A polyherbal phytogenic additive improved growth performance, health, and immune response in dairy calves

ABSTRACT

The objective was to characterize the effects of a supplemental Herbal Antibiotics Source (HAnS) on performance, blood chemistry, blood cells, and antibody counts of Holstein calves. Forty calves (initial BW 45.8 ± 7.2 kg) were randomly assigned to treatments: 0, 3, 4, and 5 g d⁻¹ HAnS. The additive improved hip height and thoracic girth. The reduction of starter diet intake among individuals varied greatly, while milk replacer intake increased. HAnS at intermediate doses reduced the cases of pneumonia. It also linearly reduced the blood serum glucose and B-OH butyrate. Urea and monocytes showed a quadratic response, and basophils decreased. HAnS improves calf development at intermediate doses and reduces the incidence of diseases.

KEYWORDS:

• Calves
• polyherbal
• phytogenic
• health
• antibody

Introduction

Rearing calves is among the most important and sensitive cattle management activities, as calves are a major source of profit for farms (Seifzadeh et al., 2017). During calf rearing, respiratory and digestive problems occur that can cause high morbidity and mortality (Beaver et al., 2019). Because of health issues, the use of antibiotics, anabolic products, and growth promoters has been limited. Legislation in many countries is restricting the use of antibiotics. Therefore, it is urgent to identify other sources of antimicrobial properties and immunostimulant effects. In recent years, research in the use of herbal supplements has reported beneficial effects during the suckling period in ruminants (Crosby et al., 2017; Roque-Jiménez et al., 2020). Assan (2018) reported that supplementation with herbal formulas could lead to antibiotic-free feeds (Assan, 2018), and during the suckling period, they could maintain or improve calf health and productive indicators (Frankič et al., 2009; Wall et al., 2014).

In dairy and beef production systems, there is interest in using herbal formulas as alternatives to antibiotics during disease (Mendoza et al., 2019) and as enhancers of feed efficiency (Wall et al., 2014). Herbal formulas have mainly aimed to modify rumen fermentation and improve nutrient utilization in ruminant production (Frankič et al., 2009). Herbal feed additives or supplements contain different bioactive compounds, including alkaloids, polyphenols, isothiocyanates, tannins, saponins, and terpenoids, all of which can stimulate the immune system (Frankič et al., 2009). Standardized herbal antibiotic sources can contain Adhatoda vasica, Solanum xanthocarpum, Curcuma longa, Hedychium spicatum, Boerrhavia diffusa, Piper longum, and Albezia lebbek. These plants have been related to higher phytogenic contents that have been shown to have positive effects on ruminant production and health (Mendoza et al., 2019). Herbal formulas differ from extracts and essential oils in that they usually contain several molecules with one or more predominant active substances or nutrients responsible for positive biological effects that compromise diverse metabolites and mechanisms of action. In ruminant livestock production, herbal formulas have been reported to have positive effects: stimulating a non-specific immune response (Kumar & Pandey, 2014), epigenetic changes (Roque-Jiménez et al., 2020), antioxidant effects (Toghyani et al., 2015), inhibiting replication of specific pathogenic microorganisms (Rivera-Méndez et al., 2017), stimulating the immune response or providing nutrients with or without nutraceutical properties.

We hypothesized that the herbal compounds of Herbal Antibiotics Source (HAnS) have positive effects on the immune system, growth performance, blood biochemistry, and biometry of dairy calves. Therefore, the objective of this experiment was to characterize the natural compounds in HAnS and determine their effect on the immune system, growth performance, blood biochemistry, and biometry of dairy calves.

Material and methods

Ethics

The animal procedures were reviewed and approved by the Committee for Ethical Use of Animals in Experiments of Universidad Autónoma Metropolitana, in compliance with the regulations and standards issued by the Mexican government for the use of animals in diverse activities. Federal law on technical specifications for the care and use of laboratory animals and livestock on farms and production, reproduction, and breeding centres, and zoo and exhibition animals must meet the basic principles of animal welfare (NOM-062-ZOO-1995).

Location

The current experiment was conducted on a Mexican dairy farm in Torreon, Coahuila (25°39′14.4″ N 103°27′27.8″ W; altitude 1,139 m) in a semiarid climate (mean temperature 20.21°C).

Herbal Antibiotics Source characterization

The herbal formula used in the current experimental study (HAnS; Animunin Powder®, Nuproxa, LTD, Switzerland) is a labelled commercial standardized herbal formula that contains Adhatoda vasica, Solanum xanthocarpum, Curcuma longa, Hedychium spicatum, Boerrhavia diffusa, Piper longum and Albezia lebbek. Bioactive compounds were extracted using an ultrasonic processor (GEX130, 115 V 50/60 Hz) equipped with a 3 mm titanium tip and mechanical stirrers (Cole-Parmer, IL, USA). One gram of HAnS was mixed with 10 mL of hexane. Subsequently, the organic phase was separated, concentrated to 1 mL of extracted mixture, and evaporated (Zymark, Turbovap LV Concentration Evapotarot, NB, USA) for the final analysis.

HAnS was characterized by gas chromatography (GC-HP 6890) coupled with mass spectrophotometry (MSHP 5973), equipped with a capillary column 60 m long, 0.255 mm in diameter, and 0.25 µm film thickness (HP 5MS, Agilent). The temperature programme was 70°C for 2 min, which was then increased to 250°C at the rate of 20°C/min, then to 290°C at the rate of 5°C/min, then increased to 300°C at the rate of 1°C/min, then to 310°C at the rate of 5°C/min and held for 36 min. The injector temperature was 250°C in splitless mode. The helium flow rate was 1 mL/min. The mass spectrophotometry was programmed in SCAN mode (50–500 m/z) to identify compounds.

Animals, treatments, and sampling

Forty female Holstein calves (initial body weight (IBW) 45.8 ± 7.2 Kg), 20.4 ± 2.9 days of age were randomly assigned to one of four treatments. The treatments (10 calves per treatment) consisted of oral doses containing 0, 3, 4, or 5 gd⁻¹ of HAnS. Treatments were top-dressed on the feed using gelatin as the medium. Gelatin was prepared following the protocol described by Sánchez-Hernández et al. (2019). The calves were housed in individual pens equipped with steel buckets for feed and milk. All calves received colostrum after birth and the initial total serum protein was confirmed with a refractometer (Quigley, 2011).

Calves were fed twice a day (07:00–17.00) with milk (56%) and milk replacer (44% Milk replacer Nu-3 Group, México), reconstituted in hot water (65°C; 130 g/L). The milk mix was served at a temperature of 39°C in buckets. Milk replacer was offered twice per day (4 L/feeding) and reduced to once on day 25 as starter diet intake augmented. The starter diet concentrate was offered as of the third day of age and was gradually increased (Started Premium, Nuplen, México).

Experimental procedures

Calves were weighed on days 1, 20 and 90 of the experiment to calculate the average daily gain (ADG). Also, on day 1 and day 10, all the calves received a bovine rhinotracheitis vaccine (Pyramyd-5; Boehringer Ingelheim). Later, on day 50, all the calves were vaccinated with a clostridial bacterin-toxoid (Covexin 10; MSD). On day 90, hip, wither height, and thoracic girth were measured to evaluate growth and development (Maciel et al., 2016 Parish et al., 2012;). Feed intake (milk and starter diet concentrate) was recorded daily and based on the dry matter (DM) content of the starter diet and milk replacer. The feed efficiency ratio was estimated (feed efficiency = kg gain/kg feed) (Kazemi-Bonchenari et al., 2018) and the coefficient of variation of DM intake between days was calculated as a stability indicator (Britton et al., 1990). Calves were examined daily for disease and given veterinary treatment when needed (Pardon et al., 2013). On day 90, three blood samples (5 mL per sample) were collected from each of the calves from the jugular vein by puncture with blood collection tubes. The first sample without anticoagulant was centrifuged to obtain blood serum which was later used to determine contents of cholesterol, glucose, total protein, urea, uric acid, albumin, globulin, bilirubin, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), Ca, P and creatinine using an autoanalyzer (Kontrolab QS EasyVet, Italy). The second sample without anticoagulant was centrifuged at 1500 rpm for five minutes to obtain blood serum, which was collected in vials and stored at −80°C until ELISA analysis. Finally, the last blood sample collected in tubes with anticoagulant was used for complete blood count, haematocrit, and leukocyte differential count in a haematology analyser (Kontrolab QS EasyVet, Italy).

Quantification of immunoglobulins (IgG) by ELISA

An ELISA was performed using plates with antigen (Clostridium spp) obtained from Covexin 10. The antigen was incubated for 24 h at room temperature; then 200 µL was added to each well to decrease non-specific binding sites. Later, 100 µL of the serum sample was added to each well and incubated for 1 h at 37°C. Plate contents were decanted, and 100 µL of the bovine IgG specific secondary antibody was added and allowed to incubate for 1 h at 37°C. Subsequently, 100 µL of TMB (3,3’, 5,5'-liquid tetramethylbenzidine) was added to each well. Finally, 100 µL of buffer solution (H2SO4) was added to each well to stop the reaction. The plate was read at 540 nm.

Statistical analysis

Shapiro–Wilk tests were used to ascertain normal distribution, and data were analysed under a completely randomized design with R software, testing for linear and quadratic effects of HAnS supplementation dose by orthogonal polynomials (Mirman, 2014). The initial body was tested as a covariate. For main effects, a significant difference was established at p ≤ 0.05, trends were determined at p ≥ 0.05 and p ≤ 0.10.

Results

Herbal Antibiotics Source (HAnS) characterization

Gas chromatography coupled with mass spectrophotometry revealed various chemical compounds present in the HAnS formula (Figure 1). The main components detected were a variety of alkanes such as hepacosane, heneicosane, hexacosane, eicosane, octacosane and octadecane. Alkaloids, such as piperine, as well as a type of allylbenzene called eugenol, terpenes such as thymol and vitamin E, and other components, were also detected.

Figure 1. Total ion chromatogram of the volatile components and chemical composition in HAnS. Chemical composition of Herbal Antibiotics Source (HAnS) by GC-MS with retention time^(rt): 0. 3-Hexanol (standard mixture solvent); 1. Thymol; 2. Eugenol; 3. Benzene, 1-methyl-2-(1-methylethy); 4. Phenol, 2-methoxy-5-(1-propenyl)-(E)-; 5. Cyclobutane, 1,2-dipropenyl-; 6. 2-Cyclohexen-1-one, 5-methyl-2-(1 methylethyl); 7. Cyclohexane, bromo; 8. 3-Hexen-1-ol, 2,5-dimethyl-, formate,(Z)- 9. (-)-E-Pinane 10. s-Indacene, 1,2,3,5,6,7-hexahydro-1,1,7,7-tetramethyl; 11. 6-Methoxy-2,3-dihydro-1-benzofuran-3-acetic acid; 12. 9-Octadecenoic acid, (E)-; 13. Hexacosane; 14. Heptacosane; 15. Silicic acid, diethyl bis(trimethylsilyl) ester; 16. Piperine; 17. Vitamin E; 18. Heneicosane, 11-decyl-; 19. Eicosane; 20. Octacosane; 21. Octadecane.

Effects of an HAnS on calf productive performance and health

The productive performance and health of calves supplemented with HAnS was evaluated (Table 1). We observed no differences among the treatments in ADG and final BW, while final hip height tended to be greater (p = 0.08) for calves supplemented with HAnS. Also, there was an increase in final thoracic girth (quadratic, p = 0.02) with supplementation of 4 g d⁻¹ of HAnS. HAnS supplementation to calves resulted in a lower starter intake g d⁻¹ than the treatment without HAnS (p = 0.01). A similar response of calves without HAnS supplementation was observed for feed intake (p < 0.01) compared with the calves with different doses of HAnS. However, milk and milk replacer intake was higher in calves that received supplementation with HAnS. Calves that consumed 3 g d⁻¹ of HAnS showed the highest consumption of milk and of milk replacer (linear p < 0.01; quadratic p < 0.01). Interestingly, when the health status of the calves was evaluated, a positive effect was observed during the experimental period (Table 1). Although no changes were observed in the incidence of diarrhoea, cases of pneumonia significantly decreased in calves that consumed HAnS (linear, p = 0.05), relative to the group without supplementation.

Table 1. Effect of an Herbal Antibiotics Source (HAnS) on calf productive performance and health.

Item	HAnS^‡ gd^−1				^¥SEM	p-value
	0	3	4	5		Linear	Quadratic
ADG, g/d	0.672^b	0.657^b	0.713^c	0.602^a	0.04	0.37	0.22
Final BW, kg	86.7^b	85.7^b	89.3^c	82.22^a	3.53	0.22	0.34
Final wither height, cm	90.0	91.0	92.5	90.6	1.22	0.42	0.13
Final hip height, cm	95.4	95.5	97.9	97.7	1.11	0.08	0.77
Final thoracic girth, cm	101.5	101.9	104.5	99.8	1.70	0.61	0.02
Starter intake, g/d	829	684	774	613	62.7	0.01	0.87
Feed intake variation among individuals, %	107.3	117.7	118.0	122.2	2.45	< 0.01	0.39
Milk + replacer intake, L/d	2.454	2.812	2.807	2.806	0.03	< 0.01	< 0.01
Feed conversion ratio	2.15	2.14	2.09	2.23	0.09	0.63	0.38
Feed efficiency ratio	0.471	0.474	0.483	0.458	0.01	0.68	0.42
No. diarrheas	0.29	0.09	0.17	1.05	0.42	0.19	0.26
No. pneumonias	3.61	2.33	0.60	0.60	1.00	0.05	0.77
No. otitis	5.66	3.22	0.40	4.50	2.23	0.32	0.11
Antibiotic doses	5.35	6.53	0.81	5.19	1.93	0.42	0.37

^a,b,c p < 0.05.

^‡ Animunin Powder®, Nuproxa, LTD, Switzerland.

^¥ SEM, standard error of the mean.

Effects of an Herbal Antibiotics Source (HAnS) on metabolic parameters of calves

The evaluation of metabolic parameters (Table 2) in serum samples from calves supplemented with HAnS showed considerable reduction in levels of glucose (linear, p < 0.01), B-OH butyrate (linear, p < 0.05) and urea (quadratic, p < 0.05). A decrease in serum albumin was also detected (linear, p = 0.05; quadratic, p < 0.05) in calves treated with 3–4 g d⁻¹ of HAnS and, thus, there was also a reduction in the albumin/globulin ratio (quadratic, p < 0.05). Even though the serum bilirubin concentration increased slightly in calves that consumed HAnS (quadratic, p = 0.05), a decreasing trend was observed in aspartate aminotransferase levels (linear, p = 0.10). No changes were observed in the other evaluated parameters.

Table 2. Effect of an Herbal Antibiotics Source (HAnS) on blood serum biochemistry of calves.

Item	HAnS^‡ gd^−1				^¥SEM	p-value
	0	3	4	5		Linear	Quadratic
Glucose, mg/dl	73.88	51.55	55.70	45.77	3.39	< 0.01	0.07
B-OH Butyrate, mmol	0.320	0.300	0.280	0.220	0.02	0.01	0.74
Urea, mg/dl	22.05	15.88	18.30	19.00	1.38	0.26	0.02
Uric acid, mg/dl	0.505	0.422	0.650	0.533	0.04	0.25	0.81
Creatinine, mg/dl	1.012	1.033	0.980	1.044	0.04	0.92	0.80
Protein, g/dL	6.98	7.28	6.94	6.97	0.11	0.55	0.26
Globulin, g/dL	3.03	2.87	2.99	3.18	0.13	0.44	0.12
Albumin, g/dL	3.95	4.30	3.85	3.67	0.15	0.05	0.01
Ratio Albumin/Globulin	1.33	1.49	1.29	1.11	0.10	0.07	0.02
Cholesterol, mg/dL	93.29	99.88	91.80	96.33	4.77	0.92	0.87
Bilirubin, mmol/L	0.370	0.488	0.530	0.477	0.04	0.08	0.05
ALP, U/L	25.7	24.55	27.4	30.55	1.89	0.35	0.64
LDH, U/L	79.64	73.22	83.1	72.33	4.78	0.60	0.75
AST, U/L	25.52	22.55	18.90	20.00	2.57	0.10	0.46
Calcium, %	9.24	9.03	9.28	8.85	0.27	0.76	0.64
Phosphorous, %	5.14	4.77	4.74	4.84	0.23	0.51	0.33

^‡ Animunin Powder®, Nuproxa, LTD, Switzerland.

^¥ SEM, standard error of the mean.

Effects of an Herbal Antibiotics Source (HAnS) on blood cells of calves

The haematic biometry study (Table 3) of blood cells from calves supplemented with HAnS showed no differences in the proportion of erythrocytes, platelets, lymphocytes, and eosinophils nor in the concentrations of hemoglobin and plasma proteins among the experimental groups. On the other hand, when other cells that participate in the immune response were measured in calves supplemented with HAnS, a quadratic increase in monocytes (p < 0.05) was detected with the dose of 5 g d⁻¹ of HAnS and a linear increase in the number of basophils (p < 0.05) with the dose of 3 g d⁻¹ of HAnS.

Table 3. Effect of an Herbal Antibiotics Source (HAnS) on blood cells of calves.

Item	HAnS^‡ gd^−1				^¥SEM	p-value
	0	3	4	5		Linear	Quadratic
Haematocrit, %	35.13	37.72	34.20	36.80	0.92	0.72	0.99
Haemoglobin, g/dl	11.91	12.74	11.62	12.35	0.29	0.88	0.87
Erythrocytes, 10^6/ml	5.39	5.63	5.10	5.52	0.18	0.82	0.56
MCV, fl	65.73	67.22	67.01	66.61	1.69	0.64	0.44
MCH, pg	22.27	22.68	22.78	22.39	0.57	0.80	0.35
MCHC, g/dl	33.90	33.78	33.99	33.58	0.16	0.35	0.46
Platelets, 10^3/ml	407.22	370.67	475.80	394.70	35.86	0.60	0.47
WSR, ml/h	0.27	0.00	1.00	0.00	0.42	0.91	0.31
Leukocytes, 10^3/ml	11.40	12.88	11.56	11.86	1.54	0.99	0.59
Lymphocytes, 10^3/ml	45.88	40.88	45.60	45.90	3.56	0.74	0.45
Monocytes, 10^3/ml	4.55	4.00	3.60	5.40	0.59	0.37	0.04
Segmented neutrophils, 10^3/ml	41.94	48.33	45.40	42.40	3.93	0.92	0.21
Neutrophils in band, 10^3/ml	4.33	3.11	2.90	3.10	0.79	0.11	0.22
Eosinophils, 10^3/ml	3.00	3.22	2.40	3.20	0.59	0.93	0.65
Basophils, 10^3/ml	0.27	0.44	0.10	0.00	0.13	0.01	0.25
Plasma proteins, g/dL	9.09	9.35	9.40	9.61	0.31	0.13	0.91

^‡ Animunin Powder®, Nuproxa, LTD, Switzerland.

^¥ SEM, standard error of the mean.

Serum titres of antibodies against Clostridium spp in calves supplemented with Herbal Antibiotics Source (HAnS)

To further evaluate the immunological status of calves supplemented with HAnS, we performed antibody titres for antigens of Clostridium spp (Figure 2), one of the pathogens that frequently affect claves during their growth. A linear increase in serum antibodies was detected in calves supplemented with 4 (p = 0.01) and 5 g d⁻¹ (p < 0.05) of HAnS, relative to the control group.

Figure 2. Effects of an Herbal Antibiotics Source (HAnS) on antibodies in calf blood plasma. Linear p-value = 0.01; Quadratic p-value = 0.76; SEM = 0.08.

Discussion

HAnS is an herbal extract composed of different medicinal plants native to India and East Asia (Kumar & Pandey, 2014). In the present study, twenty-one organic compounds were detected in HAnS, including thymol, eugenol, fatty acids, and vitamin E (Figure 1). Previous studies (Silva-Júnior et al., 2020; Wall et al., 2014) provided solid evidence that several of the compounds in HAnS improve growth performance and feed intake and increase livestock immune response.

Results from in vitro (Castillejos et al., 2006; Fraser et al., 2007) and in vivo (Cardozo et al., 2006; Benchaar et al., 2006) studies on effects of the active component of thymol have been contradictory, and more research is needed to understand its effects on rumen fermentation and metabolism in dairy cattle. However, thymol has been found to have antimicrobial effects. Thymol has been attributed to disruption of the plasma membrane of bacteria and reduction in glucose uptake (Benchaar et al., 2008; Calsamiglia et al., 2007). It has been suggested that this occurs through the interaction of its carbonyl group with proteins in the periplasm, inactivating microbial enzymes (Vakili et al., 2013).

Based on in vitro rumen fermentation experiments, eugenol from herbal formulas improves the production of volatile fatty acids and nitrogen utilization by the rumen (Calsamiglia et al., 2007). Among the essential oils, eugenol has attracted much attention because of its potential antimicrobial activity against rumen microbes (Benchaar et al., 2012). Also, it has been reported in dairy cows that eugenol from herbal sources can increase dry matter intake and milk production (Wall et al., 2014). The vitamin E (α-tocopherol) status of dairy cattle is an important component of a well-functioning immune system of cows and young dairy calves because of its antioxidant effects (Kafilzadeh et al., 2014). Experimental reports of vitamin E from herbal formulas are few. Vitamin E content is high in fresh grass, but it decreases markedly during forage storage and conservation. For this reason, NRC (2001) recommends that the entire vitamin E requirement be given via dietary supplements when conserved forages are fed. Extra supplementation may be useful during immune suppression periods, such as around calving (Kafilzadeh et al., 2014).

There was no significant effect of HAnS supplementation on ADG, final BW, or final wither height in the current study (Table 1). In contrast with our results, previous studies indicated that daily gain in feedlot lambs could be improved by 5% with dietary concentrations equivalent to 0.1 g kg⁻¹ DM of HAnS (Hernández-Reyes et al., 2017). Other experimental tests showed that growth can increase by 11.1% with 1 g kg⁻¹ DMI. However, 2 g kg⁻¹ had reduced beneficial effects on different performance variables (Dorantes-Iturbide et al., 2019; Orzuna-Orzuna et al., 2019). This could indicate that the concentration of plant metabolites, such as terpenes, phenols, polyphenols, carotenoids, oligosaccharides, and vitamins, must be precise to obtain benefits from these phytogenic growth promoters (Cardinali et al., 2015; Dalle et al., 2016). Nevertheless, Jouany and Morgavi (2007) concluded that these metabolites in greater amounts could cause negative or toxic effects in the organism (Jouany & Morgavi, 2007). Some of the plants that constitute HAnS have been evaluated individually with positive responses in lactating ewes (Jaguezeski et al., 2018) and growing lambs (Alade et al., 2010; Awohouedji et al., 2013).

There is no clear explanation of why HAnS decreased starter diet intake and increased milk consumption, as HAnS supplementation was offered through gelatins. In lambs, Hernández-Reyes et al. (2017) did not observe changes in dry matter intake using 1.5 g d⁻¹, while Orzuna-Orzuna et al. (2019) reported an increment of 8% in dry matter intake in lambs supplemented with 1 or 2 g kg⁻¹ DM. However, digestibility was not reported in either study. In non-ruminants, Shon et al. (2004) observed an increase in feed digestibility using a herbal source similar to HAnS. The difference in results could be related to flavour and palatability, which could depend on the type and dose of the phytogenic source (Upadhaya & Kim, 2017). A few experimental studies have reported positive effects against bovine respiratory disease using herbal essential oils. In our experiment we observed fewer cases of pneumonia in the calves supplemented using HAnS. Amat et al. (2019) observed that essential oils from an herbal formula (0.025%) reduced Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni in dairy calves. Spisni et al. (2020) stated that oral administration of a herbal formula containing eugenol, thymol, eucalyptol, and menthol, may be related to an increase in antibacterial activity in the immune system of non-ruminants. However, not all bio-compounds from plants have been described, and data reported in the literature regarding antimicrobial activity of herbal formulas are minimal. Continued research with new studies focused on these interesting compounds is needed.

Several medicinal plants from India have shown hypoglycemic effects (Khosla et al., 2000) similar to those of other herbal products evaluated in calves (Sánchez-Hernández et al., 2019). El Barky et al. (2017) reviewed the different hypoglycemic effects of different levels of plant saponins on insulin (modifying signalling, rejuvenation, release), glycogen synthesis, gluconeogenesis inhibition and reduction of mRNA gene expression of glycogen phosphorylase and glucose 6-phosphatase, and increased Glut4 expression. Other researchers interpret the lower glucose values in calves supplemented with phytogenics as an improved capacity to metabolize glucose (Lakhani et al., 2019). Like these experimental studies, our results showed lower serum glucose concentration in calves that received supplementation with HAnS, relative to calves without the supplement (Table 2).

The reduction in blood butyrate may indicate that phytogenic secondary metabolites could affect rumen microbiota development or have an indirect effect by reducing solid intake and causing slow rumen development. It has been recognized that VFA, particularly butyrate, affect papillary development (Mentschel et al., 2001). Differences in rumen development with milk only versus milk supplemented with forage or concentrates has been documented (Heinrichs & Lesmeister, 2004). Using a microarray approach, Connor et al. (2013) have demonstrated that changes in feeding the growing calf can affect several gene networks involved in lipid metabolism, molecular transport, and cell proliferation, growth, and morphology of rumen epithelium (Connor et al., 2013). Additionally, secondary metabolites from the phytogenic could affect gene expression of proton-linked monocarboxylate transporters (MCT) (Nakamura et al., 2018; Wang et al., 2014). MCT 1 and 2 have been reported in rumen epithelial cells to transport short-chain fatty acids. Kirat et al. (2006) stated that additives that can alter the gene expression of MCT1 in rumen might directly increase butyrate in the bloodstream.

The quadratic response in blood serum urea suggests that at intermediate doses protein efficiency and utilization were improved (Kohn et al., 2005), and there was better utilization of N in the rumen (Bruno et al., 2009) presumably as a result of greater ruminal microbial protein synthesis (Razavi et al., 2019). However, at the highest doses of phytogenics, blood urea nitrogen again increased, indicating negative effects associated with the lowest daily gain, even when the values were within the normal range (Issi et al., 2016; Nozad et al., 2012). Uric acid, a primary product of protein metabolism, was not affected (Eggum et al., 1982), and creatinine, a blood parameter of kidney function, remained unaffected (Karagul et al., 2000).

The changes observed in albumins and the A/G ratio cannot be related to liver damage or chronic or inflammatory response to HAnS because globulins were not elevated, calves were healthy, and HAnS reduced respiratory diseases (Table 1). Some secondary metabolites from herbal supplements containing polyphenolic compounds caused inhibitory effects on UGT1A9, the gene responsible for glucuronidation of bilirubin (Mohamed & Frye, 2011), which may alter the bilirubin concentrations, and other secondary metabolites could cause cross-reactions responsible for increasing bilirubin, rather than liver injury (Dufour et al., 2000).

The changes in monocytes and basophils were within the reference limits for calves (Roland et al., 2014) (Table 3). Therefore, our results do not indicate a pathologic process (Aslinia et al., 2006), bone marrow disorder, or autoimmune disease but rather suggest that some metabolites of the herbal mixture send stimuli to the immune system that are being interpreted as stress. This could be considered positive given that cattle have a small bone marrow reserve for granulocytes (Roland et al., 2014), and some leukocyte subpopulations may be stimulated. The AST activity we measured in calf blood serum tended to be greater in calves without HAnS. AST activity increased during the pathological processes associated with necrosis and subclinical acidosis in calves during solid feed intake. Following this increase in hepatic activity, AST is released into the bloodstream. AST concentration has been related to liver damage (Quezada-Tristán et al., 2014). An AST value between 5 and 40 U/L is considered normal (Huang et al., 2006). Our results fall within the normal range; however, it is interesting to observe a lower AST level in the calves with 3 g d⁻¹ of HAnS.

In most of the little research on the effects of herbal sources on dairy calves, attention is focused on secondary metabolites (Frankič et al., 2009; Mendel et al., 2017), but herbal formulas also contain volatile compounds (Mendoza et al., 2019). In any case, herbal formulas are neutraceutical mixes that can reduce infections and diseases such as pneumonia. Another study reported a decrease in other diseases that most commonly occur during calving and calf growth (Gutiérrez et al., 2019). It may be that the herbal formulas act in multiple ways through nonspecific mechanisms of the phytogenic on immunomodulation, antioxidants, anti-inflammatories, antimicrobial compounds, intestinal microbiota modulation, and others that are being detected in gene expression modulation (Frankič et al., 2009; Upadhaya & Kim, 2017; Wang et al., 2014). Combining plants has additive effects since evaluations of individual plants do not show changes in haematological parameters (Bharti et al., 2004) or immune response (Chand et al., 2014).

Finally, the increase in antibodies by HAnS supplementation could be related to immune-stimulation through many pathways (Figure 2). In contrast to antibiotics, herbal formulas and their essential oils contain different antibacterial agents that possibly employ several inhibitory mechanisms, making it difficult for pathogens to initiate resistance (Aljarallah, 2017). In non-ruminants, thymol has been reported to suppress pathogens in the small intestine without damaging beneficial commensal colonic bacteria (Aljarallah, 2017). Saleh et al. (2018) reported that thymol negatively affected Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, and Clostridium perfingers. These results suggest that the combination of antimicrobials contained in herb mixtures controls microflora in the gastrointestinal tract, which enhances feed digestibility and growth performance.

Regarding the plants included in the herbal formula HAnS, antihelmintic properties have been recognized in Adhatoda vasica, Piper longum, and Albizia lebbeck (D'Cruz et al., 1980; Khan et al., 2013; Ravikumar et al., 2009; Singh et al., 2008, 2009); Curcuma longa also has antihelmintic effects as well as anti-inflammatory and antioxidant properties (Cervantes-Valencia et al., 2016; Molosse et al., 2019). Boerhavia diffusa extracts have hypoglycemic and anti-inflammatory properties (Khan et al., 2013) and are hepatoprotective, nephroprotective, and immunomodulating (Khan et al., 2013; Kumar et al., 2018). Albizia lebbeck is analgesic, anti-infection, and anti-inflammatory (Duke & Bogenschutz, 1994). Solanum xanthocarpum has been reported to be antibacterial, antioxidant (Kumar & Pandey, 2014; Nithya et al., 2018), hypoglycemic, and hepatoprotective (Parmar et al., 2010). Hedychium spicatum is used as an analgesic, anti-inflammatory, antimicrobial, antioxidant, and antifungal, and has cytotoxic activity (Sravani & Paarakh, 2011).

Conclusion

We evaluated the effects of including HAnS in the early-life diet of calves on productive performance, health, blood serum biochemistry, blood cells, and antibodies. Significant changes in milk intake, cases of pneumonia, blood serum butyrate, blood serum albumin, and AST were attributed to the herbal formulas containing bioactive substances with phytogenic properties, which have a possible role in decreasing diseases. Supplementation with HAnS had no impact on growth, but it increased health parameters and antibody count and decreased disease incidence. Future research should focus on elucidating the effect of varying amounts of individual bio-compounds from herbal formulas on calving and calf early life, their metabolomic effect, and the subsequent health outcomes in adult life.

Acknowledgements

We are grateful to the owners and management of the dairy farms where we conducted this study. We are also grateful to Nuproxa for providing the Herbal Antibiotics Source.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The first author thanks the PRODEP-SEP for her financial support through the postdoctoral [511-6/2019-10950] project.