Known as an aphrodisiac, Eruca sativa is a medical plant. In order to improve acceptability and reduce side effects, medicinal plants are a crucial component of complementary and alternative medicine treatment. Eruca sativa is regarded as a medicinal plant with numerous documented benefits in folklore management, including its potent aphrodisiac effects [1]. According to a number of studies, the presence of the flavonoids saponine and alkaloids in rocket extract enhanced spermatogenesis and significantly increased sperm activity [2,3]. Homady et al. [4], El-Missiry and El-Gindy [5], Hetta and Aly [6], Shalaby and Hammouda [7], Yehuda et al. [8] have reported hypoglycemic qualities of the plant. Diabetic properties have also been reported. 

Eruca sativa has been shown by Hussien et al. [9] to have strong antioxidant activity and improve liver function [10,11]. As far as we are aware, there is no evidence on how Eruca sativa affects the reproductive system of male oxidatively stressed rats. Therefore, the purpose of the current study was to find out how Eruca sativa leaves, a herbal plant, might enhance certain elements of male reproductive and hormonal profile in rats that were under oxidative stress due to hydrogen peroxide. Since frozen semen has extremely poor fertility, fresh semen is typically used for Artificial Insemination (AI) in tiny ruminants. 

The preservation of endangered species, the use of valuable sires' semen to address male infertility issues and the exchange of semen between subpopulations that may become biologically or geographically isolated are all made possible by sperm cryopreservation in conjunction with various Assisted Reproductive Techniques (ARTs), such as artificial insemination [12,13]. Since then, various extenders and freezing techniques have been reported in various animal species, particularly in the bull [14], goat [15-18] and ram [19,20] to reduce the negative effects of cryopreservation on sperm motility, viability, morphology [21], plasma membrane [22] and acrosome [23]. Artificial Vagina (AV) and Electro Ejaculation (EE) are two more popular methods of collecting semen from tiny ruminants.

Aims of Study

Given this, along with the fact that there isn't much information in the literature about the cryopreservation of Ram sperm using antioxidants (Eruca sativa) and how they affect the sperm's physical characteristics, the current study was conducted with the following objectives in mind: to find out how plant leaves of Eruca sativa can improve the quality of Ram sperm after the cryopreservation process; and to compare the effects of various concentrations of Eruca sativa of Ram sperm.

Experimental Animals

The collection of ram semen was place between November 2022 and March 2023. The rams utilized in the trials ranged in age from 2-4 years and in weight from 40-56 kg. As stated by Mishra et al. [24], semen was collected twice a week using an artificial vagina (AV; Minitube, Germany) after two consecutive false mounts. For a brief period, Rams were permitted to mount homosexually in order to ejaculate into an AV that was filled with warm (50-52°C) water. immediately upon collection, once a week for four months, with each ram yielding a total of two ejaculates. Straight into graduated test tubes, the semen was collected and kept in a thermo flask (which held water at 35°C). Next, the gathered semen was moved to the laboratory for one-hour semen processing and microscopic examination of sperm. The sperm motility of each ram was assessed using its raw, undiluted and diluted semen sample. The sperm motility analysis was conducted using the Computer Assisted Sperm Analysis (CASA) system [25]. Following the first assessment of the unprocessed semen samples, all ejaculates were combined and subsequently diluted using a citrate extender derived from egg yolk.

Preparation of Eruca sativa

An specialist in plant classification from a nearby medical plant market produced the Eruca sativa. The Awan et al. [26] methods were followed in the preparation of the aqueous extract. After being cleaned in distilled water, Eruca sativa were crushed and dried at 50°C. Subsequently, 50 mL of distilled water was combined with 10 g of powdered Eruca sativa and left for 15-20 minutes. After that, the mixture rested for half an hour. Following a 15 minute centrifugation (1,340×g). Two filtrations were done in order to improve accuracy. Before being used, the extract sterilization was kept for a few days at 4°C in specific sterile containers. Lastly, based on earlier research, the desired concentrations of the extract were created. Awan et al. [26].

According to Awan et al. [26], the concentrations of 1 and 2%, respectively contained 0.5 µL and 5 mL of extract and 1 µL and 5 mL of distilled water.

Experimental Design

Twelve of Ram's semen samples were split into three groups, T1, T2 and Control and they were treated as follows:

• Group C: Tris-citric egg yolk-glycerol (TCEG) is the only ingredient in the control group

• Group T1: Dilute 5 mL of semen and add 0.5 µL of Eruca sativa extract

• Group T2: Dilute 5 mL of semen and add 1.0 µL of Eruca sativa extract

Assessing the Quality and Collecting Semen

Collection of Semen: An artificial vagina was used to help gather the semen from each individual ram. Semen was extracted from one ejaculate, immediately transferred into five milliliter tubes and stored at 35°C in a thermo flask. Within one hour, the ram semen sample was collected and sent to the lab for microscopic sperm analysis. First, the computerized Sperm Class Analyser® (CASA system) was used to assess the raw semen samples macroscopically for the ejaculate Spermatozoa parameters (motility and velocity).

Assessment of Sperm Motility with the CASA System

Sperm motility was analyzed using the Computer Assisted Sperm Analysis (CASA) system with assistance from a Sperm Class Analyzer®-SCA® (V.4.0.0.1 Animal/ Veterinary Microptic S.L, Barcelona, Spain). 5 μL of a sample was put on a pre-warmed microscope slide, covered with a pre-warmed cover slip (done for each sample) and then subjectively loaded under a phase-contrast microscope using a sperm analyzer to assess the sperm at a 10X magnification [27]. The proportion of motile sperm, curved (VCL), straight-line (VSL), average path (VAP), linearity (LIN), straightness (STR) and wobble (WOB) velocities were the motility characteristics that were examined. The smoothed trajectory of the sperm head is crossed at the Beat Cross Frequency (BCF).

Storing Ram Semen in Liquid

Semen samples were diluted 1:1 and used in experiments to compare different storage times for the egg yolk-citrate extender. For the experiment, the ultimate glycerol inclusion level was 4.7% due to the egg yolk-citrate diluent comprising 14% diluted with 1:1, v/v of fraction A. Evaluations of sperm motility and velocity were conducted after 24, 48, 72, 96 and 120 hours of storage cooling, as well as after 10 days and 1 month of freezing.

Freezing of Semen

Following the laboratory inspection of the semen under a microscope, the semen samples were diluted 1:1 with Egg Yolk Citrate (EYC) and cooled to 5°C for 24, 48, 72, 96 and 120 hours. Then, after the semen samples had cooled, they were put into 0.5 mL straws and frozen. To do this, they were positioned 5 cm above liquid nitrogen (LN2) for ten minutes, after which they were submerged in LN2 and kept in an LN2 storage tank at -196°C for later analysis.

Semen Thawing in Preparation for Post-Thaw Analyses

After being cryopreserved, semen straws were thawed by immersing them in a water bath at 37°C for 30 seconds, which was done 10 days and 1 month later and Purdy. Semen was transferred into 15 mL test tubes after the sealed straws' two ends were chopped off with scissors. After diluting 10 μL of semen, the semen samples were assessed similarly to raw semen in terms of sperm motility and velocity parameters.

Examinations of Statistics

The Statistical Application Program's approach was used to an ANOVA using data from version 22 (SPSS 22). The percentage of motile sperm, curvilinear (VCL), straight-line (VSL), average path (VAP), linearity (LIN), straightness (STR) and wobble (WOB) velocities, beat cross frequency (BCF) and abnormal sperm were judged to be significant at p<0.05. These differences were expressed as the Mean±Standard error (SE) [28].

Storage Period (24 h) 

The CASA system was used to measure the rams' sperm motility and velocity rates, which are shown in Table 1. The percentage of motile sperm in total varied from 99.42±0.065 to 93.91±0.37% in this investigation. Ram as a whole had an impact on the total amount of motile sperm, however the T2 group generally recorded higher sperm motility rates than the control group. After a 24 hour cooling period, the results indicated that T2 had the most significant value (p<0.05) at 99.42±0.065%%), while C had the lowest significant value (p<0.05) at 93.91±0.37%. This was in contrast to T1's 96.48±0.075%). Between the values T1 and T2 on the ALH (2.78±0.021 and 2.69±0.080) and WOB (75.39±0.66 and 78.80±0.95) Table 1. There was no statistically significant difference.In addition, the CASA system examined the linearity (LIN) and aberrant sperm and found that there was no significant difference between the values T1 and T2 (58.56±0.28, 61.28±0.69 and 2.98±0.094, 2.92±0.020) when compared to other controls.

Storage Period (48 h)

Following 48 hours of cooling, the CASA system measured the sperm motility and velocity characteristics of the ram semen, which are presented in Table 2. T1 and T2's total motile sperm percentages (86.28±0.050 and 89.97±1.47) were impacted by the proportion of Eruca sativa concentration inclusion. During 48 hours of storage, the present trial's VAP (average path velocity) and VSL (straight-line velocity) were measured using the CASA system to determine the impact of storage temperature (5°C) between T1, T2 and control.

Sperm that had been held for 48 hours had VAP and VSL values that were considerably (p<0.05) higher at T2 (45.94±1.02 and 37.32±0.53) than at T1 (33.88±0.93, 18.40±0.054) and control (38.66±0.85, 28.48±1.29). The control group exhibited a significantly larger percentage of LIN (linearity), WOB (wobble), ALH (amplitude of lateral head displacement) and BCF (beat cross frequency) features at 48 hours of storage as compared to T2 and T1.

A numerically (statistically) larger percentage of control (57.49±1.09%) was obtained from the semen held for 48 hours in LIN (linearity) compared to T2 (45.38±0.56%) and T1 (37.43±0.085%). Significant variations exist in the percentage of abnormal sperm across all treatments, with T2 and T1 values being non-significant (p<0.05) and the control group having the highest significant (p<0.05) value (3.50±0.062%) display as seen in Table 2.

Table 1:  Effect of Different Eruca sativa Concentrations During Cooling on Ram Semen Sperm Motility and Velocity Parameters as Determined by CASA after 24 hours (Mean±SE)

Curvilinear velocity is represented by VCL, Straight-line velocity by VSL, Average path velocity by VAP, Linearity by LIN, Straightness by STR, Wobbling by WOB, Amplitude of lateral head displacement by ALH and beat cross frequency by BCF, *A and B Superscript differences between values in a row indicate a significant difference (p<0.05)

Table 2:  Effect of Different Eruca sativa Concentrations During Cooling on Ram Semen Sperm Motility and Velocity Parameters as Assessed by CASA after 48 hours (Mean±SE)

Curvilinear velocity is represented by VCL, Straight-line velocity by VSL, Average path velocity by VAP, Linearity by LIN, Straightness by STR, Wobbling by WOB, Amplitude of lateral head displacement by ALH and beat cross frequency by BCF, *A and B Superscript differences between values in a row indicate a significant difference (p<0.05)

Table 3: Effect of Different Eruca sativa Concentrations During Cooling on Ram Semen Sperm Motility and Velocity Parameters as Assessed by CASA after 72 hours (Mean±SE)

Curvilinear velocity is represented by VCL, Straight-line velocity by VSL, Average path velocity by VAP, Linearity by LIN, Straightness by STR, Wobbling by WOB, Amplitude of lateral head displacement by ALH and beat cross frequency by BCF, *A and B Superscript differences between values in a row indicate a significant difference (p<0.05)

Table 4: Effect of Different Eruca sativa Concentrations During Cooling on Ram Semen Sperm Motility and Velocity Parameters as Determined by CASA after 96 hours (Mean±SE)

Curvilinear velocity is represented by VCL, Straight-line velocity by VSL, Average path velocity by VAP, Linearity by LIN, Straightness by STR, Wobbling by WOB, Amplitude of lateral head displacement by ALH and beat cross frequency by BCF, *A and B Superscript differences between values in a row indicate a significant difference (p<0.05)

Time of Storage (72 h)

After storing semen for 72 hours at 5°C, there was a non-significantly (p<0.05) difference in WOB between control and T1 (54.53±0.53, 55.56±0.60%) compared to others in T2 (43.38±0.103%). After being held for 72 hours at 5°C, the amount of motile sperm in T1 (76.23±o.53%) increased to T2 (84.86±0.44%) when semen was added. Table 3. The VAP (average path velocity), VSL (straight-line velocity) and curvilinear (VCL) data demonstrated that T2 had the most significant (p<0.05) value, while T1 and control had the lowest non-significant (p<0.05) values.

According to the data, T2 had the lowest significant value (p<0.05) at 29.47±0.06%, whereas the control group had the highest significant value (43.40±0.131%) and T1 had the lowest (37.43±0.085%) (Table 3). The results showed that there was no significant difference (non-significant) between the control group (67.43±0.076%) and the test group (67.25±0.19%). The highest significant value (p<0.05) was found in T1 (65.51±0.087%) (Table 3). Based on the data, it was shown that there was no significant variation in the abnormality percentage of sperm between T1 (3.48±0.08%) and T2 (3.25±0.55%).

Time of Storage (96 h)

After 96 hours of storage, the measurable properties of the semen were recorded. Again, the percentage of VCL, VSL, STR, WOB and BCF characteristics for semen preserved at control was only numerically larger than that of T1 but the differences were not statistically significant. In comparison to the other control group, T2 had a larger percentage of total motile and VAP (83.56±0.37%, 23.09±0.43) while T1 had a higher percentage (74.87±0.35%, 17.96±0.31) of both. After cooling for 96 hours, the values of the Abnormality percentage with varying doses of Eruca sativa were as follows: Control (10.21±0.38%), T1 (7.16±0.29%) and T2 (4.10±0.17%), as shown in Table 4. In comparison to other T1 (7.16±0.29%), the control group (10.21±0.38%) had the greatest significant (p<0.05) value, while T2 (4.10±0.17%) had the lowest (p<0.05) value.

Table 5: Effect of Different Eruca sativa Concentrations During Cooling on Ram Semen Sperm Motility and Velocity Parameters as Determined by CASA after 120 hours (Mean±SE)

Curvilinear velocity is represented by VCL, Straight-line velocity by VSL, Average path velocity by VAP, Linearity by LIN, Straightness by STR, Wobbling by WOB, Amplitude of lateral head displacement by ALH and beat cross frequency by BCF, *A and B Superscript differences between values in a row indicate a significant difference (p<0.05)

Table 6: Effect of Different Eruca sativa Concentrations at Freezing on Ram Semen Sperm Motility and Velocity Parameters as Determined by CASA after 10 days (Mean±SE)

Curvilinear velocity is represented by VCL, Straight-line velocity by VSL, Average path velocity by VAP, Linearity by LIN, Straightness by STR, Wobbling by WOB, Amplitude of lateral head displacement by ALH and beat cross frequency by BCF, *A and B Superscript differences between values in a row indicate a significant difference (p<0.05)

Table 7: Effect of Different Eruca sativa Concentrations at Freezing on Ram Semen Sperm Motility and Velocity Parameters as Determined by CASA (Mean±SE) after One Month

Curvilinear velocity is represented by VCL, Straight-line velocity by VSL, Average path velocity by VAP, Linearity by LIN, Straightness by STR, Wobbling by WOB, Amplitude of lateral head displacement by ALH and beat cross frequency by BCF, *A and B Superscript differences between values in a row indicate a significant difference (p<0.05)

Following a 120 hour storage period for semen, T2 had considerably (p<0.05) larger proportions of total motile or VAP (average path velocity), at 71.98±0.83% and 12.25±0.17, than both Control and T1 (51.14±0.62%, 62.65±0.84%, 8.14±0.13 and 10.10±0.15). Between T1 and T2, there were no discernible variations in the percentages of VCL, LIN, STR, ALH, BCF and abnormal sperm features (Table 5). Values recorded generally tended to be greater for the semen preserved at T2 than at T1. This is because it's thought that sperm cells maintained at 5°C lose their motility while still remaining viable. Although a linear decline in semen quality is typically anticipated with time, the pattern that the sperm motility rates followed throughout time was not constant.

Ten Days is the Storage Duration

In Table 6 presents a comparison of the CASA-measured sperm motility and velocity characteristics before and after freezing and thawing. Overall, the freezing process affected every sperm motility characteristic, with T2 semen freezing at a higher rate than any other. Sperm frozen in control had significantly (p<0.05) lower VSL, VAP, LIN, ALH (amplitude of lateral head displacement) and BCF than semen frozen with 0.5 and 1 µL. Nonetheless, all of these differences were significant. T2 had a greater proportion of total motile sperm (91.69±0.468%) than T1 (86.07±0.53%) and control (75.90±6.17%). It is important to include Eruca sativa in the semen extender added to sperm because the proportion of progressive motile sperm cells showed the same tendency, as shown in Table 6. After thawing, the findings for WOB (wobble) and abnormality percentage showed that the values in T2 (68.74±1.12%, 5.05±0.071) and T1 (64.29±0.33%, 5.78±0.089) were non-significant (p<0.05) when compared to the other control group Table 6 the findings of this investigation.

One-Month Storage Duration

Table 7 presents the total motility percentage values at different concentrations of Eruca sativa following post-

thawing. The results indicate that the control group (38.65±3.09%) had the lowest significant values (p<0.05) compared to the other group (t1) (±63.830.54%). The highest significant value was found in T2 (±77.100.67%).

The findings of the post-thawing analysis showed that the control Table 7 had the lowest significant values of VCL curvilinear velocity, VAP average path velocity, WOB wobble, ALH amplitude of lateral head displacement and abnormality %, while T2 and T1 had the lowest non-significant values of these variables. In contrast to the other control group, T2 had the highest significant value (p<0.05) for LIN linearity and STR straightness (±74.200.48 and±52.100.35%), whereas T1 had the lowest significant value (p<0.05) for these two characteristics (±64.090.67 and±41.240.63%) (Table 7).

Our findings demonstrated a substantial difference in sperm motility at various times (24, 48, 72, 96 and 120 hours) of storage at 5°C between the T1 and T2 groups and the control group. Leaf extracts are thought to raise testosterone hormone levels, boost sperm activity and lessen sperm death and abnormalities [29,30]. The presence of glucosinolates (Erucin is the main glucosinolate) and other stimulant materials in Eruca sativa has been shown by Al-Tohamy et al. [31] to have several biological activities and may be able to shield cells from oxidative stress, which improved the fertility and semen characteristics in male rabbits [32,33].

Ansari and Ganaie [34] state that Eruca sativa's medicinal and therapeutic characteristics are utilized as a "aphrodisiac" to enhance sexual activity for both sexes by promoting fertility and sperm production. When Reactive Oxygen Species (ROS) are produced at an unbalanced rate and the antioxidant system is not working properly to neutralize and eliminate them, the result is oxidative stress [35]. The presence of vitamins E and C in Eruca sativa is the cause of these outcomes [36]. This could be because vitamin E is the primary antioxidant system component of spermatozoa and is essential in shielding the sperm membrane from lipid peroxidation and reactive oxygen species because it contains high amounts of polyunsaturated fats (PUFA) in the phospholipids [37,38]. Sitohang et al. [39] stated that vitamin E is the most potent antioxidant in their research because it is fat-soluble and crucial because cell membranes and low-density lipoproteins, which are both composed of fat molecules, sustain the majority of the damage caused by free radicals. 

Lubis and associates, in order to avoid damage to lipid peroxidation, which affects the viability, motility and fertility of spermatozoa, vitamin C can fend against free radicals and prevent the occurrence of a free radical reaction chain. Alkaloids and saponins in the (Eruca sativa) extract increase sperm activity [29,30]. Eruca sativa has flavonoids in it. According to Caamaño et al., taxifolin is advantageous as a plant flavonoid for cryopreserving goat semen. Concentrate supplementation increased sperm production and semen quality in Sardinian rams, according to Tufarelli et al. [40]. In this investigation, both groups' mean values were within a conventional range. Moreover, it was noted that feeding rams, buck and rabbit buck [41-44]. With supplements of Eruca sativa enhanced the quality of their semen. The observations made by Barillari et al. [3] and Hussein [45], who observed that Eruca sativa stimulated sperm activity in rats, are consistent with our findings. Although the motility of spermatozoa in the control group was lower than in the supplemented group, our data demonstrate that sperm motility was adequate until 120 hours in both groups (Table 3) Even after Eruca sativa addition, cryopreservation generally results in a decrease in sperm motility and viability after injury (irrespective of the inclusion level). Fresh ejaculate sperm cells often have a high rate of metabolism for a few hours before becoming viable. The metabolic rate will rise at a high temperature and this will shorten the lifespan of sperm cells. The sperm were 37°C, the typical physiological temperature. Low sperm motility and a large percentage of immotile sperm detected before freezing are signs of this. The current study's results concurred with those of Dorado et al. [46], which demonstrated that a significant percentage of sperm cells lost their motility during chilling, with various percentages of 93.91±0.37%, 86.24±2.41, 72.35±0.630, 56.37±0.58 and 51.14±0.62%. Because of this, CASA has been utilized more frequently in domesticated animal production facilities to evaluate the quality and function of sperm, including quality control of ram semen sold for artificial insemination [47,48]. Sperm VCL, VSL, VAP, ALH and STR are indications of potential fertility, hence most researchers concentrate their findings on them. According to Verstegen [25], there is a substantial correlation between ALH and sperm-oocyte fusion and reflects the intensity of flagellar beating, which influences the result of in vitro fertilization.

The general characteristics of ram semen and the quality of the semen after adding Eruca sativa were revealed by this investigation. Sperm motility results that were unbiased, trustworthy and provided by the CASA system. The majority of the sperm motility and velocity characteristics showed a substantial change upon cooling and post-thawing in semen that had been diluted with an Eruca sativa extender.

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