of variance (ANOVA) was used to compare the means of the three groups. Statistical significance at the 95% probability levels was set at P < 0.05.
Results
Effects of fulvic acids on sperm quality
The effects of FA addition to the extender on the parameters (i.e., sperm motility, acrosomal integrity, sperm-membrane integrity, and sperm morphology) of frozen–thawed goat buck semen are shown in Table 1. Although there were no significant differences in semen quality between the 0.4% and 0.6% FA groups (P > 0.05), FA addition improved semen quality when compared with the control group (P < 0.05).
Effect of fulvic acids (FAs) on post-thawed progressive motility, acrosome integrity, membrane integrity and percent abnormality
Values are expressed as mean ± standard deviation (SD) (n = 3). Means with different superscripts within a column indicate significant differences (P < 0.05).
Effects of fulvic addition on oxidative status
The effects of FA treatment on the oxidative status of MDA, SOD, and CAT of frozen–thawed goat buck sperm are shown in Table 2. FA addition improved SOD and CAT activities and decreased MDA levels compared with the control group (P < 0.05). Excessive FA addition (>0.4%) also did not improve the efficiency of addition (P < 0.05).
Effect of fulvic acids (FAs) on sperm malonaldehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) activity
Values are expressed as mean ± standard deviation (SD) (n = 3). Means with different superscripts within a column indicate significant differences (P < 0.05).
Discussion
Fulvic acids are involved in increasing cell membrane permeability, photosynthesis, oxygen uptake, respiration and phosphate uptake and control hormone levels and enhance secondary metabolites, and therefore are used as a plant growth regulator (Çimrin et al., Reference Çimrin, Türkmen, Turan and Tuncer2010; Che et al., Reference Che, Huang, Xu, Zhao, Li, Ma and Yu2017). Moreover, FAs promote animal growth performance, e.g. for juvenile loach Paramisgurnus dabryanus (Gao et al., Reference Gao, He, He, Li, Zhao, Mu, Lee and Chu2017), growing-finishing pigs (Bai et al., Reference Bai, Chang, Shi and Shan2013) and Litopenaeus vannamei (Gutiérrez-Dagnino et al., Reference Gutiérrez-Dagnino, Fierro-Coronado, Álvarez-Ruí, del Carmen Flores-Miranda, Miranda-Saucedo, Medina-Beltrán and Escamilla-Montes2015). However, data regarding the cryoprotective ability of FAs on goat buck are limited.
FA addition to the extender improved sperm quality, including sperm motility, acrosomal integrity, plasma-membrane integrity, and sperm morphology at the appropriate supplement amount of 0.4–0.6%. Thus, FAs protected goat buck sperm against the freezing–thawing process. Moreover, sperm SOD and CAT levels increased and MDA level decreased, indicating that FAs could inhibit the peroxidation of sperm components. Data regarding the cryoprotect mechanisms of FAs on sperm are currently limited. Fulvic acids presumably protected frozen sperm in two ways: (1) its strong antioxidant activities, which could inhibit the lipid peroxidation of goat buck sperm during freezing–thawing; and (2) its high carboxyls, hydroxyls, quinones and phenols content offer a great number of hydrogen bonds, which improve the water holding capacity of sperm proteins and subsequently inhibited the denaturation of sperm proteins.
In conclusion, addition of FAs to the extender showed a cryoprotective effect on frozen–thawed goat buck sperm. Results indicated that FAs were a promising cryoprotectant in freezing goat buck semen. However, the cyoprotective mechanisms of FA addition on cryopreserved goat buck sperm require further investigation.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Ethical standards
Not applicable.
Conflict of Interest statement
The authors have declared that no competing interests exist.
