The Addition of Opuntia ficus-indica Ethanolic Extract to a Skimmed Milk-Based Extender Impacts Ram Sperm Quality

Recently, researchers have focused on the use of natural antioxidants to improve semen quality as a key element for successful artificial insemination. In this context, the first aim of this study was to determine the antioxidant activity and composition (minerals, vitamins, and sugars) of Opuntia ficus-indica cladode ethanolic extract (ETHEX). A further purpose of the study was to investigate the effect of ETHEX supplementation on the quality of liquid ram semen extended with skim milk (SM) at 5°C. The antioxidant activity of ETHEX was studied using free radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH•) assay. The mineral composition and the sugar and vitamin contents of ETHEX were determined using an inductively coupled plasma optical emission spectrometry (ICP-OES) and HPLC-DAD-RID analytical instruments. As a second part, semen was collected from five Boujaâd rams with an artificial vagina. The ejaculates with more than 70% motility were pooled, extended with skim milk (SM) extender without (control) or supplemented with 1–8% of ETHEX (37°C; 0.8 × 109 sperm/mL). Sperm quality parameters were assessed at 8, 24, 48, and 72 h. The results showed that ETHEX had a higher antioxidant activity compared to those of ascorbic acid and butylated hydroxytoluene (BHT). Furthermore, ETHEX contains a considerable amount of minerals, vitamins, and sugars. The inclusion of 1 or 2% ETHEX in SM increased the sperm motility, viability, and membrane integrity and decreased the abnormality of spontaneous and catalyzed lipids peroxidation (p < 0.05) up to 72 h. In addition, semen diluted with 1 and 2% ETHEX decreased the level of DNA fragmentation compared to the control group (p < 0.05). In conclusion, the ETHEX could be recommended to improve the quality of liquid ram spermatozoa. However, its effects on artificial insemination should be further studied.


Introduction
Artifcial insemination with cryopreserved rams is not common in the sheep industry [1] because cryopreservation leads to a decrease in fertility. Acceptable fertility results have only been obtained with laparoscopic surgery. In fact, the cryopreservation process extremely decreased all sperm parameters compared to the liquid storage. Besides, semen cryopreservation is an expensive process, and for all cited reasons, liquid-stored semen can be an alternative to frozenthawed semen for artifcial insemination [2]. Terefore, some studies have motivated the use of liquid semen preservation for diferent species [3,4]. One of the main factors associated with low sperm quality during liquid storage and cryopreservation is the production of reactive oxygen species (ROS), which occurs as an abnormal consequence of spermatozoa metabolism [5]. To prevent such stress, the semen needs to be diluted with suitable extenders and conditions. However, the addition of extenders signifcantly reduces the antioxidant capacity of spermatozoa and seminal plasma. Te spermatozoa and seminal plasma system comprise taurine, reduced glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) to prevent oxidative damage. Te antioxidant system of spermatozoa is compromised during semen processing [6]. For that reason, the need for extender supplementation becomes necessary. Many additives with antioxidative properties have been reported to reduce the impact of ROS-induced and cold shock damage on sperm cells [7] and thus improve sperm ram quality [8][9][10].
Research has shown that plant extracts can be efective in improving semen quality in many animal species [11][12][13][14]. One of the key properties of plant extracts is their antioxidants activity, which is responsible for their ability to scavenge free radicals [15]. Opuntia fcus-indica, commonly known as the cactus, is a plant cultivated in many parts of the world, including North and South America, as well as Mediterranean countries such as Morocco and South Africa [16]. Te ethanol extract from Opuntia fcus-indica cladodes is a natural source of antioxidants, which makes it efective in scavenging free radicals. Tis extract contains various constituents, such as tocopherol, polyphenols, favonoids, quercetin, phenolic acids (cafeic and gallic acid), minerals, and sulfur amino acids (taurine, methionine, arginine, and cysteine) [17,18]. Several studies have reported that this extract has anti-infammatory and antioxidant properties [19].
Opuntia fcus-indica extract has been found to be benefcial for improving semen quality. Its antioxidant properties help to reduce oxidative stress, which is known to have a negative impact on sperm quality [20,21]. Terefore, the use of Opuntia fcus-indica extract may be a natural and efective approach to improving semen quality in animals. Opuntia fcus-indica is a plant that contains a range of compounds, including polyphenols, favonoids, quercetin, phenolic acids, and sulfur amino acids [22]. Both acetone and ethanolic extraction methods are commonly used to extract these compounds from the plant [23]. However, the choice of extraction method may depend on the specifc compounds of interest. Acetone extraction is more efective in extracting nonpolar compounds such as lipids and pigments, while ethanolic extraction is better suited for polar compounds such as polyphenols and favonoids due to its ability to extract a wider range of polar compounds. In a previous study, we utilized acetone extraction method to improve ram sperm quality [24], whereas in the current study, we are using ethanolic extraction to extract compounds from Opuntia fcus-indica. To the best of our knowledge, this is the frst report focusing on the efect of Opuntia fcus-indica cladode ethanolic extract (ETHEX) on Boujaâd ram sperm quality parameters. Te objective of this study was to determine the antioxidant properties, mineral, vitamin, and sugar composition of ETHEX and to assess its inclusion in SM on sperm total and progressive motility, viability, abnormality, membrane integrity, lipid peroxidation levels, and DNA fragmentation during liquid storage at 5°C up to 72 h.

Ethical Approval for Animal Study.
Animal studies were conducted following the protocols of Animal Use and Care of the University of Hassan 1 st , Settat, Morocco.

Chemicals and Reagents.
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Merck (Merck, Schuchardt, OHG, Germany). Ultrapure water was obtained from a Millipore Milli-Q system (Bedford, MA, USA).

Cactus Extraction and DPPH Inhibition Method.
Nopal cactus cladodes were collected from the experimental station of Regional Center-INRA Settat, Morocco. Tey were washed with distilled water, dried at 55°C in an oven, and mechanically milled. Te obtained powder was stored in a closed container at room temperature until use. To prepare the ethanol extract, a fne dried powder (5 g) was extracted by stirring at room temperature with 100 mL of ethanol/ water mixture (70 : 30 v/v) for 4 days at 4°C [25]. After the solution was fltered, then the solvent was evaporated using a vacuum rotary evaporator (Buchi R-210) at 38°C. Te remaining part was lyophilized and stored at 4°C until used.
Radical scavenging activity (RSA) of ETHEX was measured using the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) [26]. A known amount of the dry ETHEX (0.1 mL) was centrifuged in methanol (2.9 mL) at 5000 rpm for 15 minutes followed by fltration through flter paper (Whatman No. 1). Methanolic solution of DPPH (0.5 mL, 100 μM) was added to the tubes including the supernatant of each concentration. After vigorous shaking of the tubes, they were incubated at room temperature for 45 minutes in the darkness. Ascorbic acid and BHT were used as reference compounds. Te lower absorbance of the reaction mixture indicated a higher free radical scavenging activity.
Te percentage of free radical scavenging activities of the samples was calculated using the following formula: where A D and A E is the absorbance of the DPPH blank sample and the absorbance of the test solution, respectively. A E was the diference between the absorbance value of the test solution and its blank.

Total Sugar Content.
Te sample (2 g) was extracted with ultrapure water (20 mL) in an ultrasonic bath (Elmasonic, Germany) for 10 minutes at room temperature. Ten it was fltered through a 0.45 µm PTFE flter. Te total sugar content of the sample was determined using a highperformance liquid chromatography (HPLC) system (Agilent Technologies, USA) equipped with a refractive index detector (RID). Te sugars were separated by NH 2 column (Inertsil, 5 μm, 4.6 mm × 250 mm, GL Sciences) maintained at 30°C. Te injection volume was 10 μL. Te mobile phase was acetonitrile (80%) with a fow rate of 1.0 mL·min −1 . Standard solutions were injected to get the retention time and standard curve for each sugar. Te results were evaluated with the ChemStation Software and expressed as mg·g −1 dry weight.

Vitamin Content.
Te sample (1 g) was extracted with ultrapure water (10 mL) in an ultrasonic bath (Elmasonic, Germany) for 10 minutes at room temperature. After, 1 mL of 2 M NaOH and 12.5 mL of 1 M phosphate bufer (pH � 5.5) were added and fulflled to 25 mL with ultrapure water. Ten, the sample was fltered through a 0.45 µm PTFE flter before injection. Te vitamin content of the sample was determined using a high-performance liquid chromatography (HPLC) system (Agilent Technologies, USA) equipped with a diode array detector (DAD). Te vitamins were separated on an ODS column (5 μm, 4.6 mm × 250 mm, Inertsil, GL Sciences) maintained at 30°C. Te injection volume was 10 μL. Te mobile phases were the aqueous solution of trifuoroacetic acid (0.025%, v/v), solution A; and acetonitrile, solution B. Te elution program was the gradients of solvent B as following: 0.0% solvent B (0-5 th min), 25% solvent B (6−11 th minutes), 45% solvent B (11−19 th minutes), 40% solvent B (19−20 th minutes), 0% solvent B (20−22 nd minutes). Standard solutions of vitamins (Vitamins C, B2, B3, B5, and B9) were injected to get the retention time and standard curve for each vitamin. Te results were evaluated with the ChemStation Software and expressed as mg/100 g dry weight.
2.6. Mineral Content. Te digestion of the sample was performed on a microwave digestion system (CEM MARS5 (USA)). Approximately 0.5 g of dry sample was transferred to a PTFE digestion tube containing 6 mL of nitric acid (65%) and 2 mL of hydrogen peroxide. Te operating conditions of the microwave oven were as follows temperature (150-200°C), ramp (20 min), time (2 min), and power (100%) for each step. Te digested sample was cooled to room temperature, fltered, and the fltrate was diluted by adding 100 mL of ultrapure water. Te mineral concentration was determined by the inductively coupled plasma optical emission spectrometry (ICP-OES), (Agilent 5100, USA). Te nitrogen content was estimated by the Kjeldahl method [27].

Animals, Semen Collection, and
Processing. Five Boujaâd mature rams (weight: 80-85 kg, aged between 3 and 4 years) were used as semen donors. Tey were maintained at the station of animal reproduction biology at the Research Regional Center, Settat INRA-Morocco. A total number of 50 ejaculates were collected from the rams using an artifcial vagina, during the breeding season (July to September) and then the semen was pooled to minimize individual variation. After semen collection, the semen (10 µL) was placed on a glass slide without a coverslip, and the wave motion of semen was evaluated (0-5 scale) after judging fve diferent microscopic felds. Te sperm concentration was determined by a spectrophotometer. Ejaculates, which met the following criteria: the volume of 0.5-2 mL; good wave motion (≥3 on a 0-5 scale); ≥2.5 × 109 spermatozoa/mL; and ≥70% motile sperm were evaluated and used for the next step. All ejaculates were pooled to eliminate individual diferences and diluted with skim milk (SM) as the base extender. It was prepared by diluting skim milk (11 g) in distilled water (100 mL) and then heated at 95°C for 10 minutes. Penicillin and streptomycin (0.05 mg/mL) were added to the extender. Te semen was diluted with the base extenders at 37°C, containing 0 (control), 1, 2, 4, and 8% of ETHEX to reach a fnal concentration (0.8 × 109 sperm/mL) (single step dilution). Te semen samples were then cooled from 37 to 5°C. Sperm total motility, progressive motility, viability, abnormality, membrane integrity, and lipid peroxidation were determined at 8, 24, 48, and 72 h. Te combination of extender × antioxidant concentration giving the best protective efects on sperm progressive motility was selected to assess DNA fragmentation at 8, 24, 48, and 72 h using the tunnel technique.

Evaluation of Semen
Characteristics. Sperm total and progressive motility were assessed by a computer-assisted sperm analysis system (ISAS, version 1.0.17, Proiser, Valencia, Spain) by analyzing fve felds by sample (200 sperms/feld). Te semen was diluted with PBS-BSA to reach 20 × 106 sperm/mL at 37°C. Te sperm motility was assessed using a 10× negative phase contrast objective on a UB203 microscope (UOP/Proiser, Paterna, Valencia, Spain). Spermatozoa were defned as nonmotile if average path velocity (VAP) was lower than 10 µm/s and sperm cells were considered as progressive motile when VAP was higher than 75 µm/s and straightness index (STR) was higher than 80%.
For viability assessment, eosin-Y (1.67 g), nigrosin (10 g) and sodium citrate (2.9 g) were dissolved in distilled water (100 mL), and then nigrosin-eosin stain (10 µL), and sperm dilution (5 µL) were mixed on a glass slide [28]. Te mixture was smeared and examined with bright-feld microscopy (400x). A total number of 200 spermatozoa were counted. Sperm showing partial or complete purple heads were Veterinary Medicine International considered nonviable or dead, and only sperm showing strict exclusion of the stain were considered alive.
Te hypoosmotic swelling test (HOST) was used to evaluate the functional integrity of the sperm plasma membrane [29]. Te solution for the HOST assay consisted of fructose (9 g) and sodium citrate (4.9 g) per liter of distilled water. For sperm tail assessment, semen (30 µL) was mixed with hypoosmotic solution (300 µL, 100 mOsM) and incubated for 50-60 minutes at 37°C. After incubation, 10 µL of the mixture was spread with a coverslip on a warm slide. A total number of 200 spermatozoa were counted at least using fve diferent microscopic felds at 400x. Te percentage of spermatozoa with swollen and curved tails was recorded.
Te morphology of sperm was evaluated using the Dif-Quik kit (Diagnostic Systems S.L. Barcelona, Spain). Briefy, 3 µL of diluted semen was smeared on a glass slide and allowed to air-dry. Te slide was then dipped into a fxative solution for 1 min and the frst and second solutions seven to ten times. Between the fxing step and each of the staining steps, the excess solutions were dried from the slides by placing them vertically on absorbent paper. At least 200 sperms were evaluated under light microscopy at 1000x magnifcation using UB203 microscope (UOP/Proiser, Paterna, Valencia, Spain).

Measurement of Lipid Peroxidation. Malondialdehyde
(MDA) concentrations, as indices of lipid peroxidation (LPO) in the semen samples, were measured using the thiobarbituric acid reaction [30]. Te thiobarbituric acid reactive substances (TBARS) were measured in the semen spontaneous LPO or after incubation with 0.24 mM of FeSO4 at 37°C in a water bath for 60 minutes (iron-catalyzed LPO). Te TBARS concentration was determined by comparing the sample's absorbance at 532 nm with a standard curve prepared using MDA. Te results were expressed in nmol TBARS/10 8 sperm.

Assessment of DNA Fragmentation.
For the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique, the In-Situ Cell Death Detection Kit with fuorescein (Roche Diagnostics GmbH, Mannheim, Germany) has been used according to Nur et al. [31]. Briefy, diferent aliquots of semen samples were diluted with phosphate-bufered saline (PBS) and centrifuged at 400 × g for 10 minutes. One drop of resuspended spermatozoa was smeared on a glass slide and fxed with 10% formaldehyde for 30 minutes at room temperature.
Te slides were washed three times with PBS (5 minutes each), treated in a humidifed chamber with proteinase K for 10 minutes, washed with PBS, treated with 3% H 2 O 2 in distilled water for 10 minutes at room temperature, and washed again with PBS. Te slide was permeabilized with 0.1% Triton X-100 for 5 minutes on ice. Te slides were incubated in dark at 37°C for one hour with the TUNEL reaction mixture. After labeling, samples were washed with PBS and analyzed immediately using fuorescence microscopy (Zeiss Eurostar, Germany 100×). Te percentage of TUNEL-positive sperm was determined by the evaluation of at least 100 sperm.

Statistical Methods.
Te data were analyzed statistically using JMP15.0 (SAS Institute Inc., Cory, NC, USA) program. All dependent variables were submitted to the Shapiro-Wilk test normality and homogeneity analysis. Dependent variables with normal distribution were evaluated using variance analysis with the comparison of means using Tukey's test. Te results were expressed as the mean ± SEM (standard error meaning). Te diferences with values of p < 0.05 were considered to be statistically signifcant.

Radical Scavenging Activity.
Te scavenging ability of antioxidant substances was evaluated by the DPPH assay. Te results of the scavenging activities of ETHEX, ascorbic acid, and BHT are presented in Table 1. Te results indicate that the antioxidant activity of the ETHEX was higher than that of ascorbic acid and BHT in all tested concentrations. Tables 2-4, the major sugars presented in ETHEX were saccharose (2.85 mg·g −1 ) and glucose (2.67 mg·g −1 ). Te fructose content was found to be 0.56 mg·g −1 ( Table 2). As seen in Table 3, vitamins C, B2, B9, and B3 were detected in ETHEX. Among them, the major vitamin was B2 (1384 ± 5.04 mg/100 g), followed by vitamin C (963.1 ± 3.04 mg/100 g), and vitamin B3 (91.79 ± 0.19 mg/ 100 g). Te vitamin B5 was undetected in the ETHEX.

Microscopic Sperm Parameters.
Te efects of ETHEX on sperm motility, viability, membrane integrity, and abnormality of ram semen during diferent storage times at 5°C are presented in Figures 1-3.
Te efect of ETHEX on sperm total motility and progressive motility was signifcantly higher in SM supplemented with 1 and 2% compared to the control groups ( Figure 1). However, no diference was found between SM with 4% ETHEX and the control group (Figure 1).
Regarding viability, a signifcant diference was recorded between the control and the SM with 1 and 2% ETHEX (p < 0.05) after 48 h of storage ( Figure 2). In contrast, no diference was found between the control and other samples (SM with 4% and 8% ETHEX) (Figure 2). Whereas the enrichment of the basic extender with 1% ETHEX resulted 4 Veterinary Medicine International in a higher percentage of membrane integrity compared to the control groups ( Figure 2). Te results for abnormalities up to 72 h of incubation with diferent concentrations of ETHEX in SM are presented in Figure 3. Tese results showed that the addition of 1% of ETHEX to SM decreases the percentage of abnormality compared to the control (p < 0.05).

Malondialdehyde Concentration.
Te spontaneous and catalyzed lipid peroxidation levels in ram semen containing diferent concentrations of ETHEX for diferent storage periods at 5°C are given in Table 5. Te level of spontaneous and catalyzed LPO in control groups was signifcantly higher when compared to the experimental groups supplemented with 1 and 2% of ETHEX during the storage period at 5°C in SM. Te skim milk with 1% of ETHEX maintained a better efect when compared to the other concentrations. While no efect (p < 0.05) was observed when the concentration exceeded 2% compared to the control group.

DNA Fragmentation Levels.
Te efect of ETHEX on DNA fragmentation of liquid-stored ram semen at diferent periods is shown in Table 6. It was determined that using the selected concentrations was statistically signifcant in sperm DNA damage of ram semen during liquid storage. Te DNA fragmentation was signifcantly reduced, in comparison to the controls, with the addition of 1% ETHEX after 8 h of storage and 2% ETHEX to SM after 24 and 72 h of storage.

Discussion
To overcome the loss of semen quality during storage, several extracts from plants have been listed as natural antioxidants that could target such efects [32][33][34]. Even, to the best of our knowledge, there is no report in the literature on the supplementation of ram semen with ETHEX. Tus, the present study was carried out to test the ability of ETHEX at four concentrations (1, 2, 4, and 8%) to prevent the harmful efect of handling liquid ram semen in SM at 5°C up to 72 h. Te main fnding that emerged from this study was that the benefcial infuence of ETHEX resulted in higher overall quality of ram semen. Te inclusion of 1 and 2% ETHEX to the ram semen extended in SM at 5°C increases sperm motility, viability, and membrane integrity and decrease sperm abnormality. In contrast, concentrations above this threshold (2%) showed no signifcant results on sperm quality.
Te benefcial efects recorded while using 1% and 2% ETHEX are probably due to synergistic efects of multiple constituents of natural extracts and not to the single purifed active compounds [35]. Te ETHEX characterization showed a considerable radical scavenging activity exceeding that of BHT and ascorbic acid (Table 1). Besides, the benefcial efects of ETHEX on lipid peroxidation and DNA fragmentation were also recorded.
Numerous researchers have already pointed out that ETHEX contains an important antioxidant compound ranging from favonoids to phenols [36]. Some of them have the potential to act as scavengers, superoxidase, and hydroxyl and peroxy radicals released from oxidative phosphorylation. Together they can avoid the free radical's formation and protects the mitochondria, DNA, and plasma membrane of sperm [37][38][39]. Prevent oxidative damage could be achieved by a variety of diferent mechanisms, such as free radical scavenging, transition metal chelation and interactions with lipid membranes, proteins and nucleic acids [40]. In addition to the previously mentioned compounds, ETHEX contains sugars (glucose, fructose, and sucrose) and vitamins (C, B2, B3, and B9). Te inclusion of such compounds in extenders has been shown to improve semen quality [41,42]. More precisely, sugars and vitamins have benefcial antioxidative and protective efects against      the cold shock and freeze-thaw damage of sperm [43][44][45][46]. Sugars act as plasma membrane protectors and increase the integrity percentage in several animals' species [47,48]. Furthermore, the sugars provide the main energy source required for metabolic processes during spermatozoa development [49]. Regarding the vitamins, an improvement of ram semen quality observed in the current study could be attributed to the presence of Vitamin C. Previous studies stated the improvement of sperm viability and motility and a decrease in the percentage of abnormality [50,51]. Coming back to minerals, the characterization of the ETHEX revealed that it contained calcium, magnesium, sodium, and zinc (Table 4). Especially, zinc has been reported to protect spermatozoa against oxidative stress and subsequently improve male fertility [52]. Furthermore, Zn showed a positive outcome regarding antioxidant activity [53,54]. It plays an important role in controlling motility by controlling the use of energy through adenosine triphosphate systems and by regulating the energy stores of phospholipids [55]. Additionally, to zinc, calcium triggers the acrosomal reaction in mammalian spermatozoa and is also involved in sperm motility [56] and inhibits the enzyme phosphodiesterase, which prevents cAMP degradation and enhances sperm motility [57].

Conclusion
In conclusion, the fndings of the present study imply that supplementation of SM with ETHEX at 1 and 2% level has a benefcial efect on quality parameters of liquid ram semen stored up to 72 h, which may be due to the antioxidant efects of one or more active compounds presented in ETHEX.
However, additional studies are required to reveal the active components existing in ETHEX and test the impacts of this extract's inclusion in the freezing media and the success of AI in sheep.

Data Availability
Te data for this article are made available when needed.

Conflicts of Interest
Te authors declare that they have no conficts of interest. Te DNA fragmentation index (%) was analyzed for semen stored during 72 hours at 5°C in skim milk supplemented with selected ETHEX concentrations. Values are expressed as mean ± SEM. Values are expressed as mean ± SEM. Diferent superscripts (a,b) within the same column indicate a signifcant efect of the extender within each duration of storage.