Trial 1: The descriptive statistics of chemical composition and in vitro gas production rumen fermentation parameters for RRTR
Chemical composition of RRTR
Our current study is the first report on the nutrient level of RRTR mainly because it is a regional and almost unused fruit residue. Analyzing the chemical composition is the basis for further exploitation and utilization of any variety of feed. Compared to other commonly used fruit residues in previous studies, the CP level of RRTR (11.99%) was greater than that of apple residue (5.47–9.02%) (Kosmala et al., 2011; Afshar et al., 2011), pineapple residue (5.56–8.10%) (Gowda et al., 2015; Hu et al., 2023; Dieu et al., 2023), and citrus residue (6.17–7.3%) (Tayengwa and Mapiye, 2018). The NDF level of RRTR (53.71%) was lower than that of apple residue (61.2%) (Afshar et al., 2011) and higher than that of pineapple residue (19.58%) (Dieu et al., 2023) and citrus residue (24.2%, NRC, 2001). The ADF level of RRTR (47.22%) was greater than that of pineapple residue (9.40%) (Dieu et al., 2023) and citrus residue (22.2%, NRC, 2001) and was similar to that of apple residue (46.70%) (Afshar et al., 2011). Based on the calculated CF level (37.06%) through the model (CF=-2.512 + 0.838×ADF) (Zhou et al., 2019), we speculated that RRTR is classified as forage according to the international feed classification. From the chemical composition comparison between RRTR and other reported fruit residues, it seems that RRTR has a medium proximate nutrient level. To date, fruit residues have been widely used as forage for cattle (Gowda et al., 2015); thus, we argue that RRTR can also serve as a forage alternative for cattle. This speculation is verified by the following in vitro rumen fermentation and animal feeding experiments with cattle.
In vitro gas production and dynamics of RRTR
The in vitro gas production method is known for its ability to precisely evaluate the nutritional value of feed by calculating gas production through simulating rumen peristalsis in vitro to reflect the digestion of feed in the rumen. Gas production is a key index for measuring the extent of fermentation of substrates. In our experiment, the gas production of RRTR rapidly increased with time within 24 h and gradually tended to plateau. This profile is associated with the amount of substrate degradation during fermentation. GP dynamics are the result of regression analysis of gas production based on a classical mathematical model. This model is used to reflect the potential and utilization degree of a substrate. Our results showed that the rapidly degraded section (a), slowly degraded section (b), potential gas production section (a + b), and gas production rate (c) were 11.66 mL, 252.01 mL, 263.67 mL, and 0.0395%/h, respectively. Afshar (2011) also conducted an in vitro rumen fermentation experiment using 3 cattle as rumen fluid donors and apple residue as fermentation substrate, and their (a + b) values were lower than ours (263.67 vs. 77.82 mL), indicating that RRTR may be utilized more than apple residue for digestion. Margarette et al. (2023). reported that supplementation with proteolytic enzymes increased the in vitro nutrient degradation and fermentation characteristics of pineapple waste silage and that the (c) value was 0.45%/h without the addition of enzymes, which is higher than that in our study. Morteza et al. (2012). used citrus pulp silage as an in vitro gas fermentation substrate and reported that (c) was 0.05%/h, which was slightly greater than that in our study. Our findings showed that when RRTR was utilized as a fermentation substrate, aberrant data for in vitro gas production dynamics parameters were not observed, whereas RRTR had no negative effect on the utilization of substrate.
In vitro rumen fermentation parameters of RRTR
Studies have shown that a pH range of 6.2 ~ 6.6 is conducive for rumen microbes to produce VFAs as an energy supply, and a pH range of 5.7 ~ 6.2 is conducive to internal metabolism via the utilization of NH3-N (Schmitz et al., 2018). The normal ruminal NH3-N concentration ranges from 5.0 to 30.0 mg/dL (Thao et al., 2014). If the NH3-N concentration is lower than 5.0 mg/dL, rumen microorganisms would have a reduced ability to synthesize protein due to insufficient N, which would result in rumen ammonia poisoning when the NH3-N concentration is greater than 30.0 mg/dL. In this experiment, the pH and NH3-N concentration of the RRTR were 5.86 and 9.25 mg/dL, respectively, which fall within the normal range. Our results indicate that the RRTR did not negatively affect rumen fermentation or nitrogen metabolism.
Cellulase activity is an important indicator reflecting the ability of rumen microorganisms to degrade cellulose in feed. The main cellulase enzymes in the rumen are carboxymethyl cellulase, xylanase and cellulobiase, which work together to degrade cellulose, hemicellulose, lignin and other substances that are difficult to absorb in feed. To the authors’ knowledge, data on the cellulase activity of the RRTR have been insufficient until now. Therefore, our RRTR results are also the first to be reported. However, further studies are needed to compare cellulase activity among fruit residues.
VFAs are the products of carbohydrate degradation by rumen microorganisms and can provide 75% of the energy required by ruminants, and acetate, propionate, and butyrate account for 95% of the total VFAs produced in the rumen. The acetic acid in the rumen can be converted into lactate, and high lactate concentrations decrease the abundance of some microbes, including protozoa, methanogens, and cellulolytic bacteria, in the rumen; propionate can inhibit the growth of lactic acid bacteria in the rumen, reduce the production of lactic acid, increase the rumen pH, facilitate the activity of digestive enzymes, and promote the digestion and absorption of food (Kajikawa et al., 1990). The VFA content has been shown to be positively correlated with gas production (Fitri et al., 2021). Dieu et al. evaluated the in vitro fermentation (with 3 adult ewes as rumen donors) of 2 different varieties of pineapple residue (Smooth Cayenne and Sugarloaf) and reported that the TVFA level was 90.28 and 71.70 mmol/L (Dieu et al., 2023), which is lower than that in our study (114.78 mmol/L). This result may indicate that the RRTR has a greater ability to provide energy than pineapple residue for ruminants. The value of A/P is a key index for determining the rumen fermentation pattern. When the A/P ratio was greater than 2.5, acetic acid-type fermentation was dominant in the rumen, which was conducive to body fat synthesis. When the A/P ratio is less than 2.5, propionic acid-type fermentation is dominant, and more propionic acid is converted to glucose to provide energy. In this study, the in vitro rumen fermentation A/P ratio of RRTR was 2.45, which was slightly lower than the 2.5 observed for the propionic acid fermentation pattern.
Overall, the authors suggest that RRTR has the potential to serve as forage for cattle, and the following animal experiments were subsequently carried out to evaluate the feeding effect.
Trial 2: Effects of RRTR on growth performance, blood metabolites, and in vivo rumen fermentation parameters in cattle
Growth performance
Growth performance can reflect the growth state and metabolic health of animals and is commonly assessed based on the DMI, ADG, and F/G. The DMI can directly indicate feed palatability, the ADG can be used to evaluate feed conversion efficiency, and the F/G ratio can indicate the feed utilization efficiency. Oduguwa et al. (2020) reported that pineapple residue (supplemented with cassava peel) improved feed intake and growth performance in West African dwarf sheep and that sheep fed the same diet had greater ADG than animals in the other treatment groups. These authors concluded that feed containing ensiled pineapple fruit waste and cassava peel could be recommended to sheep farmers to improve feed intake and performance, especially during periods of austerity. In a study on pigs, Hsu et al. (2004) reported that the dietary inclusion of 15% citrus residue did not affect the ADG and that whole citrus residue silage and pressed citrus residue silage had greater (P = 0.04) feed conversion efficiency than did dried citrus residue. The results of the current study showed that neither the DMI nor the ADG differed significantly between the two groups of cattle, suggesting that RRTR has palatability and nutritional value similar to those of whole corn silage. There are 2 possible reasons for this phenomenon. First, RRTR is rich in flavor compounds and saccharine, which makes it highly palatable. Second, RRTR contains a variety of organic acids, which can promote gastrointestinal peristalsis, improve digestive capacity, and maintain acid‒base balance, resulting in improved appetite. Unexpectedly, cattle in the treatment group had a significantly greater F/G ratio than did cattle in the control group. We are unsure why RRTR supplementation reduces the feed conversion ratio because the DMI and ADG are unaffected. A possible explanation might be attributed to the limited number of cattle in each group (n = 8). Accordingly, a large-scale trial involving more cattle is needed to evaluate the precise effect of RRTR on feed conversion efficiency.
Plasma metabolites
Plasma metabolites are important parameters for assessing metabolic status and body health. Plasma Glu is the primary indicator of the relative homeostasis of glucose catabolism and anabolism in the body. Plasma TG and TC are indicators of lipid catabolism and nutrient absorption in animals. Plasma LDL-C and HDL-C are the 2 forms of lipoprotein-incorporated cholesterol and function as cholesterol carriers and transporters, respectively; accordingly, they reflect cholesterol transport and lipid metabolism in the body. Plasma TP and ALB reflect the absorption and transformation of dietary protein in animals. Plasma ALT is mainly distributed in hepatocyte plasma, while AST is present in hepatocyte plasma and the mitochondria of hepatocytes; both indices are key indicators of amino acid metabolism and hepatocyte damage (Biasi et al., 1991; Parola et al., 1992) and play a critical role in the evaluation of animal health, growth, and development. Gowda et al. compared 2 TMRs comprising either 62% pineapple residue silage or maize green fodder silage with 38% compound concentrate feed in a 75-day feeding trial with sheep (n = 10 per group) and found no difference in the serum total protein, urea, creatinine, glutamate oxaloacetate transaminase (SGOT), or glutamate pyruvate transaminase (SGPT) activities between the two groups (Gowda et al. 2015).
In our study, there were no differences in plasma Glu, TG, TC, LDL-C, HDL-C, TP, ALB, AST, or ALT concentrations or in the AST/ALT ratio between the 2 groups of cattle, but the plasma urea level was 71.1% and 53.8% lower in the treatment group than in the control group for the first (day 35) and second (day 70) blood sampling time points, respectively. As a product of protein catabolism, plasma urea can reflect dietary protein utilization efficiency. A reasonable plasma urea concentration range is generally suggested to be 2.0 ~ 7.1 mmol/L (Coma et al., 1995). If dietary protein is fully utilized by the body, it will be excessively catabolized into urea and excreted by the kidney, resulting in nutritional losses. The utilization of nitrogen is negatively correlated with the plasma urea level. In the present study, the plasma urea concentration in the treatment group was significantly lower than that in the control group in both stages, indicating that the inclusion of RRTR in the diet can enhance the plasma nitrogen utilization efficiency of cattle. This result may be associated with the compound. However, further investigations are needed to determine the exact underlying mechanism involved.
In vivo rumen fermentation parameters
Ruminal fermentation characteristics are largely affected by the composition of the diet, and it is reasonable to evaluate the feasibility of the RRTR diet based on rumen fermentation characteristics (Ma et al., 2024). Rumen pH is an important measurement for ruminants because it is associated with acid‒base balance, especially for the survival of ruminal microbes. The normal rumen pH is reported to be in the range of 5.5 to 7.5. Sparkes et al. (2009) reported that replacing 30% of lucerne hay with fresh citrus pulp resulted in a significantly lower rumen pH (5.90 vs. 6.53) for ewes, indicating that citrus pulp affected rumen fermentation. In our experiment, there was only a slight difference in rumen pH (7.26 vs. 7.30), indicating that the inclusion of RRTR had no impact on the ruminal acid‒base balance. Our results were supported by those of Fang J et al. (2019) and Soares et al. (2014), who reported that feeding apple pomace and citrus pulp had no significant effect on rumen pH in sheep and cows, respectively.
NH3-N is an important source of nitrogen for rumen microbe growth and bacterial protein synthesis in ruminants. Devant et al. (2000) reported that after supplementation with a low-degrading protein and/or when the CP concentration was low, the ruminal NH3-N concentration decreased to less than 5 mg/100 mL. In the review of Tayengwa and Mapiye (2018), the authors found that citrus residue (30% for ewes and 26% for lambs) resulted in varying ruminal VFA (154 mmol/L for ewes, 72.8 mmol/L for lambs) and NH3-N (40 mg/dL for ewes, 6.09 mg/dL for lambs) concentrations, illustrating that the inclusion of citrus residue in ruminant diets improves the individual and total VFA profile by providing more lipogenic metabolizable nutrients. In our study, the NH3-N concentration in the treatment group was significantly lower than that in the control group, indicating that RRTR can decrease the degradation of protein by ruminal microorganisms and increase the efficiency of protein utilization.
The ruminant diet contains a substantial amount of fibrous substances that rely on cellulase (carboxymethyl cellulase, fibrinosaccharase, and xylanase) secreted by microorganisms in the rumen for degradation and utilization. In a previous study, we found that the activities of carboxymethyl cellulose and cellobiase were unaffected, but the xylanase activity was 17.4% lower in the treatment group than in the control group throughout the trial period, possibly because xylanase activity was decreased in fermented RRTR.
VFAs are the main energy source for ruminants and an important carbon source for ruminal microbes, and their concentration and composition are important indicators of ruminal digestion and metabolism. Khaled et al. (2023) reported that increased VFA concentrations were associated with increased fermentable energy content and improved DMI and DM and OM digestibility. Ahn et al. (2022) reported that when 30% apple residue was supplemented in the diet of goats, the TVFA concentration was 144.8 mmol/L, which was significantly greater than that in a control group (80.6 mmol/L) without apple residue. In our study, there was no difference in TVFA, acetate, propionate, or butyrate levels between the control and treatment groups. This result indicated that there was no significant difference in overall digestibility between the 2 groups of cattle. Moreover, cattle in the control and treatment groups exhibited A/P values greater than 2.5, indicating an acetic acid-type rumen fermentation pattern.
Economic benefit
The economic benefit index can reflect whether RRTR is cost-effective when it is used as roughage and can, to some extent, provide an understanding of the feasibility of large-scale or long-term RRTR utilization in practice. Currently, RRTR is less expensive than conventional roughage because it has similar nutrients and can, in theory, replace conventional roughage in terms of feed cost. In this study, there were no differences in feed cost, net weight gain, or gross profit between the two groups of cattle. This result indicates that replacing whole corn silage with RRTR does not negatively affect feed cost or economic benefit and provides a good feed alternative for cattle.