Evaluation of Soy Lecithin Efficacy in Comparison with Egg Yolk on Freezing of Epididymal Sperm in Dogs

Introduction

High economic value, zootechnical, and affective character of some individuals increase the advances of reproductive biotechnologies for future preservation (Thomassen & Farstad 2009). In the case of azoospermia or when a donor male accidentally dies or undergoes orchiectomy, the retrieval of epididymal spermatozoa opens new possibilities to generate progeny. Spermatozoa can be collected by different techniques from ex vivo or in vivo testicles and cryopreserved for future use (Luvoni & Morselli, 2017).

Collecting sperm from the epididymis allows the use of genetic material post-mortem or after orchiectomy from high-value animals or endangered species (Ortiz et al., 2017), and there are several situations in which epididymal sperm for artificial insemination may be used. The most obvious reason for AI is the perceived inability of the male and female to breed (for example: weakness, arthritis, back pain, premature ejaculation, etc.). For many years Egg yolk was widely used as a cryoprotectant in dog semen extenders. Still, there are some concerns and risks with the use of egg yolk, including the risk of bacterial contamination and the potential risk of causing disease (Hermansson, Johannisson & Axnér, 2021). Soy lecithin is a promising option for egg yolk substitutes due to ease of component standardization, availability, and a reduced potential risk of contamination. It has a similar composition (i.e., low-density lipoprotein) as egg yolk and may provide protection to the sperm plasma membrane during cold shock (Dalmazzo et al., 2018). Some of the studies have shown the effect of soy lecithin as a suitable alternative to egg yolk on ejaculated sperm in dogs (Beccaglia, Anastasi & Luvoni, 2009^a; Beccaglia et al. 2009^b; Kmenta et al., 2011; Kasimanickam et al., 2012), and so far, its effect on epididymal semen in dogs has not been studied. The aim of the present study was to compare five different concentrations of soya bean lecithin, with egg yolk as a control, in tris extender for cryopreservation epididymal sperm of dogs.

Materials and Methods

Soybean lecithin (L-a-phosphatidylcholine (product number: P3644)) in this study was prepared from Sigma (St. Louis, MO, USA), and other chemicals were purchased from Merck (Darmstadt, Germany). Straws used were from IMV Co. (France).

Animals

Ten intact male dogs aged between 1 - 8 years old, with different breeds (Golden Retriever, Pomeranian, Terrier, Shitzu) and body weights (between 3 kg and 25 kg were used for this study. The dog testes were temporarily preserved at 4°C in a plastic can filled with a physiological solution (0.9% saline solution) supplemented with gentamycin at a concentration of 10mg/mL, a physiological solution. The testes were processed within 2 hours of castration.

Sample Collection

Sperm samples were collected by repeated incision of the epididymal tail and proximal vas deferens in extruded (tris-based) medium without glycerol at 37°C. Sperm motility (total and progressive) was assessed by microscopic examination. Briefly, 5 µL of each sample was deposited on microscope slides previously warmed at 37°C and covered by coverslips, after that Eosin-Nigrosin staining and HOST (Hypo Osmotic Swelling Test) were performed. Sperm concentration was assessed using a standard counting chamber (Neubauer Lam); the final concentration of sperm was (50 × 10 ⁶ /mL). The extenders (Tris-egg yolk and tris-lecithin) with glycerol were prepared and put in the fridge at 4°C. The extender tris-lecithin with glycerol was vortexed for 30 minutes. Sperm extracted from the testes was divided into six groups: G₁: egg yolk 20% (control), G₂: lecithin 0.4% (L0.4), G₃: lecithin 0.8% (L0.8), G₄: lecithin 1.2% (L1.2), G₅: lecithin 1.6% (L1.6), and G₆: lecithin 2% (L2). Then, the extender and spermatozoa mixer were transferred to the beaker (containing 500 mL of water at 25°C) and stored in the refrigerator for 2 hours at 4°C for cooling. Then extended sperm was loaded into 0.5 mL straws. Samples with motility <50% were removed from the study.

Extender Preparation

The basic extender was made of TRIS-buffer (3.025 g + citric acid 1.7 g + fructose 1.25 g + Penicillin 100IU/ mL +Streptomycin 100 µg /mL, which was added to 100 mL of distilled water). The pH was set at 6.8-7. Then egg yolk 20% or different concentrations of lecithin (0.4, 0.8, 1.2, 1.6, and 2%) was added to the medium. Glycerol 7% was added to Tris buffer at one step.

Freezing and Thawing

After two hours of refrigeration, the samples reached 4°C. They were placed in the vapor of liquid nitrogen (4 cm above the liquid nitrogen) for 10 minutes to reach -102°C. Straws were subsequently plunged into liquid nitrogen and stored until thawing. Straws were placed in a warm water bath at 37.5°C for 30 seconds for thawing. The sperm released from each straw was stored in a test tube for 5 minutes at 37.5°C. Then, all tests (CASA, Eosin- Nigrosin staining, HOST, MMP, ROS) were carried out on freeze-thawed spermatozoa.

Tests on Spermatozoa

Eosin-Nigrosin Staining

Eosin- nigrosin staining was performed to determine the vitality and morphology of sperm. Briefly, an aliquot of semen (5 µL) was placed on a slide and mixed well with (5 µL) eosin stain 1% for 30 seconds. Then, 5 µL of nigrosin stain 10% was added for 30 seconds on a 37°C heat plate, and a smear was made on a microscope slide. Finally, slides were examined under oil by a lens (×100) of Nikon –Japan microscope.

Hypo-Osmotic Swelling Test (HOST)

The hypo-osmotic swelling (HOS) is prepared by mixing 75 mmol of fructose and 25 mmol of sodium citrate with distilled water. Briefly, 25 µL sperm thawed + 25 µL (HOS) solution in a microtube and incubate semen/HOS solution mixture for at least 30 min, at 37°C. Then 5 µL of the mixture was placed on a warm slide and mounted with a coverslip. The sperms (n=200) were evaluated using a phase-contrast microscope, and sperms with coiled tails were recorded (Ramu & Jeyendran, 2013).

Computer Assisted Semen Analysis (CASA)

Sperm motility was evaluated with a Sperm analyzer CEROS II™ Hamilton Thorne. Five micro-liters of semen samples were pipetted onto a warm microscope slide and a coverslip placed on top. Sperm motility was analyzed in eight fields using a software program (Sperm Vision) with settings adjusted for dog spermatozoa. Total motility and the following parameters were evaluated: VCL (track velocity), VAP (path velocity), VSL (straight line velocity), LIN (linearity), STR (straightness), BCF (beat cross frequency), and ALH (amplitude of lateral head displacement).

Mitochondrial Membrane Potential (MMP):

MMP was evaluated using JC-1, a lipophilic cationic dye. JC-1 in spermatozoa with high MMP forms aggregates emitting red fluorescence, while in spermatozoa with low MMP remains as monomers emitting green fluorescence. Initially, semen samples were centrifuged for 5 minutes at 500 × g. After removing the supernatant, the spermatozoa were diluted with phosphate-buffered saline at the concentration of 1 × 10⁶ sperm per mL. Then, 1 mL of JC-1 (200 mmol dissolved in DMSO; Sigma-Aldrich, MO, USA) was added to 1 mL of the diluted sample, which was further incubated at 38°C for 40 minutes. Green and red fluorescence of JC-1 was monitored with FL1 (530 nm) and FL2 (585 nm) detectors, respectively (Akbarinejad et al. 2018).

Evaluation of intracellular ROS (Reactive Oxygen Species)

Frozen–thawed semen was re-suspended with phosphate-buffered saline (PBS) at a final concentration of 1-3×106 mL spermatozoa. The intra-cellular ROS was determined by 2,7-dichlorodihydro fluorescein diacetate (DCFH-DA) (25 μm), separately added to 1–3×106 sperm/mL fractions and incubated at 25°C for 40 min, respectively, in the darkroom. Each sample was analyzed using a flow cytometer with a 488 nm argon laser (Becton Dickinson FACScan, San Jose, CA, USA). Green fluorescence of DCFH-DA (500–530 nm) was evaluated with excitation wavelength at 488 nm and emission wavelength at 525–625 nm in the FL-2 channel. Propidium iodide PI was used as a counterstain dye for DCFH to distinguish dead sperm. Data were expressed as the percentage of fluorescent spermatozoa.

Statistical Analysis

All data were evaluated using GLM procedure. The LSMEANS statement was used to perform multiple comparisons. All analyses were conducted in SAS version 9.4 (SAS Institute Inc., Carry, NC, USA). Differences at P-value< 0.05 were considered statistically significant.

Results

Fresh Epididymal Sperm Assessment

The maximum and minimum limits of spermatozoa's initial and progressive motility were 90-95 and 85-80, respectively. The average percentage of total and progressive motility were 93.3 ± 2.9 and 83.3 ± 2.9, respectively. Initial values for the HOST and Eosin-Nigrosin were 88.1 ± 2.9, 82.7 ± 2.5 respectively. Table 2 shows the evaluation of motility, HOST and Eosin-Nigrosin tests. Table 3 shows the morphology of the epididymal sperm.

Table 1. Components of extender Tris – egg yolk, Tris- lecithin

DW: Distilled water

Table 2. Assessment of fresh epididymal spermatozoa

Table 3. Morphology of epididymal sperm before freezing.

Freeze-thawed Epididymal Sperm Assessment

Motility of freeze-thawed spermatozoa

In general, the values of total and progressive motility decreased post-freezing-thawing in cryoprese-rved samples in all treatment groups compared with the fresh semen specimen (P<0.001). The best total and progressive motility of sperm after thawing among groups were in the egg yolk group (46 ± 8.5, 26.3 ± 15.8, respectively). However, among the soy-lecithin groups, the higher total and progressive motility were seen in L0.4. In frozen samples, total and progressive motility of spermatozoa were lesser in all groups treated with different concentrations of lecithin than in the control group (P≤0.05). Table 4 shows the evaluation results of the motility of spermatozoa between groups after freezing and thawing.

Table 4. Evaluation of the motility of sperm among groups before freezing and after thawing

BF: Before freezing, AF: After Freezing, TM: Total Motility, PM: Progressive Motility. ^ab Significant differences within column for each parameter.

HOST After Thawing

The proportion of sperm with intact plasma membrane integrity based on the HOS test decreased the following cryopreservation in all treatment groups compared with the fresh semen specimen (P<0.001). The percentage of sperm with the plasma membrane integrity between groups is illustrated in Table 5. The egg yolk extender was able to preserve the integrity of the plasma membrane of the spermatozoa better than the other groups (65.1 ± 8.1). However, L 0.4 achieved better results than the different concentrations of lecithin in other groups (18.4 ± 3.4), the egg yolk was the best.

Table 5. Hypo Osmotic Swelling Test after thawing

BF: Before freezing, AF: After Freezing. ^ab Significant differences within a row for each parameter.

Eosin- Nigrosin Staining After Thawing

The percentage of live sperm among groups was listed in Table 6. The results showed the percentages of live sperm in groups L 0.4, L 0.8, egg yolk was 52.5±16.2, 46.1±11.3, 43.2±10.4, respectively; there was no significant difference among them. The proportion of sperm with morphological defects did not differ between fresh and frozen samples and among various experimental groups (P>0.05). The results are listed in Table 7.

Table 6. Eosin-Nigrosin test after thawing.

BF: Before freezing, AF: After Freezing. ^ab Significant differences within row for each parameter.

Table 7. Morphology of spermatozoa before freezing and after thawing

^aa No significant differences within column for each parameter.

CASA

CASA analysis was performed on frozen-thawed semen samples to determine the effect of soy lecithin and egg yolk on motility parameters (Tables 8 and 9). Parameters of VAP, VSL, VCL, STR, LIN, and ALH did not differ among various experimental groups (P>0.05). However, BCF was higher in all groups treated with different concentrations of lecithin compared with the control group (P≤0.05).

Table 8. Assessment of motility in freeze-thawed spermatozoa by CASA

^ab Significant differences within column for each parameter.

Table 9. Evaluation of motility in freeze-thawed spermatozoa by CASA

^aa No significant differences within column for each parameter.

MMP

The MMP level of spermatozoa in EY or LS 0.4 after the post-thawing was presented in Figure 1. A comparison was made between the control and LS 0.4 groups, which revealed better results in the control group than in the lecithin groups. Depending on the dye JC-1, the results showed a significant difference between the control and LS 0.4 group (P=0.026).

ROS (Superoxide) Production

The proportion of sperm positive for ROS in EY or LS 0.4 after thawing was presented in Figure 2. The level of H₂O₂ was higher in LS 0.4 than in the control group (P=0.049). Production of hydrogen peroxide decreased in the EY group after freezing-thawing compared with LS 0.4.

Figure 1. Mitochondrial membrane potential after thawing

Figure 2. Reactive oxygen species (ROS) after thawing

Discussion

Egg Yolk is not a defined entity but a complex biological compound containing proteins, vitamins, phospholipids, glucose, and antioxidants which are all potentially useful for cell membrane integrity. Unfortunately, it is also a biologically hazardous compound (Farstad, 2009). Research in recent years has focused on finding a suitable alternative to egg yolk from a non-animal source for global trade. Since lecithin in egg yolk plays a crucial role in preventing cold shock, and the latter can be obtained from vegetable sources such as soy and sunflower, the researchers focused on finding the ideal type and concentration of lecithin to protect sperm from cold shock. Egg Yolk extender has been shown efficient for maintaining a good level of total and progressive motility of sperm as well as the integrity of the plasma membrane, while soy lecithin did not provide the same level of efficacy. When analyzing the movement parameters (VAP, VSL, VCL, STR, LIN, and ALH) according to CASA, there was no significant difference between the experimental and control groups except for the BCF values, which were high in all the experimental groups compared to the control group. This indicates severe damage to the plasma membrane of the sperm, which results in severe tail injuries. Soy lecithin 0.4 and 0.8, respectively, had the highest total and progressive movement ratios (31 ± 16.4, 19.3 ± 14.4/ 22 ± 7.2, 13 ± 6.1) among the rest of the different concentrations of lecithin. Soy lecithin, in different concentrations, was able to maintain a good percentage of sperm life; in contrast, it had a very negative impact on the integrity of the plasma membrane, as the percentage of sperm with a healthy plasma membrane was very low. In general, the results of lecithin 0.4 % were superior to the rest of the lecithin concentrations, and the groups L 1.6 and L2 had the worst results. Assessment of ROS level and mitochondrial activity in the present study revealed that the adverse effect of lecithin on canine sperm during cryopreservation was at least partly due to excessive elevation of ROS and impairment of mitochondrial function in sperm treated with lecithin compared with sperm treated with egg yolk. Sperm generate the physiological amounts of reactive oxygen species (ROS) that are important for sperm capacitation, acro-some reaction, and the ability to fertilize the oocyte (Agarwal et al., 2006). However, when the ROS production is excessive, an imbalance occurs between the ROS-generating system and enzymatic and non-enzymatic antioxidants responsible for ROS removal, which leads to oxidative stress. This type of stress causes structural damage to biomolecules, DNA, lipids, carbohydrates, proteins, and other cellular components, including mitochondria (Dalma-zzo et al., 2018), and it may also compromise both the genetic integrity and the fertilizing capacity. There are several companies that extract lecithin, whether from soybeans or sunflowers, and there are several concentrations of it, and some of it is used for research and other nutritional purposes. For example, Sigma company has soy lecithin in many forms: (P3644, P5638, P7443, P3782), and other companies such as Swanson Health Products, Fargo, ND, USA, Minitube®, Tiefenbach, G), Solae Company, St.Lo-uis, MO, EUA, General Nutrition Corporation, Pittsburgh, PA. Differences have been found among canid species in the ability of their spermatozoa to withstand freezing. There are differences in sperm membrane fatty acid composition among species, which may explain part of these differences. Suppose the presence of long-chained polyunsaturated fatty acids contributes to increased membrane fluidity. In that case, this relationship may be biphasic, i.e., either too much membrane fluidity or too little could compromise successful sperm cryopreservation. An increase in fluidity of the outer leaflet of the plasma membrane has been shown in frozen-thawed dog spermatozoa (Farstad, 2009).

The results of this study were consistent with several studies which studied the effect of lecithin on the ejaculated semen of dogs (Axnér and Lagerson 2016; Dalmazzo et al., 2018; Hermansson, Johannisson & Axnér, 2021) and opposed to some of other studies which also studied the effect of lecithin on the ejaculated semen of dogs (Beccaglia, Anastasi & Luvoni, 2009^a; Beccaglia et al., 2009b; Kmenta et al., 2011; Kasimanickam et al., 2012; Sánchez-Calabuig et al., 2017; Zakošek Pipan et al., 2020). Researchers have studied the role of soy lecithin in protecting against cold shock among different animals and found different results. The results of some researchers' studies have shown the positive effects of soy lecithin on protecting sperm from cold shocks, such as in cats (Vick et al., 2012; Vansandt et al., 2021), in goats (Salmani et al., 2014), in rams (Forouzanfar et al., 2010), in bulls (Aires et al., 2003). Also, some other studies have shown the negative effects of Lecithin such as in buck (Sarıözkan et al., 2010; Roof et al., 2012; Salmani et al., 2013; Tabarez, García & Palomo, 2020), in black rhinoceros and Indian rhinoceros (Wojtusik, Stoops & Roth, 2018), in Japanese white rabbits (Nishijima et al., 2015), in brown bears (Alvarez-Rodríguez et al., 2013). For epididymal sperm in dog Nöthling et al. (2007) studied the effect of adding prostate fluids to frozen epididymal sperm in dogs and compared it with two prepared extenders (BilEq & Andromed) and found that BilEq extender was more suitable than Andromed as a freezing medium for epididymal sperm in dogs. Also, prostate fluids should be added before freezing of epididymal spermatozoa extended in BilEq and after thawing because such addition results in better motility, longevity, and sperm morphology (Nöthling et al., 2007). Lopes et al. (2015) compared the Tris-egg-yolk-glycerol extender with the commercial extender AndroMed for freezing of epididymal sperm in bulls and they found that the egg yolk extender was superior to the commercial extender AndroMed (Lopes et al., 2015). Other researchers applied nanotechnology on soy lecithin to reduce the size of its particles and used it as a nano extender for cryopreservation of animal semen; it had a positive effect, for instance, in bulls (Mousavi et al., 2019), in goats (Nadri et al., 2019), and humans (Mutalik et al., 2014). Other investigators have suggested adding enhancers to lecithin extenders, such as adding bovine serum albumin (Alcay et al., 2019) which had a positive effect, or the antioxidant glutathione (Zhandi & Sharafi, 2015) which had bad effects. In human semen, Reed et al. (2009) concluded that soy lecithin can successfully replace egg yolk as a supplement for cryopreservation medium, without adverse effects on sperm post-thawing, warranting further research into this and other phospholipids (Reed et al., 2009). The same result was obtained by Jeyendran (2008), who concluded that an effective medium for freezing human sperm that does not involve the supplementation of animal products may be developed by using phospholipids derived from soybean oil, along with DMSO and glycerol (Jeyendran et al., 2008).

Conclusion

The results of our current study showed that lecithin, which was used in different concentrations, was not a suitable substitute for egg yolk extenders for preserving epididymal sperm in dogs. The need to develop a specific medium without animal proteins is obvious. Lipids or lipoproteins in natural or synthetic form may be able to substitute standard whole EY-based diluents in preserving sperm survival during cooling and freezing. Some lipids of lipoproteins may be able to replace the EY and protect the sperm membranes completely. Still, it may be challenging to obtain the benefits of the entire EY with all its compounds by adding single substances.

Acknowledgments

The authors gratefully acknowledge the support provided by the Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.

Conflict of Interest

The authors declared no conflict of interest.