Addition of increasing doses of ricinoleic acid from castor oil ( Ricinus communis L . ) in diets of Nellore steers in feedlots

The aim of this study was to evaluate the performance and blood parameters of feedlot Nellore cattle fed increasing doses of ricinoleic acid (RA) in the diet. Ninety-six Nellore steers divided into 12 groups of 8 animals were used. The animals were randomly assigned to four treatments: 0, 1, 2, or 4 g of RA/animal/day, with three replicates per treatment. The experimental period consisted of 84 days divided into three 28-day periods preceded by three step-up diets. A quadratic effect was found for average daily gain and final body weight, as well as for leukocyte and lymphocyte counts, and for urea and blood urea nitrogen. A linear effect was observed for albumin, alkaline phosphatase, and gamma glutamyl transferase. The inclusion of 2 g of RA daily improved the performance of feedlot Nellore steers.


INTRODUCTION
With the largest commercial herd in the world numbering 176 million heads, Brazil has been undergoing significant changes in the system of livestock beef cattle production.It has been observed that in the feedlot system, more concentrates are fed in relation to forage, and that in most Brazilian feedlots, more than 60% of the diet is concentrates.
The use of large amounts of concentrates in beef cattle diets can lead to metabolic disorders such as acidosis, laminitis, hyperparakeratosis, and liver abscesses, resulting in a decrease in dry matter intake and performance (Nagaraja and Chengappa, 1998).Thus, it is common to include additives of rumen fermentation modulators in the diet in order to prevent these disorders when feeding diets with a higher proportion of concentrates.The main additives used are ionophores, such as monensin and lasalocid, and salinomycin antibiotics that are widely employed to modulate rumen fermentation (Gandra et al., 2009).The use of antibiotics in animal feed has been rejected by consumers in some countries, since they believe that residues of these additives may be present in the meat of beef cattle and may cause problems for human health.
Essential oils are secondary metabolites of some plants, responsible for odour and colour, and can obtained by evaporation or distillation.In the rumen they act on the structure of the bacterial cell wall, altering the permeability of the cytoplasmic membrane for hydrogen and potassium ions.The alteration of ion gradients results in deterioration of essential cell processes such as electron transport, protein translocation, phosphorylation steps, and other enzyme-dependent reactions.The resulting loss of control over chemiosmosis by the affected cell consequently leads to bacterial death (Dorman and Deans, 2000).
Many essential oils have been studied for this purpose, however, standing out among them is castor oil, which is obtained by pressing the seeds.It contains 90% ricinoleic acid (RA), which gives the oil its unique features, enabling a wide range of industrial use.The cultivation of castor beans is thus important for the economic potential of Brazil.RA is very similar to oleic acid, the only difference being a hydroxyl group present in RA and absent in oleic acid.For this reason, RA is also called hydroxyoleic acid.
Although the toxicity of the castor bean has been known since ancient times, castor oil is not toxic because ricin, a toxic protein present in the seeds, is not lipid soluble, so the toxic component is restricted only to the castor bean.Ricin specifically and irreversibly inactivates eukaryotic ribosomes, preventing protein synthesis (Dorman and Deans, 2000).The oil obtained by pressing the seeds is a polymer precursor and a solution of RA esters, and is known as castor oil (Ferreira et al., 2002).
The detergent derived from castor oil, in turn, shows antimicrobial properties in the treatment of necrosis of tooth pulp; its bactericidal activity is similar to sodium hypochlorite (Ferreira et al., 2002).
Given the antimicrobial properties of RA and growing demand for nonantibiotic animal performance improvers, it has been proposed to include RA in the diet of feedlot Nellore steers in order to observe its effects as a possible modulator of ruminal fermentation on the basis of possible performance improvement without harm to animal health.In order to explore the antimicrobial properties of RA, the objective of this study was to evaluate the performance and blood parameters of feedlot Nellore steers fed increasing doses of RA in the diet.

Animals and management
The trial was conducted in the experimental center of OuroFinoAgribusiness Ltd., located in the city of Olimpia, Sao Paulo (Brazil).Ninety-six Nellore steers, initial weight 385 ± 14 kg, were divided into 12 groups of 8 animals.The animals were randomly assigned to four treatments: 0 g RA/animal/day, 1 g of RA/animal/ day, 2 g of RA/animal/day, or 4 g of RA/animal/day, with three replicates per treatment.
The diets were formulated according to the Cornell Net Carbohydrate Protein System 5.0, with a forage: concentrate ratio of 45:55 in order to meet the requirements of Nellore steers in the growing and finishing period.The forage used was maize silage.All animals received the same diet differing only in the amount of RA per day and were fed once daily in the morning.The composition and concentrate ingredients of the diet and the nutritional composition of the concentrate and the silage are presented in Tables 1 and 2. Ricinoleic acid (RA) is produced from castor oil, which is obtained by pressing the seeds.The oil is then complexed with a polymer to give a solid consistency to the RA.
The experimental period was composed of 84 days divided into three periods of 28 days preceded by three step-up diets of 15 days.They were: 5 days at a forage:concentrate ratio of 70:30, 5 days at a ratio of 60:40, and 5 days at a ratio of 50:50.
Before the rations were given to the animals, the orts from the pens were weighed daily in order to estimate dry matter intake.The animals were fed according to the dry matter intake of the previous day in order to maintain the percentage of daily orts between 5% and 10% of the supplied diets.

Sample collection and analysis
On days 14-21 of the experimental periods, orts, silage, and concentrate ingredients were collected for analyses of dry matter (DM), organic matter (OM), ash, crude protein (CP), fatty acids (FA), and lignin in accordance with the methods described by AOAC (2000).Crude protein (CP) was obtained by multiplying the total nitrogen content by 6.25.
Neutral detergent fibre (NDF) and acid detergent fibre (ADF) contents were obtained according to the method described by AOAC (2000), using α-amylase without the addition of sodium sulphite in the determination of NDF in an AnkonSystem®.
On days 28, 56, and 84 of the trial period, before the supply of the diets in the morning, the animals were weighed and blood samples were collected by vein or coccygeal artery puncture.
After 84 days of the experiment, the animals were transported to a commercial packing plant under federal inspection, where they were slaughtered by stunning and later by the pneumatic bleeding method.Blood samples were collected (vacutainer) for measurement of blood parameters: total protein, albumin, urea, aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), and alkaline phosphatase (ALP) in plasma in addition to the haemogram (red series), that measured erythrocytes, haematocrit, haemoglobin, MCV (mean corpuscular volume), MCH (mean corpuscular haemoglobin), MCHC (mean corpuscular haemoglobin concentration), and WBC (white series) that measured leucocytes, neutrophils, lymphocytes, eosinophils, monocytes, basophils, and fibrinogen.
Immediately after collection, the samples were cooled and centrifuged at 2000 g for 15 min to separate the serum or plasma, and then stored at 2°C until the laboratory procedure, using commercially available kits (LABORLAB® and CELM®) for measuring total protein, albumin, urea, AST, GGT, and ALP.Enzymatic colorimetric and end-point methods were used; the readings were performed in an automated blood biochemistry analyzer (CELMSBA-200®).Blood samples collected for haemograms were analysed fresh on the same day of weighing animals and biochemical analyses.

Statistical analysis
The data were analysed by PROC MIXED according to the following model: Yij = μ + Di + Tj + Tj(Dj) + eij where: Yij -dependent variable; μ -overall mean; Di -effect of diet (i=1-4); Tj -effect of days in confinement; Di (Tj) -effect of interaction between diet and day of confinement; eij -error.
The calculated degrees of freedom were performed according to the Satterth waite method (ddfm -satterth).The data were subjected to analysis of variance and polynomial regression by the command PROCMIXED of SAS version 9.0 (SAS, 2004), adopting a significance level of 5%.

Performance
No linear or quadratic effect was observed (P>0.05) for dry matter intake.The difference between the control diet and inclusion of dietary RA was +0.41; -0.04; +0.74 kg/day, for 1, 2, and 4 g of RA/day, respectively (Table 3).The mean dry matter intake in this trial was 12.7 kg/day, this value is appropriate for the breed and animal growth stage.There was no linear or quadratic effect (P>0.05) for feed conversion or feed efficiency in relation to the inclusion of RA in the diets.The control diet had a feed conversion ratio that was 8.15%, 6.52%, and 11.28% higher than in the diets with the addition of 1, 2, and 4 g of RA/day, respectively.
For the variables of final body weight and average daily gain (ADG), a quadratic effect (P<0.05) was observed, and therefore the lowest ADG and final body weight were obtained in the group receiving 4 g of RA.The regression equation presenting the ADG is described by Y = 1.82 + 0.05X -0.017X2; R2 = 0.47.Analysis of this function showed that the optimal level of inclusion of RA was 1.47 g/day and the ADG estimated for this level of inclusion was 1.87 kg/ day.

Haemogram and leucogram
There was no effect (P>0.05) on the RBC (red blood cell count) due to the addition of RA in the diet, suggesting that the use of RA did not affect the red blood cells and the health of animals.
In general, the addition of RA to the diets did not affect most variables in the WBC (P>0.05).There was, however, a quadratic effect (P<0.05) for leukocytes and lymphocytes and the lowest value was found for the group receiving the control diet.The regression equation obtained for the absolute values of leukocytes is provided by Y = 13.359+ 2.656X -694X2; R2 = 0.45, where the optimal level of inclusion of RA was 1.91 g/day and the estimated value of leukocytes was 15.903 cells.The absolute values for lymphocytes was observed at the equation Y = 8.802 + 1.436X -290X2; R2 = 0.44, where the optimal level of inclusion of RA was 1.78 g/day and the estimated value of lymphocytes was 10.458 cells.

Blood metabolites
There was no difference (P > 0.05) for total protein and aminotransferase (AST) activity (Table 5).For urea and blood urea nitrogen (BUN), a quadratic effect (P<0.05) was observed and the lowest value was found for the group receiving 2 g of RA/day.The regression equations that represent the concentrations of urea and BUN are given by Y = 29.87-5.55X + 0.83X2; R2 = 0.64 and Y = 13.94 -2.58X + 0.38X2; R2 = 0.64, respectively.The optimum addition level RA to the diets for urea and BUN was 3.40 g of RA/day, where the critical concentration of urea was 20.48 mg/dl and of BUN, 9.60 mg/dl.

DISCUSSION
Performance.With regard to dry matter intake, some data from the literature show that essential oils have attractive and palatable properties that influence intake by animals (Wallace, 2004) (Table 3).Other authors, however, observed no effect on the dry matter intake of beef cattle supplemented with essential oils (Meyer et al., 2009).Hence, reports on the effect of essential oils on dry matter intake are contradictory.Benchaar et al. (2008), supplementing beef cattle with 2 and 4 g/day of essential oil observed an increase in dry matter intake when compared with non-supplemented animals.Likewise, Alçiçek et al. (2004) claimed that the addition of essential oils maximized dietary intake by animals.Cardozo et al. (2008), when testing the effects of the essential oil of pepper (Capsicum annuum) also observed an increase in the intake of water and dry matter.
With regard to the feed and conversion ratios in a similar experiment, Meyer et al. ( 2009) observed no effect on feed efficiency in cattle supplemented with essential oils compared with the control diet.Moreover, Benchaar and McGinn (2008) when evaluating beef cattle fed 0.2 or 4 g/day of essential oils found a quadratic effect on feed efficiency, with it being better in the animals fed with 2 g of essential oil.
The results for ADG and BW in this trial disagree with a large number of studies with essential oils, since Beauchemin et al. (2006) and Meyer et al. (2009) when supplementing beef cattle with essential oils, observed no difference in ADG compared with the control diets.Both authors emphasize that the lack of effect on performance is due to the similarity in dry matter intake of animals in different treatments.After all, according to NRC (2001), dry matter intake accounts for 60-90% of the variations in animal performance.
The effect of RA on dry matter intake and ADG could be related to the selection of microorganisms in the ruminal flora that produce propionate, which might reduce the dry matter intake, while maintaining or even increasing animal performance, as noted in the tests.Other studies should be conducted, however, in order to establish the actual mechanism of RA action in the ruminal environment.The use of RA in ruminant nutrition has only recently been attempted, but according to its chemical characteristics and physical mechanism of action, it seems similar to most of the essential oils extracted from plants.The actual mechanism of action has not been completely established, however, requiring new trials in order to obtain concrete results.
Haemogram and leukogram.Haematological evaluations in cattle are used to assess disease in an animal or to evaluate groups of animals within a herd and also to detect hidden diseases and to guide clinical decisions (Weiss and Perman, 1992).The values of variables observed in this study for red blood cells, haematocrit, and haemoglobin are in accordance with the reference values obtained by Benatti et al. (2011) in a study on the blood profile of cattle.Furthermore, according to the values reported by Biondo et al. (1998) , the MCV and MCHC valued in our study are within the normal range for the breed and age of Nellore steers.
The increase in the absolute count of leukocytes and lymphocytes as the dose of RA increased may be related to some cumulative residual toxic effect of the plant (Ricinus communis L.), but throughout the experimental period no clinical signs characteristic of infectious, neurological, or metabolic disease were observed in any animal.A quadratic response was observed, however, for all of the variables mentioned above, indicating that the maximum count was found for the level of 1 g of RA in animal diets, a fact that may not reflect a deleterious change in the immune system of the studied animals.A linear increase better characterizes any deleterious changes in the immune system, but this did not occur.Perhaps this increase in the white blood cell count was only a physiological adaptation to supplementation with RA.
According to Costa et al. (2000), leukocyte and lymphocyte counts are widely correlated, so the variation in total leukocytes reflects in particular the change in the total number of lymphocytes.The results for leukocyte counts are in agreement with Paes (2003), who studied the WBC of clinically healthy Nellore cattle subjected to confinement.The leukocyte and lymphocyte counts observed in this study for all treatments were within the ranges considered normal for Nellore steers in this age group (Costa et al., 2000).
The measurements of RBC and WBC in this trial were aimed at monitoring the health of animals and observing the behaviour of these cells upon supplementation with RA, due to its only recent introduction into ruminant nutrition.The results found in all groups are in accordance with the reference values for Nellore steers in this phase of growth.The quadratic effects seen for the leukocyte and lymphocyte counts can be linked to the better ADG observed for the same level of addition of RA.When the data for WBC and ADG are analysed together, the results suggest a better immune status of animals in the RA group.
Blood metabolites.According to Swenson et al. (1996), the BUN values found in this experiment are considered normal for cattle, and ranged from 10 to 30 mg/dl.Putrino et al. (2006) affirmed that BUN is used as an indicator of protein balance, and the limits indicating adequate protein intake are 9-12 mg/dl.Valadares et al. (1997) concluded that BUN concentrations from 13.52 to 15.15 mg/dl corresponded to maximum microbial efficiency and probably represent the limit above which there is loss of protein for Nellore steers fed diets containing 45% concentrate and, on average, 62.5% TDN.Bortoli (2007), working with heifers fed a commercial product (Rumex®) consisting of several essential oils, saponins and yeast, observed plasma urea nitrogen values close to those found in this experiment (15.83 mg/dl).Likewise, Coneglian (2009) using the essential oil of castor and cashew in the diet of beef cattle, did not observe any effect on BUN levels, and the average value for all treatments was 14.72 mg/dl higher than those obtained in this study.
The values found for albumins showed an increase only for the addition of 4 g of RA/day.This increase did not constitute a clinical or nutritional concern, since no effect was observed on total protein concentrations.The concentrations of gamma glutamyl transferase and alkaline phosphatase observed in the trial are within the reference values for Nellore.Despite the effects found by the inclusion of RA in the diet, no clinical changes were observed in the animals (Swenson et al., 1996).
In general, the biochemical profiles observed as a function of the addition of RA do not compromise the health and, especially, the performance of animals in feedlots.The changes found in BUN should be better established in further metabolism trials to elucidate the mechanism of RA action on the ruminal protein metabolism in beef cattle.

CONCLUSIONS
The inclusion of 1 to 2 g of ricinoleic acid (RA) in the diets of feedlot Nellore steers gave better performance without damaging the health of the animals.The results support the use of RA as a performance-modifying additive for feedlot cattle on high concentrate diets, replacing ionophores, with the reservation that more trials should be performed to confirm the mechanisms of RA action on ruminal metabolism.

Table 3
1 inclusion of 0, 1, 2 or 4 g of ricinoleic acid from of castor oil (Ricinus communis L.) per animal/day in total diet; BW -body weight; 3 ADG -average daily gain; 4 FCR -feed conversion ratio

Table 4 .
Red blood cells and white blood cells counts