Alginate Silver Nanoparticles and Their Effect on Sperm Parameters of the Domestic Rabbit

Abstract

Silver nanoparticles possess valuable physical, chemical, and biological properties, rendering them widely applied as bioactive agents in the industry. Nonetheless, their influence on the natural environment and on living organisms remains unclear. Therefore, this study aims to investigate the impact of polymer composites containing silver nanoparticles on sperm cells. The nanosilver polymer composites were chemically synthesized, employing sodium alginate as the stabilizer. The reducing agents employed were solutions comprising sodium borohydride and xylose. The concentration of silver nanoparticles obtained after synthesis was 100 parts per million. The examined biological species were rabbit sperm cells. The impact of nanosilver on the sperm was assessed through the elucidation of the toxicity profile, comet test, and analysis of morphological characteristics of the animal cells. The results of the study demonstrate a twofold impact of polymer composites infused with silver nanoparticles on domestic rabbit sperm when obtained through chemical synthesis using two reducing agents (xylose and sodium borohydride) at a 10 ppm concentration. The comet test showed no harmful effect on the DNA integrity of rabbit sperm by the tested compounds. Twenty-four-hour exposure of rabbit spermatozoa to silver nanoparticles, obtained by reducing xylose and borohydride, induced significant secondary changes in the morphological structure of male reproductive cells. These findings indicate the potential reproductive toxicity of silver nanoparticles.

1. Introduction

The 21st century is a time of significant development in various industries and scientific fields. Nanotechnology is one of the most frequently described and analyzed areas of science in terms of applications. Achievements related to the dynamic development of research on structures smaller than 100 nm find many fascinating applications in biotechnology, medicine, modern food technology or cosmetology [1,2,3,4]. The successes of scientific work on nanotechnology are the result of reflections on the idea of miniaturizing and modifying particles, one of the most famous popularizers of which was the scientist Richard Feynman. The introduction of technology for manipulating matter at the atomic scale allowed for the development of scientific concepts that, ultimately, led to the emergence of nanotechnology [5,6,7].

Scientific literature increasingly demonstrates the potential of metal nanoparticles, particularly silver nanostructures, due to their fascinating physicochemical properties and strong biological activity. These particles have a wide range of applications in various areas of human life. Nanosilver has a valuable feature in significantly limiting the development of microorganisms [8,9,10,11,12,13,14,15,16]. The antimicrobial properties of silver nanoparticles allow for their use in many industries and, more recently, in animal breeding and artificial insemination methods [17]. One of the representatives of breeding animals also used as an animal model in laboratory research are rabbits [18]. These animals are characterized by high fertility and a relatively fast generation replacement [19,20]. Over the past few years, there has been a significant increase in the production of rabbit meat for the consumer market. This high demand for rabbit meat has led to the introduction of the artificial insemination technique in the breeding of these animals to increase the number of litters [18]. This technique can generally use fresh semen, as well as frozen sperm [21]. However, due to its practical use in large-scale breeding, the chilled semen has found greater use in rabbit insemination. As a consequence, recent years have seen a significant increase in research on the use of this assisted reproduction technology in domestic animals, including rabbits [22]. The main focus of these studies has been on improving the quality of semen after cooling, and, thus, on developing the best possible diluent mixture to use in the cryopreservation technique for rabbit semen [23]. An important factor affecting the possibility of using semen for storage and insemination is the presence of bacteria that naturally enter the ejaculate during collection [24,25]. Despite the use of strict hygienic practices, the problem of bacterial infections was so serious that the World Organization for Animal Health (OIE) recommended the addition of antibiotics to the diluents used for semen cryopreservation to control bacterial infection [26]. As a result of the numerous recommendations, antibiotics have become necessary and mandatory additives in semen diluents for the preservation and storage of liquid semen of several animal species (cattle, swine, rabbits) [27]. Although contamination of semen with opportunistic microorganisms usually does not pose a threat to females, it can affect the quality of spermatozoa. The ability of sperm to fertilize can be directly affected by bacteria, which can impair sperm motility and adhere to it, inducing the acrosome reaction or affecting the structure of the DNA molecule in the acrosome [28,29]. Currently, due to the widespread use of antibiotics in various industries related to medicine, pharmacy, veterinary medicine, and animal breeding, one of the most significant problems is the increasing resistance of microorganisms [24,30]. Numerous studies related to semen storage and analysis show critical patterns of bacterial resistance found in semen samples of various species, including boars, cattle or humans [25,31,32]. The increase in bacterial resistance to antibiotics as a result of their widespread use in various industries is forcing the search for other substances, agents with antibacterial properties that could replace antibiotics in certain processes [25,30]. One of the potential agents with antibacterial properties for use in the dilution and storage of animal semen are silver nanoparticles [17].

Research on obtaining composites containing silver nanoparticles suggests that the chemical synthesis method, which involves reducing silver ions using various substances such as sugars, is an effective way to obtain nanostructures. It is also possible to embed these particles in the structures of non-toxic carriers, which act as a stabilizer for the resulting structures. For instance, polysaccharide sodium alginate can be used as an ecological stabilizer [33]. Sodium alginate is commonly obtained from natural sources. The chemical structure of alginate consists of numerous subunits composed mainly of β-d-mannuronic and α-1-guluronic acid residues. Sodium alginate has a number of valuable biological properties, including biodegradability and biocompatibility with the natural environment. Importantly, from the point of view of the conducted research, this substance easily undergoes the gelatinization process and, thus, creates plastic gels [33,34,35,36]. Alginate is widely used in various industries, such as pharmaceuticals, tissue engineering, and agri-food, due to its low toxicity [37,38,39,40,41].

Interdisciplinary industrial applications of polymer composites with metal nanoparticles result in the need to assess their safety, and, therefore, the degree of their toxicity, towards organisms inhabiting the natural environment, including farm animals. Due to the fact that these animals are particularly exposed to the undesirable effects of xenobiotics entering the ecosystem, it is necessary to directly verify the new substances against cells that are particularly important for the proper growth and development of these organisms.

Spermatozoa are mature male reproductive cells with half the number of chromosomes reduced; in addition, they are characterized by an almost 90% smaller size than somatic cells. Through evolution, spermatozoids have evolved a mechanism for condensing genetic material that results in sperm chromatin that is 6–20 times more packed and condensed than nuclear chromatin in somatic cells [42,43]. The transformation and packing of chromatin are linked in the process of sperm formation, which is spermiogenesis. During this process, only about 5–15% of histone proteins, which are part of the nucleosome, remain in the sperm nucleus and undergo chemical transformations [44]. Other chromatin proteins, such as protamine 1 and 2, are also bound to the sperm’s DNA; they are proteins rich in cysteine and arginine, which allow for stable association with DNA. Due to the presence of protamines, the process of protamination and transformation of DNA strand structure takes place [43,44,45]. As a result of the remodeling of sperm chromatin, these cells achieve a high degree of DNA condensation in the sperm head, and this allows for a reduction in cell size, as well as the development of a more hydrodynamic shape [42]. In addition, this strong degree of chromatin packing protects the genetic material from damage, which is important because repair processes are usually not observed in spermatozoa [42,43,44]. The processes of chromatin reorganization occurring in cells as a result of endogenous and exogenous factors can be disrupted, resulting in abnormal chromatin condensation, and, thus, the occurrence of single and double DNA strand breaks, their fragmentation, or abnormalities in the process of converting histone proteins into transition proteins and further into protamines [46,47]. The consequences of these changes can lead to the destabilization of the genetic material of male germ cells [45,46]. In the ejaculate, sperm naturally produce small amounts of reactive oxygen species, which play an important role in several processes, including capacitation or the acrosome reaction [48]. A higher amount of ROS in the ejaculate can lead to the induction of oxidative stress, causing damage to DNA strands in sperm [48]. Damage to the sperm’s nuclear DNA or changes in its structure is a cause of infertility in men, and the presence of these disorders is also an important reason for failures in treatments using artificial insemination methods [48].

Replacing or supplementing antibiotics with AgNPs in animal breeding practices could offer numerous advantages. These include reducing the risk of bacterial antibiotic resistance development [49,50], improving wound healing and preventing infections in animals [50,51], enhancing animals’ immune systems and natural defenses [51,52], and providing a cost-effective and environmentally friendly alternative to antibiotics [50,52].

In the present study, an innovative composite of silver nanoparticles suspended in sodium alginate was used as a safe, non-toxic, antibacterial component in a diluent for rabbit semen storage. The purpose of this study was to evaluate the effect of silver nanoparticles, stabilized with sodium alginate, and obtained by chemical synthesis using two reducing agents, xylose and sodium borohydride, on the morphology and integrity of nuclear chromatin in rabbit spermatozoa.

2. Materials and Methods

2.1. Materials

The chemicals used in the experiment were sodium alginate (with a molecular weight of approximately 1.565 × 10⁵ Da), AgNO₃ (99.99%), LMP agarose (low melting point agarose), NMP agarose (normal melting point agarose), alkaline buffer, EDTANa₂ (ethylenediaminetetraacetic acid disodium salt dihydrate), TRIS (Trizma base), Triton X-100, NaOH buffer, Tris, ethidium bromide, xylose, sodium borohydride, and glycerol (99.5%). All reagents were purchased from Sigma-Aldrich (Poznań, Poland).

2.2. Synthesis of Polymer Gels with Silver Nanoparticles

The synthesis of silver nanoparticles (AgNPs) was carried out in a manner similar to our previous work [53,54]. A 1.5% gel was obtained by dissolving 4.5 g of sodium alginate in 295.5 g of distilled water. The resulting suspension underwent gelation at 60 °C for 24 h with the addition of glycerol as a plasticizer in a weight ratio of 1:2 to the weight of alginate.

Table 1. Content and concentration of AgNPs in samples.

	Water [g]	Sodium Alginate [g]	AgNO3 Solution [g]	Glycerol [g]	Xylose Solution, 4%, [g]	NaBH4 Solution, 1%, [g]	Total Mass of Gel [g]	AgNPs Concentration [ppm]
Alg	130.85	1.50	-	0.75	-	-	133.1	-
AgNP AlgX	98.5	1.50	12.35	0.75	20.0	-	133.1	100
AgNP AlgB	98.5	1.50	12.35	0.75	-	20.0	133.1	100

provides the quantities of each component. The gel was divided into three equal parts. To ensure identical alginate concentration, 32.35 g of water was added to part one (Alg). In parts two (AgNP AlgX) and three (AgNP AlgB), a solution of AgNO₃ with a concentration of 0.0100 M was added. The resulting suspensions were stirred continuously on a magnetic stirrer at 60 °C. Then, a solution of Xylose was introduced into part two (AgNP AlgX), and sodium borohydride was introduced into part three (AgNP AlgB). In each case, 133.1 g of gel were obtained, and the final concentration of AgNPs was 100 ppm in both samples, AgNP AlgX and AgNP AlgB.

For Fourier Transform Infrared Attenuated Total Reflectance (FTIR-ATR) measurements, 25 g of gel from each sample were poured into Petri dishes and dried at 37 °C. Thin films were obtained.

2.3. Attenuated Total Reflection–Forier Transform Infrared Spectroscopy (ATR–FTIR)

The spectra for the control sample and the films containing AgNPs were recorded in the range of 4000–700 cm⁻¹ with a resolution of 4 cm⁻¹. ATR–FTIR spectroscopy analysis was conducted using a MATTSON 3000 FT-IR spectrophotometer (Madison, WI, USA) with a 30SPEC 30° reflective cap and a MIRacle ATR accessory from PIKE Technologies Inc. (Madison, WI, USA).

2.4. Ultraviolet–Visible (UV–VIS) Spectroscopy

The gels obtained in Section 2.2 were diluted 10-fold with distilled water for UV–Vis measurements. UV–Vis absorption spectra were obtained using a Shimadzu 2101 scanning spectrophotometer (Shimadzu, Kyoto, Japan) in the range of 200–800 nm. A 10 mL quartz cuvette was used, with distilled water as a reference.

2.5. Scanning Electron Microscopy (SEM)

Samples for electron microscope analysis were prepared by drop-coating 10 µL of the sample onto carbon-coated 200 mesh copper (100) grids (TAAB Laboratories, Aldermaston, Berkshire, UK). Prior to analysis, the samples underwent a coating process which involved the application of a 20 nm layer of Cr using a K575X Turbo Sputter Coater (Emitech, Ashford, UK). The morphology and size distribution of the silver nanoparticles were characterized using a high-resolution scanning electron microscope (SEM) JEOL 7550 (Akishima, Tokyo, Japan) with a transmission electron detector (TED).

2.6. Toxicity Profile

The study was conducted on semen obtained from 10 male New Zealand White domestic rabbits. The experimental animals were sexually and somatically mature individuals of 1 year and >1 year of age. Animals with constant access to water were fed ad libitum, and maintained individually in standard cages according to EU requirements. The breeding facilities ensured optimal zoohygienic conditions provided for this animal species. The temperature inside the hall varies, depending on the season, from 15 °C to 21 °C. Gravity ventilation ensures regular air exchange in the room. The animals were kept in a 16L:8D lighting system, with a light intensity of 60 lux and a relative humidity of about 60–75%. In order to improve the conditions of the breeding animals, cages were used, equipped with boxes to ensure the expression of their natural behavior and environmental enrichment elements, such as teethers and high-quality meadow hay. Semen was collected on an artificial vagina after the males were prepared for semen donation. Decision No. 335/2019 of the 1st LKE for Animal Experiments in Cracow, dated 30 October 2019.

The collected ejaculates suspended in Ringer’s liquid were separated into Eppendorfs, into 4 groups: negative control (fresh semen 0 h—control 0 h and control 24 h—semen storage for 24 h without additional components), positive control, which was sodium alginate only, experimental group I and II. In groups I and II, rabbit semen was exposed to silver nanoparticles suspended in sodium alginate, but obtained with different reductants, respectively. For group I, it was xylose (AgNP AlgX), and for group II, it was sodium borohydride (AgNP AlgB). Before the exposure, dilutions of silver nanoparticles and control sodium alginate were prepared using phosphate-buffered saline (PBS, Sigma Aldrich, Poznań, Poland) to a concentration of 10 ppm. Ejaculates were mixed with the test compounds at a volume ratio of 1:1 (50 µL each), 50 µL of PBS (Sigma Aldrich, Poznań, Poland) was added to the semen in the negative control, and the semen was exposed for 24 h at room temperature.

After exposure, analysis of sperm DNA or chromatin integrity abnormalities was carried out using an alkaline comet assay, and changes in morphological structure were assessed by eosin and nigrosin staining.

2.7. Alkaline Comet Assay

The evaluation of changes in nuclear DNA integrity in rabbit sperm was performed according to the comet assay protocol of Singh et al. [55], with modification. The 20 µL of semen suspension after exposure was mixed with 75 µL of LMP agarose and applied to basal slides coated with NMP agarose. Lysis of the slides was carried out for 24 h in an alkaline buffer (2.5 M NaCl, 0.1 M EDTANa₂, 10 mM TRIS and 1% Triton X-100, pH = 10) at +4 °C with limited light. Electrophoresis was conducted under alkaline conditions in 30 mM NaOH buffer with 2 mM EDTANa₂, pH = 12.5, under limited light for 20 min at 0.6 V/cm. Neutralization was carried out in 0.4 M Tris. For detection, slides were stained with ethidium bromide at a concentration of 200 µg/mL. Microscopic documentation was performed using a Zeiss Imager A2 epifluorescence microscope with AxioCam MRc5 software (NIS-Elements image analysis software ver. F2.31, Carl Zeiss, Jena, Germany). Sperm damage assessment was performed using CASP 1.2.3b software (CaspLab, Wroclaw, Poland). For each animal, 100 comets were analyzed. The parameter determining the toxicity profile in the comet assay was the percentage of DNA in the tail (% of DNA in the tail, TD %) and tail moment (TM).

2.8. Analysis of Sperm Morphology

From each semen sample, 2 smears were created on a basic slide and then stained with 5% eosin blue and 10% nigrosine (Sigma-Aldrich, Poznan, Poland) [56]. For this purpose, 20 µL of the test sample was spotted on the slide, 2 drops of eosin and 4 drops of nigrosin were added and a smear was created. After drying at an increased temperature, the smear was subjected to microscopic analysis using an Olympus CX43 microscope under immersion (Olympus Evident, Shinjuku Monolith, 3-1 Nishi-Shinjuku 2-chome, Shinjuku-ku, Tokyo, Japan). Dead sperm were stained pink, while live sperm remained unstained. The smear was analyzed for the percentage of normal spermatozoa and secondarily altered spermatozoa. Among the secondary changes were the presence of a protoplasmic droplet, the presence of a looped or coiled tail, the acrosomal defect, damaged sperm, and their agglutination.

2.9. Statistical Analysis

Data obtained from the comet assay (% DNA in the comet tail and TM) on the effect of the tested compounds on semen DNA integrity, for each group, were presented as mean with standard error. The normality of the distribution of traits was checked using the Shapiro–Wilk test. Due to the lack of a normal distribution, the Willcoxon U-Man Withney non-parametric test was applied. Probability at the p < 0.05 level was considered significant.

In the morphological analysis (features: normal spermatozoa, presence of droplets, coiled or looped tail, damage to the acrosome or whole spermatozoa, or aggregation of spermatozoa), the normality of the distribution of the analyzed features was checked by the Shapiro–Wilk test. Due to the lack of a normal distribution, the non-parametric Kruskal–Wallis test and the Dwass–Steel–Critchlow–Fligner method were used to check differences between groups. Probabilities at the p < 0.05 level were considered as significant. All analyses were performed using the SAS package [57].

3. Results and Discussion

3.1. Scanning Electron Microscopy

Figure 1 displays the distribution and microscopic images of AgNPs, which confirms the synthesis and presence of AgNPs in the sodium alginate matrix. The Alg AgX sample (Figure 1a) shows a lower degree of aggregation compared to the Alg AgB sample (Figure 1b). Spherical Ag nanocrystals are well separated and particle size distribution ranges between 5–30 nm Alg AgX) and 7–55 nm (Alg AgB). The obtained results are in accordance with the UV–Vis spectrum, where an additional band at 510 nm is observed [53,58]. This is primarily due to the kinetics of the reduction reaction. We observe a better distribution of AgNPs and a smaller size in the case of reduction with xylose (Alg AgX).

Obtaining composites containing structures less than 100 nm in size with significant bioactive effects is one of the main topics of modern diagnostic research on the practical application of innovative biological materials. The work on the deposition of nanoparticles in polysaccharide structures allows for the expansion of information on the possibilities of interdisciplinary application, along with the evaluation of toxicity of these materials. The results presented in the work of the team of Rutkowski et al. [53] showed that the efficient synthesis of silver nanoparticles in the structure of sodium alginate depends significantly on the type of reducing agent applied. Significant physicochemical and biological diversity was documented between the particles obtained using xylose, maltose and glucose. The size, shape and biological activity of each object differed significantly from each other. These observations remain comparable to the parameters reported in this manuscript. Characterized in the work of Rutkowski et al. [53], silver nanoparticles in the structure of silver alginate with xylose as a reducing agent were characterized by limiting and inhibiting the growth of bacteria, such as Escherichia coli and Staphylococcus aureus, taken from horses, dogs and cats. Also, antibacterial activity against these strains was shown by other silver nanoparticles obtained using maltose and glucose as reductants.

3.2. Spectroscopic Analysis (UV–Vis and ATR–FTIR)

Figure 2 displays the UV–Vis spectra of composites containing silver nanoparticles and a control sample. Characteristic absorption bands were observed for silver nanoparticles at 430 nm for the sample with xylose-reduced AgNPs and at 417 and 510 nm for the composite with sodium borohydride-reduced AgNPs. The band above 510 nm is created by the coupling of surface plasmons to the aggregated particles. Several studies [53,54,59,60,61,62] have demonstrated the effectiveness of UV–Vis spectroscopy in detecting the presence of metallic nanoparticles. Furthermore, these studies demonstrate that the size, shape, and distribution of particles significantly affect the shape and position of the absorption band in the UV–Vis spectra. Therefore, it is crucial to consider these factors when analyzing the spectra [54]. Typically, an increase in nanoparticle size results in a shift of the absorption band towards longer wavelengths, while an increase in polydispersity is indicated by a broadening of the band. These findings partially align with our previous studies [53,54]. For silver nanoparticles obtained through the reduction reaction with sodium borohydride, there was a significant increase in polydispersity, nanoparticle size, and aggregation. These results were also confirmed by electron microscopy.

The ATR–FTIR spectra for the samples in Figure 3 were analyzed in the spectral range of 700–4000 cm⁻¹. The vibrations ranging from 3500–3000 cm⁻¹ are related to the hydroxyl groups present in the polymer and glycerol. A set of overlapping bands with moderate intensity appear around 2941 and 2879 cm⁻¹, signifying the stretching vibrations of CH and CH₂ groups in the alginate and glycerol structures. The bands observed at 1600 and 1399 cm⁻¹ were attributed to the asymmetric and symmetric stretching vibrations of the carboxylate salt ion, respectively. A number of vibrations in the range 1100–1000 cm⁻¹ assigned to the glycoside bonds in the polysaccharide (C-O-C stretching). The obtained results are consistent with the data found in the literature [63,64,65]. No significant changes were observed in the spectrum of the composites compared to that of sodium alginate, which may be due to the very low concentration of metallic nanoparticles.

Further studies identified the possibility of modifying the activity of nanoparticles by enriching them with additional substances. The results of the research work of the team of Nowak et al. [65] made it possible to obtain polymer composites, also based on sodium alginate, with silver and gold nanoparticles additionally enriched by the presence of graphene oxide. Similar to the present work, the evaluation of the physicochemical properties of these composites through the use of methods such as UV–VIS spectroscopy, SEM electron microscopy and the evaluation of Fourier transform infrared spectra allowed us to confirm the occurrence of the reduction reaction of nanoparticles from the salt containing the respective metals gold and silver. Obtaining spherical particles with sizes close to 20 nm is in dialogue with the biometric values of the silver structures presented in this manuscript. A strong antibacterial effect, inhibiting the growth of 11 bacterial strains, was demonstrated by silver nanoparticles enriched with graphene oxide compared to gold nanoparticles with graphene oxide, which did not show the ability to limit microbial growth [65].

Modern biopolymer technology also makes it possible to obtain innovative composites containing such active substances as quantum dots. In the work of a research team led by Grzebieniarz [66], the possibility of depositing biologically active quantum dots in polysaccharide carriers (starch/chitosan) was documented, which is in dialogue with the chemical synthesis for the formation of active composites presented in this work. Physicochemical parameters indicate that quantum dots are obtained in the polysaccharide matrix. The width of the bands for UV–VIS analysis indicates the size diversity in the obtained nanoparticles. Also, the presented microscopic images obtained by electron microscopy confirm the validity of the chemical synthesis method using the studied biodegradable polysaccharides, which is in agreement with the methods used in this work. The possibilities of innovative modifications of natural polymer technology also allow the formation of nanometer-sized capsules. In the results of the scientific and research work of the team of Woszczak et al. [67], it was reported that a polysaccharide-based biomaterial containing micellar nano/microstructures with extracts of turmeric and hibiscus was obtained. The evaluation of the physicochemical parameters of the tested biocomposites by using methods such as Fourier transform infrared spectroscopy (FTIR), ultraviolet–visible spectroscopy (UV–VIS) and SEM electron microscopy also in this case allowed us to document the efficiency of the method of synthesis of nanostructures, which also confirms the method of obtaining polysaccharide composites presented in this manuscript.

3.3. Alkaline Comet Assay

Changes in DNA integrity in the chromatin of spermatozoa from the rabbits tested were analyzed by two parameters, based on the percentage of DNA in the comet tail and an auxiliary parameter, in this case the tail moment (TM). This is a unitless parameter, calculated as the result of the tail length and the percentage of DNA content in the comet tail. For each individual in each of the analyzed groups, 100 spermatozoa were measured, and an example microscopic image of the cells evaluated is shown below in Figure 4.

Before analyzing the effect of the factors studied on the level of DNA damage in rabbit semen, the degree of chromatin integrity in fresh (0 h) semen was compared with semen stored for 24 h at room temperature, i.e., from the negative control group. Freshly collected ejaculates were characterized by a sperm head DNA loss of 2.19 ± 0.11%, while 24 h later, the value of this parameter was 0.56 ± 0.04%. Differences in %Tail DNA values between control groups were significant (p < 0.05). The level of semen damage observed after exposure to silver nanoparticles is shown in

Table 2. Toxicity of the analyzed AgNP solutions on rabbit semen evaluated by comet assay.

	% Tail DNA	TM
Control 0 h	2.19 ± 0.11 a	0.30 ± 0.03 a
Control 24 h	0.56 ± 0.04 ab	0.09 ± 0.01 ab
Alg 24 h	1.36 ± 0.11 bcd	0.29 ± 0.05 bcd
AgNP AlgX 24 h	0.82 ± 0.07 c	0.13 ± 0.02 c
AgNP AlgB 24 h	0.55 ± 0.06 d	0.05 ± 0.00 d

The values represent the mean and standard error; ^a–d—averages between groups marked with the same letters are significantly different (p < 0.05).

.

The loss of DNA from the sperm head in the Alg 24 h positive control was the highest among all experimental groups at 1.36 ± 0.11% of DNA in the comet tail. The observed level of damage was significantly different from the negative control after 24 h in % Tail DNA value (p < 0.05). Also, significant differences in % Tail DNA values were found between the AgNP solutions tested and the positive control. Silver nanoparticles with xylose as a reducing agent generated damage at the level of 0.82 ± 0.07% of DNA in the comet tail, which was an increase of 0.26 percentage points with respect to the negative control and a decrease of 0.54 percentage points with respect to alginate alone (Table 2). In contrast, AgNP with borohydride as a reducing agent (AgNP AlgB 24 h) after 24 h exposure did not induce damage to rabbit semen, as the changes observed in this group were at the level identified for the negative control. There were no significant differences between the percentage of DNA in the comet tail of sperm from the AgNP AlgX 24 h and AgNP AlgB 24 h groups.

The values of the auxiliary parameter, tail moment, between the control groups depending on storage time showed significant differences and were 0.30 ± 0.03 for the 0 h control and 0.09 ± 0.01 for the 24 h control, respectively (Table 2; p < 0.05). Sodium alginate alone, the positive control, showed damage similar to that analyzed in fresh semen, as the TM for this group was 0.29 ± 0.05. This damage was 0.20 points higher than in the 24 h control, and these averages differed significantly. Also, for this parameter, the damage value observed in the ejaculate after exposure to AgNP AlgX 24 h was at an average level, as the TM was 0.13 ± 0.02. This value was significantly different from the damage shown in the positive control (Alg 24 h). As with the first parameter, the semen of rabbits after exposure to AgNP AlgB 24 h showed the lowest level of tail moment damage, at 0.05 ± 0.00 TM. The value for this group showed significant differences compared to Alg 24 h (p < 0.05).

Due to the compactness of the cell nucleus and the highly condensed structure of nuclear chromatin in mammalian spermatozoa, including rabbits, it is difficult to determine changes in DNA integrity. The condensed structure of chromatin makes it impossible to adequately unwind the DNA strand to make it possible to visualize its breaks and, following electrophoresis, to measure them [18,68]. Therefore, the results obtained do not indicate a clear level of damage to nuclear chromatin generated by silver nanoparticles, or the additional effect of the used reductants. The identified damage to DNA strands after exposure to the tested solutions was minimal, less than 1% DNA loss, a value lower than the physiological changes occurring in rabbit reproductive cells, which were at the level of 2.19% of DNA in the comet tail. The results indicate a protective effect of silver nanoparticles, in both solutions, on the stability of rabbit sperm.

The effect of AgNP on spermatozoids in the rat epididymis was evaluated by Gromadzka-Ostrowska et al. [69]. The studied doses of AgNP (5 or 10 mg/kg) were injected into the tail vein of rats by a single injection and their effects were analyzed 24, 7 and 28 days after administration. In this team’s study, morphologically abnormal spermatozoids were analyzed after maceration of the epididymis, as well as DNA damage using a comet assay. After 24 h exposure, abnormal germ cells were found at 20‰, and after 7 days between 35–45‰. Analysis of the comet assay showed a significant increase in damage, in terms of % of DNA in the comet tail, after 24 h exposure to the tested doses of AgNP (damage amounting to 12–15% tail DNA, control 7% tail DNA), and a decrease in fragmentation after 7 and 28 days [69].

3.4. Analysis of Morphological Changes in Rabbit Sperm

Rabbit sperm is characterized by the small size of the cell nucleus and, thus, the head, which can cause difficulties in assessing its morphological structure. Morphological changes are an important factor affecting the assessment of fertility or the possibility of using semen for artificial insemination, which is often used in rabbits [18]. Before evaluating the effect of two silver nanoparticle solutions on sperm morphology, we analyzed the damage caused in the negative control after the storage period of the ejaculates (

Table 3. The average damage and morphological changes in rabbit spermatozoa after exposure to tested silver compounds.

	Control 0 h	Control 24 h	Alg 24 h	AgNP AlgX 24 h	AgNP AlgB 24 h
Intact spermatozoa	89.70 ± 1.23	88.79 ± 2.41 cd	82.76 ± 2.29 cd	75.43 ± 3.34 c	65.91 ± 3.42 d
Protoplastic droplet	2.03 ± 0.21	1.43 ± 0.23 e	2.80 ± 0.41	2.53 ± 0.44	2.90 ± 0.40 e
Looped tail	2.83 ± 0.92	3.57 ± 1.34 f	5.43 ± 1.53 g	8.10 ± 1.84 f	16.37 ± 1.98 fg
Coiled tail	0.47 ± 0.12	0.57 ± 0.22 h	1.03 ± 0.28 hi	2.93 ± 0.51 hi	4.53 ± 0.92 i
Acrosomal defect	1.23 ± 0.16	1.30 ± 0.24 j	1.90 ± 0.20	2.30 ± 0.30 j	2.37 ± 0.35
Damaged sperm	3.10 ± 0.29 a	3.80 ± 0.63 a	5.10 ± 0.71	7.57 ± 1.41	6.67 ± 1.14
Agglutination	0.63 ± 0.08 b	0.53 ± 0.16 b	0.97 ± 0.20	1.13 ± 0.27	1.27 ± 0.22
Secondary changes	10.30 ± 1.23	11.20 ± 2.41 k	17.23 ± 2.30 l	24.57 ± 3.35 l	34.10 ± 3.42 kl

The values represent the mean and standard error, expressed %; letters ^a to ^l in superscript—averages between groups in the columns marked with the same letters are significantly different (p < 0.05).

). In the 0 h and 24 h negative control, spermatozoa with normal structure accounted for 89.70 ± 1.23 and 88.79 ± 2.41% of the ejaculate, respectively. The most common defects identified in these groups were the occurrence of protoplasmic droplet, looped tail or generally damaged spermatozoa. The differences in the incidence of the last type of damage between the groups were significant (p < 0.05), as well as for the average incidence of agglutinated cells. Detailed results are shown in Table 3. Storage of rabbit ejaculates for 24 h resulted in an increase in two types of abnormalities: looped tail and damaged cells, relative to freshly collected material.

The fraction of normal spermatozoa in the experimental groups differed significantly from the negative control, and was 82.76 ± 2.29% in the positive control and 75.43 ± 3.34% after treatment with AgNP with xylose as a reducing agent (Table 3). The value of this parameter decreased even more significantly with respect to the control samples after the treatment of AgNP with borohydride as a reducing agent (65.91 ± 3.42). Analyzing the effect of sodium alginate alone on sperm morphology, there were significant differences with respect to the negative control in the case of the coiled tail lesion; this was an increase in damage of 0.46 percentage points.

Silver nanoparticles obtained in alginate with xylose were characterized by an increase in all types of damage analyzed compared to the negative control, and with the exception of protoplasmic droplet, these were also more frequent changes than those generated by alginate alone. A significant increase in damage in the form of looped and coiled tail, damaged spermatozoa was recorded between the material from this experimental group and the 24 h negative control (p < 0.05). With respect to the positive control, significant differences were found in the occurrence of secondary damage and coiled tail.

The second of the tested silver nanoparticle solutions, synthesized with sodium borohydride, induced the most morphological changes in rabbit semen compared to the other groups analyzed. A significant increase in damage with respect to the 24 h negative control was found in the presence of protoplasmic droplet, looped tail, or the total number of secondary changes (p < 0.05). There was a higher frequency of looped and coiled tail and secondary lesions generated by AgNP AlgB 24 h compared to the positive control. Both silver nanoparticle solutions tested, AgNP AlgX 24 h and AgNP AlgB 24 h, showed significant differences for the induction of secondary lesions or both modifications of the sperm tail (p < 0.05, Table 3).

Another area of research focuses on the development of a diagnostic method to evaluate changes in the morphology and morphometry of preserved sperm and to clarify the difficulties associated with cryopreservation [18,70]. The semen parameters of all animal species, including rabbits, in addition to the semen collection technique itself, are affected by many factors, including those related to the breeding environment (such as the cage housing system), nutrition, health status, time of year, season or age of the males [71]. There are many exogenous factors that have a detrimental effect on the quality of germ cells in mammals. According to Castellini et al. [72], environmental contaminants can accumulate in the testes, or epididymides, impairing their reproductive and endocrine function. Metals, or rather their specific chemical forms, have a greater ability to penetrate the blood–nucleus barrier, increasing the probability that these compounds will accumulate in the organs of the reproductive system, and later in semen. An example of a metal compound with the ability to penetrate the blood–nucleus barrier is methyl mercury or silver nanoparticles [73,74]. It has been shown that AgNPs can affect spermatogenesis, reduce the amount of sperm stored in the epididymis or increase the level of germ cell damage [69,75]. A study by Ema et al. [74] showed that silver nanoparticles can induce changes in the levels of sex hormones and modify the morphology or viability of sperm, as observed in rats and rabbits. Sperm damage after prolonged exposure (7–126 days) in vivo to silver nanoparticles was found in ejaculated sperm obtained from rabbits after a single intravenous injection of AgNP at a dose of 0.6 mg/kg. AgNP was found in the cytoplasm of Sertoli cells and in the elongating nuclei of spermatids. Analysis using TEM showed damage to the ultrastructure of spermatids, mainly focused on the acrosome and mitochondrial integrity. The highest percentage of abnormal sperm was found after 7 days of exposure (more than 50%) [76]. Olugbodi et al. [77] found a dose-dependent decrease in total sperm motility, as well as their progressive movement, in rats after dermal injection of AgNP at doses of 10 and 50 mg/kg body weight and 7- and 28-day exposures, to levels significantly below the acceptable 50%. The inability of such a large percentage of cells to move indicates full disruption of their biological role.

The problem of increasing the resistance of microorganisms to antibiotics results in a search for other substances, agents with antibacterial activity, which could replace antibiotics in semen diluents. Viudes-de-Castro [24] in their study identified the antibacterial properties of EDTA, bestatin and chitosan-based metal nanoparticles as alternatives to antibiotics. They also evaluated the effects of these three agents on rabbit semen parameters. In addition to their antibacterial properties, the compounds showed no effect on total sperm motility, acrosome integrity, membrane functionality or rabbit spermatozoid viability. Also, after 24, 48, or 72 h of exposure, they found no significant changes in these parameters of rabbit semen. Silver nanoparticles suspended in sodium alginate, analyzed in this work, can be considered as a potential alternative to antibiotics in semen diluents. However, these compounds induce a relatively high level of secondary changes in the morphology of rabbit spermatozoa after 24 h of treatment; AgNP AlgX 24 h generated >24% changes and AgNP AlgB 24 h generated 34% changes, despite the small effect of both solutions on chromatin integrity in the acrosome demonstrated by the comet assay.

Achi et al. [78], in their study of modified pellet composition, found in a control sample of rabbit ejaculate pH 6.75, sperm viability of 88.75%; and, in a morphological analysis, normal spermatozoids accounted for 82.75%, and spermatozoa with a coiled tail accounted for 2.00%, while the presence of spermatozoa with a looped tail was at 3.75%. Bodnar et al. [79], examining the abnormality of spermatozoa obtained from three breeds of rabbits, including New Zealand Whites, showed the presence of 83.50 ± 0.20% normal spermatozoa and 16.5 ± 0.20% abnormal spermatozoa. Within the altered spermatozoids, acrosome lesions accounted for 1.71 ± 0.18%, the presence of protoplasmic droplet was at 0.42 ± 0.15%, and 1.03% represented spermatozoa with a coiled tail. In addition, it was found that the pH of the ejaculate was significantly dependent on the percentage of primary and secondary abnormalities.

Sodium alginate is not harmful to health, which has been confirmed by opinion institutions, including FAO/WHO experts and the U.S. FDA have recognized the compound as a safe food additive, including for children [80]. Above that, alginate is not absorbed into the body. Taken in the recommended amounts, as a rule, it does not cause any side effects. Some harmfulness can be spoken of only in the case of non-compliance with the recommended doses. When significantly exceeded, this compound can impair the absorption of proteins and some elements [81,82]. Despite the lack of toxic properties, it was sodium alginate that showed the strongest effect after 24 h in altering the stability of rabbit sperm chromatin, relative to the other compounds tested. In the comet assay, Alg 24 h showed the highest %Tail DNA and TM values after 24 h exposure compared to the negative control 24 h, AgNP AlgX 24 h and AgNP AlgB 24 h.

A factor influencing the effectiveness of the silver nanoparticles used in the study, in addition to sodium alginate as a carrier, was the reductant used—xylose. This is a monosaccharide obtained by hydrolyzing hemicellulose-rich plants, such as straw or corn cobs [83]. One of the primary components of semen diluents and cryopreservatives is sugar, usually glucose or fructose. In that medium, sugars have several important functions, as they provide an energy substrate for sperm during storage, maintain the appropriate osmotic pressure of the diluent, and act as a cryoprotectant. Yildiz et al. [84] studied the effects of various sugars added to the diluent on the viability, motility and rate of acrosomal reaction in canine spermatozoa during dilution, standardization and freezing. At the stage of assessing the quality of the material, they showed that xylose was one of those sugars, along with fructose, galactose and maltose, that provided higher total rates of active spermatozoa compared to the control without added sugar. Samples with xylose addition had higher survival rates and lower percentages of dead and acrosome-damaged cells than the control. The opposite effect of xylose on water buffalo spermatozoa was demonstrated by Arshady et al. [85], who found a significant decrease in all semen parameters analyzed by CASA in semen samples supplemented with xylose at a dose of 1 to 15 mM, after 24 h of treatment, compared to the control sample. In our study, silver nanoparticles produced using xylose as a reducing agent showed negligible damage to rabbit sperm in the comet assay, both in % Tail DNA and TM. In the morphological analysis, AgNP AlgX 24 h induced more primary and secondary damage compared to the Alg 24 h positive control and the negative control, the semen without the additional agent.

Silver nanoparticles, or other nanomaterials, have been obtained for many years through chemical reduction methods of AgNO₃. The most commonly used reducing agent for obtaining nano metal forms is sodium borohydride (NaBH₄), as indicated by numerous literature reports [86]. In addition to its strong reducing properties, including Ag⁺ to Ag⁰ during the synthesis of AgNP, it is an agent that additionally possesses properties to stabilize the nanostructure, and its use allows for the control of the size and shape of the obtained nanoparticles. This inorganic compound, NaBH₄, has also found application in other processes for obtaining ceramic materials, and it is a good hydrogen store, so it is used for the production of hydrogen gas [87]. Due to the general harmful effects of sodium borohydride, other reducing agents or methods for synthesizing AgNP are being investigated. The AgNP AlgB24h used in the comet assay showed no harmful effects on rabbit spermatozoa after 24 h exposure, as the observed damage in this group in % DNA content in the comet tail and tail moment values was similar to the negative control after 24 h, and significantly lower than the control alginate solution (Alg24h). Cytotoxic effects were demonstrated in the analysis of morphological changes within germ cells. AgNP AlgB24h induced the most secondary changes within the structure of the spermatozoa, >34% of secondary changes, and the proportion of normal cells after treatment with this agent fell to 65.91%, compared to the control groups and sodium alginate alone.

4. Conclusions

The silver nanoparticles analyzed in this paper, suspended in sodium alginate, and obtained by chemical synthesis with two reducing agents (xylose and sodium borohydride) at a concentration of 10 ppm, showed dual effects on domestic rabbit spermatozoa. Based on the results of the comet assay, no toxic effect of the tested compounds on DNA integrity in rabbit spermatozoa was demonstrated. The 24 h in vitro exposure of rabbit sperm to AgNP AlgX and AgNP AlgB induced a significant level of secondary changes in the morphological structure of male germ cells.

Further studies will be required to determine the potential use of the tested AgNPs in the sodium alginate structure as substitutes for antibiotics used in the semen diluents of breeding animals. In the next stage, it will be important to directly confirm the antibacterial activity of all the analyzed nanomaterials, as well as to verify if the application of lower concentrations of AgNP AlgX and AgNP AlgB of 1 or 5 ppm will induce changes in sperm morphology, and see what the level of these disorders might be.