Ruminant farming is an important pillar industry of the country, however, the utilization of nitrogen by ruminants is relatively low under general feeding conditions, and the inefficient use of nitrogen not only affects the ruminant production performance and economic benefits, but also increases nitrogen emissions and causes serious environmental pollution. An important reason for low nitrogen utilization in ruminants is the extensive degradation of nitrogenous compounds in the diet by rumen microorganisms, resulting in the production of ammonia nitrogen in the rumen that oversteps the ability of micro-organisms to utilize the ammonia nitrogen, which is ultimately excreted through urea nitrogen (Tan et al., 2021; Niyigena et al., 2024). Tannin is a plant extract from a wide range of sources, with a variety of biological activities. Some studies have shown that tannin has a strong affinity for proteins, and adding the right amount of tannin to animal feed can increase feed utilization and improve animal health (Aboagye et al., 2018; Battelli et al., 2024). Naturally derived tannins are generally categorized as condensed tannins (CT) or hydrolyzable tannins (HT) based on their chemical structure (Nuamah et al., 2024).
In recent years, HT as a safe and effective feed additive in animal husbandry has attracted much attention, which mainly exists in the bark and fruit of various trees, such as chestnut and oak trees (Oroian and Escriche, 2015). HT can regulate many physiological activities, which could alter nitrogen metabolic pathways, and reduce nitrogen losses and ammonia emissions (Grainger et al., 2009; Aboagye et al., 2019; Zhu et al., 2020). HT can also interact with proteins to form hydrogen bonds between the phenolic groups of tannins and the carboxyl groups of the protein chain bonds, and the strength of the bond affects protein digestibility (Yusiati et al., 2018). Moreover, relevant studies have shown that the addition of 6 g/kg chestnut HT in rabbit diet can increase the utilization efficiency of proteins (Mancini et al., 2019). Furthermore, Wang et al. (2021) reported that dietary chestnut HT supplementation can reduce ammonia nitrogen. Relevant studies have shown that HT can reduce urea nitrogen in the urine of dairy cows and affect the nitrogen metabolism process in ruminants (Aguerre et al., 2020).
Tannic acid (TA) is a typical HT, which may be toxic to ruminants, especially when ingested at a high concentration, and is easily decomposed into gallic acid, pyrogallic acid and resorcinol by microorganisms using the tanilacylhydrolases and esterases (Singh et al., 2001; Fonseca et al., 2023). However, when consumed at low to moderate levels, TA can provide beneficial effects. Research has found that the dietary 2 g/kg TA could significantly improve average daily gain of beef cattle (He et al., 2023). Based on the work of Choi et al. (2022), the addition of 0.25 g/kg, 0.5 g/kg, 1 g/kg or 2 g/kg dry matter (DM) TA to broiler chicken diets could linearly increase the expression of protein-related genes and improve the gut microbial community. Dietary 10 g/kg DM TA supplementation to pigs can reduce the crude protein (CP) digestibility and promote gut microbial metabolism and short-chain fatty acid production (Koo and Nyachoti, 2019). In summary, dietary HT supplementation may have effects on protein metabolism in different species of animals.
Liaoning cashmere goat is a precious genetic protection resource in China, which has the advantages of high cashmere yield, superior quality and stable genetic performance. With the transformation of its feeding method, it is likely to cause unnecessary protein waste. Therefore, how to feed Liaoning cashmere goat scientifically and rationally without wasting protein resources has become an urgent problem to be solved. However, there is a limited number of papers about the influence of TA on the protein metabolism of Liaoning cashmere goat. This study aimed to investigate the effect of TA on the growth performance, apparent digestibility, blood indices, nitrogen metabolism and rumen fermentation in Liaoning cashmere goat.
This experiment was conducted at the goat farm of Liaoning Agricultural Technical College, Yingkou City, Liaoning Province, China. All experimental procedures were performed according to the Institutional Animal Care and Use Committee of Shenyang Agricultural University.
Twenty-four healthy Liaoning cashmere goats with body weight of 23.78±1.25 kg were selected and divided into four treatment groups (n=6) using a one-way completely randomized design. Goats in the control group (TA0) were fed only basal diet and did not receive any supplementation, while goats in the three other groups, TA2, TA4 and TA8, received basal diets supplemented with 2, 4 and 8 g/kg DM TA, respectively. Experimental basal diets were formulated to meet the nutritional requirements of Liaoning cashmere goats with estimated weight gain of 30 g/day following the Agricultural Industry Standard of the People's Republic of China (NY/T4048-2021). The formulations and chemical analyses (%DM basis) of the basal diet are shown in Table 1. Commercial TA (purity 98%, Sinopharm Chemical Reagent Co., Ltd., China) was added to the concentrate and homogenized immediately prior to feeding. Prior to the feeding experiment, the experimental equipment was sterilized. Diets were offered twice a day at 7:00 h and 16:00 h as total mixed ration with 66% forage and 34% concentrate (DM basis). Goats were allowed ad libitum access to feed and water throughout the experiment. The feeding trial lasted for 32 days after an adjustment period of 7 days.
Ingredients and chemical composition of the basal diet (DM basis)
| Item | Content % |
|---|---|
| Ingredients | |
| corn | 19.60 |
| wheat bran | 5.30 |
| soybean meal | 5.60 |
| DDGS | 1.40 |
| NaCl | 0.20 |
| CaHPO4 | 0.50 |
| CaCO3 | 0.50 |
| premix1 | 1.00 |
| peanut seedling | 39.50 |
| corn stalks | 16.50 |
| oat grass | 9.90 |
| total | 100 |
| Nutrient levels | |
| digestible energy (MJ/kg)2 | 10.20 |
| ash | 7.10 |
| crude protein | 11.37 |
| ether extract | 4.73 |
| neutral detergent fiber | 45.63 |
| acid detergent fiber | 28.07 |
| calcium2 | 0.61 |
| phosphorus2 | 0.31 |
Supplied per kilogram of diet: vitamin A 15000 IU; vitamin D3 5500 IU; vitamin E 110000 IU; Cu 200 mg; Fe 1600 mg; Mn 1100 mg; I 30 mg; Zn 1500 mg; Se 6 mg; Co 15 mg.
Digestible energy, calcium, and total phosphorus in the nutrient levels are calculated values, and the rest are measured values.
The test goats were weighed before the early morning feeding on the 1st day of the main feeding period for the initial body weight (IBW), and before the morning feeding on the 28th day of the main feeding period for the final body weight (FBW). Body weight gain (BWG) was calculated as the difference between the FBW and IBW, and average daily gain (ADG) was calculated as BWG divided by the number of days (28 days). The feed conversion ratio (FCR) was calculated by dividing average daily feed intake (ADFI) by ADG.
Before the end of the feeding experiment, a digestion and nitrogen balance trial was conducted lasting for 4 days. Liaoning cashmere goats from each treatment group were housed individually in metabolic cages for feces and urine separation. The dietary intake was collected every 24 hours prior to feeding in the morning. The total amount of feces and urine excreted per goat was measured at intervals of 24 hours and treated with 10% sulfuric acid. An aliquot corresponding to 10% of the total amount per goat was collected, and stored at −20°C. The feces samples were oven-dried at 65°C for 48 h, then ground and stored for further chemical analysis.
The DM, ash and CP of feed and feces samples were determined according to methods 934.01, 920.39 and 924.05 of AOAC (1990), respectively. Ether extract (EE), acid detergent fiber (ADF) and neutral detergent fiber (NDF) were analyzed using an Ankom A200i fiber analyzer (ANKOM Technologies, USA) following the method described by Van Soest et al. (1991).
Rumen fluid samples were obtained before morning feeding on the 28th day. A stomach tube attached to a vacuum pump was used to collect approximately 100 mL of rumen fluid from the central area of the rumen. A portable pH meter was used to measure the pH value of the rumen fluid immediately. The rumen fluid samples were filtered through two layers of cheesecloth and stored at −80°C. The rumen fluid volatile fatty acid (VFA) was analyzed by gas chromatograph (Agilent 7890B, USA). Ammonia nitrogen was analyzed according to the method of Broderick and Kang (1980). The rumen microbial crude proteins (MCP) were determined by assay kits (Nanjing Jiancheng Bioengineering Institute, China).
On the 28th day, blood samples were collected through the jugular vein using vacuum blood collection tubes containing procoagulant. The blood samples were then centrifuged at 3000 rpm for 15 min at 4°C. The supernatant was retained and stored at −20°C for measurement of urea nitrogen and total protein (TP), albumin (Alb), creatinine (CREA), uric acid (UA), glucose (Glu), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The above indices were determined using kits purchased from the Beijing Sino-Uk Institute of Biological Technology according to the instructions.
The data were analyzed by one-way ANOVA in SPSS v20 statistical software (SPSS Inc., 2010, Chicago, USA). Duncan's multiple comparisons were conducted to compare statistical differences among different treatments. Values in the tables are presented as means and pooled sample standard error of the mean (SEM). When a significant difference was observed, the difference among the groups was assessed (P<0.05).
There were no significant differences in IBW, FBW, BWG, ADG, ADFI and FCR among the dietary TA groups (P>0.05) (Table 2). Table 3 shows the effect of TA on the nutritional apparent digestibility of Liaoning cashmere goats. As can be seen after detailed analysis, dietary TA supplementation increased the CP digestibility significantly (P<0.05). Compared to the control group, the CP digestibility was increased by 10.2%, 5.7% and 8.6% in TA2, TA4 and TA8, respectively. The addition of 2 g/kg DM TA could increase the digestibility of EE and NDF (P<0.05). However, no difference was observed in the digestibility of DM, ash and ADF between the four groups (P<0.05).
Effect of tannic acid on growth performance in Liaoning cashmere goats
| Items | Tannic acid supplementation (g/kg DM) | SEM | P value | |||
|---|---|---|---|---|---|---|
| 0 | 2 | 4 | 8 | |||
| IBW (kg) | 23.25 | 24.08 | 24.06 | 23.80 | 0.27 | 0.676 |
| FBW (kg) | 24.35 | 26.23 | 25.64 | 25.27 | 0.36 | 0.283 |
| BWG (kg) | 1.10 | 2.15 | 1.58 | 1.47 | 0.22 | 0.406 |
| ADG (g/d) | 41.66 | 76.66 | 56.00 | 55.00 | 7.98 | 0.180 |
| ADFI (g/d) | 658.3 | 662.27 | 702.37 | 715.02 | 13.00 | 0.173 |
| FCR | 23.86 | 11.63 | 16.04 | 26.01 | 3.82 | 0.254 |
IBW: initial body weight, FBW: final body weight, BWG: body weight gain, ADG: average daily gain, ADFI: average daily feed intake, FCR: feed conversion ratio, SEM: standard error of mean.
Effect of tannic acid on apparent digestibility (%) of nutrients in Liaoning cashmere goats
| Items | Tannic acid supplementation (g/kg DM) | SEM | P value | |||
|---|---|---|---|---|---|---|
| 0 | 2 | 4 | 8 | |||
| Dry matter | 69.88 | 69.72 | 70.30 | 71.83 | 0.91 | 0.884 |
| Ash | 60.38 | 56.97 | 62.70 | 63.19 | 1.54 | 0.795 |
| Crude protein | 80.83 b | 89.10 a | 85.44 a | 87.80 a | 1.19 | 0.047 |
| Ether extract | 86.48 bc | 93.90 a | 91.54 ab | 84.32 c | 1.30 | 0.022 |
| Neutral detergent fiber | 56.79 b | 64.66 a | 63.96 ab | 67.65 a | 1.44 | 0.046 |
| Acid detergent fiber | 51.47 | 53.54 | 52.40 | 57.78 | 1.53 | 0.564 |
– different letters indicate significant differences (P<0.05).
SEM: standard error of mean.
No significant effects related to TA inclusion levels were observed for the nitrogen intake and fecal nitrogen (FN) in Table 4 (P>0.05). It shows a significant decrease in urine nitrogen (UN) and urinary urea nitrogen (UUN) in the 4 or 8 g/kg DM TA supplementation group compared to the control group (P<0.05). The 4 g/kg DM TA supplementation group showed a significant increase in nitrogen deposition and nitrogen deposition rate (P<0.05), reaching increases of 75.4% for nitrogen deposition, 64.3% for nitrogen deposition rate.
Effect of tannic acid on nitrogen metabolism in Liaoning cashmere goats
| Items | Tannic acid supplementation (g/kg DM) | SEM | P value | |||
|---|---|---|---|---|---|---|
| 0 | 2 | 4 | 8 | |||
| Nitrogen intake (g/d) | 96.74 | 98.51 | 102.72 | 104.63 | 1.47 | 0.202 |
| Nitrogen excreted (g/d) | ||||||
| feces | 17.29 | 16.49 | 18.39 | 18.24 | 0.94 | 0.922 |
| urine | 54.55 a | 48.20 ab | 40.67 b | 39.29 b | 2.27 | 0.021 |
| Nitrogen deposition (g/d) | 24.88 b | 33.81 a | 43.65 a | 47.08 a | 3.16 | 0.020 |
| Nitrogen deposition rate (%) | 25.72 b | 34.32 ab | 42.26 a | 44.99 a | 2.81 | 0.025 |
| UUN (mmol/L) | 582.13 a | 518.87 ab | 501.64 ab | 421.38 b | 21.68 | 0.043 |
– different letters indicate significant differences (P<0.05).
UUN: urinary urea nitrogen, SEM: standard error of mean.
The results of the rumen fermentation from Liaoning cashmere goats are shown in Table 5. Compared to the control group, the concentrations of propionic acid, butyric acid, valeric acid and MCP were increased in the 4 g/kg TA group significantly (P<0.05). No significant change was observed in the concentrations of total VFA, acetic acid, isobutyric acid and isovaleric acid (P>0.05). Moreover, there were no significant differences in the pH value and ammonia nitrogen between the four groups (P>0.05).
Effect of tannic acid on ruminal fermentation in Liaoning cashmere goats
| Items | Tannic acid supplementation (g/kg DM) | SEM | P value | |||
|---|---|---|---|---|---|---|
| 0 | 2 | 4 | 8 | |||
| pH | 6.89 | 6.92 | 7.05 | 6.98 | 0.02 | 0.247 |
| Total VFA (mmol/L) | 71.17 | 71.01 | 85.74 | 81.57 | 3.16 | 0.239 |
| Individual VFA (mmol/L) | ||||||
| acetic acid | 52.30 | 51.49 | 59.33 | 57.02 | 2.15 | 0.540 |
| propionic acid | 10.33 b | 9.04 b | 13.67 a | 12.44 ab | 0.66 | 0.045 |
| butyric acid | 6.33 b | 6.59 ab | 10.05 a | 9.79 ab | 0.65 | 0.048 |
| isobutyric acid | 0.49 | 0.98 | 0.82 | 1.86 | 0.26 | 0.382 |
| valeric acid | 0.41 c | 0.49 bc | 0.78 a | 0.71 ab | 0.05 | 0.026 |
| isovaleric acid | 0.57 | 0.72 | 1.05 | 0.79 | 0.07 | 0.159 |
| Ammonia nitrogen (mg/100 mL) | 8.78 | 8.69 | 8.76 | 8.91 | 0.16 | 0.980 |
| MCP (mg/mL) | 12.84 b | 23.09 ab | 26.82 a | 22.12 ab | 1.96 | 0.036 |
– different letters indicate significant differences (P<0.05).
VFA: volatile fatty acids, SEM: standard error of mean, MCP: microbial crude proteins.
The serum samples of Liaoning cashmere goats were collected and analyzed biochemically, and the results are shown in Table 6. The content of TP, Alb and CREA was significantly increased in the 4 g/kg TA group compared to the control group (P<0.05). The content of blood urea nitrogen (BUN), UA, Glu, AST and ALT in serum was not affected by the TA treatment (P>0.05).
Effect of tannic acid on serum parameters in Liaoning cashmere goats
| Items | Tannic acid supplementation (g/kg DM) | SEM | P value | |||
|---|---|---|---|---|---|---|
| 0 | 2 | 4 | 8 | |||
| Serum protein | ||||||
| total protein (g/L) | 65.06 b | 74.56 a | 72.73 a | 66.02 b | 1.59 | 0.047 |
| albumin (g/L) | 18.80 b | 20.86 ab | 23.67 a | 19.87 b | 0.68 | 0.040 |
| Serum metabolites | ||||||
| creatinine (μmol/L) | 73.41 c | 77.88 bc | 106.14 a | 87.75 b | 4.16 | 0.002 |
| blood urea nitrogen (mmol/L) | 7.35 | 6.34 | 6.93 | 6.20 | 0.34 | 0.683 |
| uric acid (μmol/L) | 2.27 | 2.51 | 0.81 | 0.57 | 0.36 | 0.113 |
| glucose (mmol/L) | 3.19 | 2.44 | 2.81 | 2.93 | 0.13 | 0.297 |
| Enzymes (U/L) | ||||||
| aspartate aminotransferase | 83.53 | 90.09 | 89.14 | 92.28 | 3.73 | 0.898 |
| alanine aminotransferase | 20.74 | 26.63 | 30.70 | 25.81 | 1.51 | 0.119 |
– different letters indicate significant differences (P<0.05).
SEM: standard error of mean.
Growth performance is the most critical indicator of how fast or slow an animal's growth is, and it is also the final performance of the animal's digestion and absorption of nutrients in the ration. There are some controversies about the effect of tannins on animal growth performance. Aguerre et al. (2016) reported that supplementation with TA would linearly reduce DM intake in Holstein cows. Adding HT at moderate levels (16 g/kg DM or 32 g/kg DM) to the diet could increase the body weight of goats (Pimentel et al., 2021). However, Liu et al. (2011) found that the addition of chestnut HT to the diet had no effect on the growth performance of sheep. Likewise, there were no differences in IBW, ADG and FCR on sheep by feeding 40 g/kg DM CT (Jacondino et al., 2022). Our study found that dietary TA supplementation did not affect the growth performance of Liaoning cashmere goats, which could be due to that the goats were fed the experimental treatments for a relatively short time (32 days).
Tannins and proteins usually form protein-tannin complexes in the weak acidity of the rumen (pH=6–7), preventing degradation by rumen microorganisms. In the abomasum, dissociation occurs when the pH<3.5, thus releasing more proteins for protease digestion (Yang et al., 2017). At present, there was no significant change in rumen pH between the four groups, and the pH value was within a reasonable range, which indicated that the addition of TA would not change the rumen environment of Liaoning cashmere goats. In addition, our study discovered that the apparent digestibility of CP was generally increased in the dietary TA supplementation groups. The main reason probably being caused by that the appropriate amount of TA added to the ration of Liaoning cashmere goats leads to the complete dissociation of the tannin-protein complex, thus improving the CP digestibility. But the result is contrary to the results of some previous studies by Pimentel et al. (2021) and Wang et al. (2022), who reported that addition of TA or CT to the goat diet would decrease CP digestibility. On the other hand, Silva de Sant'ana et al. (2022) found that the inclusion of 5 g/kg CT in the goats diet promoted a significant increase in the digestibility of CP. The above information suggests that the inconsistencies in these results are most likely due to differences in the types and the dosage of supplementation, as well as differences in test animals feeding conditions.
The rumen fluid pH intuitively reflects the stability of the rumen internal environment and the level of rumen fermentation (Castillo-Lopez et al., 2021). At present, it was found that the rumen pH value was in the normal range in all groups and dietary supplementation with TA had no effect on rumen pH. A similar trend in lambs was reported by Yildiz et al. (2005), who found no effect of CT on rumen pH. Fiber is an essential source of nutrients for rumen microorganisms and ruminants themselves, especially NDF, which has an important role in regulating normal physiological processes in ruminants (Tjardes et al., 2002). The results showed that adding TA increased the digestibility of NDF. Our results are not quite the same as a study by Wischer et al. (2014), who observed that TA had no effect on the NDF digestibility of sheep. VFA is produced primarily from the breakdown of carbohydrates, including cellulose, hemicellulose, and various sugars. The addition of TA increased the concentrations of propionic acid, butyric acid and valeric acid in rumen fluid, and the reason for this result is most likely related to the increase in the apparent digestibility of NDF. Meanwhile, the biotransformation and fermentation of TA metabolites especially pyrogallol to VFA is by whole rumen microorganisms or bacterium isolated from rumen (Krumholz and Bryant, 1986; Ephraim et al., 2005).
TA will be decomposed into phenolic substances during digestion, and phenolic substances need to be detoxified by the liver to reduce the harm to the organism (Makkar et al., 2007). Dietary TA supplementation had no effect on serum AST and ALT levels, which indicates that the concentration of TA added in this experiment is appropriate and will not affect the health of the animals. TP and Alb levels in blood are important indicators of animal protein metabolism (Bühler et al., 1998). The addition of 4 g/kg DM TA could increase the TP and Alb levels in the blood of Liaoning cashmere goats in this experiment. Proteins are usually broken down by microorganisms in the rumen to form ammonia nitrogen. Part of the ammonia nitrogen will be utilized by microorganisms to produce MCP, while the other part will be absorbed into the bloodstream by the rumen wall and form BUN, the concentration of which can also indirectly reflect the nitrogen utilization. Baldwin et al. (1987) illustrated that the lower BUN in the normal range, the higher nitrogen utilization. However, the BUN of Liaoning cashmere goats was not influenced by the TA added in this experiment.
CP is digested in the rumen of ruminants and exported from the rumen mainly in the form of ammonia, undegraded protein and MCP (Bach et al., 2005). Ammonia is the nitrogen source for microbial growth in rumen fluid. The concentration of rumen ammonia nitrogen is closely related to the rate of microbial utilization of proteins in the feed, and it is a dynamic indicator reflecting protein degradation and microbial utilization of ammonia in the rumen. The concentration of ammonia nitrogen in the rumen fluid of Liaoning cashmere goats in all groups was within the normal range (6–30 mg/100 mL), which indicated that dietary TA supplementation had no adverse effect on rumen fermentation. Moreover, dietary TA supplementation had no effect on ammonia nitrogen. A similar trend in beef was reported by He et al. (2023), who found that adding 2 g/kg TA to the diet had no effect on ammonia nitrogen. Our results are also in good agreement with the report by Liu et al. (2011), who observed that adding 10 g/kg chestnut HT to sheep diets had no effect on ammonia nitrogen. In contrast, Mohammadabadi et al. (2010) found that the addition of 25 g/kg DM TA reduced ammonia nitrogen concentrations in rumen fluid. The reason for the different findings is likely that TA alters nutrient metabolic pathways, and affects the rumen nitrogen cycle, and thus more nitrogen is recycled and reused (Makkar et al., 1997). MCP can provide 40%–80% of the protein requirement for ruminants, and supplementation of TA at a level of 4 g/kg DM has been shown to increase rumen MCP concentration in goats, which is consistent with the study of Makkar et al. (2003). The results of this study pointed out that the dietary TA supplementation had no effect on the FN excretion of Liaoning cashmere goats, but supplementation of TA at levels of 4 g/kg DM or 8 g/kg DM has been shown to decrease UN excretion. A significant increase in nitrogen deposition rate was found in goats fed a diet supplemented with 4 g/kg DM TA or 8 g/kg DM TA as compared to the unsupplemented group. In general, the effect of TA on Liaoning cashmere goat is consistent with previous studies by Pathak et al. (2017) and Wischer et al. (2014), who demonstrated that the addition of 1.5 g/kg DM CT or 15.3 g/kg DM chestnut HT to sheep diet could elevate FN excretion and nitrogen retention, and decrease UN excretion. The reason for this result is likely to be that TA alters the nitrogen excretion pathway in goats, prompting a shift from urine to feces, and previous studies have also confirmed that the addition of CT or HT would cause changes in the nitrogen excretion pathway (Powell et al., 2009; Pathak et al., 2017).
In fact, the efficiency of nitrogen utilization is usually low in ruminants (Du et al., 2020), leading to high feeding costs and high levels of nitrogen being excreted into the environment. The above results show that adding TA can inhibit the degradation of protein in the rumen by forming protein-TA complexes, reduce the circulation of ruminal nitrogen, thus changing the excretion route from urine to feces, and improving the utilization rate of nitrogen in Liaoning cashmere goats, which might have an environmental benefit, because UN is more easily hydrolyzed to ammonium than FN, and it accelerates the generation of nitrous oxide, a potent greenhouse gas (Kronberg et al., 2018; Orlandi et al., 2020).
4 g/kg DM TA can be used as a strategy to improve nitrogen utilization by Liaoning cashmere goat, as shown by the reduced UN excretion and the increased rate of nitrogen deposition and MCP. However, it is recommended to carry out further studies by an accurate microbiological examination in order to understand in detail the mechanism of TA action on the process of nitrogen metabolism in the rumen.