*2.2. Effect of Herbal Supplements on Honey Bee Strength and Production Traits* 2.2.1. Strength of the Honey Bee Colonies (Estimated Based on Mass)

The strength of the bee colonies was evaluated based on colony mass 12 times during the first pre-treatment (August 2019) and after that 4 times at 12-day intervals posttreatment until September 2020. After the winter period October 2019–February 2020, the same investigations were performed before the second pre-treatment (March 2020) and after that 4 times at 12-day intervals post-treatment until June 2020 (Figure 2). The first difference between the experimental groups (NH and NHP) and the control C group was observed about 25 days after the first treatment (at the end of August 2019). Then, the mass of the bee colonies in the NH and NHP groups was estimated to be 1.65 ± 0.05 kg and 1.63 ± 0.12 kg, respectively, while in the control C group it was 1.25 ± 0.10 kg (*F* = 6.818, *df* = 6, *p* = 0.040; *F* = 6.943, *df* = 6; *p* = 0.038, respectively). As the autumn period approached, there was a gradual decrease in the studied indicator of bee colony strength in both the control and the two experimental groups, reaching almost full equalization at the end of September(Figure 2). During the second pre-treatment (9 April 2020) the average mass values indicating the strength of the honey bee colonies were significantly higher in the experimental groups than in the control group (ANOVA with Tukey's HSD post hoc test, *F* = 26.727, *df* = 8, *p* = 0.002; *F* = 30.857, *df* = 8, *p* = 0.001, respectively) (Figure 2). The most significant difference was observed in the NHP group, where the mass exceeded 1.2 times that of the control group. This is an indicator of the successful wintering of honey bee colonies and a good start for spring development.

**Figure 1.** *N. ceranae* spore counts (±SD) pre- and post-treatment with NOZEMAT HERB® and NOZEMAT HERB PLUS® during 2019 (**a**) and 2020 (**b**). The number of *N. ceranae* spores was counted by microscopic examination. Asterisks indicated the level of significance as determined by an analysis of variance followed by Tukey's multiple comparison test. \*\*: *p* < 0.01 compared to the Nosema spore during pre-treatment.

**Figure 2.** Average (±SD) values in colony strength in honey bee colonies treated with NOZEMAT HERB® and NOZEMAT HERB PLUS® and in an untreated (control) group during 2019 and 2020. Asterisks indicated the level of significance as determined by an analysis of variance followed by Tukey's multiple comparison test. \*: *p* < 0.05, \*\*: *p* < 0.01 compared to the untreated control group.

Similar to the first treatment in the previous year (August 2019), the first significant difference between the control and the experimental groups was observed around the 10th day after the second treatment (9 April 2020). Then, the mass (and, accordingly, the strength) of the colonies from the NH group was 1.8 times higher than that of the control C group (*F* = 31.176, *df* = 6, *p* = 0.001). The difference between the NHP and the control C was about 0.150 kg bees (*F* = 32.666, *df* = 6, *p* = 0.001). About two months after the second treatment, the difference between the two experimental groups and the control group was about 0.450 kg bees. The mass (and, accordingly, the strength) of the bee colonies of the NH group (1.60 ± 0.07 kg) and NHP group (1.35 ± 0.10 kg) was, respectively, about 1.5 and 1.2 times higher than that of the control group (1.06 ± 0.09 kg) (*F* = 19.091, *df* = 8, *p* = 0.002; *F* = 16.90, *df* = 8, *p* = 0.003, respectively). These significant differences persisted during the next measurement period (middle of May 2020), after which there was a less pronounced advantage of the experimental groups over the control group until the last reporting period (June 2020) (Figure 2).

#### 2.2.2. Sealed Worker Brood Area within the Hives

A lot of factors determine the queen's egg-laying rate: age, genotypes, colony size, colony nutrition, etc. It is difficult to quantify the performance of queens relative to workers in the field, and there are not many laboratory assays on queen performance. For this reason, the obtained results on sealed worker brood areas within the hives may not be completely comprehensive, yet this is a parameter that needs to be considered. In contrast to the achieved results regarding the strength of the honey bee colonies, the parameter of sealed worker brood area showed fewer differences after the treatment of the honey bee colonies with herbal supplements (Figure 3). After the first application of NH and NHP (August 2019), the sealed worker brood area gradually decreased both in the experimental (NH and NHP) and in the control C group, which is a biologically determined process in the late autumn period (September 2019). After the winter period and the second pretreatment (April 2020), the sealed worker brood area in the hives increased; however, only in the NH group (925 ± 29.3 cm2) there were statistically significant differences, compared to the NHP (200 ± 31.7 cm2) and the control C group (120 ± 26.9 cm2) (*<sup>F</sup>* = 6.241, *df* = 6, *p* = 0.037; *F* = 0.303, *df* = 6, *p* = 0.610, respectively) (Figure 3). The second statistically significant difference between the experimental groups and the control group was observed after the fifth month following the second treatment (June 2020). Then, the NH and NHP groups had a significantly larger sealed worker brood area, compared to the control C group (ANOVA with Tukey's HSD post hoc test, *df* = 8, *F* = 9.424, *p* = 0.018; *F* = 13.256, *df* = 8, *p* = 0.008, respectively).

**Figure 3.** Average (±SD) worker sealed brood area (cm2) in honey bee colonies treated with NOZEMAT HERB® and NOZEMAT HERB PLUS® and in an untreated (control) group during 2019 and 2020. Asterisks indicated the level of significance as determined by an analysis of variance followed by Tukey's multiple comparison test. \*: *p* < 0.05 compared to the untreated control group.
