1. Introduction
Artificial insemination is a widely used technique for spreading genetic progress and improving herd productivity. However, in goats, its use is relatively limited and does not offer the same level of fertility as in cattle [
1]. The successful use of this technique depends mainly on the quality of the stored semen [
2]. It is therefore essential to optimize sperm storage protocols by extending sperm life without compromising its functionality. Deterioration of sperm quality is inevitable during storage, with bacterial proliferation and oxidative stress being the main causes of this degradation [
3].
Bacterial proliferation is an important factor affecting sperm quality, because semen is routinely collected and processed under non-sterile conditions. Moreover, the preservation of diluted semen at moderate temperatures, with nutrient-rich extenders, creates favorable conditions for considerable bacterial multiplication [
4]. Bacterial proliferation leads to sperm agglutination, decreased sperm motility, viability, membrane integrity and acrosomal integrity acrosome damage [
5].
Oxidative stress is another important factor affecting semen quality. Sperm has defense mechanisms against free radicals, including endogenous antioxidants [
6]. However, despite these protective systems, sperm cells remain highly sensitive to free radicals. Recent research has highlighted the role of oxidative stress, resulting from an imbalance between antioxidants and reactive oxygen species (ROS), on the deterioration of sperm quality [
7]. All cellular constituents, such as proteins, lipids, and nucleic acids, are vulnerable to oxidative stress caused by ROS, with serious consequences for sperm membrane integrity and motility [
8]. This ultimately accelerates the process of sperm degradation, reducing its viability and ability to fertilize [
9].
In recent years, natural compounds extracted from plants have attracted great interest in semen preservation due to their varied biological activities, notably their antibacterial and antioxidant mechanisms [
10,
11]. Essential oils have specific properties linked to their main constituents. For example, carvacrol content in
Thymus vulgaris (46%) contributes to its many properties [
12], while
Lippia thymoides essential oil owes its effects mainly to its thymol content (62%) [
13]. Although many compounds are present, one or two often dominate the oil’s physiological action. This chemical complexity helps explain the marked effects of essential oils, even when certain components are present in low concentrations, such as bergapten in bergamot oil (around 0.3%) [
14].
This paper focuses on Beni Arouss goats, an autochthonous breed of Northern Morocco officially recognized by the Moroccan Ministry of Agriculture. The Beni Arouss breed is a mixed-purpose goat known for its red coat and good build and reared under an extensive production system in the Rif mountains. Milk yield is estimated at 55 kg per lactation and used for suckling kids and making traditional cheese [
15]. This breed is under a specific breeding program carried out at the farm level by the Sheep and Goat National Association. Improving Beni Arouss buck semen conservation is essential to support this program by improving the success rate of artificial insemination. A previous study showed that the addition of 0.01% of
Thymus satureioides essential oil to skim milk extender effectively preserves buck semen for 48 h at 4 °C; this essential oil contains mainly carvacrol and thymol, representing 31% and 28% respectively [
16]. In the present study, we selected a concentration of 200 µM for carvacrol and thymol to reflect the proportions found in
Thymus satureioides essential oil at 0.01%, aiming to assess their impact on Beni Arous buck semen quality during 48 h storage at 4 °C. The use of these specific compounds allows for the avoiding of any variability in essential oil composition due to the variety, crop management, or climatic and edaphic conditions.
3. Results
3.1. Antioxidant Activity
Free radical-scavenging activity by DPPH showed different results between sperm-free extenders, with values of 49.58%, 56.44% and 54.35% in control skim milk and skim milk supplemented with carvacrol and thymol, respectively.
3.2. Sperm Motility
Results for total and progressive motility showed significant variations according to treatment and storage duration (
Table 1).
All treatments showed a decrease in total and progressive motility over 48 h of storage. Semen treated with carvacrol and thymol showed a less pronounced decline compared to the control group in total motility (p < 0.05). Additionally, the carvacrol-treated group showed higher levels of progressive motility than the control and thymol group after 48 h of storage (p < 0.05).
Kinetic parameters showed significant variations depending on the treatments applied and storage duration (
Table 2).
There were no significant differences in kinetic parameters between treatments at 0 h; variations appeared after 6 hours’ storage, with carvacrol affecting all kinetic parameters and thymol influencing VCL and VAP (p < 0.05). After 48 hours’ storage, carvacrol and thymol showed higher values than the control group for VCL, VSL, and VAP (p < 0.05).
3.3. Viability and Abnormalities
Viability decreased for all treatments (Control, carvacrol, and thymol) at all storage durations (
p < 0.05;
Table 3).
Treatments with carvacrol and thymol improved viability compared to the control at 48 h (
p < 0.05). The percentage of abnormal spermatozoa revealed a significant increase over time for all treatments at all storage times (
p < 0.05;
Table 3). No significant variation between treatments was observed over the entire sperm storage period (
p > 0.05).
3.4. Membrane Integrity and Lipid Peroxidation
The membrane integrity showed a significant decrease during storage for all treatments (carvacrol, thymol, and control) (
p < 0.05). However, no significant differences were detected between treatments (
Table 4).
The MDA concentration increased significantly during storage for all treatments (
Table 4). The carvacrol-enriched treatment showed the lowest MDA levels, followed by thymol and control at 24 h and 48 h storage at 4 °C (
p < 0.05).
3.5. Bacterial Growth
Bacterial growth showed a significant increase in CFU/mL during storage for all treatments. Moreover, it should be noted that bacterial growth was generally less pronounced with the carvacrol and thymol treatments than with the control group (
p < 0.05). Remarkably, the antimicrobial efficacy of carvacrol was superior to that of thymol at 6 h, 24 h, and 48 h storage (
p < 0.05,
Table 5).
3.6. Correlation between Sperm Quality Parameters
Correlations between the different sperm quality parameters are shown in
Table 6.
The results reveal significant positive correlations between total motility, progressive motility, kinematic parameters, viability, and sperm membrane integrity (p < 0.001). Abnormality showed a negative correlation with total motility, progressive motility, kinematic parameters, viability, and membrane integrity (p < 0.001), while positive correlations were found with lipid peroxidation and bacterial growth (p < 0.001). Furthermore, bacterial growth was negatively correlated with total motility, progressive motility, kinematic parameters, viability, and membrane integrity (p < 0.001).
4. Discussion
To the best of the authors’ knowledge, this is the first study analyzing the effect of thymol and carvacrol on goat semen conservation. Previous studies were conducted on boar [
26,
27], stallions [
28] and humans [
29], while others have investigated the effect of orally administered thymol and carvacrol on rat semen [
30,
31]. The results obtained in these studies showed varied trends. For example, the highest motility of boar spermatozoa after 24 h of storage was 25% with thymol and 40% with carvacrol compared to other substances such as ethylgallate, hydroquinone, and cnicin, but with no improvement compared to the control [
26]. Furthermore, the addition of carvacrol did not influence porcine sperm motility, but at low concentrations it decreased ROS production, while 30 μM of carvacrol reduced membrane integrity and 25 μM decreased mitochondrial membrane potential [
27]. Regarding thymol, (50 μM, 100 μM, and 150 μM) maintained total and progressive motility and kinematic parameters in stallion semen during all storage times (0, 24, and 48 h) without affecting fungal growth after 48 h of storage compared with the control [
28]. Conversely, human spermatozoa’s percentage motility and viability decreased from a 200 μg/mL thymol concentration in a dose-dependent way [
29].
Our results showed that the addition of carvacrol and thymol to skim milk improved its antioxidant activity and the highest value was observed by adding carvacrol with 56.44%. The antioxidant and antimicrobial properties of thymol and carvacrol have already been reported [
32]. Al-Mansori et al. [
33], examining the antioxidant activities of thymol and carvacrol, suggested that carvacrol had the highest DPPH radical scavenging activity. David [
34] reported that carvacrol demonstrated strong antimicrobial activity against
Acinetobacter calcoacetica,
Aeromonas hydrophila,
Bacillus subtilis,
Clostridium sporogenes, and
Pseudomonas aeruginosa. In contrast, the same author stated that thymol was more effective against
Citrobacter freundii,
Enterobacter aerogenes,
Klebsiella pneumoniae, and
Escherichia coli.
As expected, our findings showed that during storage there was a decline in total and progressive motility, kinematic parameters, viability, and membrane integrity, coupled with an increase in abnormalities, lipid peroxidation, and bacterial growth for all treatments (
p < 0.05). After 48 h of storage, total and progressive motility, kinematic parameters, viability, and lipid peroxidation were better preserved by carvacrol and thymol. These results are in accordance with our previous findings [
16] using 0.01% of
Thymus satureioides essential oil (carvacrol 31%, thymol 28%). In contrast, and unlike
Thymus satureioides essential oil, no significant effects on membrane integrity or sperm morphology were observed between treatments, despite a lower MDA concentration in the carvacrol and thymol group compared to the control. This result is most likely due to the limited antioxidant activity of carvacrol and thymol at the tested level protecting the sperm membrane and morphology against oxidation during storage. It appears that
Thymus satureioides essential oil exhibits a higher antioxidant activity, likely as result of a synergistic interaction between carvacrol and thymol and other minor compounds [
35].
The correlation results highlighted that carvacrol and thymol’s antimicrobial activity was responsible for the beneficial effect on sperm quality during storage, rather than their antioxidant properties. Indeed, lipid peroxidation, an indicative marker of oxidative stress, had no significant correlation with motility and viability, while bacterial growth showed a significant negative correlation with these parameters. This suggests that the high bacterial presence played a role in causing damage to sperm. The bacteriostatic effect of carvacrol and thymol at reduced concentrations succeeded in minimizing bacterial proliferation, thus preserving motility and viability compared with the control group after 48 h of storage. This result agrees with the findings of Mazurova et al. [
36], who reported that carvacrol and thymol are the most effective natural substances for decontaminating boar semen against Gram-negative and Gram-positive bacteria. The reduced CFU concentration in the carvacrol group was probably responsible for a higher progressive motility compared to the thymol group. During the first 24 h, no significant change was noted between the different treatments for all parameters except for lipid peroxidation. This observation could be attributed to the low bacterial load compared to 48 h of storage, exceeding 20,000 CFU/mL. Previous studies highlighted that when the bacterial load exceeds this threshold, a deleterious impact on viability and motility occurs [
37,
38].
For successful fertilization, motility and viability remain the most crucial and visibly impacted parameters [
39]. Our results showed that over the storage period, bacterial concentration increased while total motility, progressive motility, and viability decreased. It is pertinent to point out that in vitro studies have revealed that the mechanism of action of bacteria on sperm motility may result from attachment to sperm acrosome or flagellum receptors [
40], secretion of toxic factors such as lipopolysaccharide endotoxin [
41], or mitochondrial membrane damage and acrosome membrane rupture [
42]. Reduced sperm viability due to bacterial contamination can occur through at least two mechanisms: (1) certain bacteria produce soluble spermatotoxic agents, such as sperm immobilization factors, which may lower viability by inhibiting mitochondrial ATPase activity [
43], and (2) inflammation caused by bacteria can result in excessive reactive oxygen species (ROS) production, leading to sperm DNA damage and apoptosis [
44].
Our study showed that the abnormality rate increased, and membrane integrity decreased over the storage time, and both parameters were positively correlated with bacterial growth and lipid peroxidation. Meanwhile, previous studies have highlighted the detrimental effects of various bacterial strains, including
Escherichia coli,
Ureaplasma urealyticum,
Chlamydia trachomatis,
Mycoplasma hominis, and
Neisseria gonorrhoeae, on sperm morphology and membrane integrity [
45]. Oxidative stress resulting from the overproduction of ROS can have detrimental consequences on the sperm membrane and its acrosomal region, leading to potential sperm morphological abnormalities [
46,
47].