**3. Results**

#### *3.1. Histopathology of the Liver of Gilts and Their Suckling Piglets*

In gilts, seven assessed histopathological changes were observed on H&E-stained liver sections: fibrosis, sinusoidal leukocytosis with inflammatory infiltrates of hepatic lobules, portal tract inflammatory infiltrates, hepatocytes with vacuolar or granular cytoplasm, hepatocellular necrosis and apoptosis, hepatocellular megalocytosis, and hepatocellular megakaryosis. Overt fibrosis was only observed in the liver of one gilt from the control group, but even this was mild based on its final score of 1 and was not statistically significant in comparison with the experimental group (*p* = 0.3681). The remaining six histopathological changes also had low final scores with a maximum score of 2, and in one experimental gilt, hepatocytes with vacuolar or granular cytoplasm reached a score of 4 (Table 1). Final scores for hepatocellular necrosis and apoptosis (*p* = 0.0318) and sinusoidal leukocytosis with inflammatory infiltrates of hepatic lobules (*p* = 0.0004) were significantly higher in the experimental group compared with the control group (Figure 1A,B), whereas portal tract inflammatory infiltrates (*p* = 0.4539) showed no statistically significant differences. Additionally, hepatocellular necrosis and apoptosis and sinusoidal leukocytosis with inflammatory infiltrates of hepatic lobules were strongly correlated (Spearman's ρ = 0.73, *p* = 0.0226). The inflammatory infiltrates were composed of different proportions of lymphocytes, plasma cells, neutrophils, eosinophils, and, occasionally, macrophages. Hepatocytes with vacuolar or granular cytoplasm (*p* = 0.3681), hepatocellular megalocytosis (*p* = 1.0000), and hepatocellular megakaryosis (*p* = 1.0000) did not show statistically significant differences between both groups.

**Table 1.** The final scores for each histopathological change in the liver of control and experimental gilts presented with basic descriptive statistics.


\* Statistically significant difference between the control and experimental groups (*p* < 0.05). N—number of animals, Min—minimum, Q1—lower quartile, Q3—upper quartile, Max—maximum.

> In piglets, only three assessed histopathological changes were observed on H&Estained liver sections, these being hepatocytes with vacuolar or granular cytoplasm, hepatocellular megalocytosis, and hepatocellular megakaryosis. Hepatocellular megalocytosis and hepatocellular megakaryosis had low final scores that did not exceed a final score of 1, whereas hepatocytes with vacuolar or granular cytoplasm had a final score ranging between 0 and 12 (Table 2). Similar to what was observed in gilts, hepatocytes with vacuolar or granular cytoplasm (*p* = 0.1981), hepatocellular megalocytosis (*p* = 1.0000), and hepatocellular megakaryosis (*p* = 1.0000) showed no statistically significant differences between both groups (Figure 1C,D).

**Table 2.** The final scores for each histopathological change in the liver of control and experimental piglets presented with basic descriptive statistics.


N—number of animals, Min—minimum, Q1—lower quartile, Q3—upper quartile, Max—maximum.

**Figure 1.** Representative microphotographs of the liver of gilts and their piglets: (**A**) gilt from the control group, (**B**) gilt from the experimental group with increased sinusoidal leukocytosis with inflammatory infiltrates of hepatic lobules (inset depicting the latter), (**C**) piglet from the control group with hepatocytes diffusely exhibiting vacuolar or granular cytoplasm, (**D**) piglet from the experimental group with hepatocytes diffusely exhibiting vacuolar or granular cytoplasm and a single hepatocyte exhibiting megalocytosis and megakaryosis (arrow). H&E, bar = 50 μm.

Several assessed histopathological changes, namely, irregularity of hepatic cords, biliary hyperplasia, dilatation and thickening of blood vessels, and thrombosis of blood or lymphatic vessels, did not occur in any of the liver samples from either the gilts or their piglets.

#### *3.2. Apoptosis and Proliferation Index in the Liver of Gilts and Their Suckling Piglets*

The cumulative number of apoptotic hepatocytes was significantly higher in the experimental group of gilts compared with the control group (*p* = 0.0224) and an even more significant difference was observed for the apoptotic cells in hepatic sinusoids (*p* = 0.0007) (Figure 2A,B). Moreover, the apoptotic cells in hepatic sinusoids were marginally significantly but strongly correlated with sinusoidal leukocytosis with inflammatory infiltrates of hepatic lobules (Spearman's ρ = 0.69, *p* = 0.0535). The apoptotic cells in hepatic sinusoids were most likely lymphocytes, based on their morphology, but double immunohistochemical labelling was not attempted to further clarify this. There was no statistically significant difference in the proliferation index of hepatocytes between the two groups of gilts (*p* = 0.6901) (Table 3).

**Figure 2.** Representative microphotographs of apoptosis in the liver of gilts: (**A**) gilt from the control group with a single apoptotic cell in a hepatic sinusoid (arrow), (**B**) gilt from the experimental group with increased numbers of apoptotic cells in hepatic sinusoids (arrows) and an apoptotic hepatocyte (inset). TUNEL, counterstained with Mayer's hematoxylin, bar = 50 μm.

**Table 3.** The cumulative number of apoptotic cells and the proliferation index of hepatocytes in the liver of control and experimental gilts presented with basic descriptive statistics.


\* Statistically significant difference between the control and experimental groups (*p* < 0.05). N—number of animals, Min—minimum, Q1—lower quartile, Q3—upper quartile, Max—maximum.

> In piglets, there were no statistically significant differences in the cumulative number of apoptotic hepatocytes (*p* = 0.1265) and apoptotic cells in hepatic sinusoids (*p* = 0.8581) between the two groups. No statistically significant difference in the proliferation index of hepatocytes was seen when comparing both groups of piglets (*p* = 0.1069) (Table 4). Nevertheless, the proliferation index of hepatocytes was found to be strongly correlated with hepatocytes with vacuolar or granular cytoplasm (Spearman's ρ = 0.74, *p* = 0.006).

**Table 4.** The cumulative number of apoptotic cells and the proliferation index of hepatocytes in the liver of control and experimental piglets presented with basic descriptive statistics.


N—number of animals, Min—minimum, Q1—lower quartile, Q3—upper quartile, Max—maximum.

#### *3.3. Interlobular Connective Tissue in the Liver of Gilts*

Morphometrical analysis of interlobular connective tissue was only implemented on liver samples from both groups of gilts. Subjectively, the interlobular connective tissue in the control group of gilts appeared to be of the expected thickness, whereas in the experimental group, it appeared mildly decreased; therefore, automated detection was important to decrease subjective bias. The interlobular connective tissue in the experimental group appeared to have decreased amounts of collagen fibers, therefore forming narrower bands of fibrous connective tissue among hepatic lobules (Figure 3). The amount of interlobular connective tissue proved to be significantly lower in the experimental group compared with the control group of gilts (*p* = 0.0232) (Figure 4).

**Figure 3.** Representative microphotographs of hepatic lobules and surrounding interlobular connective tissue of gilts: (**A**) gilt from the control group with an expected amount of interlobular connective tissue and (**B**) gilt from the experimental group with a decreased amount of interlobular connective tissue in comparison with the control group. Goldner's Masson trichrome stain, bar = 100 μm.

**Figure 4.** Distribution of interlobular connective tissue in the liver of the control and experimental groups of gilts with a statistically significant difference.
