Research Article

The Potential of Using Ocimum gratissimum Leaf Essential Oils as a Supplement in Extender to Improve Chilled Canine Sperm Quality by Assessing Its Antioxidant Effects

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

1Tra 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 | March 16, 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: [email protected]

Citation | Nguyen VV, Ponchunchoovong S, Kupittayanant S, Kupittayanant P (2023). The potential of using Ocimum gratissimum leaf essential oils as a supplement in extender to improve chilled canine sperm quality by assessing its antioxidant effects Adv. Anim. Vet. Sci. 11(8): 1338-1347.

DOI | https://doi.org/10.17582/journal.aavs/2023/11.8.1338.1347

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

Sperm preservation and cryopreservation are prevalent practices in canine breeding and play a significant role. Before being stored through cooling or freezing, appropriate semen extenders are mixed with the sperm (Thomassen and Farstad, 2009). Cooling is more popular due to its convenience and effectiveness compared to freezing (Linde-Forsberg, 1991; Linde-Forsberg, 1995; Eilts, 2005), and canine sperm is capable of being maintained at cooling temperatures without adverse effects before undergoing cryopreservation (Santana et al., 2013). According to our study, the incorporation of egg yolk in Tris buffer and mineral salts extender yielded high-quality chilled dog sperm (Vui et al., 2019). However, the presence of oxidative stress poses a significant risk to the viability of chilled dog sperm due to the abundance of polyunsaturated fatty acids in their plasma membrane, which makes them vulnerable to lipid peroxidation upon exposure to reactive oxygen species (ROS) (Lamirande et al., 1997; Vieira et al., 2017). This lipid peroxidation can impair sperm cell structures and cause apoptosis (Moustafa et al., 2004; Lucio et al., 2016). Fortunately, there is a constant production of antioxidant enzymes in the living organisms to combat oxidative stress (Ighodaro and Akinloye, 2017). In canine semen, almost antioxidant enzymes are mainly found in seminal plasma (Angrimani et al., 2014), but it is typically removed before mixing with semen extenders to avoid harmful impacts during preservation (Hori et al., 2017). This creates a decreased antioxidant capacity and increased sensitivity to oxidative effects. Hence, supplementing with antioxidants is necessary to neutralize free radicals and inhibit the effects of ROS during preservation, prolonging sperm lifespan. In addition, various antioxidant substances have been studied in canine sperm to prevent lipid oxidation during storage time (Neagu et al., 2009; Michael et al., 2009; Monteiro et al., 2009; Thiangtum et al., 2012), however, the outcomes were variable depending on the level and type of antioxidant substances.

Furthermore, the medicinal plant Ocimum gratissimum is known for its leaves that possess a substantial concentration of essential oils, accounting for 3.5% of their composition (Trevisan et al., 2006). Studies have identified eugenol, β-selinene, thymol, 1,8-cineole, and α-bisabolene as constituents of Ocimum gratissimum essential oils (Trevisan et al., 2006; Prabhu et al., 2009), all of which possess potent antioxidant properties (Ouyang et al., 2012; Mahapatra and Roy, 2014). However, there have been no studies assessing the impact of these essential oils on animal sperm quality. Incorporating essential oils into canine semen extenders might prevent sperm oxidation during chilling preservation, thus improving sperm lifespan. The aim of this investigation was to assess the effects of Ocimum gratissimum essential oils on the quality of chilled dog sperm over a 12-day period of storage in Tris buffer and mineral salts extender.

MATERIALS AND METHODS

Extraction Of Essential Oils

To acquire the essential oils, fully grown leaves of Ocimum gratissimum were subjected to drying at a temperature of 40°C in a hot air oven for an entire night. Subsequently, 100 g of the dried leaf powder was immersed in 1 liter of absolute ethanol for a duration of five days. The resultant solution underwent filtration, and the ethanol was removed by employing a rotary evaporator. The concentrated mixture was then subjected to centrifugation at 3500 x g for a duration of 10 minutes, leading to the separation and preservation of the essential oils present in the lower layer for utilization in this study.

Essential Oils Antioxidant Capacity

The antioxidant activity of the essential oils was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, as previously described by Blois (1958). Vitamin E served as the reference standard. In this process, the essential oils and standard solutions (100 µl) were dissolved in methanol and placed in a 96-well microplate. Subsequently, 100 µl of DPPH (200 μM in methanol) was added to the mixture of essential oils and standard solutions. The control group consisted of methanol and DPPH, while the blank group contained methanol with the essential oils or standard. After a 30-minute incubation period in darkness, the absorbance of the solution mixtures was measured using a spectrophotometric plate reader. By plotting the inhibition proportion against the concentration, a linear graph was utilized to calculate the IC50 value of the essential oils and standard, indicating the concentration at which 50% inhibition of DPPH activity was achieved.

Semen Collection

Over a period of one week, the digital manipulation technique outlined by Linde-Forsberg (1991) was employed to collect canine semen samples from five American Bully dogs. The research exclusively focused on semen samples of exceptional quality, adhering to specific criteria such as a sperm concentration of ≥0.2×107 sperm/ml, progressive motility of ≥70%, sperm viability of ≥90%, and sperm abnormal morphology of ≤5%. The assessment of sperm progressive motility and concentration was conducted using an automated sperm analyzer system. Furthermore, the viability and morphology of the sperm were evaluated using eosin-nigrosin staining, following the method outlined by Tamuli and Watson (1994).

Preparation Of Extenders

Sigma-Aldrich (Singapore) provided all the chemicals required for semen extender preparation. In this study, the semen extender employed consisted of Tris buffer and mineral salts supplemented with 20% egg yolk. Various treatments were examined by incorporating different concentrations of essential oils extracted from Ocimum gratissimum leaves into the base semen extender. The essential oil concentrations ranged from 0 to 800 µg/ml. Table 1 shows the main components of the semen extender solutions used. To dilute the essential oils, dimethyl sulfoxide (DMSO) was used as a solvent, and each extender consisted of 0.8% DMSO.

Semen Processing And Experimental Design

A repeated measures design was employed in this study for a period of 12 days, with four replicates and five ejaculates for each replicate. Following semen collection, the collected semen was divided into eight groups, and the seminal plasma was separated through centrifugation, as described by Rijsselaere et al. (2002). Subsequently, the sperm was diluted with extender solutions in the appropriate ratio to achieve a concentration of 100 x 106 cells/ml. The extended sperm samples were then subjected to chilling in cold water until reaching a temperature of 5°C, following the proced-

 

Table 1: The composition, pH, and osmolality of the extenders

Ingredients	Extenders
	T0	T25	T50	T100	T200	T400	T600	T800
Tris (mg)	900	900	900	900	900	900	900	900
Citric acid (mg)	500	500	500	500	500	500	500	500
Fructose (mg)	1250	1250	1250	1250	1250	1250	1250	1250
NaCl (mg)	450	450	450	450	450	450	450	450
KHPO4 (mg)	60	60	60	60	60	60	60	60
KCl (mg)	60	60	60	60	60	60	60	60
CaHPO4 (mg)	20	20	20	20	20	20	20	20
MgCl2 (mg)	10	10	10	10	10	10	10	10
Egg yolk (mL)	20	20	20	20	20	20	20	20
Essential oils (µg)*	0	25	50	100	200	400	600	800
Gentamicin (mg)	200	200	200	200	200	200	200	200
DMSO (µl)	80	80	80	80	80	80	80	80
Distilled water (mL)	To 100	To 100	To 100	To 100	To 100	To 100	To 100	To 100
pH	6.57	6.56	6.55	6.54	6.54	6.54	6.53	6.53
Osmolality (mOsmol/kg)	453	454	455	457	460	469	472	476

T0: control; T25, T50, T100, T200, T400, T600, and T800: 25, 50, 100, 200, 400, 600, and 800 µg/ml essential oils, respectively.* Essential oils extract from Ocimum gratissimum leaves.

-ure outlined by Bouchard et al. (1990). The samples were maintained at this temperature throughout the evaluation period of sperm quality, with assessments conducted once every three days over a total duration of 12 days.

Sperm Motility

For the assessment of sperm motility parameters, the automatic sperm analyzer system (CASA; HTR-IVOS 14.0; USA) was employed. Prior to analysis, the sperm samples were incubated in a water bath at 38°C for a duration of 15 minutes. Five randomly selected fields were chosen, and approximately 200 sperm were evaluated from each chamber. The analysis involved recording several sperm motility parameters, including the total motility proportion (TM%), progressive motility proportion (PM%), curvilinear velocity (VCL, µm/s), straight line velocity (VSL, µm/s), and average pathway velocity (VAP, µm/s).

Sperm Lipid Peroxidation

The evaluation of canine sperm lipid peroxidation was conducted by measuring the production of malondialdehyde (MDA) using a thiobarbituric acid reactive substance (TBARS) assay, as described by Maia et al. (2010). To induce lipid peroxidation, the sperm samples were treated with 0.24 mM FeSO4 and incubated for 15 minutes at a temperature of 38°C in warm water. Subsequently, 0.5 ml of the sperm sample was mixed with 1 ml of TBA reagent, followed by boiling for 20 minutes. After cooling and centrifugation to remove the supernatant, the absorbance of the supernatants was measured at 535 nm using a microplate spectrophotometer. The MDA level was determined by comparing the absorbance of each sample to a standard curve of MDA, and the resulting values were presented in nmol/50x106 sperm.

Sperm Membrane Integrity

The evaluation of canine sperm membrane integrity was conducted using a fluorescent staining technique that involved a combination of Pisum sativum agglutinin, Hoechst 33342, 5,5’,6,6’-tetrachloro-11’,3,3’-tetraethylbenzimidazolyl-carbocyanine iodide, and propidium iodide, following the protocol outlined by Vui et al. (2019). The stained sperm samples were immediately examined using a confocal microscope. Sperm exhibiting an intact acrosome and plasma membrane, along with a high mitochondrial membrane potential, displayed a distinct bright red-orange color in the mid-piece region and appeared blue-stained in the nucleus. Conversely, sperm with damaged acrosomes and plasma membranes, along with a low mitochondrial membrane potential, exhibited a bright green color in the mid-piece area, a yellow-green color in the acrosome region, and a red color in the nucleus. The stained sperm samples were identified and presented in Figure 1.

Statistical Analysis

The statistical analysis was performed using IBM SPSS Statistics, version 22, and the results were presented as mean ± standard deviation. To assess the impact of individual factors and the interaction between two factors (period and extender), a two-factor mixed analysis of variance (ANOVA) was conducted. For comparing means among groups within each factor, the Tukey method was applied. A significance level of P<0.05 was considered statistically significant.

 

RESULTS

Essential Oils Antioxidant Capacity

At a level of 203.00 µg/ml, the essential oils derived from Ocimum gratissimum leaves were found to inhibit 50% of DPPH radical, whereas vitamin E exhibited the same inhibitory capacity at a concentration of 11.03 µg/ml. Based on the findings, it can be concluded that the antioxidant effectiveness of vitamin E, at an equivalent concentration level, exceeded that of the essential oils derived from Ocimum gratissimum leaves.

Sperm Motility

Table 2 and Table 3 present the outcomes of the canine sperm motility analysis. In general, sperm quality in all extenders reduced gradually in total motility and progressive motility indicators during 12 days storage. There was no significant distinction observed in total motility and progressive motility measures among the extenders containing essential oil concentrations below 200 µg/ml during the chilling storage period. However, it should be noted that beyond day 9 of storage, the total motility and progressive motility indicators of sperm exposed to extenders with essential oil concentrations exceeding 400 µg/ml displayed a substantial decrease and exhibited a noticeable disparity compared to the other extenders (P<0.05).

In addition, canine sperm velocity served as a crucial parameter for assessing sperm motility characteristics. The results of sperm velocity values exhibited a similar pattern to that of total motility and progressive motility indicators. Notably, a significant decline in sperm velocity values was observed on day 12 for essential oil supplementation exceeding 400 µg/ml and on day 9 for supplementation exceeding 600 µg/ml (P<0.05).

Sperm Membrane Integrity

Table 4 presents the data on sperm membrane integrity. In general, these parameters showed a progressive decrease during sperm preservation in all essential oil concentrations. Sperm quality demonstrated a progressive enhancement in extenders containing essential oil concentrations below 100 µg/ml, whereas a consistent decline was observed in extenders with essential oil concentrations exceeding 200 µg/ml. Particularly noteworthy is that the extender supplemented with 100 µg/ml of essential oils exhibited the highest level of sperm membrane integrity. This difference was statistically significant compared to the extender without essential oils, as evidenced by both intact plasma and mitochondrial membrane indicators from day 3 to day 9 of preservation (P<0.05). Specifically, the sperm acrosome membrane values were highest in the extender with 100 µg/ml of essential oils, and this was significantly different from the extender without essential oils as well as the extenders with higher 400 µg/ml levels of essential oils during the 12-day preservation period (P<0.05).

Table 5 presents the percentages of healthy sperm, categorized based on the integrity of their acrosome, mitochondrial membrane, and plasma membrane. Similar to the aforementioned indicators, a gradual increase in the rates of healthy sperm was observed with the addition of essential oils to the semen extender, with the optimal outcomes achieved at a concentration of 100 µg/ml. However, it should be noted that the rates of healthy sperm declined at higher essential oil concentrations of 200 µg/ml. While the healthy sperm rates at 100 µg/ml did not show significant differences compared to the rates at 50 and 200 µg/ml, they were notably higher compared to the other extenders (P<0.05).

Sperm Lipid Peroxidation

Table 6 presents the concentrations of malondialdehyde (MDA) in canine sperm that underwent chilling and storage. The MDA levels in all extenders exhibited a consistent

 

Table 2: Effects of various levels of essential oils supplementation in extender on total motility (TM) and progressive motility (PM) parameters of chilled canine sperm.

Parameters	Extenders	Day1	Day3	Day6	Day9	Day12
TM (%)	T0	92.9±0.7A	91.4±0.9A	87.0±2.0B	83.2±1.0aB	61.2±4.9aC
	T25	93.7±0.8A	92.2±1.5A	88.7±1.5B	86.1±1.9aB	69.2±6.8aC
	T50	93.8±1.4A	92.2±0.8AB	90.2±1.0B	85.5±4.2aB	71.6±3.7aC
	T100	94.4±1.3A	92.3±1.5B	90.2±2.5B	88.0±3.3aB	64.5±6.7aC
	T200	94.1±1.0A	91.5±1.9B	89.0±2.1B	84.5±2.4aB	43.4±9.7bC
	T400	94.0±1.2A	91.2±1.2B	88.5±2.9B	66.0±8.4bC	8.3±1.1cD
	T600	93.6±0.5A	91.0±0.6B	88.8±1.5B	53.6±5.8bcC	4.9±0.9cD
	T800	93.2±0.8A	90.9±0.9B	86.3±2.4C	48.3±8.5cD	3.4±0.9cE
PM (%)	T0	70.1±0.7A	68.4±4.7A	63.6±2.2B	55.9±9.7aB	27.9±3.6aC
	T25	70.7±3.1A	68.7±3.5A	64.4±2.3B	59.2±7.1aB	31.2±4.9aC
	T50	72.4±2.0A	69.9±3.6AB	66.3±3.0B	62.7±3.2aB	33.2±4.8aC
	T100	74.5±1.2A	72.8±2.1A	68.9±1.4B	59.5±6.1aB	26.8±7.4aC
	T200	74.7±3.9A	69.6±4.2B	66.7±2.4B	50.9±9.5aC	23.6±8.2aD
	T400	73.0±1.9A	69.4±3.7B	65.9±2.1B	30.1±8.4bC	4.0±0.8bD
	T600	72.9±1.8A	69.2±2.0B	65.4±2.5B	23.2±5.8bC	0.0±0.0bD
	T800	72.6±3.1A	68.7±1.7B	64.6±2.6C	20.1±4.1bD	0.0±0.0bE

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 five ejaculates. Lowercase superscript letters (a, b, or c) in the same column indicates significant difference among extenders (P<0.05) and uppercase superscript letters (A, B, C, or D) in the same row indicates significant difference within extenders with different storage time (P<0.05).

 

Table 3: Effects of various levels of essential oils supplementation in extender on average pathway velocity (VAP), straight line velocity (VSL) and curvilinear velocity (VCL) parameters of chilled canine sperm.

Parameters	Extenders	Day1	Day3	Day6	Day9	Day12
VAP (µm/s)	T0	84.0±3.0A	78.5±2.6B	73.5±3.4BC	67.7±5.9abCD	57.9±8.2aD
	T25	84.4±3.0A	80.9±2.2A	73.6±4.2B	68.8±3.3abBC	62.0±6.9aC
	T50	86.0±2.5A	80.8±2.3AB	75.6±4.7BC	69.8±2.7abCD	62.3±4.0aD
	T100	86.1±2.5A	81.3±1.4AB	76.9±1.6BC	70.1±4.3aCD	60.3±7.1aD
	T200	86.9±2.8A	83.7±1.1A	74.3±4.2B	70.5±0.6aB	57.1±8.1aC
	T400	86.4±1.2A	82.3±1.7AB	76.6±3.3B	58.9±2.8abcC	38.8±8.4bD
	T600	86.3±1.0A	82.2±2.0A	72.7±7.2B	57.9±7.3bcC	0.0±0.0cD
	T800	86.1±2.3A	81.8±2.1A	71.9±4.4B	51.3±8.6cC	0.0±0.0cD
VSL (µm/s)	T0	78.0±2.3A	72.2±3.9AB	67.7±4.9BC	59.6±7.3abC	45.4±5.6abD
	T25	78.7±3.3A	72.7±1.0AB	67.3±5.4BC	61.4±4.4aC	51.4±6.0aD
	T50	79.0±3.2A	73.0±1.7AB	67.7±3.1B	62.6±4.5aB	49.9±6.8aC
	T100	79.2±3.2A	74.8±2.9A	70.5±4.2AB	61.6±5.6aB	46.8±7.9aC
	T200	82.3±2.0A	76.7±1.3A	68.9±2.3B	60.7±8.9aB	44.4±8.4abC
	T400	79.6±1.2A	77.0±2.1A	68.1±1.6B	46.8±4.5abC	30.3±8.2bD
	T600	79.2±1.1A	73.5±7.3AB	66.6±9.9B	45.7±7.3abC	0.0±0.0cD
	T800	78.3±3.4A	72.0±7.1A	63.8±7.5B	44.6±7.4bC	0.0±0.0cD
VCL (µm/s)	T0	116.5±6.1A	111.9±3.3AB	110.5±3.7B	108.6±5.4aB	100.9±6.8aC
	T25	117.7±5.5A	111.4±6.5B	109.6±5.9BC	107.6±4.8aBC	102.5±6.6aC
	T50	117.0±5.3A	112.2±2.0AB	110.3±2.0BC	104.4±6.0abCD	102.1±5.2aD
	T100	118.0±6.0A	113.7±3.2A	112.3±2.6AB	111.2±2.4aAB	105.7±4.5aB
	T200	116.9±7.1A	111.9±2.5AB	109.0±3.9B	107.6±3.6aB	99.7±2.6aC
	T400	116.7±4.3A	111.2±3.7A	104.6±3.5B	100.7±1.5abB	84.2±3.3bC
	T600	116.3±1.7A	111.2±5.1A	106.7±3.2B	93.4±6.5bcC	0.0±0.0cD
	T800	116.1±1.1A	112.5±1.1A	106.4±2.4B	86.3±4.3cC	0.0±0.0cD

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 five ejaculates. Lowercase superscript letters (a, b, or c) in the same column indicates significant difference among extenders (P<0.05) and uppercase superscript letters (A, B, C, or D) in the same row indicates significant difference within extenders with different storage time (P<0.05).

 

Table 4: Effects of various levels of essential oils supplementation in extender on high mitochondrial membrane potential, intact plasma and acrosome membrane of chilled canine sperm.

Parameters	Extenders	Day1	Day3	Day6	Day9	Day12
Plasma membrane (%)	T0	72.4±5.6abA	67.1±5.8bcB	59.2±9.2bC	52.1±0.9bCD	45.5±5.6abD
	T25	73.2±2.1abA	70.2±4.3abcA	63.8±5.6abB	56.7±5.1abB	48.4±6.0abC
	T50	78.5±5.6aA	74.9±3.2abA	65.7±5.3abB	60.9±6.4abB	52.3±3.3aC
	T100	80.0±3.5aA	77.5±2.2aAB	74.3±3.7aB	65.1±5.1aC	56.4±3.5aD
	T200	73.4±2.0abA	71.4±1.2abcAB	66.9±2.1abBC	59.7±3.7abC	50.2±6.0abD
	T400	71.5±2.3abA	69.1±2.4abcAB	64.1±1.9abBC	56.9±3.0abC	39.0±3.2bcD
	T600	69.1±2.9bA	67.2±2.2bcA	60.5±5.8bB	55.4±2.6abB	33.3±3.7cC
	T800	68.7±3.3bA	64.4±3.8cAB	58.9±2.8bBC	50.5±4.9bC	31.8±4.9cD
Mitochondrial membrane potential (%)	T0	78.7±1.3aA	70.3±4.3bB	62.5±5.9cC	54.1±3.2cD	47.9±1.7abD
	T25	79.6±4.8aA	76.2±3.9abA	68.5±6.1abcB	61.9±6.1abcC	52.9±8.6abD
	T50	82.4±5.9aA	80.0±6.7abA	73.3±4.5abB	67.6±5.5abB	58.8±5.7aC
	T100	86.2±1.6aA	82.2±2.5aB	78.0±3.0aC	73.7±3.0aC	62.5±3.5aD
	T200	81.6±3.2aA	78.3±4.5abA	73.2±2.8abcB	65.9±5.0abcC	53.9±7.2abD
	T400	78.1±4.2aA	72.9±4.0abB	69.1±3.3abcC	63.3±3.8abcC	40.1±3.8bcD
	T600	78.7±1.9aA	72.0±3.4abB	67.6±4.0abcC	60.7±3.0bcD	28.6±9.1cE
	T800	77.6±1.9aA	70.0±2.7bB	66.0±3.5bcC	57.0±7.1bcD	25.3±4.4cE
Acrosome membrane (%)	T0	63.6±3.4bcA	57.1±4.6cB	48.3±3.9eC	39.4±4.4cD	24.8±3.7cdE
	T25	66.7±2.1abcA	64.7±1.9abA	53.8±1.7cdeB	45.1±5.0bcC	34.7±7.3bcD
	T50	70.1±2.5abA	67.2±2.5aA	59.0±2.1bcB	50.5±5.5bC	43.7±7.0abC
	T100	73.0±1.8aA	70.2±1.6aAB	66.4±2.8aB	60.1±2.8aC	50.2±3.4aD
	T200	67.8±1.5abcA	65.8±1.8aA	60.4±1.9bB	52.1±1.9abC	36.4±6.1abcD
	T400	62.5±1.4cA	59.0±1.3bcAB	57.9±1.3bcB	48.2±1.6bcC	26.8±6.8cdD
	T600	62.4±4.3cA	57.9±0.8cB	54.0±1.7cdB	45.1±1.9bcC	22.2±4.9cdD
	T800	61.4±3.8cA	54.3±2.5cB	50.1±1.7deC	39.8±4.1cD	17.3±5.6dE

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 five ejaculates. Lowercase superscript letters (a, b, c or d) in the same column indicates significant difference among extenders (P<0.05) and uppercase superscript letters (A, B, C, D or E) in the same row indicates significant difference within extenders with different storage time (P<0.05).

 

Table 5: Effects of various levels of essential oils supplementation in extender on healthy sperm with high mitochondrial membrane potential, intact plasma and acrosome membrane of chilled canine sperm.

Extenders	Day1	Day3	Day6	Day9	Day12
T0	60.0±3.6cdA	52.9±9.3cB	43.0±6.5eC	36.3±5.4cD	21.9±4.5cdE
T25	64.7±2.2bcA	60.9±5.6abcA	51.9±2.4bcdB	43.0±5.4bcC	33.0±6.7abcD
T50	68.1±3.7abA	64.3±4.1abA	56.2±3.6bB	46.5±3.6bC	39.9±4.9aC
T100	71.4±1.6aA	68.7±0.5aAB	65.6±2.2aB	56.3±3.5aC	44.3±5.9aD
T200	65.4±2.2abcA	64.4±1.9abA	59.3±2.7abB	49.4±1.7abC	34.1±7.4abD
T400	61.7±1.0cdA	59.1±0.7abcAB	55.7±1.4bcB	44.8±1.3bcC	22.8±3.3bcdD
T600	61.2±1.8cdA	56.7±0.6bcA	50.8±2.5cdeB	42.2±2.8bcC	15.6±3.4dD
T800	57.8±2.1dA	52.5±3.1cB	46.6±2.3deC	37.1±3.5cD	10.4±2.0dE

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 five ejaculates. Lowercase superscript letters (a, b, c or d) in the same column indicates significant difference among extenders (P<0.05) and uppercase superscript letters (A, B, C, D or E) in the same row indicates significant difference within extenders with different storage time (P<0.05).

 

Table 6: Effects of various levels of essential oils supplementation in extender on the concentration of malondialdehyde (MDA) (nmol/50x106 sperm) of chilled canine sperm.

Extenders	Day1	Day6	Day12
T0	7.03±0.47aA	6.34±0.38aB	6.85±0.20aA
T25	6.55±0.48abA	6.02±0.28abB	6.46±0.19abA
T50	6.15±0.22abAB	5.76±0.16abB	6.31±0.27abA
T100	6.02±0.24bAB	5.61±0.08bB	6.04±0.24bA
T200	5.93±0.33bAB	5.51±0.26bB	6.03±0.39bA
T400	6.06±0.32bAB	5.74±0.32abB	6.30±0.37abA
T600	6.24±0.40abAB	5.83±0.28abB	6.37±0.30abA
T800	6.46±0.48abA	5.98±0.37abB	6.31±0.26abAB

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 five ejaculates. Lowercase superscript letters (a or b) in the same column indicates significant difference among extenders (P<0.05) and uppercase superscript letters (A or B) in the same row indicates significant difference within extenders with different storage time (P<0.05).

decrease from day 1 to day 6, followed by a gradual increase from day 6 to day 12. Although there were no significant differences in this parameter among the extenders supplemented with essential oils, the extender with 100 µg/ml and 200 µg/ml of essential oils demonstrated the lowest MDA values. These values differed significantly from the control group extender throughout the 12-day storage period (P<0.05).

DISCUSSION

Overall, the findings of this study indicated that the effects of Ocimum gratissimum essential oils on chilled canine sperm quality followed a dose-dependent pattern. Lower concentrations of essential oils (25-100 µg/ml) exhibited positive effects on sperm quality, whereas higher concentrations (200-800 µg/ml) had detrimental effects. The optimal level of essential oil to protect chilled canine sperm during storage was found to be 100 µg/ml. The positive impacts of essential oils on sperm quality may be due to their antioxidant activities. Several bioactive compounds have been identified in the leaves of Ocimum gratissimum, including β-selinene, α-bisabolene, thymol, 1,8-cineole, and eugenol (Trevisan et al., 2006; Prabhu et al., 2009). These compounds have been found to support the activity of intercellular enzyme antioxidants such as catalase, glutathione peroxidase, phospholipid hydro-peroxide glutathione peroxidase, and superoxide dismutase (Strzezek et al., 2009; Neagu et al., 2011; Angrimani et al., 2014). These study findings are similar to previous research on rosemary essential oils and clove bud extract in extender for ram and ovine sperm, respectively (Motlagh et al., 2014; Baghshahi et al., 2014). In contrast, thymol and essential oils from Thymus munbyanus had negative impacts on human sperm (Chikhoune et al., 2015). The potential harmful effects of high concentrations of essential oils in the extender could be attributed to their antimicrobial properties. In addition to their antioxidant characteristics, the bioactive compounds present in Ocimum gratissimum essential oils have been shown to possess antimicrobial properties, effectively inhibiting bacterial growth (Prakash et al., 2011; Aguiar et al., 2015). Due to their lipophilic nature, these bioactive compounds can interact with the phospholipids and proteins in sperm membranes, impacting their potential, permeability, and ion influx (Visconti et al., 2002; Nazzaro et al., 2013). These findings are consistent with previous studies conducted on bulls (Shoae and Zamiri, 2008), boars (Roca et al., 2004), and rams (Arando et al., 2019). Therefore, while the use of essential oils in semen extenders can have positive effects on sperm quality, careful consideration of the concentration level is essential to prevent any adverse impacts on sperm quality.

Furthermore, the process of sperm storage can lead to the production of malondialdehyde (MDA) through lipid peroxidation (Buege and Aust, 1978). MDA serves as a crucial marker for oxidative damage in canine sperm, as demonstrated in previous studies (Maia et al., 2010; Toker et al., 2016; Vieira et al., 2017). The results of this study support the notion that extenders supplemented with 100 and 200 µg/ml essential oils exhibited lower MDA levels compared to other extenders, resulting in improved sperm quality. These findings align with the research conducted by Cassani et al. (2005), Kao et al. (2008), and Motlagh et al. (2014), which have all reported a negative association between lipid peroxidation levels and sperm quality. Additionally, the study observed that MDA concentrations were higher on day 1 compared to day 6, suggesting that the antioxidant substances derived from essential oils may not fully bind to sperm membranes during the initial day of preservation, thereby rendering the sperm more susceptible to oxidation induced by FeSO4 prior to reacting with thiobarbituric acid.

CONCLUSIONS

In conclusion, the addition of essential oils derived from Ocimum gratissimum leaves at a concentration of 100 µg/ml to a canine semen extender has shown beneficial effects on sperm motility, membrane integrity, and lipid peroxidation during the cooling storage process. Further research is required to examine DNA integrity, sperm fertility, and frozen canine sperm.

ACKNOWLEDGEMENTS

Our appreciation goes out to the America Bullies Dogs team in Thailand and Suranaree University of Technology for their generous assistance.

CONFLICT OF INTEREST

The article has been deposited onto a “preprint server”, research square DOI: https://doi.org/10.21203/rs.3.rs-26157/v1

novelty statement

This study examines a new method involving the use of Ocimum gratissimum leaf essential oil in extending canine semen. The results of the research highlight the positive impact of these essential oils on dog sperm, suggesting their potential usefulness in real-world breeding practices

authors contribution

The experiments were conceived and designed by P.K. and carried out by V.V.N. and P.K. Data analysis was performed by V.V.N. and P.K. S.P. provided materials for the study. The paper was written by V.V.N., S.K., and P.K. All authors reviewed and approved the final version of the manuscript.

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