Use of exogenous fibrolytic enzymes and probiotic in finely ground starters to improve calf performance

The present study investigated the effects of adding wheat straw treated with exogenous fibrolytic enzymes (EFE) and a probiotic supplement to finely ground starters on growth performance, rumen fermentation, behavior, digestibility, and health of dairy calves. A total of 48 Holstein dairy calves (39.8 ± 1.67 kg body weight) were randomly assigned to one of 4 nutritional treatments (n = 12 calves per treatment). The experiment was conducted in a 2 × 2 factorial arrangement of treatments consisting of two diets with or without EFE-treated wheat straw (2 g/day/calf) and diets with or without probiotics (2 g/day/calf). All calves were weaned on day 63 and remained in the study until day 84. The addition of EFE to wheat straw had no effect on starter feed intake, increased neutral detergent fiber (NDF) digestibility and recumbency, but decreased average daily gain (ADG) after weaning (240 g/d). The addition of probiotics to the diet had no effect on starter feed intake, improved feed efficiency, ADG (150 g/d), final weight (11.3 kg), and NDF digestibility, and decreased the ratio of acetate to propionate in the rumen. The addition of probiotics to starter feed for calves could improve their growth.

The ultimate goal of nutritionists is to maximize calf starter intake and rumen development prior to weaning. Current feeding strategies for young calves are based on starter diets high in non-structural carbohydrates that promote rumen microbiota establishment and short-chain fatty acid (SCFA) production and subsequently support rumen papillae development 1 , which has long-term effects on growth, health, and milk yield in adult cattle. However, some studies have clarified that excessive intake of rapidly fermentable carbohydrates can also predispose calves to rumen acidosis 2 , which can lead to cancerous proliferation and keratinization of rumen epithelial cells 3 and impairs rumen SCFA uptake 4 . The addition of dietary fiber to starter feed for calves may prevent the epithelium from building up a keratin layer 5 , especially if the particle size or texture of the starter feed is poor 6 .
It has been demonstrated that the addition of fibrolytic enzymes to the diet can improve weight gain and feed conversion in steers or calves by increasing nutrient digestibility 7 . Fibrolytic microorganisms in the rumen take a longer time to establish in the rumen than starch-and sugar-fermenting microorganisms 8 , which may reduce digestion of fibrous material and limit utilization efficiency 9 . Therefore, the addition of fibrolytic enzymes to starter diets containing low digestibility forages is expected to promote digestibility and improve performance in young calves. In support, the addition of fibrolytic enzymes has been reported to have a positive effect on SCFA production in the rumen 10 and to increase populations of Fibrobacter succinogenes and Butyrivibrio fibrisolvens in the rumen, resulting in improved performance of calves during the pre-and post-weaning periods 11 . In addition, Wang et al. 11 found that supplementation with fibrolytic enzymes increased microbial enzyme activities in the rumen after weaning, including carboxymethylcellulase, cellobiase, xylanase, and pectinase, compared with calves without supplementation. Both supplements, fibrolytic enzymes and probiotics, have the potential to modulate rumen fermentation and improve fiber digestibility, animal performance, and feed conversion efficiency. Some researchers have investigated the synergistic interaction between these two additives in in vitro and in vivo studies [12][13][14] . Despite conflicting results in in vitro studies, Malik and Bandla 12 reported that the combination of probiotics and fibrolytic enzymes had a reciprocal beneficial effect on rumen fermentation and improved growth performance and feed efficiency of growing buffalo calves. www.nature.com/scientificreports/ Statistical analysis. For the primary response variables, including starter intake, BW, and ADG, a power analysis was conducted to estimate sample size 28,29 ) based on previous literature values [30][31][32] . From the power test analysis with α = 0.05 and power = 0.80, the predicted sample size was 12 calves per treatment for starter intake, total DMI, ADG, and BW. All data were tested for normal distribution using the UNIVARIATE procedure of SAS version 9.4 (Shapiro-Wilk test; SAS Institute Inc), and variables that were not normally distributed were log-transformed (base 10) to meet assumptions of normality and homoscedasticity of residuals. Statistical analyzes were performed over 3 time periods: before weaning (4 to 63 days), after weaning (64 to 84 days), and over the entire period (4 to 84 days). Data were analyzed as a completely randomized design with a 2 × 2 factorial arrangement of treatments with EFE and probiotic supplementation as factors. Data were analyzed by repeated measures ANOVA using the PROC MIXED procedure of SAS for starter feed intake, ADG, BW, ADG, FE, structural growth, rumen fermentation, and behavior. The model included the fixed effects of probiotic, EFE, time (day or week) as repeated measures and their interactions. Calf was included as a random effect. Initial BW (day 4) and structural growth measurements were included as covariates for BW and skeletal growth analysis. Four variance-covariance structures (heterogeneous autoregressive type 1, autoregressive type 1, compound symmetry, and Toeplitz) were tested, and the heterogeneous autoregressive type 1 covariance structure was determined to be the most appropriate covariance structure for all repeated statements according to the Akaike and Bayesian information criteria. The main effects of EFE and probiotic supplementation and their interactions were tested using ANOVA. The effects of nutritional treatment on categorical responses related to fecal and health scores were analyzed using the GLIMMIX procedure of SAS version 9.4. The significance threshold was set at P ≤ 0.05; trends were reported at 0.05 < P ≤ 0.10.
Principal component analysis. PCA was applied to obtain an overview of growth performance, health and behavioral data based on the different treatments. The MetaboAnalyst 5.0 web-based metabolomics data processing tool was used for this purpose (see http:// www. metab oanal yst. ca for detailed methodology) 33 . Data were transformed using the generalized log transformation and then scaled by Pareto (mean-cantered and divided by the square root of the standard deviation of each variable) to correct for heteroscedasticity, reduce skewness, and mask effects.

Results
Intake and growth performance. Calf health-related data are presented in Table 2. No effect of EFEtreated wheat straw in the starter diet was observed on starter feed intake, NFC intake, BW, ADG, and FE. The probiotic supplement had no effect on starter feed intake or NFC intake. Calves fed a probiotic supplement had higher total ADG (P < 0.01), preweaning BW, total BW, FE (P < 0.01), weaning weight, and final weight (P < 0.01) than calves fed a non-supplemented diet. We observed no interaction between EFE-treated wheat straw and probiotic supplementation for starter feed intake, NFC intake, BW, ADG, and FE. A 2-way interaction was observed between probiotic supplementation and time to BW ( Fig. 1, P < 0.01), starter feed intake ( Fig. 2, P < 0.01), and FE ( Fig. 3, P < 0.01), suggesting that probiotic supplementation had a greater effect on starter feed intake and ADG during the postweaning period and on FE during the preweaning period. Body measurements data are presented in Table 3. No effect of EFE-treated wheat straw in the starter feed intake was observed on hip height and hip width. Calves fed EFE-treated wheat straw in the starter diet had lower body length (trend, P = 0.07), heart girth at weaning, and body barrel (P < 0.01) than calves fed untreated wheat straw in the starter diet. The probiotic supplement had no effect on heart girth, body barrel, or hip height. Calves fed a probiotic supplement had greater body length and hip width at weaning (P < 0.05) than calves fed a non-supplemented diet. We observed no interaction in body measurements between EFE-treated wheat straw and probiotic supplementation. A 2-way interaction was observed between probiotic supplementation and time to hip width (P = 0.03), suggesting that probiotic supplementation affected hip width only during the period before weaning. A 2-way interaction was observed between EFE-treated wheat straw in the starter diet and time to heart girth (P < 0.01), suggesting that EFE-treated wheat straw affects heart girth only during the preweaning period.
Health status. There were no effects of EFE-treated wheat straw and probiotic supplementation and their interactions on fecal score ≥ 2, rectal temperature > 39.2, electrolyte treatment, or medication day (Table 4).
Behavioral data. Data on calf behavior are presented in Table 5. The addition of probiotics in the starter feed had no effect on the time calves spent standing, lying, eating, and NNOB. No effect of EFE-treated wheat straw in the starter feed was observed on time spent drinking and rumination. The EFE-treated wheat straw and the probiotic supplement had an interaction effect on standing time and NNOB time, with calves fed EFE+ PRO− having more standing time and less NNOB time compared to the other groups.
Digestibility and ruminal fermentation. Nutrient digestibility data are presented in Table 6. The addition of probiotics and EFE-treated wheat straw to the starter feed did not affect the digestibility of DM, EE, and CP. NDF digestibility was improved by both EFE (P = 0.04) and the supplementation of probiotics (P = 0.001). Wheat straw treated with EFE and the probiotic supplement had an interaction effect on ADF digestibility, with calves fed EFE-PRO having lower ADF digestibility compared with the other groups.
Ruminal fermentation profile data are presented in Table 7. We observed no interaction between EFE-treated wheat straw and probiotic supplementation on rumen fermentation parameters. No effect of EFE-treated wheat straw was observed on rumen pH, total SCFA, and molar proportions of propionate, butyrate, iso-butyrate, valerate, and iso-valerate in the rumen. Calves fed EFE-treated wheat straw in the starter diet had lower molar www.nature.com/scientificreports/ proportions of acetate in the rumen (d 70 P = 0.05; overall P = 0.09) and a lower ratio of acetate to propionate (d 70 P = 0.03) than calves fed untreated wheat straw in the starter diet. The probiotic supplement did not affect the total amount of SCFA in the rumen or the molar proportions of butyrate, isobutyrate, valerate, and iso-valerate in the rumen. Calves fed a probiotic supplement had greater molar proportions of propionate in the rumen (P < 0.05) and less acetate (P < 0.05) on day 35 of the study than calves fed no supplement.

PCA.
For the growth performance, health, and behavior data, PCA revealed two principal components (PC1 and PC2) that explained 56% of the variance, and even the treatment groups overlapped and could not be separated (Fig. 4).

Discussion
Fibrolytic enzyme effects. In the present study, the dietary NFC concentration increased when wheat straw was treated with EFE, which could be explained by the partial hydrolysis of the hemicellulose but not the cellulose fraction to monosaccharides and oligosaccharides by the activity of fibrolytic enzymes 34 . In agreement with our findings, Rodrigues et al. 35 reported that pretreatment of wheat straw with enzyme extracts isolated from white rot fungi with ligninolytic and cellulolytic activities degraded hemicellulose content, resulting in lower NDF concentration 35 . They indicated that hemicellulose was preferentially degraded by the enzymatic extracts. Fibrolytic enzymes pretreating the forage can weaken and/or degrade the lignin-hemicellulose-cellulose complex of the plant 36 . This loosening effect could facilitate the attachment of microorganisms to the substrate, leading to faster growth of microbial populations and increased fiber degradation 37,38 . In addition, it has been reported that exogenous enzymes can synergistically enhance the hydrolytic potential of endogenous microbial enzymes in the rumen, resulting in improved digestion of dietary fiber in the rumen 39 , which is consistent with the higher NDF digestion when the calves in the current study received EFE-treated wheat straw. In addition, the higher NDF digestibility with enzyme pretreatment may be due to the relatively prolonged retention time of the feed in the rumen caused by the lower feed intake during the study. The prolonged retention time of fiber in the rumen could improve fiber digestion by EFE administration. Feeding EFE-treated wheat straw decreased starter feed intake before weaning and throughout the study, but had no effect on calf NFC intake, ADG, BW, or feed efficiency. To our knowledge, there is no previous report on pretreatment of low-quality forages such as straw with fibrolytic enzymes for feeding newborn dairy calves. Table 2. Least squares means for starter feed intake, NFC intake, ADG, body weight (BW), and feed efficiency in dairy calves supplemented with or without exogenous fibrolytic enzymes (EFE) and probiotic (PRO) in the starter diets (n = 12 calves per treatment). Treatments were (1) calves fed diets without exogenous fibrolytic enzymes (EFE)-treated wheat straw and probiotic supplement (EFE-Pro−), (2) calves fed diets of EFE-treated wheat straw but with probiotic supplement (2 g/d/calf; EFE-Pro+), (3) calves fed EFE-treated wheat straw (2 g/d/calf) but without probiotic supplement (EFE+ Pro−), (4) calves fed EFE-treated wheat straw and probiotic supplement (EFE+ Pro+). Exogenous fibrolytic enzymes were applied to wheat straw. www.nature.com/scientificreports/ Studies on pretreatment of forages with fibrolytic enzymes on feed intake have shown conflicting results. Some researchers reported that EFE supplementation had no effect on feed intake of growing buffaloes 12 and weaned dairy calves 40 . Wang et al. 11 showed that supplementation with fibrolytic enzymes tended to increase feed intake of bull calves aged 46 to 90 days. The discrepancy between our results and those of other researchers could be due to the method of administration of fibrolytic enzymes (direct administration into the starter feed versus pretreatment of the forage with fibrolytic enzymes), the composition of the basal feed, especially fiber sources or fiber content, which affect the activity and stability of rumen enzymes 14 . The lower concentration of NDF and higher digestibility of NDF should improve feed intake. Although the exact mechanism is not clear, the higher digestibility of forages pretreated with enzymes could result in greater amounts of oxidizable fuels being available to hepatocytes, thereby reducing feed intake.
In the current study, lower intake of starter feed and ME was not associated with poorer ADG or feed efficiency because lower feed intake was compensated by higher fiber digestibility, resulting in similar growth performance. However, skeletal growth parameters, including final body length, heart girth at weaning, and body barrel at weaning and finishing were lower in calves fed EFE-treated wheat straw. These results suggest that enzyme treatment of straw limited the supply of nutrients other than energy needed for skeletal growth. Furthermore, in addition to improved fiber digestion in calves fed EFE, lower feed intake could be related to lower intestinal filling 41 , resulting in lower body barrel as an indicator of intestinal filling 42 in calves.
Feeding diets containing EFE-treated wheat straw had no effect on rumen fermentation parameters, except that the molar proportion of acetate and the ratio of acetate to propionate in the rumen decreased with EFE supplementation. It had been noted earlier that NFC concentrations were higher in diets containing EFE-treated wheat straw, which may have contributed to a shift in rumen fermentation toward more propionogenesis, as indicated by the numerically higher molar proportion of propionate production at the expense of acetate, resulting in a lower acetate-to-propionate ratio. In agreement with our results, Ribeiro et al. 43 reported that treatment with a recombinant fibrolytic enzyme 24 h after feeding decreased the molar ratio of acetate, which was consistent with a lower protozoan population in the rumen. As a result of fermentation of starch and cellulose by protozoa, acetate and butyrate are the main end products 44 . Probiotic supplementation effects. Probiotic products generally contain a mixture of microorganisms, including S. cerevisiae and lactic acid bacteria, that optimize the intestinal health of animals by competing with undesirable microorganisms, stimulating the production of beneficial bacteria, and increasing nutrient absorption in the gut 45,46 . The effect of feed supplements such as probiotics has also been shown to depend on the type of supplementation, with solid feed supplementation having a more pronounced effect [47][48][49] . However, in the first Time (d) 10 20 www.nature.com/scientificreports/ weeks of life, when calves are exposed to stressful situations, solid feed intake is not sufficient to provide calves with adequate feed supplements. Interestingly, our results showed that probiotic addition improved the uptake of the starter feed around weaning but not throughout the study period, which may suggest that probiotic addition had an ameliorative effect on the weaning stress. Chaucheyras-Durand et al. 50 indicated that yeast products are particularly useful during periods of dietary change and stress. Milk restriction before weaning causes calves to consume significant amounts of solid feed, which can increase acidity in the rumen and lead to a decrease in rumen pH and eventually rumen acidosis 51,52 . Silberberg et al. 53 reported that during acidosis challenges, yeast supplementation stabilized fermentative parameters, promoted protozoal numbers, decreased lactate-producing bacteria, and helped normalize the animals' inflammatory status. In addition, probiotic supplementation improved NDF digestibility, which would stimulate the intake of DM. Consistent with our results, some studies have found that starter diets increased when claves were supplemented with yeast fermentation products in the starter diet 47,54 .
In addition, probiotic supplementation improved preweaning ADG and total ADG, BW, and feed efficiency in the current study. In general, the improvement in growth performance in the current study might be related to the improved rumen fermentation, nutrient digestibility, and health status of the probiotic-supplemented calves. Consistent with our findings, intensive research has reported that growth performance was improved when young beef or dairy calves were supplemented with active dry yeast 55,56 , yeast fermentation products 47,48 , or Bacillus amyloliquefaciens strain H57 19 compared to control calves. In addition, calves supplemented with probiotics exhibited higher feed efficiency, which may be associated with greater fiber digestion in the rumen and improved rumen fermentation, as indicated by the lower acetate-propionate ratio without affecting total SCFA concentration, which may improve growth performance. The lower acetate-propionate ratio provides faster and more energy to the body, which has a positive effect on animal growth performance 56 . In general, a higher acetate-propionate ratio means better digestibility of cellulose 57 . However, the results of the current study suggest that improved fiber digestion was accompanied by a shift in rumen fermentation toward propionate synthesis at the expense of acetate production. Consistent with our results, administration of yeast to weaned beef calves 56 or Lactobacillus rhamnosus GG to newborn calves improved NDF digestion and promoted propionate production 30 . Probiotics, including yeast cultures or direct feeding bacterial microbiome, are thought to improve animal performance by influencing the rumen microbiota to support the growth and activity of fibrolytic microorganisms and those that metabolize lactate 58,59 . Therefore, probiotic administration in the current study could promote the growth and metabolism of lactate-utilizing bacteria in the rumen and then support the conversion of lactate to propionate, allowing calves to obtain more energy from their diet 60 19 . Calf health is a critical factor affecting welfare and growth performance during early life and future productivity 61 . Probiotics can have beneficial effects on gastrointestinal functions in humans and animals and other health-related outcomes. It has been reported that the inclusion of yeast-based 47,48,62,63 or bacterial-based 19 live probiotics in calf starter feed improved fecal scores and some other health-related indicators in young calves. The administration of yeast-based probiotics has been shown to reduce diarrhea by modulating the composition of the intestinal microbiome, preventing the binding of pathogenic bacteria to intestinal epithelial cells, modulating intestinal mucosa and systemic immunity, improving the integrity of the intestinal barrier, and reducing the infiltration of toxic luminal antigens and bacteria 15 . However, probiotic supplementation did not improve the health status of the dairy calves in the current study.

Conclusion
The results of the study showed that wheat straw treated with EFE had no positive effects on starter feed intake, performance, and health status of calves during the preweaning and postweaning periods. Nevertheless, supplementing calf diets with wheat straw treated with EFE has the potential to improve NDF digestibility. The diet supplemented with probiotics showed a potential improvement in feed efficiency and growth performance of dairy calves, as evidenced by increased ADG and final weight. The addition of probiotics increased the digestion of NDF and altered rumen fermentation. Probiotic administration to calves requires further research to better understand the underlying mechanisms responsible for the observed improvement in feed efficiency and growth performance so that better supplementation strategies can be implemented. Given the limited data, future studies need to investigate whether probiotics should be used prophylactically or therapeutically in young calves.    www.nature.com/scientificreports/ Table 7. Least squares means for ruminal fermentation in dairy calves supplemented with or without exogenous fibrolytic enzymes (EFE) and probiotic (PRO) in the starter diets (n = 12 calves per treatment). Treatments were (1) calves fed diets without exogenous fibrolytic enzymes (EFE)-treated wheat straw and probiotic supplement (EFE-Pro−), (2) calves fed diets of EFE-treated wheat straw but with probiotic supplement (2 g/d/calf; EFE-Pro+), (3) calves fed EFE-treated wheat straw (2 g/d/calf) but without probiotic supplement (EFE+ Pro−), (4) calves fed EFE-treated wheat straw and probiotic supplement (EFE+ Pro+). Exogenous fibrolytic enzymes were applied to wheat straw.  Treatments were (1) calves fed diets without exogenous fibrolytic enzymes (EFE)-treated wheat straw and probiotic supplement (EFE-Pro−), (2) calves fed diets of EFE-treated wheat straw but with probiotic supplement (2 g/d/calf; EFE-Pro+), (3) calves fed EFE-treated wheat straw (2 g/d/calf) but without probiotic supplement (EFE+ Pro−), (4) calves fed EFE-treated wheat straw and probiotic supplement (EFE+ Pro+). Exogenous fibrolytic enzymes were applied to wheat straw.