Effect of Rubus coreanus leaf and stem extract on boar spermatozoa

The present study was performed in accordance with the guidelines provided by the Animal Care and Use Committee (ACUC) of Chonbuk National University. Boar semen was collected from Duroc boars twice a week. Sperm concentrations were estimated using a hemocytometer, and semen was diluted with Beltsville thawing solution (BTS) [21] with or without (W/O) antibiotics (penicillin G and streptomycin), to a final concentration of 1×10⁸ spermatozoa/mL. RLSE extract (with 99.9% methyl alcohol) was purchased from Korea Plant Extract Bank (KPEB, Ochang, Chungbuk-do, Korea). Different concentrations of RLSE (final concentration: 0–10 μg/ mL; dimethyl sulfoxide (DMSO) as a solvent control) were added to BTS. The diluted semen was stored in a storage unit at 17°C for 2 days. Unless otherwise noted, all other reagents used in this study were purchased from Sigma-Aldrich Chemical Co. LLC (St. Louis, MO, USA).

Sperm motility was examined using a computer-assisted sperm analysis system (Sperm Class AnalyzerⓇ, Microptic, Barcelona, Spain). Spermatozoa were incubated for 30 min at 37.5°C, and a 1 μL aliquot of sperm sample was then placed on a pre-warmed (38°C) Leja counting slide (Leja products B.V., Nieuw-Vennep, The Netherlands). Ten fields were analyzed at 37.5°C, assessing a minimum of 500 spermatozoa per sample. The proportion of total motile spermatozoa (%), progressive motile spermatozoa (%), and hyperactive spermatozoa (%) was determined. The kinetic parameters measured for each spermatozoon included: curvilinear velocity (VCL, μm/s), straight-line velocity (VSL, μm/s), average path velocity (VAP, μm/s), percentage linearity (LIN, %), percentage straightness (STR, %), and the wobble percentage (WOB, %).

Incubated spermatozoa (1×10⁸ cells/mL) were washed twice with phosphate-buffered saline containing 0.1% (w/v) polyvinyl alcohol (PBS-PVA). Sperm viability was assayed using the LIVE/DEADⓇ Sperm Viability kit (Molecular Probes, Eugene, OR, USA), which contains DNA dyes SYBR14 (100 nM) and propidium iodide (PI; 10 μM), following manufacturer’s protocol. The spermatozoa were stained, and images were acquired using a fluorescence microscope (Nikon Eclipse Ci microscope, Nikon Instruments Inc., Seoul, Korea) with camera (DS-Fi2, Nikon) and an imaging software (version 4.30, Nikon). The spermatozoa were classified and counted as viable (SYBR14) or dead (PI). The level of intracellular ROS in spermatozoa was assayed using 1 μM carboxy-DCFDA (Invitrogen, Eugene, OR, USA). The fluorescence intensity was measured using a multimode microplate reader (Spark™ 10 M, Tekan, Männedorf, Switzerland) with excitation (ex.) at 485 nm and emission (em.) at 520 nm.

E. coli (ATCC 8739) were cultured in nutrient broth for 18 h at 37°C and harvested by centrifuging the cultured broth three times with PBS (pH 7.2) at 1000 × g for 10 min. E. coli stock suspension was prepared by resuspending the final pellets. Initial E. coli population ranged 1.5 × 10⁴ CFU/mL was added by diluting the stock suspension, and the cell concentration was determined by the spreading plate method with incubation for 24 h at 37°C. During the experiments, 0.1 mL solution was withdrawn from each experimental sample and diluted 1/10 times to measure E. coli abundance. Three replicate plates were used at each dilution step.

Values are expressed as mean ± standard error of the mean (SEM). Data were analyzed using one-way analysis of variance (ANOVA) using the SAS package 9.3 (SAS Institute Inc., Cary, NC, USA) in a completely randomized design. Duncan’s multiple range test was used to compare values for individual treatments when the Fvalue was significant (p<0.05).

Boar spermatozoa were preserved with different concentrations of RLSE in BTS for 2 days. Sperm motility was determined after 30 min of incubation. Table 1 represents the sperm motility parameters across the treatment groups. The proportion of total motile spermatozoa was significantly higher in the treatment with 0.5 μg/mL RLSE than in the other groups (p<0.05; Table 1). Similarly, the percentage of progressive motile spermatozoa was significantly higher in the treatment with 0.5 μg/mL RLSE (p<0.05; Table 1). Kinetic values were not significantly different among the groups, except the VAP in 0.5 μg/mL RLSE treatment (p<0.05; Table 1). Significantly lower levels of motile spermatozoa, progressive motile spermatozoa, and VAP were seen in the DMSO treatment, a negative control (Table 1). The administration of R. coreanus fruit extract in male rodents has been shown to improve their epididymal sperm number, sperm motility, and the expression of cAMP-responsive element modulator (CREM) associated with sperm maturation [19, 22]. CREM proteins have also been detected in the connecting piece of ejaculated boar spermatozoa [23]. In this study, improved sperm motility was observed upon direct addition of RLSE to the diluent. Thus, this effect might be related to sperm function, and could prove beneficial in maintaining or improving sperm motility during liquid semen preservation.

For the assessment of sperm viability, preserved spermatozoa were stained with SYBR14 and PI, followed by the counting of viable and dead spermatozoa under a fluorescence microscope. As shown in Fig. 1, significantly higher live cells were recorded in the treatments with 0.5 and 1 μg/mL RLSE (p<0.05). In contrast, the level of viable spermatozoa gradually decreased from 2 μg/mL RLSE onwards, and a significantly high percentage of dead spermatozoa were observed at 10 μg/mL RLSE (p< 0.05; Fig. 1A). Excessive ROS levels can affect sperm viability and fertilization [24, 25]. In order to measure the intracellular ROS levels, preserved spermatozoa stained with carboxy-DCFDA were subjected to fluorometric assay. The fluorescence intensity was significantly lower in the treatments with 0.1, 0.5, and 1 μg/mL RLSE than in controls and at 2–10 μg/mL concentrations (p<0.05; Fig. 1B). A recent report demonstrated that R. coreanus seed extract effectively protects DNA and protein damage by inhibiting ROS generation, owing to its polyphenol content [26]. Similarly, increased total phenolic content in Kalopanax pictus leaves resulted in better protection against DNA damage [27]. Meanwhile, total polyphenol and total flavonoid content was higher in the water extract of R. coreanus stem and leaf than the unripe water extract [28]. Therefore, RLSE could protect sperm DNA damage and regulate ROS levels during sperm preservation.

Fig. 1. Assessment of sperm viability and intracellular ROS production after sperm preservation. Different concentrations of RLSE (0–10 μg/mL; DMSO as a control) were added to liquid boar semen, and then sperm viability (A) and intracellular ROS production (B) were examined. Experiments were repeated three times with three different boars. The values are expressed as mean ± SEM. The different superscripts (a–c) denote a significant difference between treatments at p<0.05.

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Bacteria are rarely found in the semen of healthy pigs. However, bacterial inflow may occur through the preputial diverticulum, during excretion or via dirty skin during semen collection, or due to contamination in the sampling site [29]. Bacterial contamination of semen causes sperm agglutination, acrosome damage, and decrease in sperm motility, resulting in decreased sperm viability and longevity [30, 31]. E. coli is one of the most common bacteria identified in liquid boar semen [31]. In the present study, E. coli was added to liquid semen diluted with antibiotics-free BTS. Overall, the levels of total motile spermatozoa, progressive motile spermatozoa, and sperm kinetic parameters decreased significantly in sperm samples preserved without antibiotics than in spermatozoa preserved with antibiotics (p<0.05; Table 2). Among the groups treated with different concentrations of the extract, addition of 0.5 or 1 μg/mL RLSE significantly improved the sperm motility (p<0.05; Table 2). Bacterial growth was completely inhibited in BTS containing antibiotics (Fig. 2). Although it did not differ significantly among the spermatozoa preserved in antibiotic-free BTS, the bacterial abundance was higher in groups treated with the extract than in treatments with no extract. Thus, no anti-bacterial activity of RLSE against E.coli was observed during liquid semen preservation (Fig. 2). According to Cha et al. [12], R. coreanus leaf extract with 80% methanol exhibited higher anti-bacterial activity against Bacillus cereus than unripe or ripe fruit extracts, but no anti-bacterial activity was observed against E. coli, Salmonella typhimurium, and Staphylococcus aureus, which is in agreement with our result (Fig. 2). Therefore, RLSE is expected to be effective against bacteria other than E. coli found in liquid semen, and higher concentrations of RLSE should also be tested.

Fig. 2. The levels of growth in E. coli population in liquid boar semen in the presence of RLSE (0–10 μg/mL; DMSO as a control) after 2 days of preservation. Experiments were repeated three times with three different boars. Values are expressed as mean ± SEM. The superscripts (a & b) denote a significant difference between treatments at p<0.05.

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In conclusion, the addition of RLSE in the diluent improved sperm motility and viability, and modulated the ROS level during liquid semen preservation. However, there was no inhibition of E. coli growth, and the antibacterial activity of RLSE warrants further investigation. Overall, the results suggest that RLSE is a potentially beneficial reagent for assisted reproductive technology in mammals.