Holstein calves fed a milk replacer with a direct fed microbial (DFM) and a starter containing a botanical extract or a DFM alone or in combination.

Botanical extracts ( BE ; Apex, Adisseo, North America) have demonstrated enhanced DMI and improved gut health, while direct fed microbials ( DFM ), such as Lactobacillus acidophilus fermentation product ( EX : Excell, Pacer Technology, Inc.), has demonstrated improved gut health and growth performance of growing Holstein calves. The hypothesis was this combination may be synergistic to neonatal calf growth performance and intestinal health. Eighty, 2–5-d old Holstein bull calves were blocked by BW and randomly assigned to one of 8 treatments arranged in a 2 × 4 factorial using a randomized complete block design. The main factors were milk replacer ( MR ) without (Control) and with EX added at 5 g/d fed and calf starter ( CS ). The CS containing no additives ( Control ); CS containing BE at 496 mg/ kg; CS containing EX at 2.50 g/kg; and CS containing BE and EX at the same inclusion rates. The MR were fed 2x/d at 0630 and 1800 h along with free choice CS (amounts and orts weighed d) and water. Weaning oc-curred after d 42 for the 56-d experiment. No MR by CS main effects interactions were detected for BW, ADG, CS intake, total DMI, feed efficiency or body frame gain parameters. The BW gain (38.0 and 39.3 kg for control and EX, respectively) for MR main effect was similar for calves fed both MR, while CS main effects (38.7, 39.7, 39.2, and 37.2 kg for control, BE, EX, and BE&EX, respectively) was similar among all CS. Gains in body length (10.6 and 10.8 cm), hip width (4.5 and 4.5 cm), withers height, (10.5 and 10.6 cm) heart girth (18.6 and 19.9 cm) and body length (9.1 and 7.9 cm) were similar for calves fed both MR, while CS main effects for hip height (10.5, 10.2, 10.3, and 10.9 cm), hip width (4.7, 4.6, 4.4, and 4.3 cm) withers height (10.7, 10.9, 10.3 and 10.6 cm), heart girth (19.9, 18.9, 18.9, and 19.4 cm), and body length (11.7, 9.1, 8.3, and 8.4 cm) were similar. Total days of a fecal score = 0 was greater for calves fed Control MR and BE CS compared with calves fed Contol MR and the combination of BE&EX with calves fed the remaining treatments being intermediated and similar. This study demonstrated little calf growth performance and health benefits when feeding a BE or EX alone or in combination compared with calves fed control.


INTRODUCTION
Calf health is one of the most significant livestock health issues influencing sustainable livestock farming (Reddy et al., 2020).Replacement dairy calves are extremely susceptible to enteric bacterial pathogens that can be found in their digestive tracts from the first days of life causing pre-weaning diseases (Frizzo et al., 2010;Tapki et al., 2020).So, digestive problems and intestinal health challenges leading to scours are directly attributable to economic losses and poor calf growth (Amanullah et al., 2020) combined with long-term performance effects (Reddy et al., 2020).Therefore, gastrointestinal health is an important development factor for healthy calves to prevent a detrimental microbial impact on calf growth for the successful raising of replacement stock (Arne and Ilgaza, 2021;Tapki et al., 2020).
Antibiotics have been used for decades as feed additives to minimize morbidity and mortality, as well as to encourage growth (Reddy et al., 2020).However, prolonged use of antibiotic feed additives has generated global concerns of antimicrobial resistance, which necessitates evaluating antibiotic alternatives, since antibiotic usage is being banned around the globe (Official Journal of the European Union, 2003;Ghimpeteanu et al., 2022).Encouraging the shift to alternative natural products/ additives maybe required for improving/maintaining animal health and growth (Arne & Ilgaza, 2021).
Holstein calves fed a milk replacer with a direct fed microbial (DFM) and a starter containing a botanical extract or a DFM alone or in combination.
The ongoing search for natural additives as antibiotic alternatives for livestock has identified several technologies to stimulate growth and proper body functioning (Maciej et al., 2016).The various formulated products/ additives fed to livestock have included prebiotics, probiotics or symbiotics for increasing resistance to digestive system diseases and consequently, improved animal performance.However, studies have demonstrated that the beneficial responses are largely dependent on their various natural components and supplementation with different component formulations can produce divergent results (Reddy et al., 2020).Jonova et al. (2021) emphasized the combination of 2 feed additives can improve the implantation and survival of a direct-fed microbial (DFM) in the gastrointestinal tract, which will stimulate the growth and activate the metabolism of a limited number of beneficial bacteria.
Excel (EX) is a natural lactobacillus acidophilus fermentation product and contains nutritional and microbial metabolites of fermentation.Lactobacillus products have been reported to improve calf growth performance and digestive health (Cangiano et al., 2020, Casper et al., 2021b).Therefore, a symbiotic feeding program using probiotics and prebiotics in combination has been proposed to be more beneficial and effective in their action for calf growth, health, and performance than the use of probiotics or prebiotics alone (Arne and Ilgaza;2021;Radzikowski, 2017;Casper et al., 2021b).
Current research has indicated that botanical extracts (BE) can also be valuable alternative feed additives (Stefańska et al., 2022;Tapki et al., 2020) with different BE being promoted.Reddy et al. (2020) noted challenges when using BE as feed additives, but their investigations revealed nutrient digestibility, glucose absorption, and performance benefits when used appropriately.Researchers (Reddy et al., 2020;Stefańska et al., 2022) reported a combination of probiotic and phytogenic additives improved calf health, feed intake, nutrient utilization, and performance attributes.Stefańska et al. (2022) reported that phytogenic, DFM, and probiotic combination studies, known as eubiotics, are still ongoing.An eubiotic feed additive in milk replacer (MR) improved feed intake, growth performance along with decreasing diarrhea occurrence (Casper et al., 2021b;Stefańska et al., 2022).Even so, most studies reported feed additives being incorporated either into liquid feeds, such as MR or solid feeds, such as calf starter (CS).Santos et al. (2015) recommended that dosage and administration routes, whether liquid MR or CS, be furthered studied for improving animal performance and health.Liu et al. (2020) recommended further studies to evaluate the impact of feed additives when incorporated into MR and CS at the same time.However, Stefańska et al. (2022) cited limited scientific data available when providing natural feed additives in combination in both dietary feeding situations.
Investigating the synchronization when administering feed additives in both liquid and solid feeds may be advantageous on growth performance and calf health during the pre-weaning stage.The hypothesis is that the combination of BE and EX will be synergistic to neonatal calf growth performance.Therefore, the study objective is to evaluate the synergistic effect of natural eubiotic feed additive with BE or DFM in the pelleted CS with or without EX in the MR.

MATERIALS AND METHODS
The following experiment reported here was conducted using the same or similar facilities, experimental design, procedures, treatment protocols, and sample collections as previously reported experiments by this Contract Research facility (Bai et al., 2020;Liu et al., 2020;Casper et al., 2021a;Casper et al., 2021b).The study specific materials and methods are briefly reported below.

Calf Management and Feeding
The experiment was conducted at Casper's Calf Ranch (Freeport, IL) from June 18th through August 12, 2021.The calves were managed, cared for, and fed following the guidelines published in the 4th edition "Guide for the Care and Use of Agricultural Animals in Research and Teaching" published by ADSA-ASAS-PSA (2020).The experiment started with 80 calves being 2-5-d old Holstein bull calves sourced by a commercial calf buyer (Knueppel Livestock, Shawno, WI) and delivered the night before trial initiation.Calves were purchased through several livestock auction barns and represented commingled calves sourced from numerous Wisconsin dairy farms.Calves were assumed to have been fed colostrum at their respective home dairy farms.Calves were vaccinated upon arrival with Inforce 3 (Zoetis Inc.Florham Park, NJ) and placed in a chopped wheat straw bedded calf hutch (Calf-Tel Deluxe II, Hampel, Germantown, WI) measuring 220 x 122 x 38 cm that was placed on a grass pasture in an open naturally well-ventilated area.Hutches were spaced 0.6 m apart in rows of 20 hutches.Each hutch had a 183 cm x 122 cm x 107 cm wire panel attached to the front with 2 bucket holders (0.65 m high) for 8 L plastic buckets.One bucket contained ad libitum fresh water and the other bucket contained a pelleted CS.On arrival night, the water bucket was filled with 1.9 L of water containing 120 mL of BlueLite Replenish (Tech Mix Global, Stewart MN).The water bucket was emptied at each feeding and filled with the appropriate experimental MR at the designated feeding time.Upon completion of milk consumption, the bucket was rinsed and filled with fresh water.Calves were fed the 22/20 MR with decoquinate (Deccox, Zoetis, Florham Park, NJ) and diflubenzuron (Clarify, Central Life Sciences, Schaumberg, IL) with added commercially available synthetic amino acids equaling a 24/20 MR the next morning after arrival during the night.After the morning feeding, calves were weighed and frame measurements collected.Calves were then blocked by BW and randomly assigned to 1 of 2 MR treatments and 1 of 4 CS treatments in 2 × 4 factorial arrangement using a randomized complete block design.Main effects were 1 of 2 MR: 1) MR without or with EX added to supply 5 g/calf/day fed 2 times per day (2X) at 0630 and 1800 h, and CS main effect being 1 of 4 CS of: Control: No additive added to CS; BE: botanical extract added to provide approximately 450 mg/calf/d; EX: added to CS to provide approximately 5 g/calf/day; BE&EX: BE and EX added to CS at the same inclusion rates.The 2 × 4 factorial resulted in 8 treatments.The EX addition goal was to achieve a 5 g/d per calf intake across the study as a program (MR+CS) versus only as a product addition (CS or MR) for a few wk when intake achieved 5 g/d.In addition, this approach could identify if response is intestinal (MR shunted to intestine) or ruminal (CS).All MR contained 20% fat, decoquinate (Deccox, Zoetis, Inc., Parsippany, NJ) added at the rate of 41.7 mg/kg (asis basis) for coccidiosis control, 16 mg/kg diflubenzuron (ClariFly, Central Life Sciences, Schaumburg, IL) for fly control, and encapsulated butyrate (Adisseo North America) for enhancing ruminal papillae growth based on previous studies (Casper et al., 2021b).The MR base ingredient composition is given in Table 1.
The MR feeding time was set at 0630 and 1800 h and the feeding sequence was kept consistent among the 8 treatments.The milk shuttle is rinsed after each experimental feeding mixture to minimize/eliminate cross contamination.Each MR was accurately weighed (Model ACE110, Smart Weigh Inc., Hurricane, WV) and mixed with the appropriate amount of 46°C hot water using an Urban Milk Shuttle (Urban GmbH & Co., Hamburg, Germany).The shuttle with its computerized mixing, heating, and delivery systems was calibrated to ensure an accurate delivery of a homogeneously mixed MR being fed at a temperature of 37.5°C or greater at the correct volume.The MR was fed at the rate of 0.55 kg/calf daily for the first 14 d, then increased to 0.81 kg/calf daily on d 15 through d 35 divided into 2 equal feedings (i.e., morning and evening).For 5 d during wk 5 (d 30 to 35) Amprolium (Merial, LLC., Duluth, GA) was added at the rate of 10 mg/kg BW to the MR for coccidiosis control.Starting on d 36 through d 42, the MR feeding rate was decreased to 0.41 kg/calf daily fed 1x/d at the morning feeding to facilitate transition to weaning.All MR were fed at 15% solids and if any calf did not consume its milk, the refused volume was recorded.Starting on d 1 ~4 mm mini-pellet pelleted ~22% CP CS ("As-Fed") formulated varying from 23.9 to 24.5% (DM basis) for each experimental CS (Table 2) and water was offered ad libitum throughout the study.All MR were manufactured and supplied by Milk Specialties (Eden Prairie, MN) and CS was formulated by Casper Ranch (Freeport, IL), manufactured by a commercial feed mill (Ralco, Inc.Marshall, MN).All MR and CS were manufactured in sufficient quantities in 1 lot for the experiment to complete the study using the same ingredient lots among treatments (Tables 1 and  2).At the completion of the 56-d experimental period, calves were then dehorned via hot iron and castrated using a scalpel to remove the scrotum bottom and physically removing the testes by a licensed veterinarian (Dr.s'Bardon and Morgan Scharping, Lena Veterinary Clinic, Lena, IL) and then sold to a commercial privately owned feedlot.Four calves out of 80 were lost due to a rotavirus outbreak at the onset of the study.
Blood Collection.Blood samples from the jugular vein were drawn into 10-mL Vacutainer R serum separation tubes (Becton, Dickson and Co., Franklin Lakes, NJ) using a 20 gauge 0.9 by 38 mm blood collection needle (Nipro Corporation, Osaka, Japan) at approximately 4 h after morning feeding on d 1 for measurement of total serum protein (TSP).Samples were immediately placed on ice and brought to the lab, centrifuged at 2,000 x g for 20 min at room temperature (Eppendorf 5702, Eppendorf, North America, Hauppauge, NY).Once separated, the serum was pipetted into polystyrene tubes (Fujian Jiakai Plastic Products Co., Ltd., Fujian, China).The TSP measurement was conducted using a digital refractometer Weather Data.Weather data were downloaded from a local personal weather station site for Freeport, IL located at 42.301° N, 89.665° W at an elevation of 256.9 m being approximately 5 km from the research location.The temperature-humidity index (THI) was calculated according to the equation of Vitali et al. (2009) based on the weather station data for daily minimum, average, and maximum THI values.A THI value of >78 was considered to compromise calves' welfare and calves will experience significant heat stress (HS) when THI >88.These HS ranges are based on limited calf literature data (Kovács et al., 2020), but these ranges should be applicable in the current study.The thermoneutral zone (TNZ) range of 13°C -25°C will also be considered in the study, the TNZ ranges are for 1 mo old calf according to Wang et al. (2020).
Feed Intake and Analysis.Starting on d-1, the CS amounts offered and refusal weights were recorded daily using a digital scale (Model ACE110, Smart Weigh Inc., Hurricane, WV).In case of those days that feed was wet, (i.e., rain), that day's data were eliminated and the remaining days during that week were compiled into weekly mean CS intake.There were 11 d when rain prevented feed intake measurement and 4 d was the minimum used to calculate a weekly mean.Samples of MR and each experimental CS were collected weekly and stored frozen at −20°C until composited at the study end.Samples collected during wk 1, 2, and 3 or wk  4, 5, and 6 2001) and (Quigley, 2007).
Milk replacer solubility was evaluated by mixing a typical MR feeding of 0.425 kg with 2.85 L of 110°C water and sitting for 5 -10 min (approximate consumption time by calf) and then filtered through 4 layers of cheesecloth.The cheesecloth was dried and weighed before and after filtering the individual MR samples to determine the amount of residue remaining on the cheesecloth.Each composite MR sample was replicated 3 times.

Body and Health Measurements
Body weight was measured weekly using a Wrangler Jr. digital scale (Digi-Star, LLC, Fort Atkinson, WI) placed on a 1.2 m x 2.4 m sheet of 1.9 cm thick sheet of plywood towed by a John Deere 825 Gator (John  Calf health along with fecal, nasal, and ear/eye scores were monitored daily during the milk feeding phase.Health scores were visually assessed before the evening feeding according to the University of Wisconsin calf health scoring chart (McGuirk, 2013) and were based on a scale of 0 to 3. Fecal scores were established as 0) normal, 1) semi-formed or pasty, 2) loose but stays on top of bedding, and 3) watery and sifts through bedding.Nasal scores were 0) normal serous discharge, 1) small amount of unilateral cloudy discharge, 2) bilateral or cloudy or excessive mucus discharge, and 3) copious bilateral mucopurulent discharge.Eye/ear scores were 0) normal, 1) small amount of ocular discharge with ear flick or head shake, 2) moderate amount of bilateral ocular discharge or slight unilateral droop, and 3) heavy ocular discharge or head tilt or bilateral droop.In cases of illness, body temperature was measured using a rectal thermometer (Zoe+Ruth, Portland, OR) and appropriate medical treatments as prescribed by a licensed veterinarian (Dr.Brandon Scharping, Lena, IL) were administered if needed.All health incidents and treatments were recorded during the study.

Statistical Analysis
All data were checked for normality and outliers using the UNIVARIATE procedure of SAS (version 9.4, SAS Institute, Cary, NC, 2013) before any statistical analyses were conducted.The box and whisker plots and the Shapiro Wilk Test were used to verify that the remaining data were normally distributed (P > 0.15).Four calves died (discussed later) while 76 calves finished the 56 d (8 wk) experiment with no identified outliers.All data were then subjected to least squares ANOVA for a randomized complete block design (Steel & Torrie, 1980) having a 2 × 4 factorial arrangement of treatments via the MIXED procedure of SAS with study week as a repeated measure ANOVA.Statistical analyses were performed with individual calf as the experimental unit.The statistical model used was: Where Y ijk = dependent variable, µ = overall mean, Rep i = replication, MR j = main effect of MR, CS k = main effect of CS, WK l = study week, MR j x CS k = MR j by CS k interaction, MR j x WK l = MR j by WK l interaction, CS k x WK l = CS by Wk interaction, MR j x CS k x WK l = MR j by CS k by WK l interaction, Cov = Covariate (initial measurement when appropriate), and e ijkl is the residual random error.Milk replacer, CS, WK and all interactions were considered fixed effects, while replication was considered a random effect.Calf is considered random although not explicitly coded in the model.Study week was considered a repeated measurement in time having an autoregressive covariance structure.Least squares means were separated by PDIFF statement when the Ftest for main effects and all interactions were significant (P < 0.05).All other data were summarized utilizing the model described above but excluded week.Initial BW and frame measurements were tested as a covariate for their respective parameter but did not improve statistical significance (P > 0.15) and therefore were excluded from the model.Significance was declared at P < 0.05 and trends at 0.05 < P ≤ 0.10.Daily feed intake and orts measurements were compiled as weekly averages and DM intakes were calculated.Each daily fecal, nasal, and eye/ear scores were summarized by tallying by week the number of days having a specific score, (i.e., # d of score 0), and analyzed as weekly averages and the second way was to total the number of days of a specific score for the entire 6 wk MR feeding phase.Bai et al. (2020) reported using this approach resulted in score data being normally distributed and these data were normally distributed until a score of 3 was recorded due to few observations, (P > 0.05).

Mixability and Nutrient Composition of Feeds
The MR residue amount remaining after mixing a typical MR feeding rate (~0.41 kg) with 43.3°C water was similar (P > 0.93) for calves fed MR without and with EX.Mixability is always a concern with MR because residues left in the container after feeding are a concern both to the calf for not being able to consume all of their allotted nutrients, the person feeding the calves who notices the residue remaining, and potential pathogen growth.The MR mixed easily and stayed in suspension to ensure accurate and precise feeding.
The MR was formulated according to (Jaeger et al., 2020) recommendations.The MR nutrient composition fed to the calves in this study indicated that the nutrient composition met or slightly exceeded formulated specifications for CP and minerals (Table 3), due to the presence of synthetic EAA.In accordance with (Jaeger et al., 2020), substitution of synthetic EAA to meet formulation specifications instead of whey protein concentrate reduces whey protein concentrates inclusion rates allowing for greater inclusion of carbohydrates, i.e., lactose because fat concentration is fixed.These nutrient concentrations would meet or exceed the nutrient requirement guidelines for neonatal calves (NRC, 2001).
The mini-pelleted CS met formulation specifications (~24% CP, DM basis) to provide the nutrients to meet or exceed the NRC (2001) nutrient requirement guidelines for growing Holstein dairy calves (Table 3).Although significant differences (P < 0.05) were detected among CS in various nutrient parameters, these differences were small and unlikely to influence growth performance.
Weather Data.The study time frame matched up with typical Illinois summer weather, the maximum, average, and minimum daily temperatures and calculated THI fluctuated during the period (Table 4).The average maximum THI value of 81.6 (THI >78) highlighted that calves were experiencing some HS stress according to (Kovács et al., 2020).The 81.6 THI would satisfy and expose animals to the ideal conditions for administering probiotics as recommended by (Radzikowski, 2017) for young animals that are most at risk of stress caused by environmental factors.However, calves were not experiencing severe HS (THI >88), although the THI maximum during wk 6 was 87.6, which was above the lowest upper critical THI associated with increased heart rate.In contrast, the THI minimum was 66.0 for wk 6, which is below the THI stress point.Therefore, the calves had the opportunity to cool down during the night before experiencing heat stress the following d.Therefore, growth performance, feed intake, and immune system were not expected to be negatively impacted (Wang et al., 2020).During the study period, the maximum weekly temperatures were above the thermoneutral zone (TNZ) range (i.e., 13°C-25°C) described by (Wang et al., 2020) for calves within one month of age, but the mean for each week was within the TNZ range except for wk 6.The maximum temperature for wk 6 was 32.3 (>25°C) with minimal rainfall.The mean rainfall for the week was 0.12 cm and the total rainfall for the week was 0.81 cm, which is expected to expose the calves to some discomfort and stressors which might impact calves negatively.But, maximum, average, and minimum wind speed was favorable for the calves' welfare.The amount of precipitation received during the study was 16.9 cm, indicating basically that summer 2021 was warm and wet.
Total Serum Protein.The study was initiated with 10 calves per individual treatment resulting in 40 calves per MR main treatment effect and 20 calves per CS main treatment effect (Table 6).The Control and EX treatments within the MR main treatment effect lost 2 calves each due to a rotavirus outbreak at the beginning of the study.The lost of 4 calves were spread out among the CS treatments with 1 calf on BE, 1 calf on EX and 2 on the BE&EX.It's speculative that the BE&EX combination may have resulted in some stress challenges to the calves adjusting to different mechanism of action from eubiotic additives supplied via different delivery methods.Hence, a thorough understanding of eubiotic mechanisms of action are required as recommended by Reddy et al. (2020) for effective outcomes on calf growth performance, feed efficiency and welfare.In addition, delivery methods should be included as well.
Total serum protein concentrations were similar (P > 0.10) among treatments being greater than 5.2 g/dL (Table 5), which indicated adequate transfer of passive immunity required for survival (Tyler et al., 1996;Lombard et al., 2020).But note that the disease status of calves can influence the TSP results.The TSP concentration range was from 4.4 to 7.7 g/dL which assumes that all calves received colostrum but how much is unknown.A TSP value of approximately 4 would indicate that this calf or calves would have received little or no colostrum at the Dairy operation.The dehydration degree in sick calves is known to cause substantial increases in TSP concentrations (Guzelbektes et al.,2007).Meanwhile, calf health status was compromised/challenged with a rotavirus outbreak on arrival.Geletu et al. (2021) confirmed rapid loss of fluid and dehydration in calves infected with rotavirus.Although Cruywagen et al. (1996) stated that effectiveness of probiotics can only be assessed by stressing animals with the presence of microbial population that depresses growth.Salazar et al. (2019) also confirmed the positive effect of probiotics on growth performance of calves when their health status is compromised.Thus, the rotavirus challenge should have been an experimental benefit for testing the eubiotics.Also, TSP concentration variations among treatments can be related to colostrum amounts given at the numerous dairy operations, number of feedings before transport (at least 3), colostrum quality, and farm management factors all of which are not under experimental control.
Growth Performance.Initial BW was not a significant (P > 0.15) covariate and was dropped from the model.All possible interactions among MR, CS, and WK and the MR and CS main effects were nonsignificant (P > 0.10; Table 5).Figure 1 demonstrates similar (P > 0.10) calf BW among all treatments indicating the lack of a BW response to the addition of BE, EX, or BE&EX combination.In addition, these data would support the conclusion that supplying EX in either the MR or CS demonstrated little benefit to either intestinal or ruminal health.These   a,b,c,d Means in the same row within calf starter differ, P < 0.05.results are in contrast with the findings by Amanullah et al. (2020) and Radzikowski (2017) that administering probiotics to calves will contribute to accelerated stomach development and consequently increase growth rates.Likewise, the addition of feed additives to CS is supposed to benefit rumen development and accelerate growth rates as reported by (Hill et al., 2007;Casper et al. 2021b;Palhares Campolina et al., 2021), but the MR treatments in combination with CS additives either alone or a eubiotic combination did not demonstrate an increase in growth performance when compared with Control (Table 5).The possibility exists that all treatments receiving encapsulated butyrate in the MR may have mitigated any potential disadvantages thus confounding responses to BE, EX, and BE&EX.The similar BW (P > 0.10) among all treatments is in contrast to Jonova et al. (2021) who emphasized synergistic effects when 2 feed additives were combined, but similar to results reported by Casper et al. (2021b) that demonstrated no beneficia effects to combining additives.All possible interactions among MR, CS, and WK and the MR and CS main effects were nonsignificant (P > 0.10) for ADG (Table 5).These results are in contrast with previous results reported by Stefańska et al. (2022) demonstrating enhanced growth performance when a bioactive eubiotic natural feed additive was incorporated into the MR.Although, Reddy et al. (2020) cited earlier stated that unification of different levels and types of supplemented plant metabolites can render synergistic effects.Therefore, the results from the current study suggested that this particular natural eubiotic feed additives might not be a proper combination that can promote the intestinal microbiota balance to improve growth performance of neonatal calves, such as BW and ADG.These data agree with Casper et al. (2021b) that the combination did not influence calf growth performance.However, Casper et al. (2021b) reported improved growth performance when feeding EX alone, while Hill et al. (2007) reported improved growth performance when feeding BE in both MR and CS.It's speculated that this eubiotic combination may have upset the intestinal bacteria balance making the calves more susceptible to stressors.Furter studies are recommended to investigate the present eubiotic combination as to mechanism of action for lack of response.
Dry Matter Intake and Feed Efficiency.All possible interactions among MR, CS, and WK and the MR and CS main effects were nonsignificant (P > 0.10) for CS DMI and total DMI (Table 6).The MR intake was similar (P > 0.10) across all treatments per study design.The CS and total DMI was similar (P > 0.10) for calves fed all individual treatments, except for wk 8, when calves fed EX MR and BE&EX CS consumed the least amount of CS and total DMI compared with calves fed Control MR and Control CS, with calves fed the remaining treatments being similar (P > 0.05) and intermediate.Why the BE&EX combination would reduce CS and total DMI is not known, but the possibility exists of an antagonism, which is known to occur between certain feed additives (Wu et al., 2020).These data reject the hypothesis of a benefit to feeding BE, EX, alone or in combination, which supports data by Casper et al. (2021b).This contrast with Reddy et al. (2020) and Hill et al. (2007) reporting that plant extracts would presumably add flavor to enhance ingesting competence and growth performance.Palhares Campolina et al. (2021) reported that CS intake in the first weeks of life is small.Thus, the results from the current study agree with Stefańska et al. (2022), who reported that the method of feed additive provision can influence its effectiveness because consumption of solid feed (CS) by calves in the first 4 wks of life is limited.Therefore, the limited capability of ingesting large CS amounts will result in limiting supplement intake.Therefore, the desired supplementation level may not be achieved which masks the potential positive effects of the eubiotic feed additive.However, the implications of natural feed additive research have been noted by Reddy et al. (2020) due to calves fed MR behaving as monogastric species with a non-functional rumen, but a well-developed abomasum.
All possible interactions among MR, CS, and WK and the MR and CS main effects were nonsignificant (P > 0.10) for feed conversion.Feed conversions were similar (P > 0.10) among calves fed all individual treatments.The hypothesis was that BE and EX additions to the MR  and CS to influence feed intake and improve feed efficiency, but these data demonstrate that supplementation did not enhance feed intake and feed conversions, which agrees with Casper et al. (2021b).These results are in contrast to Radzikowski (2017) reporting that probiotics contribute to the stabilization of microbial populations and increase enzymatic activity of the digestive tract for improved nutrient digestion and utilization.Hill et al. (2007) reportd improved feed comvesion of 0 to 56 d when feeding a BE in both MR and CS These data observations may have resulted from the combined eubiotic bioactive compounds impairing (antagonism) nutrient digestion, thus limiting calf growth performance.But, further studies are warranted for elucidating the synergistic effects of these products and possible mechanisms, Frame Measurements.All possible interactions among MR, CS, and WK and the MR and CS main effects were nonsignificant (P > 0.10) for frame measurements (Table 7).Gains in frame measurements were similar (P > 0.10) among calves fed all individual treatments.The starting, final, and gains for HH, HW, WH, and HG were similar (P > 0.10) for calves fed all treatments.The frame growth responses observed in this study are similar to responses observed in previous studies at this location (Bai et al., 2020, Liu et al., 2020, Casper et al., 2021a;Casper et al., 2021b).In contrast, Hill et al. (2007) reported an increase in HW when feeding a BE Fandiño et al. (2020) explained that active components in natural additives when combined may be additive, synergistic, or antagonistic.Antagonisms between certain feed additives have been reported by Wu et al. (2020).
Health Performance.All possible interactions among MR, CS, and WK and the MR and CS main effects were nonsignificant (P > 0.10) for fecal (scours) scores, nasal, and eye/ear scores (Table 8).Fecal (scours) scores, nasal, and eye/ear scores were similar (P > 0.10) among calves fed all individual treatments, except for a fecal score of 2 when expressed as total days or days/wk.Calves fed EX demonstrated a tendency for greater (P < 0.10) fecal score 2's than calves fed BE with calves fed Control and BE&RC being intermediate and similar (P > 0.10) when expressed as total study d or number of d during the week.The calves fed BE fought the rotavirus challenge at study initiation by demonstrating less scours than calves fed the remaining CS treatments (more fecal score 0 d) despite the challenges of scours (fecal score = 3).Reddy et al. (2020) reported that additives fed to calves aided in improving scours resistance when calves are not under stressful conditions, like a rotavirus challenge experienced here at study initiation.But, the eubiotic combination, fed via different delivery methods (MR & CS), may not provide the needed synergistic effect for promoting intestinal colonization of protective bacteria when competing with pathogenic bacteria responsible for causing gastrointestinal infections, i.e., scours.More so, Stefańska et al. (2022) explained that natural feed additives can stimulate the development of the immune response against pathogenic bacteria for promoting in-  testinal beneficial effects by favoring intestinal balance of beneficial microbiota.Thus, the MR eubiotic is recommended for further study.

CONCLUSIONS
The study hypothesis that the combination of a botanical extract (BE) and a DFM (EX) being beneficial to neonatal calf growth during the preweaning period was rejected.This particular combination was not beneficial to calf growth and health, despite previous research demonstrating that the individual additives enhanced calf growth performance.In addition, this study contrasts with previous reports that an individual DFM or BE can improve growth performance.In contrast to these data, reports have demonstrated several beneficial responses of increased nutrient bioavailability, efficiency, and growth performance when feeding these additives.
Olagunju et al.: BOTANICAL AND MICROBIAL PRODUCTS FOR CALVES Olagunju et al.: BOTANICAL AND MICROBIAL PRODUCTS FOR CALVES Olagunju et al.: BOTANICAL AND MICROBIAL PRODUCTS FOR CALVES Olagunju et al.: BOTANICAL AND MICROBIAL PRODUCTS FOR CALVES Table 5.Total serum protein (TSP) concentrations, BW, BW gains, and ADG for calves fed milk replacer (MR) without (Control) or Excel (EX) and fed a calf starter (CS) without (Control) or with Apex (BE), Excel (EX), or combination of BE&EX Olagunju et al.: BOTANICAL AND MICROBIAL PRODUCTS FOR CALVES Olagunju et al.: BOTANICAL AND MICROBIAL PRODUCTS FOR CALVES adjunct professor at North Carolina A&T University and has vested interest as co-owner and operator of Casper's Calf Ranch, LLC along with Mrs. Janet Casper.LIST OF ABBREVIATIONS: BE = Botanical extract; BL = Body length; BE&EC = Botanical extract and ExCel; CS = Calf starter; DFM = Direct fed microbial; EX = ExCel; HG = Heart girth; HH = Hip height; HS = Heat stress; HW = Hip width; MR = Milk replacer; THI = Temperature humidity index; TNZ = Thermal neutral zone; TSP = Total serum protein; WH = Withers height

Table 1 .
Olagunju et al.: BOTANICAL AND MICROBIAL PRODUCTS FOR CALVES Milk replacer ingredient composition 1 (MISCO Palm Abbe model PA202X; Solon, OH) and remaining samples were preserved at −20°C.

Table 2 .
Olagunju et al.: BOTANICAL AND MICROBIAL PRODUCTS FOR CALVES Ingredient composition of mini-pelleted (~4 mm) calf starter without (Control) or with botanical extract (BE; Apex), direct fed microbial (EX; Excel), or combination of BE&EX

Table 4 .
Weather data for the 8 week experimental period of June 17 through August 12, 2021 1 Temperatue humidity index.

Table 6 .
Milk replacer (MR), calf starter (CS), total DMI, and feed conversions for calves fed milk replacer (MR) without (Control) or Excel (EX) and fed a calf starter (CS) without (Control) or with Apex (BE), Excel (EX), or combination of BE&EX

Table 7 .
Body frame measurement for calves fed milk replacer (MR) without (Control) or Excel (EX) and fed a calf starter (CS) without (Control) or with Apex (BE), Excel (EX), or combination of BE&EX