INTRODUCTION
Feed additives are important compounds that can improve the efficiency of feed utilization and animal performance (Ahmed et al. Reference Ahmed, Bassuony, Awad, Aiad and Mohamed2009). Early consumption of dry feed by young calves is desirable to support rapid rumen development and enable early weaning, but calves usually consume only small amounts of dry feed during the first weeks of life (Morrill & Dayton Reference Morrill and Dayton1978). Supplementation of diet with flavouring agents may encourage calves to earlier consumption of dry feed and support full growth (Thomsen & Rindsig Reference Thomsen and Rindsig1980). The first report on the use of flavouring agents in dairy calves’ diet was presented by Wing (Reference Wing1961), who found that use of artificial flavours (sodium saccharin, sodium cyclamate, ethyl lactate, vanillin and citric acid) at a rate of 1 lb/t of concentrate increased voluntary feed intake of calves up to the age of 3 months, compared with unflavoured feed (particularly age between 31 and 60 days). Fathi et al. (Reference Fathi, Riasi and Allahresani2009) also found that adding vanilla flavour to the starter diet for Holstein dairy calves increased feed consumption and average daily gain (ADG) during the pre-weaning period. Propionic and butyric acids, which are largely metabolized in the rumen tissue during absorption, are primary drivers of tissue growth (Guilloteau et al. Reference Guilloteau, Zabielski, David, Blum, Morisset, Biernat, Wolinski, Laubitz and Hamon2009b ). Butyrate has been shown to inhibit intestinal mucosal apoptosis, induce absorption of water and sodium, improve the proliferation of intestinal cells, stimulate intestinal blood flow and promote the synthesis of gastrointestinal hormones (Mentschel & Claus Reference Mentschel and Claus2003). Most butyric acid is absorbed from the rumen so attempts have been made to protect it and pass it to the intestine using modern technologies. Protection is usually achieved through saponification with calcium (Ca) or sodium (Na). These salts are more stable and less odorous than butyric acid itself (Guilloteau et al. Reference Guilloteau, Zabielski and Blum2009a ). Galfi & Bokori (Reference Galfi and Bokori1990) were the first who showed a positive effect of Na-butyrate in growing pigs: in their study, with a diet that contained 1·7 g/kg Na-butyrate, ADG and feed intake of pigs was increased by 23·5 and 8·9%, respectively. Also it markedly reduced the number of Coliform bacteria and increased counts of Lactobacillus in the ileum and caecum. Guilloteau et al. (Reference Guilloteau, Zabielski, David, Blum, Morisset, Biernat, Wolinski, Laubitz and Hamon2009b ) found that adding Na-butyrate to dairy calf starter significantly increased their ADG, feed efficiency, length of duodenum villi and jejunum crypt depth. The hypothesis of the current study was that calves will consume more starters when Oleobiotec is added to the diet and that Ca-butyrate will increase intestinal absorption of nutrients, so these additives will synergistically improve the performance of dairy calves.
MATERIALS AND METHODS
Animals and diets
The experiment was conducted using 32 Holstein dairy calves (16 males and 16 females) for 4 months in the Moghufat Malek (Mashad, Iran) dairy farm. After birth, calves were separated from their dams, weighed and housed in individual pens with wood shavings for bedding, and fed 4 litres of colostrum over two feedings. Fresh colostrum from dams was used for calves if it had >60 mg Ig antibody/ml, measured using a colostrometer. Otherwise, high-quality (>60 mg Ig/ml) colostrum from other cows was fed. Calves with birth weight of 36–48 kg were selected to reduce errors and maintain consistency in different groups. The calves were fed colostrum and transition milk for 2 days at 100 g/kg of initial body weight (BW), then at 3 days of age (day 1 of study) they were assigned randomly to four groups, since the number of male and female calves and birth weights in each group were equal. During the pre-weaning period, calves were fed milk (via a bucket) at 100 g/kg of their birth weight (twice per day at 05.00 and 17.00 h) in addition to experimental diets. The dietary treatments were: (1) diet with no additive (CON), (2) diet containing Ca-butyrate (CAB), (3) diet containing Oleobiotec (OLE) and (4) diet containing Ca-butyrate plus Oleobiotec (CAO). The ingredients and nutrient composition of the starter diet used in the current study is shown in Table 1. The flavouring agent used in the current study was Oleobiotec, which mainly contained of spices (ginger and pepper) and essential oils (oregano, thyme and cinnamon) (Phode company, Terrsac, France). To aid accuracy of the experiment in terms of additive consumption by calves, from day 1 to day 23 of the study the manufacturer's recommended dose of additives (5 g Ca-butyrate and 0·25 g Oleobiotec per calf per day) were added to the morning milk meal of each calf and after day 23, additives were top-dressed into the starter. The starter diet, which was offered from day 1 of study, was pelleted (Table 1). Alfalfa hay was fed separately from day 24 of study until the end of the trial period. Alfalfa hay was chopped to c. 8–19 mm in length. Ad libitum water was provided for calves from 3 days of age throughout the study. Weaning was carried out for 3 consecutive days from day 44 to 46 of the experiment, with one serving of milk daily (morning meal) and the experiment ended 3 weeks after weaning. The health of calves was monitored daily using the procedure described by Heinrichs et al. (Reference Heinrichs, Jones, VanRoekel and Fowler2003) as follows: for scour scoring, 1 = normal, 2 = soft to loose, 3 = loose to watery, 4 = watery, mucous, slightly bloody, 5 = watery, mucous, bloody; for respiratory scoring, 1 = normal, 2 = slight cough, 3 = moderate cough, 4 = moderate to severe cough, 5 = severe and chronic cough; and for general appearance scoring, 1 = normal and alert, 2 = ears drooped, 3 = head and ears drooped, dull eyes, lethargic, 4 = severely lethargic. A scour day was defined when calves had a faecal score >3. Electrolyte therapy was initiated when calves had faecal score >3 and continued until signs of dehydration abated. The average air temperature was 25·4 ± 7·3 °C, and relative humidity was 41 ± 10% during the study. The animals were cared for according to the guidelines of the Iranian Council of Animal Care (1995).
Table 1. Ingredients (g/kg DM) and nutrient composition of starter diet
* Composition of vitamin/trace-mineral mix: Vit A 1 000 000 IU/kg, Vit D3 150 000 IU/kg, Vit E 2000 IU/kg, antioxidant 0·4 g/kg, sodium bicarbonate 71 g/kg, magnesium sulphate 19 g/kg, iron sulphate 3 g/kg, manganese oxide 2 g/kg, zinc sulphate 3 g/kg, copper sulphate 0·3 g/kg, calcium sulphate 0·1 g/kg.
† Calculated from NRC (2001).
Sample collection and statistical analyses
Milk, starter and alfalfa hay intake were measured daily. Calves were weighed individually before the morning feeding at birth, at the 23rd day of the experiment, at weaning (46th day of experiment) and at the end of experiment (3 weeks after weaning). Starter samples were taken weekly and composited, frozen and stored at −20 °C until analysis for crude protein (CP), ether extract (EE), calcium (Ca) and phosphorus (P) according to the procedures of AOAC (2000) and both neutral detergent fibre (NDF) and acid detergent fibre (ADF) using the method of Van Soest et al. (Reference Van Soest, Robertson and Lewis1991).
Blood samples were taken on days 0 (2 days age, as pre-treatment sampling), 23, 46 (weaning) and 67 (end day) of the study, 02.00 h after the morning feeding, via jugular venipuncture by vacuum tubes containing heparin. Samples were centrifuged at 1500 g (15 min at 25 °C) and plasma was stored at –20 °C until analysis. Plasma glucose, β-hydroxybutyrate (BHBA), blood urinary nitrogen (BUN) and creatinine concentrations were determined by auto analyser (Biochemistry Analyser, Gesan Chem 200, Italy). Enzymatic kits were used to analyse plasma glucose (Pars Azmoon glucose kit, Tehran, Iran), BHBA (D-3-hydroxybutyrate kit, Randox Laboratories Ltd, Antrim, UK), BUN (Pars Azmoon urea kit, Tehran, Iran) and creatinine (Pars Azmoon creatinine kit, Tehran, Iran).
Experimental data were analysed as a completely randomized design with a 2 × 2 factorial arrangements of treatments using repeated measures Analysis of Variance (ANOVA) by the Mixed Procedure of Statistical Analysis System (SAS) (2003) according to the model:
$$\eqalign{Y_{ijklm} & = \mu + O_i + B_j + D_k + Sex_l + (O_i \times B_j ) \cr & \quad + (O_i \times D_k )+ (B_j \times D_k ) + (O_i \times B_j \times D_k ) \cr &\quad + b(X_{ijklm} - \bar X)+ Calf_m + e_{ijklm}} $$
where Y
ijklm
was the response variable, μ was overall mean, O
i
was the effect of Oleobiotec, B
j
was the effect of Ca-butyrate, D
k
was the effect of time (day of measurement), Sex
l
was the effect of calf sex, (O
i
× B
j
) was the interaction of Oleobiotec and Ca-butyrate, (O
i
× D
k
) was the interaction of Oleobiotec and time, (B
j
× D
k
) was the interaction of Ca-butyrate and time, (O
i
× B
j
× D
k
) was the interaction of Oleobiotec, Ca-butyrate and time, b was the regression coefficient of response variable on initial weight, X
ijklm
was the initial weight of mth calf,
$\bar X$
was the average initial weight of calves, Calf
m
was the random effect of calf within treatment and e
ijklm
was the residual error. Plasma metabolite concentration data were analysed by the same model using the General Linear Model (GLM) Procedure of SAS (2003) as non-repeated measures and data obtained at day 0 of study (2 days age) were included in the model as covariate. Comparisons between means of treatments were made by the Tukey–Kramer test and significance was declared at P < 0·05 unless otherwise noted.
RESULTS
Adding Ca-butyrate and Oleobiotec to the diet did not significantly change consumption of starter, alfalfa hay or total of starters plus alfalfa hay in any of the trial periods. However, addition of Oleobiotec flavour to the diet tended (P < 0·1) to increase consumption of starter during the post-weaning period. Also, daily milk intake was not significantly different between calves fed the different experimental diets (Table 2).
Table 2. Feed intake, average daily gain and feed efficiency of calves fed different diets
* CON, diet with no additives (control); CAB, diet containing Ca-butyrate (consisted of 813·0 g/kg butyric acid and 187·0 g/kg calcium); OLE, diet containing Oleobiotec (consisted of spices [ginger and pepper] and essential oils [oregano, thyme and cinnamon]); and CAO, diet containing Ca-butyrate plus Oleobiotec; DM, dry matter.
† Standard error of means.
The effect of adding Oleobiotec flavour to the diet on ADG of calves was not significant in any of the trial periods, while adding Ca-butyrate to the diet increased ADG of calves during the post-weaning period (P = 0·03) and the whole trial period (P = 0·013), hence calves fed CAB diet had higher (P = 0·026) final BW than calves fed CON and OLE diets (Table 2).
Milk consumption of calves in addition to starter and alfalfa hay consumption was also considered in calculating feed efficiency during the pre-weaning period. Addition of Oleobiotec to the diet had no significant effect on feed efficiency of calves, while Ca-butyrate improved feed efficiency during the post-weaning period (P = 0·042) and over the whole trial period (P = 0·004).
Plasma glucose concentration was higher (P = 0·044) in calves fed CAB and CAO diets at the 67th day of study but no differences were found between diets in relation to other plasma metabolites (Table 3). There was no significant effect of diet on health parameters of calves and no sign of disease was noticed in calves fed different diets. The calves fed CON, CAB, OLE and CAO diets showed scour scores of 1·79, 1·58, 1·78, 1·88; respiratory scores of 1·25, 1·25, 1·22, 1·25; and general appearance scores of 1·05, 1·06, 1·05, 1·05, respectively, during the whole trial period.
Table 3. Plasma metabolite concentrations of calves fed different diets
* CON, diet with no additives (control); CAB, diet containing Ca-butyrate (consisted of 813·0 g/kg butyric acid and 187·0 g/kg calcium); OLE, diet containing Oleobiotec (consisted of spices [ginger and pepper] and essential oils [oregano, thyme and cinnamon]); and CAO, diet containing Ca-butyrate plus Oleobiotec; BHBA, β-hydroxybutyrate; BUN, blood urinary nitrogen.
† Standard error of means.
DISCUSSION
There have been conflicting results on the effect of flavouring agents on feed intake of calves. Osborne et al. (Reference Osborne, Odongo, Edwards and McBride2007) and Carlotto et al. (Reference Carlotto, Olivo, Viegas, Stiles, Gabbi, Brustolin, Charao, Rossarolla, Ziech, Pereira and Scaravelli2006) found that adding flavours to starter did not increase starter and alfalfa hay consumption of calves. Soltan (Reference Soltan2009) found that calves fed a flavoured agent containing some essential oils (eucalyptus, menthol crystal and mint) had lower starter intake than calves fed unflavoured starter during the pre-weaning period, while both starter and hay intake were reduced during the post-weaning period of study. Thomas et al. (Reference Thomas, Wright, Formusiak, Cant and Osborne2007) also found no effect of vanilla flavour on feed intake of calves when flavouring agent was added to drinking water. However, Wallace & Riggs (Reference Wallace and Riggs1967) found that molasses-supplemented starter significantly increased feed consumption of weaned Hereford calves. Cheeke (Reference Cheeke1991) stated that adding flavours to low-quality diets (with respect to energy and protein) will increase starter consumption of calves. Hence, the lack of Oleobiotec effect on feed intake of calves in the current study may be due to feeding a starter with high levels of energy and protein. However, different flavouring agents were used in each of the above-mentioned studies, which may also have contributed to differences in the reported results. The lack of effect of adding Oleobiotec to the diet on ADG of calves in any of the trial periods in the current study was consistent with results obtained by Carlotto et al. (Reference Carlotto, Olivo, Viegas, Stiles, Gabbi, Brustolin, Charao, Rossarolla, Ziech, Pereira and Scaravelli2006), Osborne et al. (Reference Osborne, Odongo, Edwards and McBride2007) and Meyer et al. (Reference Meyer, Erickson, Klopfenstein, Greenquist, Williams and Losa2007).
In relation to the effect of Ca-butyrate on feed intake of calves, several studies have reported that feed intake of calves was not changed by adding butyrate salts to calves’ diet, which is consistent with results of the current study (Guilloteau et al. Reference Guilloteau, Zabielski, David, Blum, Morisset, Biernat, Wolinski, Laubitz and Hamon2009b , Reference Guilloteau, Savary, Jaguelin-Peyrault, Rome, Le Normand and Zabielski2010; Ferreira & Bittar Reference Ferreira and Bittar2010). However, Slusarczyk et al. (Reference Slusarczyk, Strzetelski and Furgal-Dierzuk2010) found that Na-butyrate (at rates of 3·0, 10·0 and 30·0 g/kg dry matter (DM) of concentrate) increased feed intake of calves both during the post-weaning period and over the whole trial period. Lu et al. (Reference Lu, Zou and Wang2008) also found that weaned piglets fed diets supplemented with 1 g Na-butyrate/kg DM had higher feed intake than the control group, which was attributed to the positive effect of Na-butyrate on gastrointestinal performance and also to the low levels of tumour necrosis factor-α (TNF-α) and interlukin-6 (IL-6) found in the serum of these piglets. It was postulated that TNF-α released into cerebral ventricles potently blocks appetite, and such animals would starve to death despite free access to feed (Tracey et al. Reference Tracey, Morgello, Koplin, Fahey, Fox, Aledo, Manogue and Cerami1990). However, since the latter study was carried out on pigs the results might not be applicable to calves. Guilloteau et al. (Reference Guilloteau, Zabielski, David, Blum, Morisset, Biernat, Wolinski, Laubitz and Hamon2009b ) reported that enhanced ADG and feed efficiency of calves fed milk formula supplemented with Na-butyrate during long-term feeding could be due to its effect on increased efficiency of digestion by pancreatic enzymes and also maturation of gastrointestinal tract (enhanced villus size and modified activity of digestive enzymes). Gorka et al. (Reference Gorka, Kowalski, Pietrzak, Kotunia, Kiljanczyk, Flaga, Holst, Guilloteau and Zabielski2009) and Zabielski et al. (Reference Zabielski, Godlewski and Guilloteau2008) found that Na-butyrate supplementation of dairy calves diet significantly increased intestinal papillae width and length. Also, Guilloteau et al. (Reference Guilloteau, Rome, Le Normand, Savary and Zabielski2004, Reference Guilloteau, Savary, Jaguelin-Peyrault, Rome, Le Normand and Zabielski2010) showed that Na-butyrate supplementation increased nutrient digestibility and total daily pancreatic secretions in young calves. The improved ADG and feed efficiency of calves fed Ca-butyrate supplemented starter in the post-weaning period of the current study may be attributed to an improvement of rumen and small intestine capacity absorption, as reported in the above-mentioned studies.
Higher plasma glucose concentrations in calves fed Ca-butyrate may be due to a decrease in glucose oxidation in ruminal and intestinal mucosal cells. This increase in the concentration of circulating glucose in calves fed Ca-butyrate is one of the main reasons for higher ADG of these calves at post-weaning and over the whole trial period. Abeni et al. (Reference Abeni, Calamari, Stefanini and Pirlo2000) reported that higher plasma glucose, indicating higher energy availability especially before puberty, could result in higher BW gain, and stated that blood glucose seems to be a better index of energy intake for younger than for older heifers. Greater availability and oxidation of glucose might have partly contributed in improving the energetic efficiency (by sparing other oxidizable substrates, such as amino acids) and thus BW gain in calves fed a diet containing Ca-butyrate than those fed other diets (Quigley et al. Reference Quigley, Caldwell, Sinks and Heitmann1991; Khan et al. Reference Khan, Lee, Lee, Kim, Kim, Ki, Park, Ha and Choi2007c ). Supply of more glucose resulted in better feed conversion efficiency in Ca-butyrate fed calves. Slusarczyk et al. (Reference Slusarczyk, Strzetelski and Furgal-Dierzuk2010) also found higher glucose concentration in serum of calves fed Na-butyrate compared with the control diet at age 7, 14 and 42 days. The Volatile Fatty Acid (VFA) production at lower ages was probably not sufficient to spare glucose utilization by gut cells. Despite different proportions of Ca-butyrate in experimental diets, no differences in plasma BHBA concentrations were found. It is likely that Ca-butyrate had passed into the small intestine and used for development of intestinal mucosa and intestinal enzyme activity. Guilloteau et al. (Reference Guilloteau, Zabielski, David, Blum, Morisset, Biernat, Wolinski, Laubitz and Hamon2009b ) believed that Na-butyrate is a specific stimulant of calf growth and is largely active in the intestines, where it has a favourable effect on villus length, crypt depth, mitotic index and enzyme activity. It is also possible that the amount of Ca-butyrate (5 g/day) added to the calves’ diet was insufficient to change circulating concentration of BHBA. Since BUN concentration is a function of DM and CP consumption (Khan et al. Reference Khan, Lee, Lee, Kim, Kim, Ki, Ha, Lee and Choi2007b ), the same BUN concentration in calves fed different diets was expected. Moreover, Abeni et al. (Reference Abeni, Calamari, Stefanini and Pirlo2000) stated that blood urea concentration did not appear to be determined mainly by protein catabolism with energetic finality, but it appeared to be related more to CP: metabolizable energy ratio, which was also the same for calves fed different diets in the current study. Although BUN concentration of calves in the current study was slightly higher than dairy calves of the same ages in some other studies (Khan et al. Reference Khan, Lee, Lee, Kim, Ki, Hur, Suh, Kang and Choi2007a , Reference Khan, Lee, Lee, Kim, Kim, Ki, Ha, Lee and Choi b ) and may be an index of renal dysfunction, plasma creatinine concentration in calves fed different diets was in the safe range (Hammon et al. Reference Hammon, Schiessler, Nussbaum and Blum2002). This may also be due to higher CP content of the starter used in the current study compared with the above-mentioned studies.
CONCLUSION
The results of the current study demonstrated that adding Ca-butyrate to the diet improved ADG and feed efficiency of dairy calves especially during the post-weaning period. In contrast, adding Oleobiotec flavour to the diet of dairy calves had no effect on their performance either during pre-weaning or post-weaning periods. Although adding two additives to calves’ diet simultaneously improved some performance features such as feed efficiency, no synergistic effect was found for the additives.
The authors gratefully acknowledge the staff of Moghufat Malek dairy farm (Mashad, Iran) for their assistance throughout the study.
