The Effects of a Medical Plant Mixture and a Probiotic on Performance, Antioxidant Activity and Weaning Age of Newborn Holstein Calves

Document Type: Research Articles


Department of Animal Science, Faculty of Agricultural Science, University of Mohaghegh Ardabili, Ardabil, Iran


The aim of this study was to investigate the effects of a medical plant mixture and a probiotic on performance, antioxidant activity and weaning age of suckling Holstein calves. For this experiment, 30 newborn calves (0-10 days of age) with an average birth weight of 42 ± 8 kg were used in a factorial design (3×2) with 6 treatments and 5 replicates. Treatments were: 1) basal diet including a calves starter feed and whole milk, 2) control diet + 2 g probiotic 3) control diet + 1.5% of medical plant, 4) control diet + 1.5% medical plant + 2 g probiotic, 5) control diet + 3% medical plant and 6) control diet + 3% medical plant + 2 g probiotic. The calves were offered experimental pelleted feeds ad libitum and after one month were supplied with imported hay. Results showed that the treatments had no significant effect on dry matter intake during the experiment. However, addition of the 1.5% medical plant increased dry matter intake (P<0.05). However, the 1.5% level of medical plant reduced calves weaning age (P<0.05). Calves receiving control diet + 1.5% of medical plant mixed and 1.5% medical plant mixed + 2 g probiotic showed a higher plasma antioxidant activity (P<0.05). This study suggested that 1.5% of medical plant in calves starter feed will improve performance and the immune system and will also reduce the weaning age of calves.



In livestock production systems, antibiotics are commonly fed to animals to prevent disease and metabolic disorders, as well as improve feed efficiency. However in recent years, public concern over routine use of antibiotics in livestock nutrition has increased due to the emergence of antibiotic resistant bacteria that may represent a risk to human health. Consequently, considerable effort has been devoted towards developing alternatives to antibiotics. Plant extracts offer a unique opportunity in this regard (Wallace, 2004), as many plants produce secondary metabolites, such as saponins and tannins, which have antimicrobial properties. These compounds have been shown to modulate ruminal fermentation to improve nutrient utilization in ruminants (Wang et al. 2000; Hristov et al. 1999). Similarly, the well documented antimicrobial activity of essential oils (EO), and their active components, has prompted a number of scientists to examine the potential of these secondary metabolites to manipulate rumen microbial fermentation to improve production efficiency in ruminants. Contrary to their name, EO are not true oils (i.e., lipids) and are commonly derived from the components responsible for fragrance, or Quinta essentia, of plants. The antimicrobial properties of EO have been demonstrated against a wide range of microorganisms, including bacteria, protozoa and fungi (Chao et al. 2000). Essential oils can be extracted from many parts of a plant, including the leaves, flowers, stem, seeds, roots and bark. However, the composition of the EO can vary among different parts of the same plant (Dorman and Deans, 2000). This antimicrobial activity has been attributed to a number of terpenoid and phenolic compounds (Chao et al. 2000), as well as the chemical constituents and functional groups contained in the EO, the proportions in which they are present and the interactions between them (Dorman and Deans, 2000). In addition, antagonistic, and synergistic effects have been observed between components of EO (Burt, 2004). Thyme (Thymus vulgaris), mint (Spearmint sativum), oregano (Mentha pulegium), cumin (Cuminum cyminum), camel thorn (Alhagi persarum), garlic (Allium sativum) and Eucalyptus (Eucalyptus) are plants that because of their active ingredients are very important. Based on these effects, medical plants has been suggested as an alternative for antibiotics on livestock and especially calves (Soltan, 2009; Hosoda et al. 2006). Probiotics are another group of feed additives that are possible alternatives for antibiotics (Hume, 2011; Riddell et al. 2010). Probiotics can be defined as “live microbial feed supplements which beneficially affect the host animal by improving its intestinal microbial balance” (Fuller, 1989). Probiotics introduce beneficial microorganisms into the gut which act to maintain optimal conditions within the gastrointestinal tract and inhibit the growth of pathogenic or other undesirable bacteria. As far as we know, there are no studies on the effect of medical plants and probiotics as a combined treatment on performance and antioxidant activity in suckling calves. Therefore, this study was performed to investigate the effects of a medical plant mix and probiotic on performance, antioxidant activity and weaning age of Holstein calves.



Animals and diets

Thirty Holstein male calves (average birth weight=42±8 kg) were selected from a commercial Dairy Herd of Moghan Agro Industrial and Animal Husbandry to determine the effect of a medical plant mix and probiotic in the starter diet on feed intake, antioxidant activity and weaning age of newborn Holstein calves. Treatments were: 1) basal diet including a calves starter feed and whole milk, 2) control diet with 2 g probiotic per head per day in milk, 3) control diet with 1.5% of a commercial a medical plant mix in starter feed (Sabzineh Co, Mashhad, Iran), 4) control diet with 1.5% of the medical plant mix with 2 g probiotic, 5) control diet with 3% of the medical plant mix and 6) control diet with 3% of the medical plant mix with 2 g probiotic. The probiotics administration dosage was recommended about 0.5-2 g per day per head by the manufacturer. Also, the recommended dosage of the medical plant mix was 2% in the manufacturer’s catalog. The probiotic supplement used in our study was protexin (Probiotics International Ltd., south Petherton, UK). It contains the following strains of probiotics and prebiotics: Lactobacillus plantarum, Lactobacillus delbrueckii ssp. Bulgaricus, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium bifidum, Streptococcus salivarius ssp. thermophilus, Enterococcus faecium, Aspergillus oryzae and Candida pintolopesii. The medical plant mix consisted of 9% thyme (Thymus vulgaris), 25% mint (Spearmint sativum), 12% oregano (Mentha pulegium), 25% cumin (Cuminum cyminum), 10% camel thorn (Alhagi persarum), 7% garlic (Allium sativum), and 12% Eucalyptus (Eucalyptus). The ingredients of the starter feed are shown in Table 1. The chemical composition of starter feed, hay and the medical plant mixed (% DM) are in Table 2. The calves were housed in individual pens and fed with whole milk approximately at 10% of birth weight and they had free access to the feed starter and water. Milk was offered in two equal meals daily at 09:00 and 17:00. The medical plant was mixed with the starter feed and provided to the animals. Feed intake were recorded daily and the criterion for weaning of calves was at least 1500 g of starter feed per day for three days.


Sampling and analysis

Feed samples for chemical analysis were air-dried at 60 ˚C and ground with a 1 mm sieve prior to analysis for crude protein, ether extract, crude ash (AOAC, 2000), neutral detergent fiber and acid detergent fiber (Van Soest et al. 1991). Blood samples were collected from the jugular vein using tubes with heparin or EDTA just before morning feeding, and the tubes were immediately placed in ice. The collected samples were centrifuged at 2500 × g for 15 min at 4 ˚C, and thereafter plasma samples were stored at -20 ˚C until analysis. Plasma antioxidant activity was measured by a commercially available kit according to the manufacturer’s protocol (Total Antioxidant Status, Randox Laboratories, Co. Antrim, UK). Briefly, the standard and sample were mixed with chromogenic reagent and were incubated with H2O2 for 3 min at 37 ˚C. Thereafter, the optical density was measured for its absorbance at 600 nm using a spectrophotometer (U-2001, Hitachi). Plasma concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT) were measured by enzymatic methods (pars azmon kit, co. Iranian).


Table 1 The ingredient composition of the starter feed


1 Vitamin premix provided per kg of diet: vitamin A: 200000 IU; vitamin D3: 300000 IU; vitamin E: 10000 IU; vitamin K: 2 mg and Anti-oxidant: 1000 mg/kg.

2 Mineral premix provided per kg of diet: Cu: 3300 mg/kg; Fe: 100 mg; Zn: 16500 mg/kg; Mn: 9000 mg; I: 120 mg/kg; Co: 90 mg/kg and Se: 90 mg/kg.


Table 2 The chemical composition of concentrate, hay, and the mixed herbs (% DM)



Mallon di aldehyde (MDA) content in the blood samples was measured based on the method reported by Moore andRobert (1998).


Statistical analysis

Data were analyzed by the GLM procedure of SAS (SAS, 1998) with factorial arrangement of the treatments using following model:

Yijk= μ + Ai + βj + Aβij + εijk

µ: total average.

Ai: effect of herbal additive. 

Bj: effect of probiotic.

ABij: effect of the interaction of between herbal additive and probiotic.

eijk: random error.

The level of statistical significance was preset at (P<0.05).



Intake and gain performance

Intake and gain performance of calves were presented in Table 3. Probiotics supplementation had no effect on intake, whereas medical plant significantly increased starter intake (P<0.05). Calves on starter feed with 1.5% medical plant consumed more concentrate in comparison to the other other groups. Feeding of 250 mg day of EO from oregano plants to sheep (Wang et al. 2009), 2 g of juniper berry EO (containing 35% α-pinene) to cows (Yang et al. 2007), 0.75 or 2 g of EO mixture to dairy cattle (Benchaar et al. 2007) did not influence feed intake. Ababakri et al. (2012) reported that addition 0.05% essence sprayed on of starter feed increased alfalfa intake in suckling Holstein calves. Busques et al. (2003) observed a 12% reduction in concentrate dry matter intake (DMI) in dairy cattle fed 0.6 g of cinnamaldehyde per kg of dry matter. Also Cardozo et al. (2006) reported that the cinnamaldehyde and eugenol mixture decreased dry matter and concentrate intakes compared with controls in growing heifers. The different results about the effects of medical plant additives, that have been reported by previous studies can be attributed to the route of administration, the form of medical plant additives and the dosage of EO. For the present study, the medical plant additives was mixed with starter feed to ensuring proper mixing and the better intake in calves fed 1.5% medical plant can explained bythe better daily and final weight gain in this group. The reduced intake in 3% medical plant additive may be was due to the effects of high levels of EO on feed intake, impaired gastrointestinal microflora and could lead to compounds accumulating in animal tissues and products (Lambert et al. 2001). Generally, improvement in animal performance due to the addition of herbal additives could be attributed to the presence of different chemical compounds in these plants that can affect the digestive tract, feed efficiency and feed intake and eliminate bad microorganisms in digestive tract and feed (Ababakri et al. 2012). Riddell et al. (2010) observed that the DMI was not significantly different in calves fed bacterial probiotic in milk or starter in comparison whith a control group. But other studies showed that adding probiotic significantly increased feed intake (Chaudhary et al. 2008; Donovan et al. 2002). In terms of nutrition and toxicology, a low level of essential oils is important. The effects of experimental treatments on performance are shown in Table 3. Daily weight gain was significantly increased by the medical plant additive (P<0.05) whereas no effect of probiotics was observed. The highest body weight gains were recorded in calves fed starter with 1.5% medical plant. Riddell et al. (2010) reported that use of probiotic in calf starter had no significant effect on body weight gain. Inclusion of probiotics in the diet of young calves has been shown to improve performance characteristics including body weight gain and feed conversion as well as average daily gain in the first two weeks of life (Timmerman et al. 2005).


Table 3 The effects of a medicinal herb mix and probiotics on feed intake and growth rate of calves (g/day)


Treatments included: 1) control group (diet without herbal drugs mixture an probiotic), 2) 1.5% of medical plant mixture based on dry matter without probiotic, 3) 1.5% medical plant mixture + 2 g probiotic, 4) control diet + 3% medical plant mixture and 5) 3% medical plant mixture + 2 g probiotic and 6) 2 g probiotic without medical plant.

H: herbal anf P: probiotic.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

NS: non significant.


Other research has shown no benefit from feeding probiotics to calves (Winds-Chitl et al. 2008). Soltan (2009) reported that essential oils (mint and eucalyptus) given at different levels in milk replacer for calves had no effect on daily body weight gain when compared with controls. Cardozo et al. (2006) observed that the essential oil had no significant effect on average daily gain during the experiment. Fathi et al. (2009) reported that addition of vanilla in a starter diet, increased dairy body weight in the Holstein calves. Generally, body weight gain depends on feed intake and nutrient digestibility and animal health (Ababakri et al. 2012). The higher body weight gain in calves fed 1.5% medical plants may be due to the higher starter intake and healthier calves in this group.


Antioxidant activity

The effects of medical plant mixture on blood total antioxidant activity and mallon di aldehyde (MDA) content was significant (P<0.05) (Table 4). Blood antioxidant activity of calves fed 1.5% medical plant with 2 g probiotics was significantly higher than the other groups. No difference was observed in the AST and ALP between the other groups. Vakili et al. (2013) reported that vanilla addition in starter had no effect on AST and ALP in blood of calves. The lowest blood MDA content was measured in calves fed 3% medical plant mixed with 2 g probiotic. Concentrations of MDA, a degradation product of lipid peroxidation (Halliwell and Chirico, 1993), were used as a proxy measure for oxidative damage. Malondialdehyde is formed under oxidative stress conditions (Nielsen et al. 1997) and offers the advantage that it can be measured in blood as well as in liver tissue and in milk. The influence of probiotics on enhancing immune system were related to its attachment to intestinal epithelium and mucosa which is the key factor in host’s safety. Also, they can improve the immune system by assessment of the establishment of beneficial bacteria (Sami et al. 2001). Immune response and immunity status can be affected by the ingestion of antioxidant substances and oxidative stress (Miyazaki et al. 2001). Stabel et al. (1989) demonstrated that the administration of selenium as an antioxidant source to calves inoculated with Pasteurella hemolytica resulted in a decrease in anti Pasteurella hemolytica a titers. Chatterjee et al. (2003) reported that vitamin E supplementation as an antioxidant source increased both the antioxidant activity and IgG concentration in plasma in periparturient cows. The dietary supplementations of antioxidants such as selenium or vitamin E have been reported to decrease the risks of retained placenta, metritis and clinical mastitis after delivery in cattle (Erskine et al. 1997). These previous studies suggest that antioxidant ingestion should bring health benefits in cattle. Use of plants with anti-oxidant characteristics results in free radicals being annihilated so they will improve animal health. Polyphenolic compoundsare diverse antioxidants which inhibit these free radicals. Carvacrol and thymol are the most important antioxidant components of thyme that have powerful antioxidant characteristics. Thymol has OH group and uses it as H transporter for peroxidation and reduces hydroxyl peroxide free radicals (H2O2) (Lee et al. 2007). Hosoda et al. (2006) reported that between three herbs (mint, cloves and lemongrass), clove significantly increased total antioxidant activity of the serum. Higher antioxidant levels in this plant is the reason for this increase that is in agreement with results of the present study. These antioxidant effects in the current plants are not temporary and can guaranty the health of calves.


Weaning age

Weaning of calves was when a fixed starter intake of at least 1500 g/day was achieved for three consecutive days. Based on this criterion, calves fed the starter with 1.5% of a medical plant mix were weaned earlier compared with other groups (Figure 1). Fathi et al. (2009) observed that using a flavored starter met weaning criteria at a younger age and resulted in a shorter preweaning period and increasing DMI.


Table 4 The effect of a medicinal plant mix and probiotics on the Antioxidant activity in the Holstein calves


Treatments included: 1) control group (diet without herbal drugs mixture an probiotic), 2) 1.5% of medical plant mixture based on dry matter without probiotic, 3) 1.5% medical plant mixture + 2 g probiotic, 4) control diet + 3% medical plant mixture and 5) 3% medical plant mixture + 2 g probiotic and 6) 2 g probiotic without medical plant.

H: herbal; P: probiotic; AST: aspartate aminotransferase and ALT: alanine aminotransferase.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

NS: non significant.


Figure 1 Treatments included: 1) control group (diet without herbal drugs mixture), 2) 1.5% of medical plant mixture based on dry matter without probiotic, 3) 1.5% medical plant mixture + 2 g probiotic, 4) control diet + 3% medical plant mixture, 5) 3% medical plant mixture + 2 g probiotic and 6) 2 g probiotic without medical plant



Age of weaning was significantly decreased when peppermint oil was added to the diets, so calves receiving peppermint oil were weaned 8 and 2 days earlier at 0.05% and 0.025% inclusion levels, respectively, (Ababakri et al. 2012). It is common practice within the dairy cattle industry to restrict the consumption of milk or milk replacer to stimulate an earlier consumption of calf starter, as this decreases the age at which calves can be completely weaned from milk.



Inclusion of a medical plant additive at 1.5% of starter feed resulted in higher starter intake and daily weight gain. Based on these results, no effect of probiotic supplementation was observed. The results showed that a medical plant mixe improved calves gain performance and immune parameters, so it can be considered as a proper feed additive in calves rearing programs.



The authors thank the Manager and Staff of the Dairy cow Station of Agro-Industrial Complex of Moghan, special to Mr. A. Basiri and Mr. M. Ramezani, for their assistance in providing farm and experimental animals and for their other kindly helps.

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