**4. Discussion**

When discussing tannins, it must be noted that there are many published studies with different, even contradictory results, which report positive, negative or no effect of tannin supplementation on the growth performance of bulls. In this context, Patra and Saxena [10] reported that the growth performance was associated with various responses to tannin supplementation using different chemical structures and concentrations of tannins used in studies, different animals, and basal diet used in feeding trials. Different effects are often attributed to condensed tannins, compared with hydrolyzable tannins. Although research on these two types of tannins is important, and previous studies have reported similar effects [44–46], generalizations should be carefully considered [47]. In the present study, we focused on hydrolyzable tannins and their effect on ruminants. However, some comparisons are discussed, as many more studies associated with the effects of tannins are conducted with condensed tannins [9].

#### *4.1. Effects of Tannins on Growth Performance of Bulls*

As previously mentioned, tannins can improve the ratio of bypass proteins, which can lead to improved intestinal absorption of amino acids and thus have a positive effect on daily gain. Our results show that supplementation of the diet with HT affected the growth of bulls, and significant differences were detected in the last three months of our trial when the bulls reached 538 ± 20.3 kg BW until the slaughter maturity (677 ± 19.2 kg). In the present study, there were no differences in final ADG and BW between the groups. Furthermore, no reduction in the daily DMI was detected. This is in agreemen<sup>t</sup> with the feeding study of Aboagye et al. [48]. They studied the effects of feeding chestnut (HT) extract and a combination (50:50) of HT and quebracho (CT) extracts in a powdered form at different concentrations of dietary DM for three months on steers. There were no effects of treatment on DMI, BW, ADG in both HT groups, or a combination of both tannins and both concentrations. In the current study, total ADG and BW were not affected, although we observed a higher ADG at the end of the trial. Tabke et al. [49] supplemented tannic acid (HT) into steam-flaked corn-based finishing diets of steers. Overall, no effects were observed for ADG, carcass characteristics, hot carcass weight, *longissimus* muscle area, fat thickness, and yield grade during the study in any treatment. The study by Krueger et al. [44] used commercially available mimosa (CT) and chestnut (HT) extracts and added them to a high-grain diet to fatten crossbred steers. Including tannins in the diet resulted in similar DMI for steers in the control and both tannin-treatment groups. Tannin supplementation had no effect on animal performance or carcass, except for HCW. We conclude that the absence of an effect of tannins on animal performance observed in this study could be due to the conservative dose of tannins.

We recorded differences in ADG during the last three months of the feeding trial. The positive impacts of tannin supplementation during the last feeding period were also observed by Brus et al. [50]. Commercial HT additives in powder form were used in the diet of Simmental bulls. After month 8 of the study, ADG was significantly higher in the group with tannin wood extract and lower in the control group. The higher measured ADG in the last period of the feeding trial is in agreemen<sup>t</sup> with our findings. Tannin supplementation in the finishing feeding phase of Holstein steers was studied by Rivera-Mendez et al. [46]. Supplementation with tannins increased average daily gain (ADG, 6.8%) and dry matter intake (DMI, 4.0%) in the finishing feeding phase. The authors concluded that the mechanism responsible for the higher ADG has not been fully understood and explained.

A possible explanation for the achievement of significantly higher ADGs in the final phase of bull fattening in the present study is the nature of nutrient metabolism of ruminants and the specific effect of hydrolyzable tannins. The extent and composition of daily gain in the final phase of fattening depends on the available energy and proteins in the animal's diet, which is above the maintenance requirement [51]. In this context, ruminants can take advantage of the symbiotic effect of digestion with microorganisms in the rumen, which decompose the nutrients by using their own digestive enzymes. Microbes can form ideal nutrients for ruminants from the decomposition products. Ruminants meet their metabolic protein needs from two sources: true microbial proteins from microbial synthesis and proteins from feed (bypass proteins) [52]. The addition of HT influenced the microbial metabolic process of nutrient synthesis in the rumen, which may lead to better feed efficiency and a higher growth rate of fattening bulls. The chemical composition of the tested feed mixtures is the same in terms of energy content, thus, differences in ADG size are not due to energy alone, as the authors claim [53]. The possible explanation for the differences in ADG can be attributed to the different amounts of crude protein in the TMR. In the present study, all groups with HT had significantly higher ADG than the control

group. Therefore, we hypothesized that HT influenced the ruminal digestion of rumen degradable and undegradable dietary proteins. Consequently, increased protein production in the rumen from both sources resulted in greater availability of enzymatically degradable proteins in the small intestine. It can be assume that the addition of HT increased the availability of amino acid absorption, which could pass into the amino acid pool.

#### *4.2. Effect of Tannin Supplement on Meat Quality*

In this study, meat quality traits such as meat color, IMF, protein content, marbling, and tenderness were not affected by HT plant extract. Similar results regarding meat quality traits were observed in a study by Joo et al. [54]. The objective of this study was to examine the effects of dietary fermented chestnuts on growth performance, carcass, and meat quality parameters (cold carcass weight, back fat thickness, *longissimus* muscle area, marbling score, and fat color) in the late fattening period of Hanwoo steers. No effects on growth performance or carcass traits were observed. Moreover, differences were observed in physicochemical characteristics (cooking loss, water-holding, shear force), except meat pH. Beef meat quality in relation to added tannins was also studied by Larraín et al. [55]. They observed an effect on meat color and lipid oxidation in beef *longissimus lumborum* (LL) and *gluteus medius* (GM) muscles. They found that supplementation with a diet of high-tannin sorghum increased the rate of color change during aerobic oxygenation and modulated lipid oxidation in two ways: it reduced oxidation before aerobic storage and accelerated oxidation during aerobic display of the tissue.

In other species, supplementation with tannins or feeding tannin-rich feeds had little or no effect on meat quality, as seen when supplementing a natural extract of chestnut wood to rabbits [56] or pigs [57]. Similarly, de Jes ús et al. [58], who studied the effect of feeding dried chestnuts (15% and 25% of the formulation), found no effect on the physiochemical properties (color parameters, water holding capacity, and shear force) of *Longissimus dorsi* muscle.

Our research has shown that adding tannins to a bull's diet significantly reduces drip loss in fresh meat. However, after two weeks, meat from bulls that received tannin supplementation exhibited significantly higher meat thaw loss. Similar conclusions were also reported by Joo et al. [59]. They reported that adding chestnut meal at 30 g kg−<sup>1</sup> resulted in lower drip loss in pigs. Drip loss is a method of evaluating water holding capacity, which is an essential quality parameter for both the industry and the consumer and is related to the status of proteins that bind the water that is mainly affected by postmortem conversion of muscle to meat (pH decline) and the rate of carcass refrigeration (especially deep muscles) [60].

#### *4.3. Effect of Supplement on Fecal Clostridia Concentration*

In the present study a notable reduction was observed when the concentration of individual *Clostridium* species was compared. Differences in the concentration of *C. perfringens* (*p* = 0.004) were observed between groups at the end of the feeding trial. The results of our study are in good agreemen<sup>t</sup> with previous studies. Redondo et al. 2015 [61] found that *C. perfringens* isolated from bovine feces had difficulty forming resistance to hydrolyzable tannins. The study of Elizondo et al. [62] confirmed bacteriostatic and bactericidal activities in vitro experiments with *C. perfringens*. The bacteriostatic activity of quebracho and chestnut tannins was tested on selected *C. perfringens* strains of toxin types A, C, D, and E. The concentrations of quebracho required to inhibit the growth of *C. perfringens* were 7–85 times higher than those of chestnut tannin (0.6–1.2 mg/mL vs. 0.003–0.15 mg/mL). The bactericidal effect of quebracho occurs within 5 h of administration and, in the case of chestnut tannin, virtually immediately in 5 min. Our results are in agreemen<sup>t</sup> with the previous study on steers, in which an antimicrobial effect of hydrolyzable tannins on *E. coli* and coliform bacteria was observed [63]. The effect was limited to the rumen, whereas in present study to the entire digestive tract.
