Skip to main content

Camellia oil with its rich in fatty acids enhances post-thawed boar sperm quality

Abstract

Background

Boar sperm are highly susceptible to specific conditions during cryopreservation, leading to a significant decrease in their fertilizing potential due to damage to their membranes. Camellia oil, known for its fatty acids with antioxidant and biological properties, has not been previously explored for the cryopreservation of boar semen. This study aimed to examine the effects of camellia oil on post-thawed boar sperm quality. Boar semen ejaculates (n = 9) were collected and divided into six equal aliquots based on camellia oil concentrations (0, 0.5, 1, 1.5, 2 and 2.5% v/v) in the freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm morphology by scanning electron microscope, sperm motility using a computer-assisted sperm analyzer, sperm viability, acrosome integrity, mitochondrial function, MDA level and total antioxidant capacity.

Results

The results demonstrated that the supplementation of 1.5% (v/v) camellia oil showed superior post-thaw sperm qualities such as improved sperm morphology, motility, acrosome integrity and mitochondrial function by 14.3%, 14.3% and 11.7%, respectively, when compared to the control group. Camellia oil at a concentration of 1.5% (v/v) showed the lowest level of MDA (18.3 ± 2.1 µmol/L) compared to the other groups.

Conclusions

In conclusion, adding 1.5% (v/v) camellia oil in the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in a higher post-thawed sperm quality.

Background

Boar sperm are highly responsive to the conditions encountered during cryopreservation, with several factors coming into play. These factors include susceptibility to oxidative harm, ice formation inside as well as outside the sperm cells during the semen cryopreservation and thawing processes, an overproduction of reactive oxygen species (ROS), and exposure to osmotic stress [1, 2]. It is important to highlight that cryopreservation of boar semen leads to a significant decline in fertilization potential due to the detrimental effects on sperm cell membranes [2]. This is particularly significant because polyunsaturated fatty acids (PUFAs) are abundant in mammalian sperm [3]. Also, the lipid makeup of the sperm's plasma membrane plays a pivotal role in shaping its mobility traits, susceptibility to cold temperatures, viability, and membrane integrity [4]. Notably, boar sperm exhibits a greater abundance of PUFAs within the plasma membrane than other species [5]. The most abundant saturated fatty acids (SFAs) were C16:0 (18%) and C18:0 (16%), and the most abundant fatty acids were docosapentaenoic Acid (DPA) (15%) and docosahexaenoic acid (DHA) (16%) [6]. Fatty acids (FAs) are important in male sperm biology due to their close association with membrane fluidity, acrosome reaction, sperm motility, and viability [7]. Within sperm membranes, FAs play a pivotal role in shaping both the structure and function of sperm, facilitating crucial membrane fusion events during fertilization [4]. Among these FAs, PUFAs stand out for their ability to permeate the sperm cell membrane, bolster the flexibility of the sperm plasma membrane, uphold its structural and functional integrity, bolster the resistance of the acrosome membrane to osmotic stress, and provide safeguarding against physiological or thermal fluctuations encountered during cryopreservation [3, 8, 9]. Nevertheless, diminishing the presence of PUFAs within the sperm plasma membrane can trigger oxidative stress, suggesting a connection between lipid peroxidation and sperm motility, viability, and morphology [10]. To protect sperm cells from the oxidative stress induced by free radicals, a robust array of antioxidants and fatty acids has been used in semen cryopreservation. It has been reported earlier that those fatty acids from various sources, such as DHA from fish oil [11,12,13], DHA from different egg yolks, and olive oil, [14] influence fatty acids on the sperm plasma membrane and consequently improve post-thawed boar semen quality [15]. Besides supplementation of fatty acid into freezing extender, palm kernel meal protein hydrolysate with its bioactive peptides [16, 17], curcumin [18], and resveratrol [19] have also been applied to improve frozen boar semen quality, such as total motility, progressive motility and viability as well as minimize lipid peroxidation.

Camellia oil, also known as tea seed oil, is a versatile vegetable oil that is extracted from the seeds of the Camellia oleifera plant or the Camellia sinensis plant, which is the same plant used to make tea [20]. It has been traditionally used in various Asian cuisines, and there are trends known to all regions as applications in skincare and haircare products due to its potential benefits [21]. Camellia seed oil, which is also called oriental olive oil, is recommended as a health-care plant oil by the FAO because of its high content of unsaturated fatty acids, polyphenols, vitamin E, and carotene [21]. Camellia oil is rich in unsaturated fatty acids such as oleic acid, linoleic acid; sesamin, saturated acids, and polyphenols [22,23,24]. Many studies reported that camellia oil with its rich in FAs and other advantage compounds, has a variety of bioactivities including antioxidant, anti-inflammatory, antimicrobial, gastroprotective, and hepatoprotective bioactivity [25]. During the past decades, numerous studies demonstrated that oleic [26, 27], linoleic [28, 29], vitamin E [30], and polyphenol [31], which are also constituents in camellia oil, could improve sperm qualities and reduce lipid peroxidation during cryopreservation of boar semen and other species. However, no studies have been published on the effects of camellia oil during the cryopreservation of boar sperm. This study, therefore, aims to elucidate the cryoprotection effect of camellia oil by evaluating the post-thawed boar semen qualities.

Methods

Animals

A total of nine semen ejaculates (n = 9) were obtained from six boars, comprising both Landrace and Large White breeds, with ages ranging from 1.5 to 3 years old, from a commercial farm in Ratchaburi province, Thailand were used in this study. These boars were individually housed in pens equipped with an evaporative cooling system. They had access to fresh, clean water at all times through automated watering systems, and their daily feed intake was adjusted to fulfil the semen production demands, which amounted to 3 kg per day. These boars were regularly utilized for semen collection for the purpose of artificial insemination.

Chemicals and extenders

In this study, the commercial oil named camellia oil from the Thailand Royal Project (Camellia oleifera seed oil, FDA No. 57-2-03254-2-0001, PatPat, Bangkok, Thailand) was used. There were three extenders used for boar semen cryopreservation as follows: extender I was the commercial semen extender the Beltsville Thawing Solution (BTS, Minitube, Tiefenbach, Germany); extender II was composed of 20% egg yolk and 11% lactose solution supplemented with different concentrations of camellia oil solution (0, 0.5, 1, 1.5, 2, and 2.5% v/v) in distilled water. Each aliquot was diluted with or without (control) camellia oil substance with an equal extender volume; and extender III was composed of 89.5% extender II, with 9% (v/v) glycerol and 1.5% (v/v) Equex-STM® (Nova Chemical Sales Inc., Scituate, MA, USA).

Fatty acid determination

The fatty acid profiles of camellia oil were determined by followed the Association of Official Analytical Chemists 2005 (AOAC) [15]. The content of the total fat for 32 fatty acids was determined by calculating the area under the peak and presented as each fatty acid per 100 g of total fatty acid.

Semen collection and preparation

Boar semen samples were obtained using the glove-hand technique. Subsequently, the semen was passed through gauze to retain only the sperm-rich fraction, which was then subjected to a comprehensive evaluation based on various parameters, including semen volume, pH, sperm motility, concentration, sperm viability, and morphologically intact spermatozoa (sperm morphology was stained with William’s staining method and evaluated using a light microscope). For cryopreservation process, only sperm-rich fraction with a motility of at least 70% and a morphology of at least 80% were selected [32].

Semen freezing and thawing process

Each sperm sample underwent cryopreservation using the conventional nitrogen freezing technique. In brief, post-collection, the semen was diluted with the BTS extender in a 1:1 volume ratio. The diluted semen was then transferred into 50 mL centrifuge tubes and subjected to a stabilization period at 15 °C for 120 min. After this equilibration period, the samples were centrifuged at 15 °C, with a force of 800 g applied for 10 min. (LMC-4200R, Biosan, Latvia) After centrifugation, the supernatant was removed, and the sperm pellet was resuspended at a concentration of 1.5 × 109 sperm/mL in extender II at an approximate ratio of 1–2:1. At this stage, the sperm sample was partitioned into six groups, distinguished by varying concentrations of camellia oil (0, 0.5, 1, 1.5, 2, and 2.5% v/v) and subsequently, cooled to 5 °C for 90 min. Each group of samples was then blended with extender III to attain a concentration of 1.0 × 109 sperm/mL before being loaded into 0.5 mL straws (IMV Technologies, L'Aigle, Basse-Normandie, France). The semen straws were frozen using a traditional nitrogen by contacting nitrogen vapor at 4 cm above the liquid nitrogen level for 20 min (−  20 °C /min) in a polystyrene box and plunged into the liquid nitrogen tank (− 196 °C) for storage prior to analysis. The frozen semen had been kept for 12 h before evaluations. Before sperm evaluation, the frozen semen samples were thawed at 50 °C for 12 s and extended (1:6) with a pre-warmed BTS extender at 37 °C for 15 min [33].

Assessment of sperm motility

Sperm motility assessments were performed using Computer-assisted Sperm Motility Analysis (CASA) with the AndroVision® system (Minitube, Tiefenbach, Germany). In brief, 3 µL of the semen sample was meticulously transferred into a disposable counting chamber (Leja® 20 µM, IMV Technologies, L’Aigle, Basse-Normandie, France) and maintained at a constant temperature of 37 °C during the entire analysis. Each analysis involved the enumeration of a minimum of 600 sperm cells, derived from examining five different fields within each sample. The results were expressed as percentages, reflecting total sperm motility, progressive motility, and various motility parameters, which encompassed curvilinear velocity (VCL, µm/s), average pathway velocity (VAP, mm/s), straight-line velocity (VSL, mm/s), amplitude of lateral head displacement (ALH, mm), straightness (STR, %), and linearity (LIN, %). Motile spermatozoa were defined with VCL ≥ 24 µm/s and ALH > 1 µm. Progressive motility (PMOT) has been interpreted as the presence of a VCL ≥ 48 µm/s and a VSL < 10 µm/s. The total motility (MOT) is the summation of sperm motility subpopulations that were determined by VCL thresholds, including local motility (VCL ≥ 24 and < 48 µm/s), slow motility (VCL ≥ 48 and < 80 µm/s), and fast motility (VCL ≥ 80 µm/s) [16].

Assessment of sperm viability

Sperm viability was evaluated utilizing a staining procedure employing SYBR-14 (L7011(A); Live/Dead Sperm viability kit, Invitrogen, Waltham, MA, USA) and Ethidiumhomodimer-1 (EthD-1, E1169, Invitrogen, Waltham, MA, USA). In brief, 50 µL of semen was mixed with 2.7 µL of 0.54 µM SYBR-14 working solution in DMSO to arrive at a final SYBR 14 concentration of 0.27 µM and 10 µL of 4.68 µM EthD-1 was added to the sample to yield a final EthD-1 concentration of 2.34 µM. The resulting mixture was incubated at 37 °C for a duration of 15 min. a fluorescence microscope magnified at 400 × , 200 sperm were assessed. The SYBR-14/EthD-1 stained sperm were classified into viable and non-viable sperm. Nuclei from viable sperm with intact plasma membranes exhibited a green fluorescence, whereas those from deceased sperm or sperm with damaged plasma membranes displayed a red fluorescence. The percentage of viable and non-viable sperm were calculated [33].

Assessment of acrosome integrity

Acrosome integrity was assessed through fluorescein isothiocyanate-labelled peanut (Arachis hypogaea) agglutinin (FITC-PNA; L7381, Sigma-Aldrich Co., Darmstadt, Germany). staining. Specifically, 10 µL of diluted semen was combined with 10 µL of 4.68 µM EthD-1. The sample was processed to obtain a final concentration of EthD-1 of 2.34 µM and incubated at 37 °C for 15 min. Following this, a 5 µL sample was smeared onto a glass slide and allowed to air dry. Subsequently, the sample was fixed with 95% ethanol for 30 s and air-dried. Forty µL of FITC-PNA (100 µg/mL in PBS) was evenly distributed over the slides, which were then placed in a moist chamber at 4 °C for 30 min. The slides were subsequently rinsed with cold PBS and air-dried once more. Under a 1000 × magnification fluorescence microscope, a total of 200 sperm cells were examined. Sperm with intact acrosomes exhibited a green colour with a smooth contour on the acrosomal region, as depicted in Fig. 1. Conversely, sperm with damaged acrosomes displayed a green colour with a rough contour. The results were quantified as the percentage of sperm with intact acrosomes [33].

Fig. 1
figure 1

Assessment of frozen-thawed boar spermatozoa using specific fluorescent dyes (400 × magnification). a Intact and non-intact acrosomes stained with FITC-PNA/EthD-1 staining (b) Mitochondrial membrane potential (MMP) stained with the dyes JC-1 and PI (counted only viable sperm); high MMP with orange fluorescence in midpiece; and low MMP with green fluorescence in midpiece

Assessment of mitochondrial membrane potential

The mitochondrial membrane potential was evaluated using a staining protocol employing the fluorochrome 5,5′,6,6′-tetrachloro-L,L′-tetraethylbenzimidazolylcarbo cyanine iodide (JC-1, T3168, Invitrogen, Waltham, MA, USA). A total of 50 µL of diluted semen was combined with 3 µL of a 2.4 mM propidium iodide (PI) (L7011(B); Live/Dead™ Sperm viability kit, Invitrogen, Waltham, MA, USA) working solution in to arrive at a final PI concentration of 129 µM and 3 µL of a 1.53 mM JC-1 solution in DMSO was added to the sample to yield a final JC-1concentration of 82 µM. This mixture was then incubated for 10 min at 37 °C in an opaque container. Under a 400 × magnification fluorescence microscope, 200 viable sperm (PI-neg) were assessed. Midpiece staining revealed that sperm exhibiting a heightened mitochondrial membrane potential emitted a yellow-orange fluorescence, while sperm with a diminished membrane potential emitted a green fluorescence (Fig. 1). The percentage of sperm with a high mitochondrial membrane potential was calculated [34].

Assessment of lipid peroxidation

The levels of malondialdehyde (MDA) in the semen samples were quantified using the concentration of MDA using a colorimetric lipid peroxidation assay kit (OxiSelect™ TBARS Assay Kit, Cell Biolabs, Inc, San Diego, USA) following the manufacturer’s instructions. In brief, 250 µL of a post-thawed semen sample was lysed by freezing at − 20 °C for 12 h and then centrifuged at a speed of 20000×g for 10 min. The supernatant was collected and analysed. The MDA in the supernatant sample (100 µL) reacted with 100 µL of lysis solution and 250 µL of thiobarbituric acid (TBA) solution to generate an MDA-TBA adduct that was quantified. The MDA-TBA product was measured immediately in a microplate reader (SPECTROstar Nano, BMG LABTECH, Ortenberg, Germany) at 532 nm. For the colorimetric assay, a 2 mM MDA standard was prepared and serially diluted for the standard curve. The levels of MDA were calculated from the MDA standard curve and expressed as µmol/L [16].

Assessment of total antioxidant capacity

The total antioxidant capacity (TAC) was monitored by using a colorimetric total antioxidant capacity assay kit (ab65329, Abcam®, Cambridge, UK) following the manufacturer’s instructions. Briefly, 250 µL of post-thawed sperm in each treatment was centrifuged at 20000 g for 10 min, supernatant was collected and diluted (1:100) in double distilled water. The 100 µL of diluted samples and standards were added to the 96 well plate. All standards and samples were mixed with 100 µL of Cu2+ solution and, incubated at room temperature for 90 min. After incubation, the samples were measured immediately in a microplate reader (SPECTROstar Nano, BMG LABTECH, Ortenberg, Germany) at 570 nm. For the colorimetric assay, a 1 mM Trolox standard was prepared and serially diluted for the standard curve. The TAC was calculated from the TAC standard curve and expressed as µmol/L.

Evaluation of sperm morphology by scanning electron microscopy (SEM)

Sperm samples were evaluated for morphology under a scanning electron microscope using the typical usual approach as follows: the semen samples were preserved in PBS for 24 h with 2.5% glutaraldehyde (Electron Microscopy Sciences, UK). Following fixation, the washing process with PBS was repeated three times for 15 min each. After staining with 0.1% osmium tetroxide (Sigma-Aldrich, Germany) for 1 h, the samples were rinsed three times for 15 min with PBS. The samples were dehydrated with a graded series of ethanol concentrations of 70%, 80%, 90%, 95%, and 100% ethanol during the dehydration process. The sperm samples were treated and subsequently coated with 50 nm platinum particles on a SEM stub [35]. Finally, the morphology of the sperm was examined using a scanning electron microscope (JEOL, JSM-IT500LA, Japan).

Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics for Windows, version 26.0 (SPSS Inc., Chicago, IL, USA). The normal distribution test of the data was examined using the Shapiro–Wilk test; the sperm parameters (i.e., progressive motility and VAP) were not normally distributed, and were then transformed using the Log 10 transformation. The parameters, including total motility, progressive motility, sperm motility patterns, sperm viability, acrosome integrity, mitochondrial membrane potential, MDA levels, and total antioxidant capacity, were presented as mean ± SEM. Owing to the main aim of this experiment was to assess the cryoprotective effect of varied concentrations of camellia oil on post-thawed boar semen qualities, and not for the variation among individual boar or different breeds or boar within the same breed. Therefore, the homogeneity of variances was tested to investigate the effect of treatment by Levene’s test, and means were compared using a one-way ANOVA. The comparison of sperm parameters among treatment groups was performed by Duncan’s multiple range test. Statistically significant difference was defined as P < 0.05.

Results

Effects of camellia oil on sperm motility

Table 1 presented descriptive statistics related to the quality of fresh boar semen. All the measurements affirm that the quality of these fresh semen samples falls within acceptable standards. Figures 2 and 3 illustrated the impact of camellia oil on sperm motility. The findings indicate that a 1.5% (v/v) supplementation of camellia oil exhibited superior post-thaw sperm quality in comparison to other concentrations. Semen samples supplemented with 1.5% (v/v) improved total motility and progressive motility by 14.3% and 10.5% when compared with control (37.2 ± 3.6% vs 22.9 ± 3.2% and 28.7 ± 3.0% vs 18.2 ± 2.8%, respectively). A significantly higher percentage of sperm characteristics of movement (i.e. VCL, VSL, VAP, and ALH) was found in 1.5 and 2% (v/v) supplementation groups when compared with the control group (Table 2).

Table 1 Sperm quality of fresh boar semen (n = 9)
Fig. 2
figure 2

Effect of camellia oil on total sperm motility in post-thawed boar semen. Bars represent means ± SEM. Different letters indicate a statistically significant difference at P < 0.05

Fig. 3
figure 3

Effect of camellia oil on progressive sperm motility in post-thawed boar semen. Bars represent means ± SEM. Different letters indicate a statistically significant difference at P < 0.05

Table 2 Sperm motility parameters after thawing at different camellia oil concentrations

Effects of camellia oil on sperm viability

There was no statistically significant difference in sperm viability (P = 0.19). However, a tendency toward a higher percentage of viability in treatment groups than the control group was found (Fig. 4). Supplemented with 1.5% (v/v) camellia oil showed the highest viable sperm (41.3 ± 4.7%) and was higher than control by 9.8%.

Fig. 4
figure 4

Effect of camellia oil on sperm viability after thawing. Bars represent means ± SEM. There are no statistically significant differences (P > 0.05)

Effects of camellia oil on sperm acrosome integrity

A variation in acrosome integrity was observed among the control and treatment groups (Fig. 5). However, the highest percentage of acrosome integrity was found at a concentration of 1.5% (v/v) (47.4 ± 4.2%), which was significantly higher than the control group by 14.3%.

Fig. 5
figure 5

Effect of camellia oil on acrosome integrity after thawing. Bars represent means ± SEM. Different letters indicate a statistically significant difference at P < 0.05

Effects of camellia oil on mitochondrial membrane potential

The results of mitochondrial membrane potential are presented in Fig. 6. A significantly higher percentage of mitochondrial function in 1.5% (v/v) supplemented group (37.1 ± 3.5%) was found when compared with the control group.

Fig. 6
figure 6

Effect of camellia oil on mitochondrial membrane potential after thawing. Bars represent means ± SEM. Different letters indicate a statistically significant difference at P < 0.05

Effects of camellia oil on lipid peroxidation

Figure 7 illustrates the effect of camellia oil on lipid peroxidation during cryo- preservation. It indicated a trend toward lower MDA levels in the 1.5% and 2.0% (v/v) supplemented group compared to the control group (P = 0.3).

Fig. 7
figure 7

Effect of camellia oil on the lipid peroxidation (MDA levels) after thawing. Bars represent means ± SEM. There are no statistically significant differences (P > 0.05)

Effects of camellia oil on total antioxidant capacity

As depicted in Fig. 8, there was no significant difference between the treatments and control groups. However, there was a trend for increased TAC in the treatment groups when compared to the control group (P = 0.08).

Fig. 8
figure 8

Effect of camellia oil on the total antioxidant capacity (TAC) after thawing. Bars represent means ± SEM. There are no statistically significant differences (P > 0.05)

Effects of camellia oil on sperm morphology

The sperm morphology evaluation using scanning electron microscopy (SEM) are presented in Fig. 9. The sperm morphology in the control group (Fig. 9 a, b) showed a higher number of non-intact morphology, including plasma membrane and acrosomal damage, while those supplemented with 1.5% camellia oil (Fig. 9 c, d) showed a high number of intact sperm morphology with a lesser degree of abnormal morphology than those in the control group.

Fig. 9
figure 9

Scanning electron micrographs of post-thawed boar sperm. The post-thawed boar sperm without supplementation (control group): (a, 1,000X) showed a high number of non-intact sperm morphology; (b, 8,000X) showed acrosome damage on the acrosome region. The post-thawed boar semen with 1.5% of camellia oil supplement: (c, 2,200X) showed a high number of intact sperm morphology; (d, 3,500X) showed intact sperm morphology. The sperms (a, c) with intact morphology were marked with a green star; sperms with non-intact morphology were marked with a red star

Fatty acid determination

From 32 fatty acids determination, the omega 6, 9, palmitic acid and stearic acid are more pronounced in the composition of fatty acids in camellia oil and a high ratio of omega 6:3 was found (Table 3).

Table 3 Fatty acids composition of camellia oil

Discussion

In the course of cryopreserving mammalian sperm, the freezing and thawing procedures, referred to as cryoinjuries, induce oxidative stress and an excessive production of ROS. This, in turn, disrupts the balance of the antioxidant system, where antioxidant defences struggle to counteract the overabundance of ROS. Consequently, this imbalance has a cascading impact on semen qualities, including sperm motility, mitochondrial activity, membrane permeability, sperm functions, and ultimately, sperm survival rates [36, 37].

The results of the first report of camellia oil with optimal concentration in freezing boar semen clearly showed that adding camellia oil to the freezing extender significantly improved post-thawed boar semen parameters, including sperm morphology by SEM, total motility (14.3%), progressive motility (10.5%), acrosome integrity (14.3%), mitochondrial membrane potential (11.7%) and kinetic motility patterns such as VCL, VSL, VAP and ALH. In the present results, an increase in sperm viability, total antioxidant capacity and inferior level of MDA, indicating a lesser level of lipid peroxidation, found in the treatment groups than in the control, particularly when supplemented with 1.5% (v/v) of camellia oil concentration. This might be explained by the observation that spermatozoa absorbed and employed fatty acids from the freezing extender to counteract ROS [37]. As a result, this process led to a reduction in lipid peroxidation within their plasma membrane and internal organelles [38]. The substantial variability observed in the parameters within the current study could potentially be attributed to individual variations among boars, encompassing distinctions in sperm biochemistry, seminal plasma composition, physiological factors, polymorphisms in the testis and epididymis, as well as the presence of fatty acids, for example, palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1, n-9), docosapentaenoic acid (22:5, n-6) and docosahexaenoic acid (22:6, n-3) in the boar sperm plasma membrane which has been earlier reported [6]. This relationship between PUFAs, FAs and cryopreservation susceptibility has been previously reviewed [36]. Considering all the findings collectively, it is concluded that the inclusion of 1.5% (v/v) camellia oil represents the optimal concentration for cryopreserving boar semen. This finding suggests that fatty acids, due to their antioxidant properties, exert a protective influence on spermatozoa during the freezing process, thereby mitigating damage to their membrane, acrosome, and mitochondria. In agreement with our study, it has been reported that oleic acid supplement in rooster chilled extender improved semen quality, decreased MDA levels and increased TAC [27]. In addition, it has been documented that oleic acid and palmitic acid improved sperm motility, viability, acrosomes, mitochondrial membrane potential and ATP production in chilled boar sperm [26]. According to previous studies in boar [33] and another study in buffalo [39], fatty acid antioxidants such as omegas 3, 6, 9 and DHA improved post-thawed semen quality in terms of plasma membrane integrity and acrosome integrity. It has also been reported by many studies that supplementation of omega 3 and/or 6 in feed improve not only semen qualities in boar but also reproductive performance in sows [38, 40,41,42,43,44]. In this study, camellia oil with its high constituent of omega 6, 9, palmitic acid and stearic acid also revealed its antioxidant capacity by protecting sperm from cryodamage associated with sperm freezing and thawing. However, too high a concentration of camellia oil showed a slightly cytotoxic effect on sperm qualities, which have also been reported in other antioxidants [45, 46]. The reason might be that an excess of antioxidants may disturb the balance between free radicals and antioxidants in mammalian cells [47].

The camellia oil in this study contained palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and saturated fatty acids as 11.12, 2.43, 78.05, 8.08, 0.32, and 13.55 g/100 g of total fatty acids, respectively. According to previous studies [22, 24], they showed that in camellia oil, more than 76% of the fatty acids are oleic acid and 50% higher than those compared with olive oil. In addition, it has also shown that camellia oil has antioxidant, anti-inflammatory, antimicrobial, gastroprotective, and hepatoprotective bioactivity [25]. Many studies have revealed excellent antioxidant activities of camellia oil, such as researchers in China, who demonstrated that the methanol extract of tea seed oil exhibited the highest yield and the strongest antioxidant activity as determined by DPPH scavenging activity and trolox equivalent antioxidant capacity (TEAC) [48]. Moreover, there is another study presented that the IC50 values of tea seed oil extracted by the DPPH free radical scavenging assay ranged from 17.2 ± 0.1% to 93.4 ± 1.8% when the concentration of the extracted oil varied from 10 to 160 mg/mL [49]. However, it is important to recognize that an abundance of antioxidants may interfere with redox signalling pathways, thereby disrupting normal cell function. Over-suppression of antioxidant enzyme genes or elevated antioxidant levels can upset the delicate balance of cellular homeostasis, possibly resulting in adverse effects on cellular health [50, 51]. This observation is clarified by the current findings, which demonstrate that incorporating the optimal 1.5% (v/v) concentration of camellia oil into the freezing extender improved the quality of post-thawed semen. Conversely, if a concentration exceeding 2% (v/v) is added, it leads to a decrease in post-thawed semen quality. However, camellia oil is a natural product and might be difficult to standardize for use as a semen extender additive. Therefore, each batch of camellia oil has to be analysed for its fatty acids composition prior using in each particular experiment. The conducting additional research involving fertility tests with frozen-thawed sperm supplemented with camellia oil for artificial insemination on pig farms would not only generate greater interest, but also provide substantial credibility to the swine industry, ultimately enhancing fertility rates.

Conclusions

Considering the cumulative results, it can be concluded that camellia oil, owing to its fatty acids content, effectively diminishes ROS production during cryopreservation. This reduction in ROS contributes to the inhibition of lipid peroxidation. It enhances various aspects of sperm quality, including sperm morphology, motility, viability, intact acrosomes, mitochondrial membrane potential, and total antioxidant capacity in frozen-thawed boar sperm. Optimal supplementation of camellia oil at a concentration of 1.5% (v/v) within the freezing extender significantly enhances post-thawed boar semen quality. Conversely, it is possible that supplementing with more than 2% (v/v) have a negative impact on post-thawed semen quality.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. Peña F, Johannisson A, Wallgren M, Rodriguez-Martinez H. Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of ejaculate. Anim Reprod Sci. 2003;78:85–98.

    Article  PubMed  Google Scholar 

  2. Chanapiwat P, Kaeoket K. Cryopreservation of boar semen: where we are. Thai J Vet Med. 2020;50:283–95.

    Article  Google Scholar 

  3. Van Tran L, Malla BA, Kumar S, Tyagi AK. Polyunsaturated fatty acids in male ruminant reproduction—a review. Asian-Australas J Anim Sci. 2017;30:622–37.

    Article  PubMed  Google Scholar 

  4. Shan S, Xu F, Hirschfeld M, Brenig B. Sperm lipid markers of male fertility in mammals. Int J Mol Sci. 2021;22(16):8767. https://doi.org/10.3390/ijms22168767.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Yeste M, Barrera X, Coll D, Bonet S. The effects on boar sperm quality of dietary supplementation with omega-3 polyunsaturated fatty acids differ among porcine breeds. Theriogenology. 2011;76:184–96.

    Article  CAS  PubMed  Google Scholar 

  6. Waterhouse KE, Hofmo PO, Tverdal A, Miller RR Jr. Within and between breed differences in freezing tolerance and plasma membrane fatty acid composition of boar sperm. Reprod. 2006;131:887–94.

    Article  CAS  Google Scholar 

  7. Collodel G, Castellini C, Lee JC, Signorini C. Relevance of fatty acids to sperm maturation and quality. Oxid Med Cell Longev. 2020;2020:7038124.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Strzezek J, Fraser L, Kuklińska M, Dziekońska A, Lecewicz M. Effects of dietary supplementation with polyunsaturated fatty acids and antioxidants on biochemical characteristics of boar semen. Reprod Biol. 2004;4:271–87.

    PubMed  Google Scholar 

  9. Chanapiwat P, Kaeoket K, Tummaruk P. Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen. Asian J Androl. 2009;11:600–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kurkowska W, Bogacz A, Janiszewska M, Gabryś E, Tiszler M, Bellanti F, et al. Oxidative stress is associated with reduced sperm motility in normal semen. Am J Mens Health. 2020;14:1557988320939731.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Memon UK, Kaka A, Haron W, Leghari RA, Ismail M, Mohammed Naeem QK, et al. Effect of in-vitro supplementation of polyunsaturated fatty acids on frozen-thawed bull sperm characteristics using Bioxcell® extender. Pure Appl Biol. 2021;5:399–405.

    Google Scholar 

  12. Kaeoket K, Sang-urai P, Thamniyom A, Chanapiwat P, Techakumphu M. Effect of docosahexaenoic acid on quality of cryopreserved boar semen in different breeds. Reprod Domest Anim. 2010;45:458–63.

    Article  CAS  PubMed  Google Scholar 

  13. Castellano CA, Audet I, Bailey JL, Laforest JP, Matte JJ. Dietary omega-3 fatty acids (fish oils) have limited effects on boar semen stored at 17 °C or cryopreserved. Theriogenology. 2010;74:1482–90.

    Article  CAS  PubMed  Google Scholar 

  14. Silva E, Cardoso T, Junior A, Dutra F, Leite F, Corcini C. Olive oil as an alternative to boar semen cryopreservation. Ces Med Vet Zootec. 2016;11:8–14.

    Article  Google Scholar 

  15. Kaeoket K, Chanapiwat P. DHA analysis in different types of egg yolks: its possibility of being a DHA source for boar Semen cryopreservation. Thai J Vet Med. 2013;43:119–23.

    Article  Google Scholar 

  16. Khophloiklang V, Chanapiwat P, Aunpad R, Kaeoket K. Palm kernel meal protein hydrolysates enhance post-thawed boar sperm quality. Animals. 2023;13:3040.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Gang L, Pan B, Li S, Ren J, Wang B, Wang C, et al. Effect of bioactive peptide on ram semen cryopreservation. Cryobiolog. 2020. https://doi.org/10.1016/j.cryobiol.2020.08.007.

    Article  Google Scholar 

  18. Chanapiwat P, Kaeoket K. The effect of Curcuma longa extracted (curcumin) on the quality of cryopreserved boar semen. Anim Sci J. 2015;86:863–8.

    Article  CAS  PubMed  Google Scholar 

  19. Kaeoket K, Chanapiwat P. The beneficial effect of resveratrol on the quality of frozen-thawed boar sperm. Animals. 2023;13:2829.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Li G, Ma L, Yan Z, Zhu Q, Cai J, Wang S, et al. Extraction of oils and phytochemicals from Camellia oleifera seeds: trends, challenges, and innovations. Processes. 2022;10:1489.

    Article  CAS  Google Scholar 

  21. Miao J, Che K, Xi R, He L, Chen X, Guan X, et al. Characterization and benzo[a]pyrene content analysis of camellia seed oil extracted by a novel subcritical fluid extraction. J Am Oil Chem Soc. 2013;90:1503–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yang C, Liu X, Chen Z, Lin Y, Wang S. Comparison of oil content and fatty acid profile of ten new Camellia oleifera cultivars. J Lipids. 2016. https://doi.org/10.1155/2016/3982486.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Wang L, Ahmad S, Wang X, Li H, Luo Y. Comparison of antioxidant and antibacterial activities of camellia oil from Hainan with camellia oil from Guangxi, olive oil, and peanut oil. Front nutr. 2021. https://doi.org/10.3389/fnut.2021.667744.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Oğraş Ş, Kaban G, Kaya M. The effects of geographic region, cultivar and harvest year on fatty acid composition of olive oil. J Oleo Sci. 2016;65:889–95.

    Article  PubMed  Google Scholar 

  25. Cheng YT, Lu CC, Yen GC. Beneficial effects of camellia oil (Camellia oleifera Abel.) on hepatoprotective and gastroprotective activities. J Nutr Sci Vitaminol. 2015;61:100–2.

    Article  Google Scholar 

  26. Zhu Z, Li R, Feng C, Liu R, Zheng Y, Hoque SAM, et al. Exogenous oleic acid and palmitic acid improve boar sperm motility via enhancing mitochondrial Β-oxidation for ATP generation. Animals. 2020. https://doi.org/10.3390/ani10040591.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Eslami M, Ghaniei A, Mirzaei RH. Effect of the rooster semen enrichment with oleic acid on the quality of semen during chilled storage. Poult Sci. 2016;95:1418–24.

    Article  CAS  PubMed  Google Scholar 

  28. Jakop U, Svetlichnyy V, Schiller J, Schulze M, Schroeter F, Mueller K. In vitro supplementation with unsaturated fatty acids improves boar sperm viability after storage at 6 °C. Anim Reprod Sci. 2019;206:60–8.

    Article  CAS  PubMed  Google Scholar 

  29. Büyükleblebici S, Taşdemir U, Tuncer PB, Durmaz E, Özgürtaş T, Büyükleblebici O, et al. Can linoleic acid improve the quality of frozen thawed bull sperm? Cryo-Lett. 2014;35:473–81.

    Google Scholar 

  30. Jeong YJ, Kim MK, Song HJ, Kang EJ, Ock SA, Mohana KB, et al. Effect of α-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes. Cryobiology. 2009;58:181–9.

    Article  CAS  PubMed  Google Scholar 

  31. Kitaji H, Ookutsu S, Sato M, Miyoshi K. Preincubation with green tea polyphenol extract is beneficial for attenuating sperm injury caused by freezing-thawing in swine. Anim Sci J. 2015;86:922–8.

    Article  CAS  PubMed  Google Scholar 

  32. Kaeoket K, Tantasuparuk W, Kunavongkrit A. The effect of post-ovulatory insemination on the subsequent embryonic loss, oestrous cycle length and vaginal discharge in sows. Reprod Domest Anim. 2005;40:492–4.

    Article  CAS  PubMed  Google Scholar 

  33. Kaeoket K, Donto S, Nualnoy P, Noiphinit J, Chanapiwat P. Effect of gamma-oryzanol-enriched rice bran oil on quality of cryopreserved boar semen. J Vet Med Sci. 2012;74:1149–53.

    Article  CAS  PubMed  Google Scholar 

  34. Garner DL, Thomas CA, Joerg HW, DeJarnette JM, Marshall CE. Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa. Biol Reprod. 1997;57:1401–6.

    Article  CAS  PubMed  Google Scholar 

  35. Bonet S, Delgado-Bermúdez A, Yeste M, Pinart E. Study of boar sperm interaction with Escherichia coli and Clostridium perfringens in refrigerated semen. Anim Reprod Sci. 2018;197:134–44.

    Article  CAS  PubMed  Google Scholar 

  36. Yeste M. Sperm cryopreservation update: cryodamage, markers, and factors affecting the sperm freezability in pigs. Theriogenology. 2016;85:47–64.

    Article  CAS  PubMed  Google Scholar 

  37. Ribas-Maynou J, Mateo-Otero Y, Delgado-Bermúdez A, Bucci D, Tamanini C, Yeste M, et al. Role of exogenous antioxidants on the performance and function of pig sperm after preservation in liquid and frozen states: a systematic review. Theriogenology. 2021;173:279–94.

    Article  CAS  PubMed  Google Scholar 

  38. Yuan C, Wang J, Lu W. Regulation of semen quality by fatty acids in diets, extender, and semen. Front Vet Sci. 2023;10:1119153.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Khalil WA, Hassan MAE, Attia KAA, El-Metwaly HA, El-Harairy MA, Sakr AM, et al. Effect of olive, flaxseed, and grape seed nano-emulsion essential oils on semen buffalo freezability. Theriogenology. 2023;212:9–18.

    Article  CAS  PubMed  Google Scholar 

  40. Roszkos R, Tóth T, Mézes M. Review: practical use of n-3 fatty acids to improve reproduction parameters in the context of modern sow nutrition. Animals. 2020;10:1141.

    Article  PubMed  PubMed Central  Google Scholar 

  41. McDermott K, Icely S, Jagger S, Broom LJ, Charman D, Evans CM, et al. Supplementation with omega-3 polyunsaturated fatty acids and effects on reproductive performance of sows. Anim Feed Sci Technol. 2020;267:114529.

    Article  CAS  Google Scholar 

  42. Liu Q, Zhou YF, Duan RJ, Wei HK, Jiang SW, Peng J. Effects of dietary n-6:n-3 fatty acid ratio and vitamin E on semen quality, fatty acid composition and antioxidant status in boars. Anim Reprod Sci. 2015;162:11–9.

    Article  CAS  PubMed  Google Scholar 

  43. Eastwood L, Leterme P, Beaulieu AD. Changing the omega-6 to omega-3 fatty acid ratio in sow diets alters serum, colostrum, and milk fatty acid profiles, but has minimal impact on reproductive performance1. J Anim Sci. 2014;92:5567–82.

    Article  CAS  PubMed  Google Scholar 

  44. Andriola YT, Moreira F, Anastácio E, Camelo FA Jr, Silva AC, Varela AS Jr, Gheller SMM, et al. Boar sperm quality after supplementation of diets with omega-3 polyunsaturated fatty acids extracted from microalgae. Andrologia. 2018. https://doi.org/10.1111/and.12825.

    Article  PubMed  Google Scholar 

  45. Parham A, Arshami J, Naserian A, Kandelousi M, Azizzadeh M. Quality of bovine chilled or frozen-thawed semen after addition of omega-3 fatty acids supplementation to extender. Int J Fertil Steril. 2013;7:161–8.

    Google Scholar 

  46. Lee SH, Kim YJ, Ho Kang B, Park CK. Effect of nicotinic acid on the plasma membrane function and polyunsaturated fatty acids composition during cryopreservation in boar sperm. Reprod Domest Anim. 2019;54:1251–7.

    Article  CAS  PubMed  Google Scholar 

  47. Rahal A, Kumar A, Singh V, Yadav B, Tiwari R, Chakraborty S, et al. Oxidative stress, prooxidants, and antioxidants: the interplay. Biomed Res Int. 2014. https://doi.org/10.1155/2014/761264.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Lee CP, Yen GC. Antioxidant activity and bioactive compounds of tea seed (Camellia oleifera Abel.) oil. J Agric Food Chem. 2006;54:779–84.

    Article  CAS  PubMed  Google Scholar 

  49. Wang Y, Sun D, Chen H, Qian L, Xu P. Fatty acid composition and antioxidant activity of tea (Camellia sinensis L.) seed oil extracted by optimized supercritical carbon dioxide. Int J Mol Sci. 2011;12:7708–19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Kurutas EB. The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. Nutr J. 2016;15:71.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Alves AF, Moura AC, Andreolla HF, Veiga A, Fiegenbaum M, Giovenardi M, et al. Gene expression evaluation of antioxidant enzymes in patients with hepatocellular carcinomaRT-qPCR and bioinformatic analyses. Genet Mol Biol. 2021;44:e20190373.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

All laboratory facilities in this study were supported by the Faculty of Veterinary Science, Mahidol University and Authors would like to thank Nawapol Udpuay, Chawalit Takoon and Dr. Suwilai Chaveanghong, scientists of Mahidol University Frontier Research Facility (MU-FRF) for their kind assistance in instrumental operation and technical supports for Scanning Electron Microscope.

Funding

Open access funding provided by Mahidol University. This project is financially supported by National Research Council of Thailand (NRCT) and Mahidol University (NRCT5-RSA63015-05).

Author information

Authors and Affiliations

Authors

Contributions

KK was involved in the conceptualisation of this study. VK and PC were responsible for data collection. All authors were involved in study design, result interpretation and discussion. KK was responsible for statistical analysis and for writing of the manuscript. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to Kampon Kaeoket.

Ethics declarations

Ethics approval and consent to participate

The present study was conducted according to the Ethics of animal experimentation of The National Research Council of Thailand and approved by the Faculty of Veterinary Science Animal Care and Use Committee (FVS-ACUC-Protocol No. MUVS-2023–09-57), Mahidol University.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Khophloiklang, V., Chanapiwat, P. & Kaeoket, K. Camellia oil with its rich in fatty acids enhances post-thawed boar sperm quality. Acta Vet Scand 66, 6 (2024). https://doi.org/10.1186/s13028-024-00728-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13028-024-00728-y

Keywords