Effect of probiotics on the growth, blood profile, and nutritional-metabolic profile of feedlot cattle (original) (raw)

1. Introduction

Livestock production systems are part of a circular bioeconomy from which food is derived, with both environmental impacts and benefits [1, 2]. Supplementation of feed additives as alternatives to in-feed antimicrobials helps maintain a balance in gut microbes and thus benefits microbial ecosystem in the gut, thereby improving nutrient absorption, productivity, health, and general well-being of animals [3, 4]. High-energy diets containing grains improve growth parameters and potentially shorten the feeding period, thus increasing animal productivity in intensive systems [5]. However, to counteract the negative effects of feeding concentrated diets to cattle, ionophore antibiotics are still used as growth promoters in some countries to maximize rumen fermentation. Although these additives were proven to increase feed efficiency, the understanding of how they influence the ruminal microbial dynamics is still incomplete [6, 7]. In addition, the development and spread of antimicrobial-resistant bacteria, which may threaten the health of animals and consumers of animal products, led to the ban of antibiotics used as growth promoters in animal production since 2006 in Europe [8].

As a result, it is necessary to explore alternative methods to improve cattle performance. Probiotics (bacteria and yeasts) and phytobiotics as feed additives have been reported as desirable alternatives to antibiotic growth promoters, supporting cattle health and growth promotion [9, 10]. There is a particular research interest in the application and benefits of probiotics in the production of meat animals. The natural mechanism of probiotics is modulation of rumen metabolism and host gene expression, while reducing the disease burden [9, 11–13]. When administered as probiotics, lactic acid bacteria (LAB) have improved the metabolic-nutritional status, overall productive performance, feeding efficiency, and well-being of cattle [14–16], by enhancing the balance of the gastrointestinal microbiota [17–19]. Impacts of LAB strains from lactobacilli, enterococci, pediococci, bifidobacteria, and some specific yeast on the growth and productive performance have been widely reported. Alterations of the gastrointestinal microbiota have been reported to influence stress-related behaviors by activating neural pathways and signaling systems of the central nervous system [20]. The scientific appraisal of cattle disease and behavior by semiological and ethological descriptions has been used to determine the impact of intensivism on animals subjected to intensive animal agriculture [21, 22]. As a reservoir of Escherichia coli O157:H7, cattle can cause infections to humans. Therefore, its reduction in the fecal shedding of farms can lessen its entry into the food chain [23]. The use of probiotic LAB and yeasts to reduce the amount of pathogenic bacteria in fecal shedding has been widely reported as an intervention strategy to mitigate their transmission to humans [24]. In previous studies, LAB from feedlot cattle feces were identified and characterized as probiotics, and their effects on fecal microbiome modulation were studied [17, 25, 26] for different administration days and life periods. Based on these findings, the impacts of probiotic lactobacilli (individual or combined strains) administration on clinical, nutritional, and blood parameters, as well as on fecal microbiology, during feedlot confinement of cattle were evaluated.

2. Material and methods

2.1. Ethics statement

All the procedures and protocols used in this study were reviewed and approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL-UNT, Argentina).

2.2. Probiotic bacteria, culture conditions, and inoculum preparation

Lactobacillus acidophilus CRL2074, Limosilactobacillus fermentum CRL2085, and Limosilactobacillus mucosae CRL2069, which were previously isolated from fattening cattle and selected for their beneficial properties related to epithelial adhesion (hydrophobicity, self-aggregating, biofilm-forming), enzymes, or antagonistic substances (organic acids and/or hydrogen peroxide), were used as probiotics [26]. Inocula were prepared by transferring frozen glycerol stock culture of the strains to MRS (De Man, Rogosa, and Sharpe) broth (Biokar, France) and subcultured twice in the same media at 37°C for 18 hours. Each LAB probiotic strain was multiplied at the CERELA (CONICET) pilot plant. The obtained concentrated cell mass of each probiotic strain was distributed in plastic containers of 100 g each, with a cell concentration of 1011 CFU/100 g, and stored at −20°C until use. The viability of the microbial cells was determined before administration by the plate-counting method (through successive dilutions in 0.85% sterile physiological solution and subsequent plating on MRS agar).

2.3. Animals, feeding, and probiotic treatments

Sixty Brangus and Braford male and castrated animals were eligible for their inclusion in this study. Cattle belonged to a commercial feedlot located in the Northern Province of Santiago del Estero (Argentina). Upon their arrival, the status of animals’ health (vaccination against infectious pathogens, respiratory diseases, and parasites) was assessed according to the preventive sanitary plan developed by the veterinary staff of the livestock industry. In the feedlot, steers were fed diets consisting of three rations with different compositions in different time periods: initial/adaptation up to 15 days (T0), intermediate up to 45 days (T45), and finishing ration until 105 days (T105). The composition of the feed was designed by decreasing sorghum silage (63–17%) and soybean expeller (9.5–3%), while increasing dry corn cracked grain (16–80%). In addition, rations were also supplemented with urea (0.5%), minerals/vitamins (1.7–2%), and occasionally wheat bran (8–10%). After the adaptation period, animals with an initial average body weight between 200 and 250 kg were randomly allocated in four separate pens, containing 15 animals each (three for probiotics administration and one as control). All the animals received ionophore (monensin, 1400 ppm) treatment, and feed rations were supplied twice a day (one-half of the allowed daily rations at each feeding) and ad-libitum access to water was provided. Pens (30 × 30 m2) were designed with individual feeding trays, and watering holes were automatically refilled. The administration of probiotics was carried out twice three days per week. The steers (n = 15) in each pen received the probiotics through the feed and were distributed linearly in the feeder. In total, 100 g of frozen concentrated cells of each probiotic strain/combination was resuspended in a spray bottle containing 750 mL of tap water. The suspension was sprayed and mixed over the feed ration to obtain a final probiotic concentration between 107 and 108 CFU/animal/day. An operator checked daily that all the animals consumed the food provided. Before administration, ear tags (numbered in different colors for each probiotic group) were used for the identification of each animal.

2.4. Animals experimental design

After veterinary control and the adaptation period (day 0), animals were randomly separated into three groups (n = 15) for the administration of probiotics as follows: (A) L. acidophilus CRL2074, (B) Lim. fermentum CRL2085, and (C) L. acidophilus CRL2074 + Lim. fermentum CRL2085 + Lim. mucosae CRL2069. A control group (which was not administered with probiotics) was also included, as shown in Figure 1. At each experimental period (T0, T45, and T105), individual samples were taken from the animals for general clinical examination (evaluation of body condition and inspection of mucous membranes, presence of ocular and nasal secretions, and sensory status). Their weight (on a mechanical scale), blood parameters, and rectal feces were analyzed. Fecal samples were collected aseptically from the colon/rectum of each steer restrained in an operating stall. The anus was cleaned with disposable paper, and then the fecal samples were collected from the rectal blister by inserting the gloved hand. If no feces were found, defecation was stimulated by massaging the inside of the intestine with the fingers. Once the samples were obtained, they were stored separately in precisely marked sterile collection bottles.

2.5. Monitoring probiotics’ effect on animals

Clinical parameters

Each animal from the different probiotics-administered groups was inspected. Following the scoring method of Edmonson et al. [27], the steers’ body condition was evaluated by visually and physically examining the body fat around their tail head. The amount of subcutaneous fat of each animal was assessed by manually palpating the tail head and observing the shape of the loins, pelvis, and tail head areas of the animal from behind. Based on the assessment, overall body condition was scored on a 1–5 scale of severe under-condition (emaciated) to over-condition (very fat). The sensorium state of steers was evaluated by observing animal behavior and characterized as depressed, normal, and exited. Nasal and ocular discharges were individually inspected, and their presence/absence was recorded on a scale. A macroscopic analysis of steers’ feces was carried out for consistency, and the observations were recorded as (1) normal or pasty and (2) diarrhea.

Figure 1

Probiotics administration to feedlot cattle. Experimental protocol was applied to different groups of animals (n = 15 each). Animals receiving probiotics from day 0 to day 105 (T0–T105) and control animals (n = 15) were fed ad libitum: (A) L. acidophilus CRL 2074, (B) Lim. fermentum CRL2085, and (C) mixture of CRL 2074 + CRL 2085 + CRL2069. Samples were taken on days 0, 45, and 105.

Nutritional parameters

Growth and performance were evaluated on days 0, 45, and 105 for each probiotics-supplemented cattle group, as indicated in the experimental protocol (Figure 1). Feedlot facilities were well designed, and handlers were trained to reduce/avoid cattle stress. Animals were individually immobilized in the chute (operating box) composed of a semiautomatic stock and a lever-operated vacuum clamp, allowing for a complete animal immobilization without hurting it to work safely. At each experimental time (T0, T45, and T105), animals were individually weighed by using a mechanical livestock scale. Mean daily weight gain (DWG) per animal was calculated as follows: [FW (final weight) – IW (initial weight)]/number of days between samplings. Then, the group average ± SD was calculated. For biometrics parameters, height (cm) was determined from the highest point of the animal’s withers to the floor using a rigid tape measure. The thoracic circumference (cm) was taken with a tape that surrounded the trunk behind the knuckle, the first ribs, and the first thoracic vertebrae of the steer. The weight, height, and thoracic diameter differences were calculated for each animal by subtracting the value obtained in the previous sampling from the value obtained in the latter sampling, and then the average per group was determined.

Hematologic and metabolic parameters

Animals’ blood was collected from the coccygeal vein using a 5-mL syringe and a sterile 18 Gx1–1/2 (Neojets Ltd, UK) disposable hypodermic needle. The collected blood was fractionated in hemolysis tubes both with and without heparin. The hematocrit % was determined from the blood collected in heparinized tubes [28]. Briefly, capillary tubes with their one end sealed were filled with blood to three-fourths of their volume and centrifuged. The hematocrit % was calculated as follows: erythrocytes column length/total blood length in the capillary tubes × 100. Leukocyte percentage was also determined as previously described [28]. Different types of leukocytes in blood were counted by staining blood smear in the glass using the May-Grünwald Giemsa technique. Different types of cells, namely lymphocytes, neutrophils, eosinophils, monocytes, and basophils, were observed using an optical microscope (100×) and counted, and the cell volumes were expressed as percentages. Hematology data taken at the beginning of the study were used for comparative purposes. Serum metabolic parameters were analyzed from the blood collected in non-heparinized tubes. Liver activity and bilirubin concentration were quantified using the diazo-reagent colorimetric method. AST (aspartate transaminase) and ALT (alanine transaminase) activities were evaluated by the Reitman–Frankel colorimetric method. Lipid profile assessment, serum triglycerides, and total cholesterol concentrations were determined by using the GT Lab Liquid Plus kits (using GPO/PAP and CHOD/PAP Trinder’s color colorimetric methods). In addition, amounts of total proteins and albumin were evaluated by using the biuret and dye-binding methods. Glucose was determined by applying the GPO/PAP Trinder’s color kit, and C-reactive protein (CRP) was qualitatively assayed using the direct latex method. All kits used for serum determinations were from GT Lab (Rosario, Argentina). Results for metabolic parameters were compared with reference values [28–31].

Fecal microbiological analysis

Rectal fecal samples from each animal were collected, and 5 g of each was used to determine the number of cultivable bacteria from the different animal groups in the sampling times. Fecal samples (5 g) were aseptically homogenized in 45 mL of saline-peptone water (8.5 g/L NaCl, 1 g/L bacteriological peptone) in a sterile plastic bag using Stomacher (Stomacher Lab-Blender 400, A.J. Seward Lab., London, UK) for two minutes, and decimal dilutions were then prepared in saline (0.9 w/v NaCl). Microbial suspensions were plated in triplicate and incubated as follows: total aerobic mesophyles (TAM) were plated on Plate Count Agar (PCA, Biokar, France) and incubated aerobically (48 hours at 37°C), and LAB were plated on the MRS agar medium and incubated under microaerophilic conditions (48 hours at 37°C). Total coliforms (TC) were determined on the McConkey agar (24 hours at 37°C).

2.6. Statistics

A descriptive analysis of the evaluated weight was carried out. Inferential analysis was performed using the hypothesis test to compare the treatments at each evaluated time; the Kruskal-Wallis’ and Fisher’s exact tests were applied. The level of significance used was p < 0.05. The statistical package used was Stata15IC (Stata-UK.com).

3. Results

3.1. Clinical parameters

The effects of individual probiotic strains, namely L. acidophilus CRL2074 (A) and L. fermentum CRL2085 (B), and the multistrain formulation of L. acidophilus CRL2074 + L. fermentum CRL2085 + L. mucosae CRL2069 (C) at a final concentration of 107–108 UFC/g for each probiotics group were evaluated for a period of 105 days in beef cattle. A combination of subjective and objective analyses was applied to animals. Exhaustive clinical examination of both probiotics-administered and non-probiotics-administered cattle was conducted to identify possible pathological evidence, as shown in Table 1. Inspection of steers’ body condition initially showed values between 3 and 5. There were no significant differences when comparing the sample groups with the control group initially (p > 0.05), while increased scores were observed for body condition over time when comparing the groups undergoing probiotic treatments with the control at different experimental times. A higher number of animals (≥80%) exhibited a score of 4 (frame as visible as covering) at day 0 in probiotics-supplemented groups, while 100% of animals reached a score of 5 (severe over-conditioning) at 105 days of confinement.

When the effects of probiotics treatments were analyzed, a score of 5 was displayed by 6.6–20% of cattle at day 0, 40–100% at day 45, and 100% at the end of the trial (105 days). Lim. fermentum CRL2085 and multistrain probiotics (CRL2074 + CRL2085 + CRL2069) were the quickest to change the general body condition of cattle during the trial. Statistical analysis of cattle supplemented with probiotics groups showed no significant differences (p = 0.129 and p = 0.082) for body condition when compared between 0 and 45 days and 45 and 105 days, respectively. When the sensorium/behavior of confined feedlot cattle was evaluated, most of them were in the normal category for all the treatments (Table 1). As the administration protocol progressed, several animals showed excited behaviors compared to day 0. Supplementation of L. acidophilus CRL2074 resulted in the highest level (20%) of excitement, while a somewhat depressed behavior (6.6%) was found for the supplementation of the multistrain probiotics. Cattle sensorium was normal (between 80 and 100% of the animals) in all treatments at 105 days. In addition, during probiotics administration, nasal and ocular discharges were monitored as disease markers (Table 1).

Initially, apart from cattle administered with probiotics formulation, a certain degree of nasal discharge was observed, decreasing at the end of the experiment for control and L. acidophilus CRL2074 supplementation. The number of cattle with this sign increased (20%) after 45 days when Lim. fermentum CRL2085 and probiotic formulation, respectively, were administered. Ocular inspection of cattle showed 20% and 26.6% of animals in the control group with eye secretions at T0 and T45, although this effect was further reduced. Probiotics administration exhibited a lower percentage of affected cattle. One hundred percent of animals from the three probiotics-administered groups were free of ocular secretions on the 105th day. As a complementary clinical parameter, consistency of cattle feces was evaluated (Table 1). Although the stools from the different groups were mostly normal (pasty), the control group exhibited a greater proportion of liquid stools at day 0, which evolved to pasty at the end of the trial. Probiotics-administered cattle groups presented lower percentages of diarrhea. Administration of CRL2074 probiotics changed the consistency of feces from pasty to more liquid in 0–20% of animals at 105 days, while supplementation of multistrain probiotics maintained the pasty consistency in 100% of feedlot cattle throughout the period of experiment.

Table 1

Clinical parameters of feedlot cattle in-feed supplemented with probiotics

3.2. Nutritional parameters

To assess the improvement in feedlot cattle growth and performance, biometric parameters, mean weight, and DWG changes of cattle were evaluated. In Figure 2a, a mean height of 110.7 ± 4.6 cm for control animals was observed initially, and it increased to 117.1 ± 2.9 cm at 105 days. Mean height changes from T0 to T105 were 7.1, 9.2, and 9.3 cm for feedlot cattle administered with L. acidophilus CRL2074, Lim. fermentum CRL2085, and multistrain probiotics (CRL2074 + CRL2085 + CRL2069), respectively. When different treatments were compared, no significant differences were detected for mean heights of animals treated with CRL2074 and CRL2085 (p = 0.887), CRL2074 and probiotics mix (p = 0.113), and CRL2085 and probiotics formulation (p = 0.149). However, there is evidence that the mean heights were significantly different at 45 and 105 days compared to the control (p < 0.001). The greatest mean height increase was produced during the 0–45-day period, while total increases of mean height values (>9.0 cm) were maximum for the administration of CRL2085 and multistrain probiotics at 105 days. The mean thoracic diameter of control animals increased from 162.3 ± 8.9 to 182.4 ± 6.2 cm between 0 and 105 days (Figure 2b). During the trial, a significant difference in thoracic diameter was observed between 45 and 105 days (p < 0.0001). When treatments with probiotics were compared, supplementation of L. acidophilus CRL2074 showed the highest mean values at 0, 45, and 105 days. However, total thoracic diameter increments were maximum (25.0 cm) in animals supplemented with Lim. fermentum CRL2085 and probiotic formulation (25.1 cm), while the increment for CRL2074 was 20.6 cm. In general, as the administration protocol progressed, all the biometric values increased indicating normal growth and development of animals.

Figure 2

Biometric parameters of steers fed ad libitum, added with L. acidophilus CRL2074, Lim. fermentum CRL2085, or combined strains (CRL 2074 + CRL2085 + CRL2069) and the control group during 45 and 105 days. (a) Height and (b) thoracic diameter of animals at different sampling times. Each point represents the data obtained from each animal.

Changes in the mean weight of feedlot cattle supplemented with probiotic groups are shown in Figure 3 and Table S1, Supplementary materials. As expected, animal weight increased during the stay in feedlot. At the end of experimental time (105 days), the highest average weight (361.3 ± 23.5 kg) was found for L. acidophilus CRL2074-supplemented group compared to the control (347.9 ± 29 kg). When cattle weights were analyzed for each administration period, the average weights of all the animals increased from 245.2 kg (0 days) to 298.9 kg (45 days), and 352.3 kg (105 days). Comparison of supplementation with Lim. fermentum CRL2085 and probiotic mixture formulation (p = 0.5756), and supplementation with L. acidophilus CRL2074 and CRL2085 (p = 0.0525) indicated non-significant differences, while significant differences were found when comparing CRL2074 with multistrain probiotic mix (p = 0.0124) (from a mixed linear model in Stata/IC 15 applied). In addition, weights between day 0 and days 45 and 105, and between 45 and 105 days had significant differences among the groups. Weight increased throughout the experimental time from 100.3 kg (CRL2074) to 113.1 kg (CRL2085), and 112.3 kg (probiotic mixture). The greatest increase of live weight at 105 days was for supplementation with Lim. fermentum CRL2085. However, changes in DWG were observed around the general mean for each administration period (Table 2). When treatments were compared, significant differences were evidenced between the control and the group supplemented with Lim. fermentum CRL2085 probiotics at day 45 (p = 0.0078) and day 105 (p = 0.0178). Average DWG was maximum between 0 and 45 days, and the highest value of DWG was obtained for Lim. fermentum CRL2085 supplementation with a value of 1.44 ± 0.19 kg/day. Although a reduction of DWG was observed during the final administration stage (45–105 days), the overall highest values (0–105 days) were obtained for CRL2085 and multistrain probiotics mix with DWGs of 1.3 ± 0.12 and 1.21 ± 0.21 kg/day, respectively. As a whole, feedlot steers supplemented with Lim. fermentum CRL2085 and multistrain probiotics thrice per week for 105 days had an 11% higher average DWG than non-administered control.

Table 2

Daily weight gain of steers fed with different probiotic formulas

Figure 3

Mean body weight of feedlot cattle fed ad libitum, added with L. acidophilus CRL2074, Lim. fermentum CRL2085, or combined strains: CRL 2074 + CRL2085 + CRL2069 and the control group during 45 and 105 days. Each point represents the data obtained from each animal.

3.3. Blood and serum parameters

The effect of probiotics administration on cattle was also evaluated by hematologic parameters at the end of administration time. The obtained results were compared to those at the beginning of the study (day 0) and physiological reference values (Table 3 and Table S2, Supplementary materials). Descriptive mean values showed hematocrit % within the reference range for cattle from the control group during the trial, whereas mean values of the probiotics-administered group were higher than the reference values, indicating that the animals were not suffering from anemia. The highest final value (59.8 ± 10.2%) was reached at 105 days by the control group, while the final hematocrit % was found between 55% and 58.3% when probiotics were supplemented in the diet (Table S2, Supplementary materials). When lymphocytes were evaluated, values at 105 days were also higher than the reference range. A mean value of ≥75% was found during the whole experiment, reaching maximum values for the control (85.5%) and L. acidophilus CRL2074 probiotics groups (83.4%), whereas CRL2085 and multistrain probiotics groups normalized their values within the referential range (Table S2, Supplementary materials). At the beginning of the trail, eosinophils were close to the lower reference values in all the groups, but resulted in higher values for the probiotic groups at the end of the assay. On the contrary, lower mean values were exhibited for neutrophils at day 0, and values within and below the referential range were obtained for control and probiotics groups, respectively. The greatest values were obtained for lymphocytes, eosinophils, and neutrophils at 105 days when L. acidophilus CRL2074 probiotics was supplemented to feedlot cattle (Table S2, Supplementary materials). The highest mean values were obtained for monocytes and basophils at day 0 (out of the reference range), reaching their maximum for Lim. fermentum CRL2085 (10.5%), and then these values reduced between 0 and 0.71 ± 1.4%, respectively, at 105 days (Table 3 and Table S2, Supplementary materials).

Table 3

Descriptive mean values of blood and serum metabolic parameters of feedlot cattle

administered with probiotics for 105 days

Reference values used in this study for hematological parameters are as follows: hematocrit, lymphocytes, eosinophils, neutrophils, monocytes, basophils [28]; total protein, glucose, albumin, triglycerides, cholesterol, total bilirubin [29]; AST, ALT [30]; C-reactive protein [31].

Moreover, when metabolic parameters were assessed (Table 3), serum descriptive mean values for glucose were at the upper limit of the referential range at day 0, suggesting a hyperglycemic state. Then, at the end of the trial, values for both groups reduced to a lower limit (∼46 mg/dL). At 105 days, glucose level reached the physiological standard for the multistrain probiotics group (68.5 mg/dL), while CRL2074 and CRL2085 probiotics exhibited lower glucose values than the referential range (Table 3 and Table S2, Supplementary materials). Mean values of serum total protein were observed near the lower limit of reference at day 0, showing a low increase at 150 days. The maximum value was registered for Lim. fermentum CRL2085 supplementation (6.89 mg/dL). Mean values of serum albumin also increased at the end of the trial, exhibiting the maximum value (3.72 g/dL) when probiotics formulation was supplemented to cattle (Table 3 and Table S1, Supplementary materials). In the analysis of liver activity, mean values for bilirubin fell within the reference range throughout the trial. In addition, mean values of AST and ALT increased at 150 days compared to day 0, although they remained at a low level. Lim. fermentum CRL2085 displayed the lowest values for both liver enzymes (Table 3 and Table S2, Supplementary materials). Animals in all experimental groups exhibited lower descriptive values of triglycerides according to the reference range. The control group showed higher values (55.8 ± 6.3 mg/dL) than the probiotics-administered groups. Among all, the L. acidophilus CRL2074 group displayed the greatest value (32.4 mg/dL). Mean values of total cholesterol were observed to fall within the normal range. However, increases in the cholesterol content were observed in both cattle groups. Lower final values (∼134–135 mg/dL) were displayed by the probiotics-supplemented group at 105 days compared to the control (Table 3). Moreover, mean values of serum CRP decreased from 1.70 to 1.40 mg/dL during the whole trial, which was consistent with the referential value (Table 3).

3.4. Fecal microbiological parameters

The effect of different groups of probiotics on feces of feedlot cattle was evaluated by analyzing the changes in the total count of aerobic mesophiles (TAM), Enterobacteriaceae, and LAB at 0, 45, and 105 days (Figure 4). Results for TAM showed initial mean values in the range of 7.16–7.45 log CFU/g, whereas initial mean values for Enterobacteriaceae and LAB ranged from 5.33 to 6.66 log CFU/g and 6.13–7.00 log CFU/g, respectively (Figure 4a–c). Different bacterial growth patterns were observed for these populations. TAM showed a sustained growth reduction during the trial for the control group, while a moderate count increase was produced when probiotics were administered to cattle. Multistrain probiotics supplementation reached maximum numbers (7.72 log CFU/g) at 45 days, and then a reduction was observed for all experimental groups (Figure 4a). Enterobacteriaceae exhibited initial values between 5.33 and 6.66 log CFU/g with a reduction at 105 days only when the probiotic formulation was supplemented, reaching the lowest final value of 6.33 log CFU/g (Figure 4b). In addition, initial LAB counts ranged from 6.13 to 7.00 log CFU/g exhibiting an increasing growth trend up 45 days, with values between 6.78 and 7.19 log CFU/g, while a decreasing tendency was found for all groups at 105 days, reaching final mean values between 6.02 and 6.75 log CFU/g (Figure 4c). The reduction in the count of Enterobacteriaceae in the cattle supplied with multistrain probiotics agrees with the high LAB counts during the trial, while no reduction was observed for the remaining experimental groups (L. acidophilus CRL2074 and Lim. fermentum CRL2085).

Figure 4

Microbiological evaluation of cultivable bacteria in feces of cattle fed with L. acidophilus CRL2074, Lim. fermentum CRL2085, or combined strains: CRL 2074 + CRL2085 + CRL2069, and the control group during 45 and 105 days. The results are expressed as mean ± SD: (a) total aerobic mesophiles, (b) enterobacteria, and (c) lactic acid bacteria.

4. Discussion

Evidence of favorable changes in the activity of the digestive microbiota induced using probiotics was abundantly reported. However, information on the impact of probiotics on productivity and health parameters is scarce. Thus, the effect of probiotics on clinical parameters and hematological, nutritional, and fecal microbiological profiles of beef cattle was evaluated in this study. Among the clinical parameters, body condition and animal sensorium showed optimal muscle skeletal development and high confinement adaptation after a satisfactory objective estimation, while only a few animals presented nasal/eye discharges and diarrheic feces consistency during the experimentation trial.

Body condition scoring is a management tool designed to assess body reserves or fat accumulation of an animal, and a reliable method for critically examining the nutritional status of beef and dairy cattle [32]. In this study, probiotics administration was shown to affect the general body condition of overconditioned cattle at the end of the trial, in agreement with that reported for goats supplemented with L. acidophilus, Ligilactobacillus salivarius, and Limosilactobacillus reuteri (1011 CFU/kg as probiotics) [33]. Evaluation of cattle sensorium indicates the relationship of animals with their environment [34]. Different behavior patterns can be induced when the nervous system is alerted by a perceived sensory information [12]. Here, a normalized sensorium state at the end of experimentation time was evidenced as a confinement adaptation. Dietary probiotics, in addition to performance improvement, can exert benefits for handling cattle without affecting their welfare with respect to stress as reported for weaned-growing calves [35]. Thus, cattle-handling facilities designed and maintained to reduce cattle stress avoid alteration in temperament, while depression may be produced by chronic and excessive confinement [36]. Animals with both depressive and highly excitable temperaments are not able to reach their maximum performance potential [22].

In commercial animal farming, environmental factors such as radiation, wind, precipitation, high/low air temperature, and relative humidity should all be considered to avoid stress risk. For example, heat stress triggers the release of stress hormones such as cortisol and epinephrine [34, 37]. Indeed, probiotic administration was reported to induce calmer behavior of ewes during weighing and alleviate weaning stress in grazing yak calves through a reduction of serum cortisol level [38, 39]. Similarly, probiotics administration in weaned/growing calves and mid-lactation Holstein cows resulted in decreased stress during handling and exposure to high-temperature conditions, respectively, without negatively affecting cattle welfare [35, 40].

In this study, the presence of nasal discharges and non-purulent ocular discharges as preliminary signs of respiratory disease and kerato-conjunctivitis may be caused by transportation, climatic conditions in the feedlot location, and/or immunological condition of the animals. The lower nasal discharge in L. acidophilus CRL2074-supplemented group is in agreement with the reported potential of this bacterial species to colonize the bovine respiratory tract, thus exerting antagonistic effects against the respiratory pathogen Mannheimia haemolytica [41]. However, Lim. fermentum CRL2085 and multistrain probiotics administered to feedlot cattle were able to avoid diarrhea as stated by stool consistency evaluation. Liquid feces in cattle suggest intestinal dysbiosis caused by intoxications, infections, ruminal acidosis, or heat stress, while pasty stools indicate a balanced highly digestible diet with adequate fiber content and water–protein ratio [42].

Results from this study agree with the better fecal scores for Nelore heifers and Holstein calves administered with probiotic or symbiotic products during the first few days in the feedlot system and 90 days after weaning, respectively [15, 43]. However, the growth and development of ruminants is defined as the accretion of protein, fat, and bone, which is accompanied by body size changes. In this study, the analysis of the effect of probiotics on cattle nutritional parameters showed the height at the withers and thoracic diameter exhibiting a growing trend. At 105 days, increases in mean biometric indices were maximum for cattle supplemented with Lim. fermentum CRL2085 and multistrain formulation. This result agrees with that reported for growing goats and lambs fed probiotics-supplemented diets with a pool of lactobacilli and Bacillus subtilis, respectively [33, 44].

Animal growth is a complex process until reaching mature age mainly depending on breed, sex, and environmental conditions, during which the volumes of bone and muscle increase faster than that of adipose tissue. However, when cattle age and protein accretion declines, animals can continue to accrete fat at a faster rate than muscle [45]. When the effect of probiotics on cattle mean weight and DWG was assessed, increases of live weight and average DWG were found at 105 days by twice-a-day and thrice-a-week administration of probiotics. Dietary supplementation of Lim. fermentum CRL2085 and multistrain probiotics allowed to reach maximum values with an ∼11% greater average DWG compared to the non-administered control. Similar increment of mean weight and DWG (12.5%) was found in Nelore heifers after administration of a probiotics consortium (Complete Bio Cycle; LAB 105 CFU/mL + yeast 106 CFU/mL) for 93 days [15].

In another study, mean DWG values were in the range of 17–20% when multistrain probiotics (Bifidobacterium, Lactobacillus, Streptococcus, and Bacillus, 106–107 CFU/g) formulation was supplemented to dairy calves during the first month of life [46]. Consistently, higher average DWG was reported for calves fed multistrain probiotics (seven bacteria and two yeast strains, 2 × 109 CFU/g/day) than the control group [47]. Conversely, lower average DWG was reported when feedlot cattle were administered with a symbiotic formulation (yeast-derived prebiotic + B. subtilis, 109 CFU/steer/day) for 45 days [14]. On the contrary, no effect was evidenced on body weight and DWG of neonatal Holstein calves fed milk diet containing compound probiotics (Lactobacillus, Pediococcus, and Bacillus, 107–108 CFU/g) up to 3 months of age [16]. In this study, a reduction of DWG during the last period (45–105 days) coincided with the reported DWG values for Nelore heifers fed diet containing a probiotic consortium [15]. These results correlate with the model of rate and composition of cattle tissue accretion of feedlot animals, which at the end of their growth would allocate most of their nutrient intake into finishing, but not into protein accretion or skeletal muscle development. The rate of weight gain and accretion of protein and fat are controlled by factors such as maturity, genetics, age, and weight [45]. As probiotics are large intestinal colonizers multiplying and establishing themselves in the gastrointestinal tract, excluding harmful bacteria, stimulating the immune system, and improving animal efficiency and performance, changes in microbiology (beneficial bacteria stimulation) and chemistry (production of volatile fatty acids for energy efficiency improvement) of the gastrointestinal tract may be considered responsible for improved DWG [47].

Data of blood biochemical parameters are commonly used to assess the nutritional and physiological status of animals. Results showed higher hematocrit and lymphocytes % mean values than the referential range at 105 days. These results may preliminarily indicate some hemoconcentration due to dehydration. However, dietary inclusion of probiotics was reported to increase hemoglobin content and red blood cell counts in newborn and growing calves, as well as Barki lambs, suggesting an improvement of iron absorption from the small intestine and B vitamin production by probiotics facilitating blood cell forming process [44, 48].

Emerging evidence supports the ability of probiotics to enhance the status of micronutrients such as vitamin B12, folate, iron, calcium, and zinc. There are several possible suggested mechanisms by which probiotics can optimize the intestinal environment for better absorption of micronutrients. These mechanisms include pH decrease by increased intraluminal lactic acid production, beneficial alterations of gut microbiota populations, and inhibition of intestinal epithelial adhesion of pathogenic bacteria and consequent reduction of competition with the host for available nutrients [49]. Indeed, some species of lactobacilli and Bifidobacterium can produce B group vitamins and are able to improve the iron status [50, 51]. However, white blood cells (WBCs), as a major part of the body’s immune system, are critical in defending the body against infections.

In cattle, the total number of WBCs decreases with age, and as the dominant subpopulation percentage of lymphocytes decreases progressively in adult cattle [52]. In this study, descriptive mean values were found above the reference range at 105 days for both groups. In line with these results, higher values of WBC count were reported for newborn calves and growing cattle administered with probiotics compared to the control group [48]. Moreover, the administration of Lactiplantibacillus (lpb) plantarum LP1 with an immunomodulatory function for alleviating inflammatory responses decreased peripheral blood lymphocyte levels in cows under high-energy diets [53]. An increased number of WBCs might be involved in the production of more immune cells that play an important role in preventing different diseases in cattle [54]. However, the status of entry-metabolization-exit process of nutrients in organs and tissues will be reflected in the animal blood and serum parameters. Homeostatic equilibrium involves complex metabolic-hormonal mechanisms. When the balance of these mechanisms is broken, zootechnical performance of the body decreases and diseases occur [55]. The severity of the diseases is dependent on the degree of imbalance. Accordingly, blood parameters were used in this study to diagnose possible imbalances in animal health.

Descriptive mean values of serum biochemistry showed that most of the metabolic indicators fell within the reference range. Although the mean value in probiotics-supplemented cattle was higher than that at day 0, the serum mean value of glucose was notably lower for both cattle groups at 105 days, indicating a nearly hypoglycemic state. Similarly, glucose level was significantly decreased in growing cattle and Barki lambs dietary supplemented with multispecies probiotics and B. subtilis, respectively [44, 48]. The decreased level of serum glucose in probiotics-supplemented lambs was explained by lowered gluconeogenesis, either directly as a result of the increased acids concentration or indirectly because of the influence of insulin inhibition of phosphorylase and gluconeogenic enzymes. Gluconeogenesis in ruminants is the main source of glucose, and it has a decisive influence on the glucose level in blood [44, 56]. On the contrary, although within the referential range, slight increases in the mean values of bilirubin, triglycerides, and cholesterol were found at 105 days compared to day 0.

The level of hemoglobin degradation products, such as total bilirubin, is an important indicator for some pathological conditions (massive hemolysis of erythrocytes, obstruction of the bile ducts, or other liver diseases). In this study, a normal level of bilirubin was found for both cattle groups, indicating no hepatocellular disorders. Similar results were reported for crossbred calves fed probiotic L. acidophilus + prebiotic [57]. When lipid profile was evaluated, a lower level of cholesterol was found for probiotics-supplemented animals after 105 days compared to the control. These findings agree with those described when L. acidophilus + prebiotic and B. subtilis were dietary supplemented to crossbred calves and growing Barki lambs, respectively [44, 57]. Contrarily, higher levels or no significant changes of cholesterol were reported for growing goats and cattle, and lactating ewes when probiotics were administered [33, 48, 57, 58].

Accumulating studies have shown that probiotics, prebiotics, and symbiotics possess hypocholesterolemic effects modulating serum lipids in humans and animals [59]. Several mechanisms are being suggested to explain this finding. As previously reported [59–61], these mechanisms include enzymatic bile acids deconjugation by bile salt hydrolases of probiotics, assimilation of cholesterol by probiotics, co-precipitation of cholesterol with deconjugated bile, cholesterol binding to probiotics’ cell walls, incorporation of cholesterol into probiotics’ cellular membranes during growth, conversion of cholesterol into coprostanol, and production of short-chain fatty acids (SCFA) by probiotics upon fermentation. Indeed, the hydrolysis of bile salts agrees with the number of hydrolases reported for probiotic lactobacilli [60]. Control and treated samples of this study showed lower triglyceride values, in coincidence to that reported for young goats fed probiotic lactobacilli pool [33]. However, cattle diet supplemented with Saccharomyces cerevisiae showed increased triglycerides compared to control [62], while no changes were observed in crossbred calves, lactating ewes, and Barki lambs after L. acidophilus + prebiotic, commercial probiotic, and B. subtilis administration, respectively [44, 57, 58]. Low triglyceride levels in feedlot cattle administered with probiotics would suggest better metabolic status and positive energetic balance of animals, while elevated levels of serum triglycerides in the control group could be attributed to insulin’s effect on increased lipid synthesis in the liver [56].

In addition, similar low mean values for activities of liver enzymes AST and ALT during the trial were found. This result can be attributed to decreased gluconeogenesis as both enzymes are highly active in the liver. Similarly, low values for activities of both liver enzymes were reported for control and probiotic/prebiotic/symbiotic dietary supplementation to crossbred calves and lactating ewes [57, 58]. It is known that diets with a high concentration of grains (as in this study) could lead to ruminal acidosis and liver lesions. However, liver activity indicators exhibited normal values after probiotics treatments. Values of blood plasmatic protein were found almost within the reference range for cattle administered with probiotics, indicating a good nutritional status that should not resort to amino acid deamination to obtain energy [62]. High albumin concentrations would indicate the presence of infection, dehydration, and high-energy diets, while lower albumin levels might indicate liver, kidney, or other health disorders [63]. Moreover, acute-phase proteins encompass all the phenomena that take place in animals following tissue damage and are particularly associated with inflammation. Acute-phase response is formulated by several different proteins that vary in magnitude and type among animal species and act as part of the innate immune system for reestablishing homeostasis and promoting health [64]. Among acute-phase proteins is CRP. Although it is not considered a major acute-phase protein in cattle, it exerts biological functions involving modulation of monocytes and macrophages, cytokine production, and tissue migration of neutrophils [65]. Elevated CRP levels in bovine serum and milk is a result of infection and disorders. Thus, it can be used as a biomarker to predict diseases.

It was reported that nutritional management of beef cattle led to inflammation during transportation and at feedlot entry, and generate stressors in cattle (nutrient deprivation, physical injury, strenuous exercise), which stimulate inflammatory and acute-phase responses [66]. In this study, CRP mean values were within the reference range, and lower values were obtained for both evaluated groups at 105 days, which is still lower in probiotics-supplemented cattle. Supplementation of _B. subtilis_–based probiotic restored heat stress–related behaviors and inflammation in broilers, while attenuation of the acute-phase response following a lipopolysaccharide challenge in weaned pigs was described [67, 68].

The effect of dietary probiotic supplementation was observed to alter the microbial composition of feedlot cattle feces. In this study, populations of cultivable total aerobic mesophiles and LAB in feces collected from feedlot steers’ rectal area were similar to those reported for four-month-old calves [69]. The presence of LAB in bovine feces at 105 days after multistrain probiotic administration was somewhat higher than that reported up to 103 days for feedlot cattle feces [26]. Moreover, the growth pattern of the fecal population in this study coincided with that of a high-energy-fed Holstein cow administered with the probiotic Lpb. plantarum LP1, in which a quick growth of lactobacilli with a rapid reduction thereafter and no alteration of coliform population by probiotic lactobacilli were reported [53]. The amount of potential pathogenic enterobacteria decreased in the intestine when multistrain probiotics were supplemented to feedlot cattle in this study, which is in agreement with that previously reported by Maldonado et al. [26].

5. Conclusion

According to this study, the administration of an individual probiotic, Lim fermentum CRL 2085, and a probiotic mix formulated by L. acidophilus CRL2074 + Lim. fermentum CRL2085 + L. mucosae CRL2069 improved clinical parameters and growth performance of feedlot cattle by increasing the body structure, weight, and DWG. No nutritional and physiological imbalances were identified. Hematological and serum biochemical indicators were positively affected by probiotics. These results correlated with the recent trend to use multistrain probiotics combining two or more bacteria of the same or different genera and species as a beneficial consortium for targeting different delivery sites and complementing each other’s effect in the host. The mixed probiotics formulation administered as in-feed supplementation to cattle under a feedlot-intensive system improved metabolic-nutritional status, overall performance, and stress-related behavior. Further studies are being currently carried out based on the gene sequence of Lim. fFermentum CRL2085 and Lim. mucosae CRL2069 to identify the genes responsible for their probiotic features and reveal their metabolic attributes to fully exploit their biotechnological capabilities.