# Alginate Silver Nanoparticles and Their Effect on Sperm Parameters of the Domestic Rabbit
## 3 Results and Discussion
### 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 ejacates (Table 3). In the 0 h and 24 h negative control, spermatozoa with normal structure accounted for 89.70 \(\pm\) 1.23 and \(88.79\pm 2.41\%\) of the ejacate, 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 \(\pm\) 2.29% in the positive control and 75.43 \(\pm\) 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 \(\pm\) 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 [PERSON] et al. [72], environmental contaminants can