Preliminary Study: A New Approach for Improving the Cryopreservation of Mammalian Sperm

Gamete preservation is a necessary and routine procedure practiced in the 21st century in both humans and animals using the cryopreservation technique. However, cryopreservation methods can cause cryoinjury. Therefore, new approaches to help extend the viability of mammalian sperm in vitro are essential. This preliminary study explored the effect of reproductive fluids (RFs) and body fluids (BFs) from two species of desert snails—Sphincterochila zonata and Sphincterochila prophetarum—on mammalian cryopreserved sperm parameters. These desert snails are active only 5% of the year. Spermatogenesis occurs during the aestivation ecophysiological stage when testosterone levels are high, and sperm is preserved in the oviduct until mating during the active ecophysiological stage in winter. RFs from S. zonata and S. prophetarum during the aestivation ecophysiological stage reduced sperm motility to 0%, while sperm viability (SV) was similar to the controls. Moreover, the motility of thawed mouse sperm was 1.34and 2.02-fold higher (p < 0.05) in RF medium obtained from S. zonata in the active ecophysiological stage than in the control medium after 5and 30-min incubation. SV was higher in S. zonata RF medium than in control after 30 min incubation. Our results indicate the potential protective effect of desert snail RFs on cryopreserved and thawed mammalian sperm cells. Further study should be conducted to advance the fulfillment of RF potential in reproductive technologies.

based on reducing the medium and sperm cell temperature. Such a procedure triggers a decrease in cell activity that prolongs cell life [1] [2] [3] and can fulfill the sperm biological functions, similar to a non-solidified cell unit. Animal gamete protection is a necessity and a routine procedure used in the 21st century to prevent the extinction of organisms at different levels [4].
Cryopreservation methods are known to have negative effects on mammalian sperm-cell properties that can initiate cryoinjury effects [5] [6] [7], including changes in cell morphology caused by shrinkage or by the formation of intracellular ice. Such changes can result in macromolecule denaturation [8] [9], by damaging plasma and mitochondrial membranes [10], and by reducing sperm motility [10]. The demand for new methodologies and approaches to help extend the viability of mammalian sperm compared with previous cryopreservation processes is of great value.
Based on accumulated knowledge, activity, feeding behavior, and the reproductive cycle of desert snails [11] [12] [13], we have learned that desert snails fulfill their biological functions during a period of 22 to 25 days of the year in an unpredictable xeric environment. This feature has become our foremost objective, represented by the most common terrestrial gastropods, Sphincterochila zonata and Sphincterochila prophetarum, in the Negev Desert, Israel. [14] described the reproductive system of terrestrial and freshwater gastropods and their endocrinology regularity system, elucidating the changes in the steroid hormone testosterone (T) and 17ß estradiol (E2). These hormones were found to encourage spermatogenesis and oogenesis in the slug Arion ater rufus [15] [16], in the terrestrial snail Euhadra peliomphala [17] [18], and in the neogastropod Ilyanassa obsolete [19]. In our previous study [11] of the levels of the steroid hormones T and E2 in S. zonata and S. prophetarum, we found fluctuations throughout the year according to their different aestivating and active ecophysiological stages in the northern Negev Desert.
These two desert snail species, known as hermaphrodites, have a distinct spermatogenic cycle that is influenced by their life cycle, which is strongly linked to the abiotic conditions. The snails are found in the exposed aestivation stage 90% of the year, i.e., throughout the dry spring, summer, autumn, and dry winter seasons [12] [13]. They become active approximately 5% of the year, and during this short and fragmented time, they fulfill all their biological functions (e.g., feeding, growing, mating, and laying eggs). Their activity is restricted to the unpredictable periods of rainfall events. During the 90% of their inactive aestivation ecophysiological stage, the desert snails prepare sperm cells that are preserved in the seminal vesicle region of the hermaphrodite duct until fertilization, thus guaranteeing successful egg fertilization.
According to [20], the oogenesis stage occurs in the winter season, during the active stage. These findings are in line with our previous results [11] showing that high T levels during the aestivation stage and low T levels and high E2 levels during the active ecophysiological stage, in winter. S. zonata and S. prophetarum [23].
The integration of field observation and knowledge accumulated regarding the long-term maintenance of sperm viability (SV) in snail RF along an irregular period and the short-term mammalian SV without cryopreservation is an enigma. The unique idea is to investigate the influence of the desert snail BFs and RFs from different ecophysiological stages on thawed mouse sperm-cell parameters. The objective of this study was to determine the effect of the RFs of S. zonata and S. prophetarum from the aestivation and active ecophysiological stages on the survival of mouse sperm cells in vitro. We hypothesized that 1) the RFs obtained from the desert snails S. zonata and S. prophetarum during the active ecophysiological stage in the winter season will provide vital support that will facilitate sperm movement, and 2) the RFs obtained from S. zonata and S. prophetarum during the aestivation ecophysiological stage will reduce sperm motility without damaging their viability. Emphasis was placed on evaluating the T and E2, whose concentrations were at two different ecophysiological stages-the active stage in winter (since oogenesis occurs during this period, and individuals are active and mating), and the aestivation stage (when spermatogenesis takes place) (see methods).

Materials and Methods
Collection of snails from the field

Laboratory analysis
Snail sex-hormone determination Before the determination of T and E2 levels, the soft tissue from each animal was removed from the shell. Due to the difference in size between the two species, T and E2 levels were determined in S. zonata individuals (n = 5), while in S. prophetarum, to receive the necessary tissues that allowed detection (n = 6), tissues from two individual specimens were pooled, allowing us to obtain three replicates. The dry weight of the snails collected in the field was determined (mean value dry weight for S. zonata of 0.45 g/individual, n = 5; vs. 0.14 for S. prophetarum, n = 5). For S. zonata, the average dry weight ranged between 0.23 and 0.68 g dry wt for summer and winter, respectively, while for S. prophetarum, Open Journal of Animal Sciences it ranged between 0.08 and 0.20 g dry wt for summer and winter, respectively.
The tissues were homogenized using a glass homogenizer. Two ml-distilled water added, and the tissues extracted twice with five volumes of ethyl acetate.
The organic solvent extract was then evaporated in air till dry and redissolved in was added to different S. zonata and S. prophetarum BFs as follows: 1) control thawed semen (n = 2); 2) thawed semen added to BFs of S. zonata (n = 3); 3) thawed semen was added to BFs of S. prophetarum (n = 9); 4) thawed semen was added to RFs of S. zonata (n = 3); and 5) thawed semen was added to RFs of S. prophetarum (n = 9).

Sperm viability (SV)
Viability of mouse sperm cells examined using the supravital staining method Open Journal of Animal Sciences [25] [26]. An amount of 20 µl spermatozoa was stirred (for 30 sec at 37˚C) in 60-µl eosin-nigrosin (E&N) stain, i.e., 5% nigrosin and 4% eosin-Y at a ratio of 3:1. After that, smears were prepared, dried at room temperature, and examined with ×100 oil magnification. Finally, 100 sperm cells were counted. Sperm cells with stained cytoplasm in the head were considered to be dead [25] and the results were expressed as the percentage of live sperm cells.

Statistical analysis
All data were subjected to statistical analysis of variance using the SAS model (ANOVA and Duncan's multiple range test and Pearson correlation coefficients) to evaluate differences between separate means. ANOVA was followed by Tukey's HSD (honest significant difference) test to establish the significance of differences between mouse sperm in the RF and BF of both snail species at different ecophysiological stages using the statistical package Statistica 4.3. Differences obtained at levels of p < 0.05 were considered significant.

Sex-hormone analysis
The levels of the sex hormones T and E2 in both snail species at the different ecophysiological stages in summer and winter are presented in Figure 3. The T levels during the aestivation stage in summer were found to be significantly higher (p < 0.05) in comparison with the T levels during the active stage in winter. Concentrations of T in S. zonata were 11.67 and 1.53 ng/g dry wt in summer and winter 2014, respectively, and the T levels of S. prophetarum were two-and five-fold lower (5.34 and 0.37 ng/g dry wt, respectively), for the same periods.
The E2 levels in the two snail species showed no significant (p > 0.05) differences between the active and aestivation periods (3.31 and 2.60 ng/g dry wt for S. zonata, and 3.52 and 2.61 ng/g dry wt. for S. prophetarum), respectively. Effect of S. zonata and S. prophetarum RFs and BFs from the aestivation ecophysiological stage on mouse sperm movement There were no significant differences (p > 0.05) between the effect of fresh RFs obtained from S. zonata and S. prophetarum, and those stored at −20˚C for seven days during the aestivation stage in summer 2014-on thawed mouse sperm (Table 1, Table 2). Accordingly, the percentage of progressive motility (PM) after 30 min of incubation in fresh and stored S. prophetarum RF at −20˚C was 0%. During the same period, the PM for the control values was 20%, 15%, 25%, Table 1. Thawing mouse-semen parameters in reproductive fluids (RFs) and body fluids (BFs) of S. zonata (a) and S. prophetarum (b) from the aestivation stage.    (Table 1, Table 2). In contrast to the decrease in PM, mouse SV exhibited values similar to those of the controls, measured as 30% for fresh S. zonata RF and 35% after storage (at −20˚C) for S. prophetarum RF, while viability of control 1 was 33% and of control 2% -30%. Since there were no significant differences in sperm parameters between fresh and stored (at −20˚C) S. zonata and S. prophetarum RFs, we continued testing stored (at −20˚C) S. zonata and S. prophetarum RFs in winter. Effect of different S. zonata and S. prophetarum fluids from the active ecophysiological stage on mouse sperm Figure 4 shows that during the active ecophysiological stage in winter, S. zonata RFs significantly amplified (p < 0.05) thawed mouse sperm motility that was 1.34-and 2.02-fold higher than the control medium after 5 and 30 min of incubation (20˚C), respectively, with values of 55% and 50.50% in S. zonata RFs compared with 41% and 25% for the control treatment, respectively. Like the control treatment, S. prophetarum RFs affected mouse sperm motility (p > 0.05). Although S. zonata BFs increased thawed mouse sperm motility, they did not differ from the control treatment (p > 0.05) after both 5 and 30 min of incubation. S. prophetarum BFs had significantly lower values than the control (p < 0.05) ( Figure 5). Figure 6 presents the positive effect of S. zonata RFs on mouse sperm PM (43.75% and 45% after 5 and 30 min of incubation, respectively), which was significantly higher (p < 0.05) than mouse sperm PM incubated in S. prophetarum RFs (20% and 7.5% after 5 and 30 min of incubation, respectively). Nevertheless, PM in S. zonata RFs was higher than in the control treatment but did not differ significantly (p > 0.05). Mouse sperm PM in both S. zonata and S. prophetarum      (Table 2). SV in the control treatment decreased from 57% after 5 min of incubation to 35% after 30 min of incubation, a value lower than the SV in S. zonata, determined as 43% (p > 0.05). SV in S. zonata RFs was significantly higher than SV in S. prophetarum (31%) ( Table 2).

Discussion
The search for a better approach for protecting sperm during the cryopreservation process and for improving post-freezing mammalian sperm abilities, such as motility and progressive motility, is ongoing. According to Watson (2000) [9] and Johnson et al. (2000) [27], there are depressing implications for sperm parameters, and only about 50% of sperm in an ejaculate can survive the thawing procedure. However, many studies present different suggestions [28]. In recent years, several studies have gleaned ideas from nature, for example, Martinez et al. (2017) [29] tested the effect of venom components from the scorpion Scorpio maurus palmatus on mammalian sperm motility, and showed a positive effect on the motility of mouse and fresh human sperm. Kumar et al. (2015) [30] examined the effect of the silk protein sericin on the motility of thawed buffalo sperm. Soleimanzaheh and Saberivand (2013) [31] tested the impact of curcumin on rat sperm morphology after the freeze-thawing process.
Our study shows the potential of using natural RFs of desert snails for preserving and improving the in vitro abilities of thawed mammalian sperm. Preliminary results based on our study [32] indicate that incubated mouse sperm cells in the RFs of S. zonata and S. prophetarum during the aestivation ecophysiological stage in summer, succeed in maintaining viability on the one hand, while reducing mouse-sperm progressive motility on the other. Moreover, sperm cells in the S. zonata RFs obtained in the winter cryptobiosis stage, enabled the maintaining of higher sperm motility, progressive motility, and viability of sperm cells in comparison to RFs obtained in the aestivation stage. In the present study, the snail RFs exhibited a favorable positive response of mouse sperm cells compared with snail BFs or the control treatment. We hypothesized that different proteins existing in S. zonata and S. prophetarum RFs during the aestivation and active ecophysiological periods were responsible for the thawed-sperm reaction shown in this study. The next step is to identify and isolate the active components of the mouse sperm in the S. zonata and S. prophetarum RFs. Such a study will enable us to determine the mobility of the live but motionless sperm in summer, along with the increasing PM in winter.

Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.