Polyherbal feed additive for lambs: effects on performance, blood biochemistry and biometry

ABSTRACT

An experiment was conducted to evaluate the effect of a polyherbal mixture based on Withania somnifera, Ocimum tenuiflorum, Tinospora cordifolia and Emblica officinalis, on lamb’s growth and blood metabolites. Thirty-two lambs (initial body weight BW 25 ± 4.1 kg) were randomly assigned to the treatments which consisted of the polyherbal ImmuPlus at dietary concentrations of 0, 0.1, 0.2 and 0.3% of dry matter (DM) for 60 days. There was a quadratic response to polyherbal in average daily gain (p < 0.01) and feed conversion (p < 0.05). The optimal concentration estimated by regression for maximum ADG was 0.14 ± 0.08% DM of ImmuPlus. Ruminal DM digestibility was not affected but pH was reduced linearly (p < 0.05). Ruminal N-NH3 (p < 0.01) and total VFA concentration showed a quadratic increment (p < 0.05) whereas butyrate proportion was increased linearly (p < 0.05). There was no effect in most blood metabolites related to energy metabolism, but uric acid was linearly reduced (p < 0.01) and bilirubin showed a quadratic decrement (p < 0.05). Mean corpuscular haemoglobin (p < 0.01) increased linearly (p < 0.10), lymphocytes and segmented neutrophils showed a quadratic response (p < 0.01) and platelets showed a linear reduction (linear p < 0.05). The inclusion of ImmuPlus improved the daily gain in finishing lambs and feed efficiency at 0.1% DM without altering blood chemistry and blood biometry.

KEYWORDS:

• Polyherbal mixture
• lambs
• performance
• phytobiotic
• blood parameters

Introduction

Some evaluations of phytogenic additives in lambs had showed inconsistent response in lamb performance (Canbolat et al. 2018; Parvar et al. 2018; Ranucci et al. 2019) but some polyherbal mixtures elaborated with medicinal plants from India have shown some consistent results in feedlot lambs (Godínez-Cruz et al. 2015; Orzuna-Orzuna et al. 2019; Martínez-Aispuro, Mendoza, Cordero, et al. 2019). It is important to explore the evaluation of plants with nutraceutical properties (García-Hernández et al. 2017) as substitutes for synthetic growth promoters (Frankič et al. 2009; Khan et al. 2016) but each feed plant additive needs to be evaluated since composition and secondary metabolites can vary in addition to different action mechanisms (Mendel et al. 2017) and optimal dose for each physiological status needs to be determined (Frankič et al. 2009).

It has been reported that the secondary metabolites of several plants have positive effects on ruminant production and health (Kraszewski et al. 2002; Greathead 2003; Cardozo et al. 2006; Mirzaei and Venkatesh 2012) comprised of diverse metabolites and mechanisms of actions (Mendel et al. 2017); some inhibit the replication of specific pathogenic microorganisms (Jouany and Morgavi 2007), others modify the metabolism confirmed through gene expression (Wang et al. 2015) or stimulating the immune response (Priyadarshini et al. 2012) or providing nutrients (Mendoza et al. 2019; Martínez-Aispuro, Mendoza, Cordero-Mora, et al. 2019) with or without nutraceutical properties (Diez-Gutiérrez et al. 2020).

Polyherbal mixtures differ from extracts or essentials oils, in that they usually contain several molecules with one or more predominant active substances or nutrients that are responsible for the biological effects (Frankič et al. 2009). A standardized polyherbal mixture (ImmuPlus) made with medicinal Indian plants and containing antioxidants, polyphenols and flavonoids has improved the daily gain in goats in deworming studies (Roy et al. 2003), IgG serum levels in dairy cattle during mastitis treatment (Mukherjee et al. 2010) and it could function as a growth promoter in feedlot lambs. Therefore, the objective of this research was to evaluate the effects of the dietary inclusion of the polyherbal ImmuPlus in lambs fed a high grain ration and to estimate the optimal concentration based on productive performance and blood metabolites, indicators of energy metabolism and health status.

Materials and methods

The research was approved by the Animal Science Departmental Committee and by the Ethics and Animal Welfare Committee of the Campus Amecamea of the Autonomous University of the State of Mexico, in accordance with the regulations by the Law of Animal Protection of the State of México.

The experiment was carried out in Amecameca, State of Mexico, located at 98° 54′11″ W and 19° 27′38″ N and 2250 m altitude. Thirty-two crossed lambs Suffolk × Dorper (initial body weight BW 25 ± 4.1 kg) were randomly assigned to the treatments which consisted in dietary concentrations of the polyherbal ImmuPlus (Nuproxa Mexico, Indian Herbs) based on Withania somnifera, Ocimum tenuiflorum, Tinospora cordifolia and Emblica officinalis at 0, 0.1, 0.2 and 0.3% of dry matter (DM) for 60 days. Lambs were housed in individual crates and were adapted for 10 days to the experimental diets given and previously were consuming a diet with grains. Experimental diets (Table 1) were formulated to achieve at least 200 g average daily gain (ADG; NRC 2007). Samples of diets were analysed for dry matter (DM), organic matter, crude protein (AOAC 2005), Neutral and acid detergent fibre (Van Soest et al. 1991).

Table 1. Experimental diets and chemical composition of the experimental ration.

	Polyherbal^a, %
	0	0.1	0.2	0.3
Ingredients, %
Corn, grain	60.0	59.9	59.8	59.7
Corn, Straw	22.0	22.0	22.0	22.0
Soybean paste	7.0	7.0	7.0	7.0
Molasses	5.0	5.0	5.0	5.0
Corn, gluten	3.0	3.0	3.0	3.0
Urea	1.0	1.0	1.0	1.0
Mineral premix^b	1.0	1.0	1.0	1.0
Sodium bicarbonate	1.0	1.0	1.0	1.0
Polyherbal additive	0.0	0.1	0.2	0.3
Chemical composition, %
Dry matter	87.6	86.8	85.9	85.0
Ash	4.1	4.1	4.0	4.0
Crude protein	14.5	14.4	14.3	14.2
Neutral detergent fibre	22.1	22.0	21.9	21.9
Acid detergent fibre	12.7	12.6	12.6	12.6
Metabolizable energy, Mcal/kg	2.83	2.80	2.76	2.73

^aImmuPlus based on Withania somnifera, Tinospora cordifolia, Ocimum tenuiflorum and Emblica officinalis.

^bMineral premix: 27% calcium, 3% phosphorus, 0.75% magnesium, 4.2% sulfur, 6.5% sodium, 0.05% potassium, 978 mg/kg iron, 15 ppm cobalt, 5 ppm copper, 25 ppm zinc, 2.25 ppm molybdenum, 1 ppm selenium, 160 ppm iodine.

The growth assay lasted 60 days in which DM intake was recorded daily. The initial and final weight (two consecutive days) were recorded to estimate ADG and feed conversion ratio. Apparent DM digestibility was estimated with acid insoluble ash (Van Keulen and Young 1977) collecting faeces during 5 consecutive days starting on day 35 directly from the rectum (Sánchez et al. 2018). The acid-insoluble ash (AIA) procedure consists in a gravimetrically method, based on burning of organic matter in samples (feed and faeces) by ashing, boiling in HCl and re-ashing, gravimetry after drying (Van Keulen and Young 1977). Dry matter digestibility is estimated with the equation: = 100 – [100 (AIA concentration in diet) (AIA concentration in faeces)].

Fifty ml of ruminal fluid were sampled on day 60 with an oesophageal tube from each lamb (preprandial 06:00) and pH was recorded immediately and then fluid acidified with 25% metaphosphoric acid (1:4) for subsequent determination of volatile fatty acids by gas chromatography (Erwin et al. 1961). Ruminal fluid samples were also used to measure ammonia N (N–NH₃; McCullough 1967) and lactate (Madrid et al. 1999).

On day 59, two blood samples (preprandial 06:00) were obtained from the jugular of each lamb using vacutainer tubes with EDTA and without anticoagulant, for biometry and serum biochemistry respectively (Jones and Allison 2007). Total cholesterol, glucose, total protein, albumin, bilirubin and liver enzymes (Alkaline phosphatases, Lactate dehydrogenase, Aspartate aminotransferase) were determined in autoanalyzer Kontrolab 2017 whereas the complete blood count, leukocyte differential count, and haematocrit, was performed in a haematology analyzer QS Kontrolab EasyVet.

Normality of the data was tested and results were analysed as a Completely Randomized Design, testing linear and quadratic effects in response to the polyherbal mixture concentration using the R software (Mirman 2014).

Results

Productive performance results are presented in Table 2. There was a quadratic response to polyherbal mixture in ADG, feed conversion (P < 0.05) and final BW (P < 0.10). The optimal concentration estimated by regression for maximum ADG was 0.14 ± 0.08% DM of ImmuPlus. No effects were found on apparent dry matter digestibility whereas ruminal pH was reduced linearly (P < 0.05), butyrate was incremented linearly (P < 0.01). Ruminal N-NH₃ (P < 0.01) and total volatile fatty acids showed a quadratic effect (P < 0.05) with the higher concentration at 0.2% of polyherbal.

Table 2. Effect of a polyherbal additive dietary concentration on lamb productive performance and rumen fermentation.

	Polyherbal^a, % DM				SEM	p-Value
	0	0.1	0.2	0.3		Linear	Quadratic
Final weight, kg	32.57	37.02	35.47	33.30	1.650	0.95	0.07
Daily Intake, g/d	982.5	1147.2	1016.0	1004.1	71.30	0.80	0.24
ADG, g	241.2	334.8	282.0	246.8	0.023	0.72	0.01
Conversion ratio	4.23	4.48	3.69	4.12	0.250	0.90	0.02
Digestibility, %	83.10	85.02	85.14	85.06	0.914	0.15	0.28
Ruminal pH	7.08	7.08	6.94	6.87	0.082	0.05	0.67
Lactate, mg/mL	3.22	2.26	2.45	3.21	0.578	0.94	0.14
Ammonia-N, mg/dL	3.46	4.00	4.20	3.62	0.165	0.36	0.001
Acid Acetic, %	56.18	56.20	54.91	54.78	0.903	0.18	0.93
Acid Propionic, %	34.51	33.09	34.20	34.52	1.025	0.80	0.40
Acid Butyric, %	9.42	10.68	10.97	11.99	0.713	0.01	0.86
Total VFA, mmol/L	62.94	68.60	79.71	63.21	5.070	0.60	0.03

^aImmuPlus based on Withania somnifera, Tinospora cordifolia, Ocimum tenuiflorum and Emblica officinalis; SEM, Standard error of the mean; p, Probability; ADG, average daily gain; VFA, volatile fatty acids.

There was no effect in most blood metabolites related to energy metabolism (glucose, cholesterol), but uric acid was linearly reduced (P < 0.01) and bilirubin showed a quadratic decrement (P < 0.05). Liver Alkaline phosphatase showed a linear increase (P < 0.001); Aspartate aminotransferase showed a quadratic increment (P < 0.10) and lactate dehydrogenase tended to show a similar response (P = 0.11) (Table 3). Serum phosphorus was reduced (linear P < 0.10). Mean corpuscular haemoglobin (P < 0.01) and plasma proteins (P < 0.10) increased linearly. Lymphocytes and segmented neutrophils showed a quadratic response (P < 0.01) and platelets showed a linear reduction (P < 0.05) (Table 4).

Table 3. Effect of a polyherbal additive dietary concentration on blood serum biochemistry of lambs.

Parameter	Polyherbal^a, % DM				SEM	p-Value
	0	0.1	0.2	0.3		Linear	Quadratic
Glucose, mg/dL	57.12	57.75	60.12	53.12	2.423	0.33	0.14
Urea, mg/dL	31.12	34.25	33.75	34.00	2.646	0.47	0.56
Uric acid, mg/dL	0.43	0.60	0.52	0.75	0.057	0.002	0.59
Creatinine, mg/dL	1.07	1.20	1.27	1.32	0.123	0.15	0.77
Total protein, g/dL	7.45	7.47	7.35	7.73	0.231	0.49	0.42
Albumin (A), g/dL	4.00	4.10	4.08	4.37	0.148	0.11	0.50
Globulin (G), g/dL	3.45	3.25	3.23	3.21	0.176	0.36	0.62
Ratio A/G	1.16	1.24	1.26	1.32	0.074	0.14	0.92
Cholesterol, mg/dL	85.7	89.3	90.6	83.6	3.907	0.78	0.18
Bilirubin, mg/dL	0.512	0.400	0.400	0.500	0.053	0.88	0.05
Alkaline phosphatases, UI/L	25.12	35.62	40.50	42.87	4.494	0.007	0.37
Lactate dehydrogenase, UI/L	79.75	77.37	89.00	91.25	6.362	0.11	0.71
Aspartate aminotransferase, UI/L	21.7	26.3	29.3	25.5	2.410	0.20	0.09
Calcium, mg/dL	8.55	8.47	9.00	9.01	0.309	0.17	0.89
Phosphorus, mg/dL	4.17	4.42	4.43	4.61	0.168	0.08	0.82

^aImmuPlus based on Withania Somnifera, Emblica officinalis, Ocimum tenuiflorum and Tinospora cordifolia; SEM, Standard error of the mean; p, probability.

Table 4. Effect of a polyherbal additive dietary concentration on blood cells of lambs.

	Polyherbal^a % DM				SEM	p-Value
	0	0.1	0.2	0.3		Linear	Quadratic
Haematocrit, %	32.43	32.17	31.78	29.87	1.756	0.30	0.63
Haemoglobin, g/dL	9.45	10.075	9.471	9.451	0.454	0.76	0.48
Erythrocytes, 10^6/mL	9.03	9.21	8.72	8.41	0.417	0.21	0.56
Mean corpuscular volume, fL	34.4	35.3	36.4	31.3	1.997	0.39	0.16
Mean corpuscular haemoglobin, pg	10.38	10.91	10.81	11.25	0.2156	0.01	0.85
Mean corpuscular haemoglobin concentration, g/dL	30.38	31.57	30.17	32.12	0.9551	0.36	0.67
Platelets, 10^3/mL	712.0	751.1	700.7	581.2	39.835	0.02	0.06
White blood cell, 10^3/mL	19.4	25.9	21.3	19.9	4.012	0.83	0.32
Lymphocytes, 10^3/mL	54.1	51.1	45.4	60.8	3.369	0.34	0.01
Monocytes, 10^3/mL	4.0	4.8	3.1	4.3	0.721	0.84	0.81
Segmented Neutrophil, 10^3/mL	38.6	39.0	47.8	31.0	3.127	0.32	0.01
Band Neutrophil, 10^3/mL	2.0	1.8	1.7	2.1	0.411	0.90	0.52
Eosinophils,10^3/mL	1.25	2.37	1.87	1.25	0.757	0.88	0.27
Basophils, 10^3/mL	0.00	0.12	0.00	0.37	0.110	0.05	0.26
Plasma proteins, g/dL	8.62	8.85	8.77	9.27	0.244	0.10	0.58

^aImmuPlus based on Withania somnifera, Emblica officinalis, Ocimum tenuiflorum and Tinospora cordifolia; SEM, Standard error of the mean; p, Probability.

Discussion

The quadratic response in lamb’s productive variables indicated that metabolites from the polyherbal mixture at a certain dose have favourable effects but at high doses have detrimental effects even though toxicological evaluations indicate that its use is safe (Das et al. 2006). This hormetic response may be explained by the metabolites reported in the plants in the polyherbal; Emblica officinalis contains alkaloids, tannins and ascorbates (Ghosal 1996; Kamra et al. 2006), Withania somnifera has alkaloids, steroidal lactones, saponins (Bharti et al. 2016) and a flavonoid catechin (Alam et al. 2011) and Tinospora cordifolia has phenols and flavonoids (Premanath and Lakshmidevi 2010). Tannins in the rumen have several beneficial depending on its dietary concentrations (Frutos et al. 2004; García-Hernández et al. 2017), saponins reduce ruminal protozoa and methanogenic bacteria (Patra 2010). These metabolites give antioxidant, antimicrobial properties to the polyherbal and may partially explain the best ADG observed with intermediate doses and there are other action mechanisms reported in the gastrointestinal tract in non-ruminants (Mendel et al. 2017) that may be acting in ruminants.

The comparison of ImmuPlus with other feed plants additives in lambs cannot be done directly since each product has different secondary metabolites and the appropriate doses to obtain benefits for each feed plant additive must be identified (Frankič et al. 2009). Nevertheless, similar response has been observed in polyherbals with medicinal plants from India (Godínez-Cruz et al. 2015; Orzuna-Orzuna et al. 2019; Martínez-Aispuro, Mendoza, Cordero-Mora, et al. 2019). Other studies indicate that some plants can improve growth (Mirzaei and Venkatesh 2012) but some feed plant additives have no effect (Chaves et al. 2008; Hassan and Abdel-Raheem 2013).

In this experiment intake remained unaltered which suggests that the negative response in high concentration was not related to palatability (Piluzza et al. 2014). Although no changes in digestibility were detected (P = 0.15). It has been speculated that some saponins may improve cellular permeability in the distal section of the gastrointestinal tract, modifying nutrient absorption (Johnson et al. 1986; Jouany and Morgavi 2007); however in general feed plant additives have had no effect on total tract digestibility (Nasri and Ben Salem 2012; Sahraei et al. 2014).

There is no clear explanation of mechanisms of ruminal pH alteration by the polyherbal, but it has been reported that some feed plant additives acidify the rumen within physiological conditions (Cardozo et al. 2006). One explanation for a more acidic rumen pH could be effects on rumen protozoa; if the secondary metabolites reduced ciliate populations the rate of starch digestion could be increased, decreasing ruminal pH without necessarily affecting total digestibility digestion (Ortega and Mendoza 2003). The more acidic pH is also an indicator of greater microbial activity (total VFA) as observed in the 0.2% level of ImmuPlus. This might be explained by better antioxidant conditions in the rumen; vitamin E has improved in vitro ruminal fermentation (Naziroğlu et al. 2002; Wei et al. 2015) and natural vitamin E has increased VFA concentration, rumen protozoa and synthesis of microbial protein (Belanche et al. 2016).

Some researchers have reported that secondary metabolites of plants can decrease methanogenic archaea, deriving the hydrogen for propionate formation (Frankič et al. 2009; Holtshausen et al. 2009; Oskoueian et al. 2013; Arowolo and He 2018) and considering the plants in the polyherbal and its metabolites and an increase in propionate and a reduction in methane was expected (Kamra et al. 2006; Mirzaei and Venkatesh 2012), however results suggests that butyrate-producing bacteria were stimulated by an unknown mechanism; Butyrivibrio represent among 10–30 % of culturable rumen bacteria in ruminants; molecular studies have identified other novel bacteria which can produce butyrate (Kopečný et al. 2003).

The reduction of N–NH₃ at higher concentration of ImmuPlus indicate that proteolytic bacteria are being affected, possibly by polyphenols (Jones et al. 1994) or by other metabolites similar to beta-acids (Flythe et al. 2017) which together decreased proteolysis as has been reported with other feed plant additives (Sahraei et al. 2014; Canbolat et al. 2018). The alpha- and beta-acids found in some plants are considered phyto-ionophores (Flythe et al. 2017) that can alter the proton-transport membrane integrity of Gram-positive rumen bacteria.

Blood chemistry values are within the ranges reported as normal for sheep (Wang et al. 2015). The albumin value tended to increase, particularly with the highest dose, however higher values or hyperalbuminemia does not always have pathological significance (Brousse 2008). The increments in uric acid may be an indicator of some metabolites that could affect renal functions (Eggum et al. 1982) but creatinine an indicator of kidney function was not altered. The minor reduction in Bilirubin suggests that in low and intermediate doses the phytogenic mixture promotes a better metabolism of porphyrins and haem group but, at the highest dose, polyphenolic compounds could affect the glucuronidation of bilirubin (Mohamed and Frye 2011) as bilirubin levels began to rise to control levels. The increments in Alkaline phosphatase and the numerically higher values of Lactate dehydrogenase and aspartate aminotransferase were within the normal values (Wang et al. 2015) but the linear response indicates that some secondary metabolites in the herbal mixture may be causing some liver stress (Mahgoub et al. 2008). There was a small reduction in serum phosphorous but within normal limits (Jezek et al. 2006; Wang et al. 2015).

Red blood cells and defence cells were also within the normal ranges reported (Wang et al. 2015; Ahmadi-Hamedani et al. 2016). Although a higher mean corpuscular volume (MCV) (the highest count was for MCH, mean corpuscular haemoglobin) count was observed at the highest phytogenic concentration, the lambs did not show any symptoms of anaemia. A higher MCV value is not always associated with a pathologic process or condition (Aslinia et al. 2006) and values are below of others reported (Wang et al. 2015). The response in the platelets indicates that the secondary metabolites of the phytogenic mixture are stimulating in low doses but later reduce the levels; all values remained within the normal limits of sheep (Wang et al. 2015). Platelets are involved in immune response by direct and indirect mechanisms; platelets interact with other immune cells expressing immune receptors and, as a result, stimulate the breakdown of microbes by lysosomal granules (Sonmez and Sonmez 2017).

The increase in lymphocytes has been reported in sheep when consuming plants with condensed tannins, which has been attributed an immuno-stimulant effect (Ramírez-Restrepo et al. 2010). Tinospora cordifolia acts as an immunostimulant and it has been shown that its use decreases the incidence of disease and the use of antibiotics (Mukherjee et al. 2010). The tocopherols, ascorbates and polyphenols within ImmuPlus could function as antioxidants and as immunostimulants (Ceccini et al. 2014). Polyphenols are secondary plant metabolites and have been shown to exert antioxidative and anti-inflammatory effects; this is important in domestic animals because of the oxidative stress conditions promoted by intensive systems; in addition, polyphenols provide protection against bacteria, viruses and fungi (Gessner et al. 2017)

The quadratic changes in segmented neutrophils could be the response to some polyherbal compounds but these counts are not correlated with the presence of bacterial disease (Honda et al. 2016). Interleukin 8 has been related to the degranulative effects of neutrophils and its gene overexpression has been described when polyphenols are added to rations (Taub et al. 1996; Jung et al. 2009). Increases in plasma proteins have also been observed when adding an extract rich in phenols to lamb diets (Rivero et al. 2012); and, as observed here, these changes were not related to infectious agents or chronic inflammatory processes, confirmed by the albumin and globulin values (Russell and Roussel 2007).

Whithania somnifera contains Withanolides and steroid lactones with immunostimulatory activity which are considered responsible of the increased count of leukocytes (Davis and Kuttan 2000), as this medicinal plant has been used as antibacterial, antioxidant, adaptogen, liver tonic, and anti-inflammatory, used as a single herb or in polyherbal formulations (Bharti et al. 2016). The metabolites of Withania somnifera have antifungal, antimicrobial, antimalarial, chondroprotective and immunomodulator biological activities (Bharti et al. 2016).

Studies with lambs fed essential oils, or plants rich in tannins or with other secondary metabolites did not report negative health effects (EL-Sabagh et al. 2014; Ahmadi-Hamedani et al. 2016; Jaguezeski et al. 2018) as was observed in this experiment. The results of blood chemistry and biometry in conjunction with lamb performance indicate that inclusion of ImmuPlus in the ration must not be greater than 0.14 ± 0.08% DM in lambs ration.

Conclusion

The dietary inclusion at 0.1% dry matter of the polyherbal formulated with Withania somnifera, Ocimum tenuiflorum, Tinospora cordifolia and Emblica officinalis improves lamb performance without altering blood chemistry and blood biometry.

Disclosure statement

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