Research Article

Effects of Green Tea Polyphenols, Vitamin E, and Ocimum gratissimum Leaf Essential Oils as a Supplement to Extender on Chilled Canine Sperm Quality

Vui Van Nguyen1, Samorn Ponchunchoovong2, Sajeera Kupittayanant3, Pakanit Kupittayanant2*

1Department of Animal Science and Veterinary Medicine, Faculty of Agriculture and Aquaculture, Tra Vinh University, Vietnam; 2School of Animal Technology and Innovation, Institute of Agricultural Technology, Suranaree University of Technology, Thailand; 3School of Preclinical Sciences, Institute of Science, Suranaree University of Technology, Thailand.

Received | February 05, 2023; Accepted | April 25, 2023; Published | June 15, 2023

*Correspondence | Pakanit Kupittayanant, School of Animal Technology and Innovation, Institute of Agricultural Technology, Suranaree University of Technology, Thailand; Email: pakanit@sut.ac.th

Citation | Nguyen VV, Ponchunchoovong S, Kupittayanant S, Kupittayanant P (2023). Effects of green tea polyphenols, vitamin e, and Ocimum gratissimum leaf essential oils as a supplement to extender on chilled canine sperm quality. Adv. Anim. Vet. Sci. 11(8): 1250-1260.

DOI | https://doi.org/10.17582/journal.aavs/2023/11.8.1250.1260

ISSN (Online) | 2307-8316

 

 

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The use of artificial insemination (AI) techniques is currently popular in dog reproduction. Before using AI techniques, canine sperm quality must be maintained with a suitable semen extender during storage. Our study have illustrated that canine sperm have a high quality during chilling storage with adding of egg-yolk in the Tris-mineral salts extender (Vui et al., 2019). After dilution with extenders, preservation or cryopreservation was used to storage extended sperm (Thomassen and Farstad, 2009). However, the use of chilled canine sperm is more convenient and has higher fertilization rates, compared to that of frozen canine sperm (Linde-Forsberg, 1995; Eilts, 2005). A main problem of chilled canine sperm is the reduction of sperm quality during long periods of storage. Thus, studies to preserve the quality of chilled canine sperm is necessary.

Due to being chilled in storage, canine sperm suffers from deleterious effects from oxidative stress. This is because canine sperms’ plasma membrane contains a high level of polyunsaturated fatty acids (Darin Bennett et al., 1974), which are susceptible to lipid oxidation when interacted to reactive oxygen species [ROS] (Vieira et al., 2017). Even though the ROS has physiological roles in sperm functions, such as hyperactivation, maturation, acrosome reaction, capacitation, and fertilization (Aitken, 2017), the high level of ROS could induce the damage of sperm structures encompassing DNA, lipids, proteins, and carbohydrates (Lucio et al., 2016; Aitken, 2017). Within normal conditions, the antioxidant defence mechanisms of living organisms can counteract the generation of ROS to prevent or minimise the negative effects of oxidation (Ighodaro and Akinloye, 2017). Almost all enzymatic antioxidants that are present in canine semen are from seminal plasma, including phospholipid hydro-peroxide glutathione peroxidase, catalase, glutathione peroxidase, and superoxide dismutase (Angrimani et al., 2014). However, seminal plasma of canine semen is separated before diluting with the semen extenders due to its negative effects on canine sperm quality (Hori et al., 2017). This may reduce the antioxidant defence mechanisms of canine sperm against the attack of ROS, therefore the addition of antioxidant elements in the canine semen extenders may expand the life of chilled sperm. Several studies have previously been carried out to evaluate the effects of various non-enzymatic and enzymatic antioxidants on canine sperm with positive outcomes (Michael et al., 2009; Neagu et al., 2009; Monteiro et al., 2009; Wittayarat et al., 2013; Ogata et al., 2015; Lucio et al., 2016), or ineffective results (Beccaglia et al., 2009; Sahashi et al., 2011; Andersen et al., 2018). Consequently, determining the appropriate antioxidant substance for canine sperm chilling is imperative for future AI success.

Additives such as vitamin E was considered due to it being a lipid-soluble antioxidant which plays a major role in inhibiting the free radicals by functioning as a chain-breaking antioxidant (Wang and Quinn, 1999; Dad et al., 2006). The supplementation of vitamin E has been conducted to improve the sperm quality of stallions (Almeida and Ball, 2005; Vasconcelos et al., 2013; Vasconcelos et al., 2014; Vasconcelos et al., 2016), boars (Cerolini et al., 2000; Jeong et al., 2009; Satorre et al., 2012), bulls (Asadpour, 2011), rams (Abdi-Benemar et al., 2015), canine studs (Michael et al., 2009), and roosters (Moghbeli et al., 2016).

Another additive such as green tea polyphenols are water-soluble antioxidants which contain a great variety of bioactive compounds including epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate. These compounds have been shown to have a powerful antioxidant by removing free radicals, such as hydroxyl radicals, alkyl radicals, peroxyl radicals (Coyle et al., 2008; Lamberta and Eliasa, 2010; Forester and Lambert, 2013). Several previous studies have illustrated that the addition of green tea polyphenols in semen extenders has had useful effects on sperm quality in various animals such as stallions (Nouri et al., 2018), boar (Gadani et al., 2017), ram (Mehdipour et al., 2016), and canine (Wittayarat et al., 2013).

For further study Ocimum gratissimum is a medicinal herb in which the leaf contains high level of essential oils (3.5%) (Trevisan et al., 2006). It has been found that the essential oil extract from Ocimum gratissimum were constituents of eugenol (30-70%), and the other phytochemicals such as β-selinene, thymol, α-bisabolene, 1,8-cineole, and γ-terpinene (Prabhu et al., 2009; Monga et al., 2017; Kumar et al., 2019). These bioactive substances are almost amphiphilic compounds which have strong antioxidant functions (Ouyang et al., 2012; Chiu et al., 2013; Mahapatra and Roy, 2014). Thus, it can be inferred that essential oils can protect canine sperm against sperm lipid peroxidation during chilling storage. The objective of the present study was to assess the effects of adding vitamin E, green tea polyphenols, and essential oils to the basic semen extender of Tris-mineral salts on canine sperm quality during preservation.

MATERIALS AND METHODS

Animals

Semen samples were collected from five healthy and sexually mature American Bully dogs. All dogs were demonstrated to be fertile after natural mating. The study was implemented with the guide of the Animal Care and Use Committee Institution of the Suranaree University of Technology, Thailand with license number SUT-IACUC 17/2560.

Canine Semen Collection

Canine semen were collected one a week from five dogs by manual manipulation technique as described by Linde-Forsberg (1991). A total of twenty ejaculates were used for four replications in this study. Before semen pooling, the sperm quality of each ejaculate was determined. Only ejaculates with high sperm quality as the following criteria were included in this study: sperm concentration ≥0.2×107 sperm/ml, sperm viability ≥90%, progressive motility ≥70%, and sperm abnormal morphology ≤5%. For sperm concentration and motility, the samples were evaluated using system automatic sperm analyser, while eosin-nigrosin staining was used to detect sperm morphology and viability (Tamuli and Watson, 1994).

Semen Extender Preparation

All chemicals from Sigma-Aldrich (Singapore) were used for semen extender preparation. Essential oils, vitamin

 

Table 1: The component of the semen extenders

Extender ingredients	Extenders
	Control	Essential oils	Vitamin E	Green tea polyphenols	Eugenol
Tris (g)	0.9	0.9	0.9	0.9	0.9
Citric acid (g)	0.5	0.5	0.5	0.5	0.5
Fructose (g)	1.25	1.25	1.25	1.25	1.25
NaCl (g)	0.45	0.45	0.45	0.45	0.45
KHPO4 (g)	0.06	0.06	0.06	0.06	0.06
KCl (g)	0.06	0.06	0.06	0.06	0.06
CaHPO4 (g)	0.02	0.02	0.02	0.02	0.02
MgCl2 (g)	0.01	0.01	0.01	0.01	0.01
Egg yolk (ml)	20	20	20	20	20
Essential oils (g)*	-	0.01	-	-	-
Vitamin E (g)	-	-	0.005	-	-
Green tea polyphenols (g)	-	-	-	0.005	-
Eugenol (mg)	-	-	-	-	0.26
Gentamycin (g)	0.2	0.2	0.2	0.2	0.2
DMSO (ml)	0.8	0.8	0.8	0.8	0.8
Distilled water (ml)	To 100	To 100	To 100	To 100	To 100
pH	6.57	6.56	6.57	6.58	6.57
Osmolality (mOsmol/kg)	475	484	483	479	486

* Essential oils extract from Ocimum gratissimum leaves.

 

E ((±)-α-Tocopherol, T3251), green tea polyphenols (Polyphenon 60, P1204), and eugenol (E51791) were used as various antioxidants. The essential oils were extracted from the Ocimum gratissimum leaves by soaking leaf powder in ethanol at room temperature. The solution was filtered through filter paper (No.1) and condensed using a rotary evaporator. Then, the condensed solution was separated by centrifuging. The bottom layer of solution was used as essential oils for the remainder of the study.

The basic extender egg-yolk in Tris-mineral salts, which has known benefits in chilled canine sperm (Vui et al., 2019), was used as the basis extender in this study. Five different extenders contained one of the various antioxidants, including essential oils (100 µg/ml), vitamin E (50 µg/ml), green tea polyphenols (50 µg/ml), and eugenol (2.6 µg/ml). The concentration of essential oils was the results of our previous study, while the level of vitamin E and green tea polyphenols were the results of our preliminary study (no data given). The concentration of eugenol used in this study was equal to the level of eugenol in essential oils extraction (2.6%), and the level of eugenol was detected using GC-MS. The extender without added antioxidants was used as a control. The components of these extenders are presented in Table 1. All antioxidants were diluted in dimethyl sulfoxide (DMSO) before the addition to the extenders, and each extender contained 0.8% DMSO.

Semen Processing And Experimental Design

After semen collection and initial evaluation, the ejaculates from all dogs were merged and divided into five equal groups. Then, semen was centrifuged at 720 × g for 5 minutes to remove seminal plasma (Rijsselaere et al., 2002). The sperm pellets were resuspended with five appreciate extenders to reach 0.1 x 107 sperm/ml. After that, each extended sperm (0.5 ml) was separated into microcentrifuge tubes and immersed in a styrofoam box of water at the temperature of 25oC. Next, the sperm samples were cooled down steadily to 5oC by adding ice at a cooling rate of 0.3oC/min (Bouchard et al., 1990). After cooling, samples were stored at 5oC. Sperm quality parameters were determined once every three days over a period of 12 days.

Experimental design was a completely randomized design with repeated measurement. Four replicates and five ejaculates for each replicate were conducted.

Antioxidant Activity Of Various Antioxidant Agents

The antioxidant activity of various antioxidant agents was conducted using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assay as previously described by Blois (1958). The solutions of various antioxidants (100 µl) were diluted in methanol and placed in a 96 wells micro plate. 100 µl DPPH solution in methanol (200 μM) was added to various antioxidant solutions. A control group was methanol and DPPH solution. Methanol (100 µl) with various antioxidants (100 µl) was used as a blank. After 30 minutes of being kept in the dark, a spectrophotometric plate reader was used to measure the absorbance of the solution mixtures. The concentration of various antioxidants capable of inhibiting 50% (IC50) of DPPH solution activity were determined by a linear graph using the proportion of antioxidant activity and the concentration of various antioxidants.

Sperm Motility Evaluation

The system automatic sperm analyser (CASA; HTR-IVOS 14.0; Hamilton Thorne, USA) was applied to analyse the sperm motility parameters. The sperm samples were mixed with Tris buffer and incubated at 38oC (15 minutes) in a water bath before evaluation. After which the sperm samples (5 μl) were deposited into a warmed (38oC) 2X-CEL slide. There were 5 randomly selected fields for each chamber which were evaluated. The following sperm motility parameters were recorded: total motility percentage (TM%); progressive motility percentage (PM%); velocity curvilinear (VCL, µm/s); velocity straight line (VSL, µm/s); and velocity average pathway (VAP, µm/s).

Sperm Membrane Integrity Evaluation

To evaluate sperm membrane integrity of canine sperm, samples were stained with a fluorescent combination of fluorescein isothiocyanate–conjugated Pisum sativum agglutinin (FITC-PSA), Hoechst 33342 (H342), 5,5’,6,6’-tetrachloro-11’,3,3’-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), and propidium iodide (PI). The staining techniques were performed according to the previous technique (Vui et al., 2019). After staining, at least 200 sperm for each sample were immediately evaluated using a confocal microscope (Nikon/Ni-E, Japan). The stained sperm with high mitochondrial membrane potential, integral acrosome membrane and plasma membrane had bright red-orange in the mid-piece region and blue-stained nucleus, whereas the stained sperm with the low mitochondrial membrane potential, broken acrosome membrane and plasma membrane had bright green in the mid-piece region, yellow-green in the acrosome region, and red-stained nucleus. The identification of the stained sperm can be seen in Figure 1.

Sperm Lipid Peroxidation Evaluation

The canine sperm lipid peroxidation was determined using thiobarbituric acid reactive substance (TBARS) assay to calculate the malondialdehyde (MDA) production, according to the previous method by Maia et al. (2010). Before measuring the level of MDA, the sperm sample was induced into lipid peroxidation by adding 0.24 mM ferric sulphate and incubated for 15 minutes in a warm water (38oC). Then, 0.5 ml of the sperm sample was combined with 1 ml TBA reagent (thiobarbituric acid 0.375% (w/v), trichloroacetic acid 15% (w/v) in hydrochloric acid 0.25N) and 1% (v/v) 50 mM butylated hydroxytoluene. The mixture solution was boiled for 20 minutes. After that, it was cooled and centrifuged to separate the supernatant. The absorbance of supernatant was measured using a microplate spectrophotometer at 535 nm. The MDA level was calculated by comparing the absorbance of each sample at 535nm with a curve of MDA standard. The results were represented in nmol MDA/50x106 sperm.

 

Statistical Analysis

Statistical analysis was performed using IBM SPSS statistics, version 22. Analysis of variance (ANOVA) of two-factor mixed was used to analyse the effects of each factor and the interaction between 2 factors (period and treatment), and the Tukey method was applied for means multiple comparisons between groups in each factor. All data of the results are provided as mean ± standard deviation. A value of P<0.05 was set for significant difference.

RESULTS AND DISCUSSION

Antioxidant Activity Of Various Antioxidants

The antioxidant activity of the various antioxidants is presented in Table 2. The IC50 value of the Ocimum gratissi

 

Table 2: The antioxidant activity of the various antioxidants

Antioxidants	Essential oils	Vitamin E	Green tea polyphenols	Eugenol
IC50 (µg/ml)	263.63±11.52a	11.03±0.55b	4.25±0.33b	6.06±0.30b

IC50: the concentration of the various antioxidants capable of inhibiting 50% of DPPH activity. Values are mean ± SD. Superscript letters (a or b) in the same row indicates significant difference (P<0.05).

 

Table 3: The percentages of the total motility (TM%) and progressive motility (PM%) in the extended canine sperm with the addition of various antioxidants during 12 days at 5oC

Parameters	Extenders	Day1	Day3	Day6	Day9	Day12
TM (%)	Control	94.4±0.5A	92.4±0.2B	89.8±2.9B	76.4±2.9C	60.3±8.4D
	Essential oils	95.0±0.8A	94.1±1.1AB	91.5±2.0B	79.9±5.2C	59.9±7.2D
	Vitamin E	95.0±0.7A	93.1±1.2B	91.1±1.8B	78.0±4.9C	58.7±8.8D
	Green tea polyphenols	94.2±0.6A	92.8±1.4B	90.9±3.3B	78.6±6.6C	56.1±9.7D
	Eugenol	94.9±1.4A	92.2±1.2B	89.7±2.4B	80.1±4.1C	58.6±8.5D
PM (%)	Control	72.8±1.5A	68.8±3.1B	62.7±3.0C	43.7±6.7D	23.8±3.2E
	Essential oils	73.1±2.9A	69.0±3.8B	63.4±3.0C	43.2±6.1D	24.3±2.8E
	Vitamin E	70.8±4.0A	67.5±5.1AB	62.7±6.2B	44.6±9.9C	21.9±3.2D
	Green tea polyphenols	71.8±3.8A	67.8±3.3B	60.0±7.5C	46.1±11.1D	20.9±4.4E
	Eugenol	70.9±3.8A	68.6±4.9A	63.1±4.9B	45.2±8.3C	23.8±2.7D

Values are mean ± SD. Uppercase superscript letters (A, B, C, D or E) in the same row indicates significant difference within treatments with different periods (P<0.05).

 

Table 4: The results of the average pathway velocity (VAP), straight line velocity (VSL), and curvilinear velocity (VCL) in the extended canine sperm with the addition of various antioxidants during 12 days at 5oC

Parameters	Extenders	Day1	Day3	Day6	Day9	Day12
VAP (µm/s)	Control	93.2±7.9A	87.8±6.6AB	79.2±3.4B	72.9±3.8C	62.3±8.5D
	Essential oils	93.2±5.3A	89.4±7.0AB	79.1±4.7B	73.1±6.6C	68.2±8.4C
	Vitamin E	93.6±7.6A	87.8±10.0AB	80.1±5.6B	73.0±4.8C	67.0±6.2C
	Green tea polyphenols	94.3±7.1A	88.9±6.7AB	79.3±6.0B	73.0±5.5C	63.9±8.8D
	Eugenol	96.2±8.0A	92.2±7.5AB	82.7±5.1B	75.4±3.7C	66.8±5.6D
VSL (µm/s)	Control	86.6±7.0A	80.9±7.9A	66.9±5.6B	59.6±3.4C	48.2±7.3D
	Essential oils	86.6±5.5A	80.1±7.7B	68.4±2.9B	60.4±4.6C	54.0±9.9C
	Vitamin E	84.0±6.7A	80.9±6.5A	72.4±3.1A	64.9±4.0B	53.2±5.2C
	Green tea polyphenols	86.0±6.2A	79.3±5.3B	69.5±3.1B	62.0±3.6C	52.5±9.6D
	Eugenol	89.0±8.1A	83.5±5.1AB	71.4±6.5B	60.5±2.6C	54.2±5.1C
VCL (µm/s)	Control	136.7±0.7A	134.6±1.6A	133.3±1.5A	130.1±2.1B	122.6±2.7C
	Essential oils	138.1±2.6A	134.8±4.6B	131.6±5.3B	129.5±5.3C	122.2±6.2D
	Vitamin E	137.7±3.9A	134.7±3.1B	131.5±4.1B	127.8±4.9C	124.8±4.1C
	Green tea polyphenols	136.7±2.9A	134.5±4.3AB	131.3±5.6B	129.0±6.7C	119.6±11.8D
	Eugenol	137.5±4.8A	135.9±4.2A	133.8±4.1AB	131.9±5.0B	126.9±5.3B

Values are mean ± SD. Uppercase superscript letters (A, B, C, or D) in the same row indicates significant difference within treatments with different periods (P<0.05).

 

Table 5: The percentages of high mitochondrial membrane potential, intact plasma membrane and acrosome membrane parameters in the extended canine sperm with the addition of various antioxidants during 12 days at 5oC

Parameters	Extenders	Day1	Day3	Day6	Day9	Day12
Plasma membrane (%)	Control	76.3±2.9bA	73.4±3.5bA	63.9±2.5bB	53.0±2.9bC	40.4±3.1bD
	Essential oils	81.9±1.4aA	80.4±1.6aA	72.6±3.7aB	61.2±1.8aC	46.7±3.0aD
	Vitamin E	80.7±1.0abA	78.0±2.0abA	71.0±2.8abB	58.3±1.1abC	48.9±1.1aD
	Green tea polyphenols	80.2±1.4abA	76.1±1.7abB	68.9±3.5abC	57.4±3.9abD	47.8±1.4aE
	Eugenol	78.9±3.3abA	75.2±2.5abB	69.0±3.1abC	54.3±3.9bD	46.9±2.5aE
Acrosome membrane (%)	Control	68.5±1.8bA	64.4±2.7bB	55.3±4.8bC	39.4±3.4bD	24.1±3.6cE
	Essential oils	75.0±2.1aA	73.4±2.4aA	66.7±2.5aB	57.7±2.1aC	42.6±2.1aD
	Vitamin E	73.1±2.1aA	69.2±4.9abB	61.6±3.8abC	46.0±6.3bD	36.2±2.3bE
	Green tea polyphenols	73.9±1.6aA	70.0±2.7abB	62.2±3.7abC	46.3±5.0bD	38.5±2.8abE
	Eugenol	74.3±2.2aA	69.4±2.4abB	61.1±1.5abC	44.5±3.7bD	37.8±2.0abE
Mitochondrial membrane potential (%)	Control	80.7±2.3bA	74.8±3.9bB	65.0±3.0cC	55.5±1.8cD	40.2±0.9bE
	Essential oils	87.2±1.7aA	85.0±2.0aA	78.2±1.4aB	66.6±2.1aC	51.1±2.7aD
	Vitamin E	85.5±1.5aA	82.1±1.9aB	74.5±2.1abC	61.7±1.4bD	55.2±2.5aE
	Green tea polyphenols	85.9±0.7aA	81.5±3.3aB	72.6±2.5abC	59.7±2.5bcD	52.0±2.6aE
	Eugenol	86.0±2.3aA	81.0±1.7aB	70.1±5.1bcC	59.0±2.4bcD	50.0±2.7aE

Values are mean ± SD. Lowercase superscript letters (a, b or c) in the same column indicates significant difference among treatments (P<0.05) and uppercase superscript letters (A, B, C, D or E) in the same row indicates significant difference within treatments with different periods (P<0.05).

 

Table 6: The percentages of the sperm with high mitochondrial membrane potential, intact plasma membrane and acrosome membrane in the extended canine sperm with the addition of various antioxidants during 12 days at 5oC

Extenders	Day1	Day3	Day6	Day9	Day12
Control	66.5±1.7bA	59.0±3.9bB	48.7±4.2bC	32.1±2.5bD	19.3±1.5bE
Essential oils	73.1±3.3aA	70.1±2.2aA	60.2±5.7aB	46.4±3.4aC	32.2±1.4aD
Vitamin E	71.6±1.9abA	66.1±3.7abB	58.0±3.7abC	41.9±4.1aD	31.1±2.3aE
Green tea polyphenols	71.7±2.2abA	65.9±2.0abB	55.8±4.7abC	40.6±1.8aD	30.0±2.7aE
Eugenol	72.2±2.3abA	64.3±3.1abB	54.7±2.2abC	40.5±1.9aD	31.2±2.7aE

Values are mean ± SD. Lowercase superscript letters (a or b) in the same column indicates significant difference among treatments (P<0.05) and uppercase superscript letters (A, B, C, D or E) in the same row indicates significant difference within treatments with different periods (P<0.05).

 

Table 7: The levels of the malondialdehyde (MDA) (nmol/50x106 sperm) of the extended canine sperm with the addition of various antioxidants during 12 days at 5oC

Extenders	Day1	Day6	Day12
Control	7.38±0.23aA	6.68±0.31aB	7.26±0.51aA
Essential oils	6.29±0.40bA	5.83±0.23bB	6.28±0.46bA
Vitamin E	6.21±0.60bA	5.74±0.25bB	6.21±0.39bA
Green tea polyphenols	6.15±0.52bA	5.69±0.48bB	6.13±0.42bA
Eugenol	6.54±0.38abA	5.93±0.25bB	6.46±0.13abA

Values are mean ± SD. Lowercase superscript letters (a or b) in the same column indicates significant difference among treatments (P<0.05) and uppercase superscript letters (A or B) in the same row indicates significant difference within treatments with different periods (P<0.05).

mum essential oils (263.63±11.52 µg/ml) was noticeably higher than that of the vitamin E (11.03±0.55 µg/ml), eugenol (6.06±0.30 µg/ml), and green tea polyphenols (4.25±0.33 µg/ml). This means that the antioxidant activity of the essential oils from Ocimum gratissimum was lower than that of the vitamin E, eugenol, and green tea polyphenols in the same concentration.

Sperm Motility

Sperm motility of canine sperm are shown in Table 3. On the whole, the results of the sperm total motility and progressive motility parameters in all the antioxidant treatments reduced gradually during the time of storage (12 days). In addition, there were no substantial differences among the treatments at each period time in these parameters. Beside that sperm velocity including velocity curvilinear (VCL), velocity straight line (VSL), and velocity average pathway (VAP) are given in Table 4. The trend values of the VSL, VCL, and VAP parameters were consistent with those of the total motility and progressive motility indicators during 12 days of storage. These parameters also reduced steadily during storage, and there were parallel values among the treatments at each period of storage time.

Sperm Membrane Integrity

Table 5 summarises the results of mitochondrial membrane potential, plasma membrane and acrosome membrane integrity parameters in canine sperm during 12 days of storage. In general, the percentages of the high mitochondrial membrane potential, integral plasma membrane, and integral acrosome membrane in the essential oils extender were the highest and had a notable difference compared with those in the control extender (P<0.05). For the plasma membrane integrity, although the essential oil extender was superior to the other extenders, the values of the intact plasma membrane in the essential oils extender were not markedly different compared to that in the rest extenders. Moreover, the proportions of intact plasma membrane in the eugenol, green tea polyphenols, and vitamin E extenders were not substantially greater than that in the control treatment. For the acrosome integrity, the value of this parameter in the essential oils extender on day nine was significantly higher than that in the vitamin E (57.7±2.1 vs. 46.0±6.3, P=0.018), in the green tea polyphenols (57.7±2.1 vs. 46.3±5.0, P=0.02), and in the eugenol extender (57.7±2.1 vs. 44.5±3.7, P=0.006). Notably the percentages of this parameter in the all antioxidant extenders were significantly different as compared to that in the control extender on day 1 and day 12 (P<0.05). For the mitochondrial membrane potential, during the whole storage period, the percentages of this parameter in the control group were evidently lower than those in the other extenders (P<0.05), except on day nine in the green tea polyphenols and eugenol extenders.

The proportions of the sperm with an intact membrane are presented in Table 6. As with the former parameters, the values of the sperm with high mitochondrial membrane potential, intact plasma membrane and acrosome membrane in the essential oils extender were sizable and substantially greater than those in the control treatment (P<0.05), the percentages of this parameter in the other antioxidant extenders were also significantly different compared to those in the control group from day 9 to day 12 (P<0.05).

Sperm Lipid Peroxidation

Table 7 presents the concentration of the malondialdehyde (MDA) of canine sperm with the addition of various antioxidants during storage. The levels of the MDA in all the extenders reduced gradually during the first of 6 days and then increased steadily to day 12. The MDA values in all the antioxidant treatments, except the eugenol extender on day 1 and day 12, were significantly less than those in the control extender. Although the concentrations of the MDA in the green tea polyphenols and vitamin E extenders were the lowest, the differences were not prominent compared to those in the essential oils extender.

The overproduction of ROS in canine sperm during storage can cause oxidative stress, which leads to damage of the sperm membrane as well as a reduction in sperm longevity (Moustafa et al., 2004; Lucio et al., 2016; Aitken, 2017). In former studies, some non-enzymatic and enzymatic antioxidant substances have been used to enhance the canine sperm quality during storage against the harmfulness of oxidative stress with various outcomes (Michael et al., 2009; Neagu et al., 2009; Monteiro et al., 2009; Sahashi et al., 2011; Thiangtum et al., 2012; Wittayarat et al., 2013; Ogata et al., 2015; Andersen et al., 2018). In particular, vitamin E and green tea polyphenols usage has shown positive effects on the sperm quality of animals (Jeong et al., 2009; Michael et al., 2009; Asadpour, 2011; Satorre et al., 2012; Vasconcelos et al., 2013; Wittayarat et al., 2013; Vasconcelos et al., 2014; Abdi-Benemar et al., 2015; Vasconcelos et al., 2016; Moghbeli et al., 2016; Mehdipour et al., 2016; Gadani et al., 2017; Nouri et al., 2018), whereas Ocimum gratissimum essential oils has never been used for sperm preservation as a herbal antioxidant substance. Our previous study has showed that the basic extender Tris-mineral salts egg-yolk could significantly improve the quality of canine sperm during chilling storage (Vui et al., 2019). This study investigated the effects of these antioxidant substances supplementation in Tris-mineral salts-egg yolk extender on chilled canine sperm.

The present study showed that although there was not a significantly difference among the treatments in the sperm motility parameters, the antioxidant treatments could improve the sperm membrane integrity parameters of canine sperm during 12 days chilling storage compared with the control treatment. Remarkably, the essential oils extract from Ocimum gratissimum extender was superior to the rest of the extenders, including the eugenol extender (as a control for the level of eugenol in Ocimum gratissimum essential oils), and greatly improved the quality of chilled canine sperm compared to the control group. This means that the composition of Ocimum gratissimum essential oils contain not only eugenol, but also other substances which have a useful impact on canine sperm quality. The primary constituents of Ocimum gratissimum essential oils are discovered to contain various bioactive compounds such as eugenol, β-selinene, thymol, α-bisabolene, 1,8-cineole, and γ-terpinene (Prabhu et al., 2009; Monga et al., 2017; Kumar et al., 2019), which have the function of antioxidant activity (Ouyang et al., 2012; Chiu et al., 2013; Mahapatra and Roy, 2014). These compounds may contribute not only their antioxidant activity individually, but also create a synergistic antioxidant activity among these compounds (Sonam and Guleria, 2017). As a result, these antioxidant properties may support the sperm intracellular antioxidant system to defend sperm against lipid oxidation (Neagu et al., 2011; Angrimani et al., 2014).

In addition, the antioxidant activity of vitamin E and green tea polyphenols were notably higher than that of essential oils, but the mitochondrial membrane potential, the plasma membrane and the acrosome membrane integrity parameters of sperm in the essential oils treatment were greater than those in the green tea polyphenols and vitamin E treatments. This may be explained by the fact that Ocimum gratissimum essential oils contains various bioactive compounds which have both hydrophilic and lipophilic characteristics (Prabhu et al., 2009; Kumar et al., 2019), while vitamin E is a lipophilic substance and green tea polyphenols are hydrophilic compounds (Prasanth et al., 2019). Therefore, Ocimum gratissimum essential oils and vitamin E can insert themselves in the lipid bilayer of sperm plasma membranes to protect against ROS and lipid membrane peroxidation (Wang and Quinn, 1999; Aitken, 2017), whereas green tea polyphenols are limited in this ability. Besides antioxidant properties, the valuable impacts of Ocimum gratissimum essential oils in chilled canine sperm quality may be due to its energy and mineral sources. Previous studies in phytochemical and nutritional properties have illustrated that Ocimum gratissimum essential oils contained high carbohydrates and mineral elements such as calcium, sodium, potassium, magnesium, manganese, zinc, iron, phosphorus (Idris et al., 2011; Igbinosa et al., 2013). These compositions could support energy and protect sperm plasma membrane as well as assist sperm metabolism and sperm function (Juyena and Stelletta, 2012; Smith et al., 2018).

Moreover, the malondialdehyde (MDA) is the major by-product of lipid peroxidation, the concentration of MDA indicates the oxidative damage level in sperm (Toker et al., 2016; Vieira et al., 2017). This study summates that the addition of Ocimum gratissimum essential oils, green tea polyphenols, and vitamin E in the extender could inhibit the formation of MDA in sperm compared to the control group. Additionally, although the sperm quality in the Ocimum gratissimum essential oils group was better than that in the green tea polyphenols and vitamin E groups, the level of MDA in the green tea polyphenols and vitamin E groups were lower than that of the essential oils group. These outcomes were consistent with the results of antioxidant activity, in which the antioxidant activity of the green tea polyphenols and vitamin E were significantly higher than that of the Ocimum gratissimum essential oils. In particular, green tea polyphenols are water-soluble which may have restricted the activity in the sperm plasma membrane, but the level of oxidative damage in this extender was the lowest in comparison to the other extenders. This may be explained by the green tea polyphenols having substantial antioxidant activity in the semen extender, but it was limited in the sperm. The lipid peroxidation parameter in the present study was conducted to evaluate the lipid peroxidation of the extended sperm, therefore the evaluation was performed in both the sperm cells and semen extender. However, the lipid peroxidation occurred not only on the sperm, but also on the extender (Maia et al., 2010). Consequently, the sperm lipid peroxidation results were the total concentration of MDA in both the sperm and the extender, and he level of MDA produced from the semen extender could have an important influence in the sperm lipid peroxidation results. This study suggests that the combination of the water-soluble antioxidant compounds with the lipid-soluble or amphiphilic antioxidant compounds may provide potential antioxidant activity for both the sperm cells and semen extenders.

CONCLUSIONS

In conclusion, the results presented that the supplementation of Ocimum gratissimum essential oils, green tea polyphenols, and vitamin E in the Tris-mineral salts egg-yolk extender for chilling have positive effects on canine sperm quality. Especially, the Ocimum gratissimum essential oils extender produced the best results and has shown a significant improvement in chilled canine sperm quality during 12 days of storage.

ACKNOWLEDGMENTS

We would like to express our gratitude to Suranaree University of Technology and the American Bully Dogs group in Thailand for their kind support.

CONFLICT OF INTEREST

Authors declared no conflict of interest.

novelty statement

The utilization of essential oil derived from Ocimum gratissimum leaves in canine semen extenders is a novel approach explored in this study. The research findings demonstrate the beneficial effects of these essential oils on dog sperms, indicating their potential application in practical breeding procedures.

authors contribution

P.K. and S.K. conceived and designed the experiments; V.V.N. and P.K. performed the experiments; V.V.N. and P.K. analysed the data; S.P. contributed materials; V.V.N., S.K. and P.K. wrote the manuscript; all authors reviewed and approved the final manuscript

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