Antioxidant Effects of Ocimum Gratissimum Leaf Essential Oils as a Supplement to Extender on Chilled Canine Sperm Quality

Background:Oxidative stress during chilled storage is a major problem withcanine sperm. To improve the quality of chilled canine sperm during storage, many synthetic antioxidants have been examined, but different outcomes were investigated depending on antioxidant properties. The bioactive compounds of essential oils fromOcimum gratissimumleaves are known as a natural antioxidant source. This study aimed to evaluate the antioxidant effects of essential oils from Ocimum gratissimumleavesas a supplement in extender on chilled canine sperm during 12 days of storage. Results:The results showed thatlow concentrations of Ocimum gratissimum essential oils (25, 50, and 100µg/mL) have benecial effectson sperm quality, whereasOcimum gratissimumessential oils athigh levels (above 200µg/mL) have harmful effects. Specically, the addition of 100µg/mL ofOcimum gratissimum essential oilsto the extender had the greatestbenecial effect in improving the quality of chilled canine sperm, and had a signicant difference in all sperm quality parameters except motility when compared to the control group (p<0.05). Conclusions:Ocimum gratissimum essential oilshave an impact on chilled canine sperm quality in a dose-dependent manner, and the best results areachieved with a maximum dose ofOcimum gratissimum essential oils of 100µg/mL. T0: control; T25, T50, T100, T200, T400, T600, and T800: and µg/mL essential oils, respectively. Essential extract gratissimum


Results
Antioxidant activity (DPPH radical scavenging activity) of essential oils from Ocimum gratissimum The concentration of Ocimum gratissimum essential oils providing 50% inhibition (IC50) of DPPH activity was higher than that of vitamin E with 263.63 µg/mL and 11.03 µg/mL, respectively [dataset] [51].

Sperm motility
The results of the total motility (TM) and progressive motility (PM) are presented in Table 2 [dataset] [51].
Overall, sperm in all treatments decreased gradually in TM and PM parameters during the whole experimental period (12 days). However, the treatments with low concentrations of Ocimum gratissimum essential oils (0-200 µg/mL) were similar in TM and PM parameters during 12 days, while the TM and PM of sperm in the high level of essential oils (up to 400 µg/mL) reduced drastically and had a signi cant difference when compared to that of the low level of essential oils (p < 0.05) from day 9 to day 12. Table 2 Effects of different concentrations of Ocimum gratissimum essential oils supplementation in extender on total motility (TM) and progressive motility (PM) parameters of chilled canine sperm during 12 T0: control; T25, T50, T100, T200, T400, T600, and T800: 25, 50, 100, 200, 400, 600, and 800 µg/mL essential oils, respectively. Values are mean ± standard deviation for four replicates, each being a pool of ve ejaculates. Lowercase superscript letters (a, b, or c) in the same column indicates signi cant difference among extenders (p < 0.05) and uppercase superscript letters (A, B, C, or D) in the same row indicates signi cant difference within extenders with different storage time (p < 0.05).
Besides TM and PM parameters, sperm velocity (VAP, VSL, and VCL) was also an important parameter in evaluating the sperm motility characteristics. The sperm velocity parameters are shown in Table 3 [dataset] [51]. The results of the VAP, VSL, and VCL were parallel with that of TM and PM parameters. It was found that there was a signi cant decrease in the VAP, VSL, and VCL parameters after the addition of up to 600 µg/mL essential oils (p < 0.05) on day 9 and 400 µg/mL essential oils (p < 0.05) on day 12. Plasma membrane integrity, acrosome membrane integrity and mitochondrial membrane potential The results of the plasma membrane integrity, acrosome membrane integrity, and mitochondrial membrane potential are given in Table 4 [dataset] [51]. In general, these parameters in all the concentrations of essential oils declined steadily during chilled storage. In addition, the quality of sperm increased progressively in the treatments with the low levels of essential oils (from 0 to 100 µg/mL) and then decreased gradually with the high levels of essential oils (from 200 to 800 µg/mL). In particular, the treatment of 100 µg/mL essential oils had the highest values and was markedly different from the control (without essential oils) from day 3 to day 9 in both intact plasma membrane (77.5 ± 2.2 vs. 67.1 ± 5.8 on day 3 (p = 0.008), 74.3 ± 3.7 vs. 59.2 ± 9.2 on day 6 (p = 0.01), and 65.1 ± 5.1 vs. 52.1 ± 0.9 on day 9 (p = 0.007), respectively) and high mitochondrial membrane potential parameters (82.2 ± 2.5 vs. 70.3 ± 4.3 on day 3 (p = 0.013), 78.0 ± 3.0 vs. 62.5 ± 5.9 on day 6 (p = 0.001), and 73.7 ± 3.0 vs. 54.1 ± 3.2 on day 9 (p < 0.001), respectively). Notably, the percentage of intact acrosome membrane in treatment of 100 µg/mL essential oil supplementation was greatest and had a substantial difference when compared to the control (73.0 ± 1.8 vs. 63.6 ± 3.4 on day 1 (p = 0.002), 66.4 ± 2.8 vs. 48.3 ± 3.9 on day 6 (p < 0.001), and 50.2 ± 3.4 vs. 24.8 ± 3.7 on day 12 (p < 0.001), respectively) and compared to the treatments with a higher concentration of essential oils, 400 µg/mL (73.0 ± 1.8 vs. 62.5 ± 1.4 on day 1 (p = 0.001), 66.4 ± 2.8 vs. 57.9 ± 1.3 on day 6 (p = 0.001), and 50.2 ± 3.4 vs. 26.8 ± 6.8 on day 12 (p < 0.001), respectively) during the whole storage period of 12 days.  Table 5 presents the proportion of healthy sperm with intact plasma membrane, high mitochondrial membrane potential, and intact acrosome membrane [dataset] [51]. As with the previous parameters, the percentage of healthy sperm increased regularly after adding essential oils to the extender and reached the top value at the level of 100 µg/mL before going down at the greater levels of essential oils (200 µg/mL). Although the values of those sperm in the treatment of 100 µg/mL essential oils were not evidently different from that in the treatments of 50 and 200 µg/mL essential oils, they were superior and had a signi cant difference when compared to the other treatments (p < 0.05).  T0: control; T25, T50, T100, T200, T400, T600, and T800: 25 Sperm lipid peroxidation Table 6 summarises the level of malondialdehyde (MDA) (nmol/50 × 10 6 sperm) of chilled canine sperm during 12 days [dataset] [51]. The concentration of MDA in all the treatments decreased slowly from day 1 to day 6 and then went up gradually to day 12. Moreover, although there was not notable difference in this parameter among the treatments with essential oil supplementation, the level of MDA was lowest and had a substantial difference from the control in both the treatments of 100 µg/mL (6.02 ± 0.24 vs. 7.03 ± 0.47 on day 1 (p = 0.029), 5.61 ± 0.08 vs. 6.34 ± 0.38 on day 6 (p = 0.035), and 6.04 ± 0.24 vs. 6.85 ± 0.02 on day 12 (p = 0.015), respectively), and 200 µg/mL (5.93 ± 0.33 vs. 7.03 ± 0.47 on day 1 (p = 0.012), 5.51 ± 0.26 vs. 6.34 ± 0.38 on day 6 (p = 0.010), and 6.03 ± 0.39 vs. 6.85 ± 0.02 on day 12 (p = 0.012), respectively) essential oils during 12 days' storage.

Discussion
During chilled storage, canine sperm are susceptible to oxidative stress due to the high amounts of reactive oxygen species (ROS), which can cause damage to all sperm components [6,7]. In previous studies, different synthetic antioxidants have been tested on chilled canine sperm to improve sperm quality during storage against oxidative stress, and the results were useful [15][16][17][18] or sometimes limited [19,20]. Our previous study demonstrated the bene ts of tris-citric-fructose-mineral salts egg-yolk extender in chilled canine sperm [5]. The present study, into applying an antioxidant substance of plant origin, was carried out to evaluate the effects of essential oils from Ocimum gratissimum leaves supplementation in tris-citric-fructose-mineral salts egg-yolk extender on chilled canine sperm.
In general, Ocimum gratissimum essential oils supplementation affected all parameters studied in a dose-dependent manner. The study suggested that low concentrations of Ocimum gratissimum essential oils (25,50, and 100 µg/mL) have positive effects on sperm quality, while Ocimum gratissimum essential oils at high levels (above 200 µg/mL) have a harmful effect. A supplement of 100 µg/mL of Ocimum gratissimum essential oil in the extender was the best treatment to improve the quality of chilled canine sperm. The bene cial effects of Ocimum gratissimum essential oils in canine sperm quality may be due to its antioxidant properties. Ocimum gratissimum essential oil is known to contain important phenolic compounds comprising eugenol, α-bisabolene, β-selinene, 1,8-cineole and thymol [21][22][23], which may subsequently assist the intercellular antioxidant system, including superoxide dismutase, glutathione peroxidase, phospholipid hydro-peroxide glutathione peroxidase, and catalase [24][25][26][27]. Our results are similar to those reported by [28]. They found that a rosemary aqueous extract supplement in soybean lecithin extender in uenced ram sperm quality in a dose-dependent manner. In the same way, Baghshahi et al. [29] have also illustrated that adding a maximum clove bud extract of 75 µg/mL in semen extender could improve sperm motility, viability, and plasma membrane integrity of ovine sperm. In contrast, Thymus munbyanus essential oils and thymol had no protective effects on human sperm, instead acting as potent immobilising and spermicidal agents [30]. In addition, the negative effects on chilled canine sperm quality at high levels of Ocimum gratissimum essential oils may be explained by its antimicrobial activities. Besides antioxidant activities, the phenolic compounds of Ocimum gratissimum essential oils are also known to inhibit microbial growth [31][32][33]. Based on hydrophobic properties, these phenolic compounds can interact with phospholipids and proteins in sperm plasma membranes. As a result, they affect membrane permeability, membrane potential and ion uxes [34,35]. These results are in agreement with previous reports on rams [29,36], bulls [37], and boars [38]. They have con rmed that the high antioxidant concentrations could induce a higher plasma membrane uidity and lead to increased sperm susceptibility.
Furthermore, one of the main by-products of lipid peroxidation is malondialdehyde (MDA) [39], which is an important indicator for oxidative damage in sperm [7,40,41]. Our results have found that the treatments of 100 µg/mL and 200 µg/mL Ocimum gratissimum essential oils have a lower concentration of MDA, while these treatments have a higher sperm quality when compared to the other treatments. These results are consistent with the previous studies [28,[42][43][44]. They have demonstrated that there was a negative correlation between sperm quality and rate of lipid peroxidation. Moreover, the results of sperm lipid peroxidation in the present study have also shown that the levels of MDA on day 1 were higher than that on day 6. This may be explained by the fact that on the rst day of storage, the antioxidant substrates in the extender may absorb incompletely into the sperm plasma membrane. Thus, spermatozoa are prone to lipid peroxidation by FeSO 4 inducing before using thiobarbituric acid (TBA) assay.

Conclusions
In conclusion, the results of our investigation revealed that supplementation of Ocimum gratissimum essential oils in an extender at an appropriate level (100 µg/mL) has protective effects on chilled canine sperm without any adverse effects on sperm motility, plasma membrane, acrosome membrane, and mitochondrial membrane potential parameters, as well as a decrease in MDA concentration. Further studies are necessary in frozen canine sperm to evaluate more sperm quality parameters such as DNA fragmentation and fertility.

Plant material and preparation of Ocimum gratissimum ethanol extract
The source of Ocimum gratissimum trees was supported by School of Crop Production Technology, Suranaree University of Technology, Thailand. The formal identi cation was performed by Khanittha Kuboran, Head of Plant Science Unit, Suranaree University of Technology Farm -Thailand and voucher specimens, herbarium No. PK-012018, was recorded and deposited at the Centre for Scienti c and Technological Equipment, Suranaree University of Technology -Thailand for future reference. Ocimum gratissimum leaves were collected in the morning and dried in a hot-air oven at 40 o C for three days. The dried leaves were nely powdered using a grinder. The extraction was conducted at room temperature by soaking 100 g of leaf powder in one litre of ethanol (98%) for ve days. The mixture was passed through lter papers (No. 1) and concentrated using a rotary evaporator at 40 o C. Then, this concentrated solution was centrifuged at 3,500 x g for 15 minutes, and the bottom layer was freeze-dried to obtain the essential oils.
Antioxidant activity (DPPH radical scavenging activity) of essential oils from Ocimum gratissimum The antioxidant activity of essential oils was carried out using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assay according to the method of Blois [45]. Vitamin E was used as standard. The diluted working solutions of essential oils and standard (100 µL) were prepared in methanol with amounts ranging from 25 to 600 µg/mL and from 0.5 to 50 µg/mL in 96 wells micro plate, respectively. 100 µL DPPH (200 µM in methanol) was added to the essential oils and standard solution. Methanol (100 µL) with DPPH solution (200 µM, 100 µL) was used as a control, while methanol (100 µL) with essential oils or standard (100 µL) was used as a blank. These solution mixtures were kept in the dark for 30 minutes and optical density was measured at 517 nm using a spectrophotometric plate reader. The percentage of antioxidant activity (AA%) was determined using the following formula [46]: The concentration of essential oils and standard providing 50% inhibition (IC50) of DPPH activity were calculated from the graph plotting between the percentage inhibition and concentration.

Animals
All dogs were supported by America Bully Dogs groups in Nakhon Ratchasima Province, Thailand. Sperm samples were obtained from ve mature American Bully dogs (2-5 years old) of proven fertility. All dogs were trained to ejaculate by digital manipulation for semen collection before studying. This study was performed under the guidelines of the Institutional Animal Care and Use Committee of the Suranaree University of Technology, Nakhon Ratchasima, Thailand. Animals were returned to the groups after sample collection.

Semen collection and evaluation
Ejaculates were collected once a week from each dog by digital manipulation according to the technique as described by Linde-Forsberg [2]. The collections were performed from all 5 dogs at the same time concurrently. Sperm with the following quality criteria was used in this study: >70% progressive motility; >200 × 10 6 sperm/mL; <5% sperm abnormal morphology; and > 90% sperm viability. Computer-assisted sperm analysis (CASA) was used to determine sperm progressive motility and sperm concentration, while sperm morphology and viability were estimated using eosin-nigrosin staining [47].

Preparation of extenders
The basis extender in this study was tris-citric-fructose-mineral salts extender added to 20% egg yolk.

Semen processing and experimental design
After collection, semen from ve dogs was pooled and separated into eight sterile tubes. Then, the seminal plasma was removed by centrifuging (5 minutes, 720 × g) [48]. The sperm pellets were diluted with a su cient volume of the extenders to reach the sperm concentration of 100 × 10 6 sperm/mL. After that, the extended sperm was slowly cooled (0.3 o C/min) to 5 o C by manual [49], and stored at 5 o C for 12 days. Experimental design in this study was presented by a repeated measurement in the completely randomised design with four replicate trials.

Sperm evaluation
Sperm quality was evaluated every three days (Day1, Day3, Day6, Day9, and Day12). Evaluation of plasma membrane integrity, acrosome membrane integrity, and mitochondrial membrane potential A uorescent staining combination of propidium iodide (PI), Hoechst 33342 (H342), uorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and 5,5',6,6'-tetrachloro-1,1',3,3'tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) was carried out to evaluate the plasma membrane integrity, acrosome membrane integrity, and mitochondrial membrane potential. The uorescent staining process was prepared as described by [50] and modi ed by the method of [5]. The stained sperm was identi ed by a confocal laser scanning microscope (CLSM; Nikon/Ni-E, Japan). The stained canine sperm under a confocal laser scanning microscope can be seen in Fig. 1. The spermatozoa with the intact plasma membrane, intact acrosome membrane, and high mitochondrial membrane potential were PI-and FITC-PSA-negative, and H342-and JC-1-positive, while the spermatozoa with the damaged plasma membrane, damaged acrosome membrane, and low mitochondrial membrane potential were PI-and

Evaluation of sperm lipid peroxidation
The lipid peroxidation of spermatozoa was determined by measuring the malondialdehyde (MDA) production, using thiobarbituric acid (TBA) assay according to the method described by Buege and Aust [39] and modi ed by Maia et al. [40]. The MDA concentration of each sample was measured immediately after inducing sperm lipid peroxidation with 0.24 mM FeSO 4 at 37 o C in a water bath for 15 minutes. Then, 1 mL TBA reagent (trichloroacetic acid 15% (w/v), thiobarbituric acid 0.375% (w/v) in 0.25N hydrochloric acid) was added to 0.5 mL of each sample. The mixture was treated in a boiling water bath for 15 minutes. After cooling, the suspension was centrifuged at 1,000 × g for ten minutes. The supernatant was separated and the absorbance was measured at 535 nm using a spectrophotometric plate reader. The MDA concentration was determined by comparing the sample's absorbance at 535 nm with an MDA standard curve. The results were expressed in nmol MDA/50 × 10 6 sperm.

Statistical analysis
Two-factor mixed analysis of variance (ANOVA) was used to examine the interaction between time and extender as the main effects, and the Tukey test was applied for multiple comparisons of means among groups of each factor (time, extender). Statistical analyses were performed with SPSS software version 17.0 for Windows (SPSS Inc., Chicago, IL, USA). All data are presented as mean ± standard deviation (SD). A difference was considered signi cant for p < 0.05.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.