# Alginate Silver Nanoparticles and Their Effect on Sperm Parameters of the Domestic Rabbit
## 3 Results and Discussion
### 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 ejacates were characterized by a sperm head DNA loss of 2.19 \(\pm\) 0.11%, while 24 h later, the value of this parameter was 0.56 \(\pm\) 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. \begin{table}
\begin{tabular}{c c c} \hline  & **\% Tail DNA** & **TM** \\ \hline Control 0 h & 2.19 \(\pm\) 0.11 \({}^{\text{a}}\) & 0.30 \(\pm\) 0.03 \({}^{\text{a}}\) \\ Control 24 h & 0.56 \(\pm\) 0.04 \({}^{\text{ab}}\) & 0.09 \(\pm\) 0.01 \({}^{\text{ab}}\) \\ Alg 24 h & 1.36 \(\pm\) 0.11 \({}^{\text{bcd}}\) & 0.29 \(\pm\) 0.05 \({}^{\text{bcd}}\) \\ AgNP AlgX 24 h & 0.82 \(\pm\) 0.07 \({}^{\text{c}}\) & 0.13 \(\pm\) 0.02 \({}^{\text{c}}\) \\ AgNP AlgB 24 h & 0.55 \(\pm\) 0.06 \({}^{\text{d}}\) & 0.05 \(\pm\) 0.00 \({}^{\text{d}}\) \\ \hline \end{tabular} The values represent the mean and standard error; \({}^{\text{a}}\)– averages between groups marked with the same letters are significantly different (_p_ < 0.05).  
\end{table} 
Table 2: Toxicity of the analyzed AgNP solutions on rabbit semen evaluated by comet assay. Figure 4: Spermat analyzed in comet assay after exposure to the tested AgNPs solutions at a magnification of 400\(\times\). 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 \(\pm\) 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 \(\pm\) 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 \(\pm\) 0.03 for the 0 h control and 0.09 \(\pm\) 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 \(\pm\) 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 ejacate after exposure to AgNP AlgX 24 h was at an average level, as the TM was 0.13 \(\pm\) 0.02. This value was significantly different from the damage shown in the positive control (Alg 24 h).