*2.1. Clinical Observations*

Clinical manifestations of ZEN mycotoxicosis were not noted during the experiment. However, histopathological analyses, ultrastructural analyses, and analyses of the metabolic profile of samples taken from same gilts frequently revealed changes in certain tissues or cells. These findings have been posted in various articles [2,19,20,31–35].

## *2.2. Optical Density*

The brown background staining of the slides (Figures 1 and 2) was not specific to all intestinal segments, and it may have occurred in staining assays examining the ERα and ERβ expression in DAB-stained gastrointestinal tissues (most samples exhibited lightbrown, non-specific staining).

The effect of six-week exposure to ZEN on the expression levels of the selected ERs was determined in selected segments of the gastrointestinal tract (GI) of gilts in the control and experimental groups using a four point scale (negative—0; weak and homogeneous—1; mild or moderate and homogeneous—2; intense or strong and homogeneous—3) (Figures 3 and 4). Expression levels were compared between the dates of sample collection in specific sections of the intestines. Meaningful differences in the IE of ERα were not

observed in the descending colon in the control group and in the ascending colon and descending colon in the experimental group. Meaningful differences in the IE of ERβ were not noted in the caecum and ascending colon in group C, and in the duodenal cap, the third section of the duodenum and the caecum in group E. The intestinal sections where no significant differences were found are not presented graphically. The brown background staining of the slides (Figures 1 and 2) was not specific to all intestinal segments, and it may have occurred in staining assays examining the ERα and ERβ expression in DAB-stained gastrointestinal tissues (most samples exhibited lightbrown, non-specific staining).

*Toxins* **2023**, *15*, x FOR PEER REVIEW 3 of 18

*2.2. Optical Density* 

**Figure 1.** Scanned slides showing the IE of ERα in the descending colon in group C ((**A**)—0; (**B**)—+; (**C**)—++; (**D**)—+++) and group E ((**E**)—0; (**F**)—+; (**G**)—++;. (**H**)—+++). HE. **Figure 1.** Scanned slides showing the IE of ERα in the descending colon in group C ((**A**)—0; (**B**)—+; (**C**)—++; (**D**)—+++) and group E ((**E**)—0; (**F**)—+; (**G**)—++;. (**H**)—+++). HE.

**Figure 2.** Scanned slides showing the IE of ERβ in the descending colon in group C ((**A**)—0; (**B**)—+; (**C**)—++; (**D**)—+++) and group E ((**E**)—0; (**F**)—+; (**G**)—++; (**H**)—+++). HE. **Figure 2.** Scanned slides showing the IE of ERβ in the descending colon in group C ((**A**)—0; (**B**)—+; (**C**)—++; (**D**)—+++) and group E ((**E**)—0; (**F**)—+; (**G**)—++; (**H**)—+++). HE.

The effect of six-week exposure to ZEN on the expression levels of the selected ERs was determined in selected segments of the gastrointestinal tract (GI) of gilts in the control and experimental groups using a four point scale (negative—0; weak and homogeneous— 1; mild or moderate and homogeneous—2; intense or strong and homogeneous—3) (Figures 3 and 4). Expression levels were compared between the dates of sample collection in specific sections of the intestines. Meaningful differences in the IE of ERα were not observed in the descending colon in the control group and in the ascending colon and On each date of analysis, ERα was more highly expressed in the control group than in the experimental group, especially at absorbance level 0 (Figure 3A–D). Significant differences in ERα expression were found in the control group at different absorption levels, but absorption was significantly more pronounced on dates I, II, and VI. Significant differences in ERα expression were also observed at other absorption levels, but the noted values were much lower than at absorption level 0, and they were only found in the small intestine (Figure 3A–D). In the control group, the average ERα expression was highest at absorbance level 0, and it increased when the digesta entered the caudal segment of the small intestine.

no significant differences were found are not presented graphically.

descending colon in the experimental group. Meaningful differences in the IE of ERβ were not noted in the caecum and ascending colon in group C, and in the duodenal cap, the third section of the duodenum and the caecum in group E. The intestinal sections where

**Figure 3.** IE of ERα (based on a 4-point grading scale: negative—0; weak and homogeneous—1; mild or moderate and homogeneous—2; intense or strong and homogeneous—3) in the intestines of sexually immature gilts from the control group: (**A**) in the duodenal cap on selected dates of exposure; (**B**)—in the third section of the duodenum on selected dates of exposure; (**C**) in the jejunum on selected dates of exposure; (**D**) in the caecum on selected dates of exposure. In the intestines of sexually immature gilts from the experimental group: (**a**) in the duodenal cap on selected dates of exposure; (**b**) in the third section of the duodenum on selected dates of exposure; (**c**) in the jejunum on selected dates of exposure only in the weak(1) and mild (2) grades; (**d**) in the caecum on selected dates of exposure only in the weak grade (1). Expression was presented as ± **Figure 3.** IE of ERα (based on a 4-point grading scale: negative—0; weak and homogeneous—1; mild or moderate and homogeneous—2; intense or strong and homogeneous—3) in the intestines of sexually immature gilts from the control group: (**A**) in the duodenal cap on selected dates of exposure; (**B**)—in the third section of the duodenum on selected dates of exposure; (**C**) in the jejunum on selected dates of exposure; (**D**) in the caecum on selected dates of exposure. In the intestines of sexually immature gilts from the experimental group: (**a**) in the duodenal cap on selected dates of exposure; (**b**) in the third section of the duodenum on selected dates of exposure; (**c**) in the jejunum on selected dates of exposure only in the weak(1) and mild (2) grades; (**d**) in the caecum on selected dates of exposure only in the weak grade (1). Expression was presented as ± (confidence interval) and SE (standard error) for some samples. \* *p* ≤ 0.05 and \*\* *p* ≤ 0.01 compared with the residual groups.

**Figure 4.** IE of ERβ (based on a 4-point grading scale: negative—0; weak and homogeneous—1; mild or moderate and homogeneous—2; intense or strong and homogeneous—3) in the intestines of sexually immature gilts from the control group: (**A**) in the duodenal cap on selected dates of exposure; (**B**) in the third section of the duodenum on selected dates of exposure; (**C**) in the jejunum on selected dates of exposure only in the negative (0) and intense (3) grades; (**D**) in the descending colon on selected dates of exposure only in the negative (0) and intense (3) grades; in the intestines of sexually immature gilts from the experimental group: (**a**) in the jejunum on selected dates of exposure only in the negative grade (0); (**b**) in the ascending colon on selected dates of exposure; (**c**) in the descending colon on selected dates of exposure only in the mild (2) and intense (3) grades. Expression was presented as ± (confidence interval) and SE (standard error) for some samples. \* *p* ≤ 0.05 and \*\* *p* ≤ 0.01 compared with the residual groups. **Figure 4.** IE of ERβ (based on a 4-point grading scale: negative—0; weak and homogeneous—1; mild or moderate and homogeneous—2; intense or strong and homogeneous—3) in the intestines of sexually immature gilts from the control group: (**A**) in the duodenal cap on selected dates of exposure; (**B**) in the third section of the duodenum on selected dates of exposure; (**C**) in the jejunum on selected dates of exposure only in the negative (0) and intense (3) grades; (**D**) in the descending colon on selected dates of exposure only in the negative (0) and intense (3) grades; in the intestines of sexually immature gilts from the experimental group: (**a**) in the jejunum on selected dates of exposure only in the negative grade (0); (**b**) in the ascending colon on selected dates of exposure; (**c**) in the descending colon on selected dates of exposure only in the mild (2) and intense (3) grades. Expression was presented as ± (confidence interval) and SE (standard error) for some samples. \* *p* ≤ 0.05 and \*\* *p* ≤ 0.01 compared with the residual groups.

An analysis of the IE of ERα revealed that it was suppressed in most intestinal segments on all dates in group E (0 points on a 4-point scale), but significant differences were detected only on dates I, II, and VI (Figure 3a). ERα was more highly expressed in the ascending and descending colon at absorption level 3 in the experimental group than in the control group. However, in group E, ERα expression was suppressed at all absorption levels (Figure 3a–d). Differences in the ERα expression were noted in the control group, but only in selected segments of the small intestine, particularly in both parts of the duodenum examined in the study (Figure 3a,b). Similarly to group C, ERα expression was induced in the experimental group at absorbance level 0, whereas at absorbance level 3, the levels of ERα expression in the analysed intestinal segments were higher in the experimental group than in the control group.

In group C, the IE of ERβ was suppressed in both segments of the duodenum, jejunum, and descending colon (Figure 4A–D). The average values of ERß expression in the control group and in the experimental group followed a certain trend. In group E, ERß expression was observed at absorbance level 3, and ERβ was more strongly expressed in all analysed tissues, but its expression was more suppressed at absorbance level 0. However, these differences were not significant. An immunohistochemical analysis of ERß expression in the examined intestinal segments, compared with ERα expression, revealed completely different results. In group E, ERβ was more strongly expressed, especially at absorption level 3 and, interestingly, in the jejunum and colon (Figure 4a–c). However, significant differences between the groups were found only on dates I, II, and III, especially in the examined segments of the duodenum, which can be explained by the fact that ERβ saturation was lower in the duodenum than in the other intestinal segments.
