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Effect of dietary cobalt supplementation on plasma and rumen metabolites in Mehraban lambs
Small Ruminant Research 90 (2010) 170–173
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Effect of dietary cobalt supplementation on plasma and rumen metabolites in Mehraban lambs Shahab Bishehsari, Mohamad Mehdi Tabatabaei ∗ , Hassan Aliarabi, Daryoush Alipour, Pouya Zamani, Ahmad Ahmadi Department of animal Science, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran
a r t i c l e
i n f o
Article history: Received 6 October 2009 Received in revised form 18 January 2010 Accepted 16 February 2010 Available online 19 March 2010 Keywords: Mehraban lambs Cobalt Performance Nutrient digestibility
a b s t r a c t Two experiments were conducted to study the effects of different levels of dietary cobalt on performance, plasma and rumen metabolites and nutrient digestibility in Mehraban male lambs. Experiment 1: 28, 8–9-month-old lambs were randomly divided into four groups. Animals were fed a basal diet containing 0.088 mg Co/kg DM and were supplied with 0 (control), 0.25, 0.50, or 1.00 mg Co/kg DM as reagent grade CoSO4 ·7H2 O. The experiment lasted for 70 days. Experiment 2: four lambs from each group in Experiment 1 were randomly allocated to the individual metabolic crates to measure the effects of dietary Co on nutrient digestibility. Final body weight, average daily gain and gain efficiency were higher (p < 0.05) in the group supplemented with 0.50 mg Co/kg DM compared to other groups. Plasma glucose and vitamin B12 concentrations increased (p < 0.05) at all levels of Co supplementation on day 68 of the experiment and for vitamin B12 were higher (p < 0.05) at 0.50 and 1.00 mg Co/kg DM compared to 0.25 mg Co/kg DM. There was no significant difference among treatments for TVFA and ruminal fluid pH. Digestibility of dry matter, organic matter, crude protein and neutral detergent fiber increased (p < 0.05) by Co supplementation, but did not differ among Co supplied treatments. The obtained results showed that lambs fed the control diet containing 0.088 mg Co/kg DM had a reduced appetite and gained less than the supplemented animals, suggesting that the level of 0.088 mg Co/kg DM was inadequate for normal growth of Mehraban male lambs, and a total level of 0.58 mg Co/kg DM might be optimum level for enhancing performance. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Cobalt (Co) has been demonstrated to be an essential nutrient for ruminants and is required by ruminal microorganisms for the synthesis of vitamin B12 (McDowell, 2000). In ruminants, a Co-induced vitamin B12 deficiency results in reduced intake and ADG (Wang et al., 2007), decreased plasma, liver and ruminal vitamin B12 (Tiffany and Spears, 2005), elevated plasma methylmalonic acid
∗ Corresponding author. Tel.: +98 8114424090; fax: +98 8114424012. E-mail addresses: [email protected], [email protected] (M.M. Tabatabaei). 0921-4488/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2010.02.010
(MMA) and homocysteine (Stangl et al., 2000). Recent findings have demonstrated that 0.10 mg/kg DM of Co intake does not meet the rumen microbial requirements (Kisidayova et al., 2001; Johnson et al., 2004) and ADG, ADFI and nutrient digestibility had been increased in growing lambs when a diet with 0.086 mg Co/kg DM was supplemented with 0.50 mg Co/kg DM (Wang et al., 2007). Singh and Chhabra (1995) reported that 0.3–0.5 mg Co/kg DM enhanced ruminal microbial activity, fermentation and vitamin B12 synthesis. The effects of dietary Co concentration on growth, nutrient metabolism and ruminal microbial activity in Mehraban lambs are not well understood. Therefore, objectives of the present study were to determine effects of dietary Co levels on performance, plasma and
S. Bishehsari et al. / Small Ruminant Research 90 (2010) 170–173
rumen metabolites, nutrient digestibility and microbial protein synthesis in Mehraban lambs. 2. Materials and methods To conduct Experiment 1, 28 Mehraban male lambs 8–9-month-old with 37.1 ± 2.3 kg initial body weight were housed in individual pens (1.9 m × 1.1 m) and fed a basal diet containing 0.088 mg Co/kg DM. The basal diet was formulated to meet or exceed all nutrient requirements with the exception of Co (NRC, 1985). Diets were offered twice a day. After a 2-week adjustment period to the experimental feeding system, lambs were weighed and randomly assigned to four treatments with seven each including: control group fed the basal diet, and three supplemental groups fed the basal diet supplied with 0.25, 0.50, or 1.00 mg Co/kg DM. Co was added as CoSO4 ·7H2 O to the premix using wheat bran as a carrier. The experiment lasted for 70 days. Body weights were obtained before the lambs were fed in the morning on 2 consecutive days at the start and end of the experiment. Blood samples were collected on days 0, 34 and 68 via jugular vein in heparinized tubes before lambs were fed in the morning and their plasma were removed and frozen at −80 ◦ C until they were analyzed. On days 37 and 67, ruminal fluid, prior to the morning feeding, was collected by stomach tube and filtered through four layers cheesecloth. At the end of the growing period four lambs from each group were randomly selected and allocated to individual metabolism crate to study the effects of dietary Co on apparent nutrient digestibility for the Experiment 2. The digestibility trial lasted for 12 days with 6-day adaptation period and subsequent 6-day collection period during which daily feed intake, residual of diet and feces were collected and recorded. Lambs were fed the same diet as in Experiment 1. Daily fecal output was weighed and 20% was kept for subsequent analysis. The Co concentrations in feeds and feces were determined using an atomic absorption spectrophotometer (PHILIPS Model PU9100, single beam) according to the procedures of AOAC (1990). Feed and fecal samples were analyzed for dry matter (DM), organic matter (OM), crude protein (CP), ether extract (EE) and non-fiber carbohydrate (NFC) according to AOAC (1990) while the neutral detergent fiber (NDF) was measured according to Van Soest et al. (1991). Plasma concentration of vitamin B12
171
was determined using a competitive binding radio-immunoassay kit (ICN, Costa Mesa, CA, USA) and plasma glucose concentration by a commercial kit (Pars Azmon, Tehran, Iran). Content of volatile fatty acids was estimated in the laboratory by markham apparatus. All traits were analyzed according to a completely randomized design. The model used for analysis was: Yij = + ci + eij ; where , ci and eij are overall mean, level of cobalt and residual effects, respectively. Initial body weight was considered as a covariate for analysis of final body weight and average daily gain. Duncan’s multiple range test was used for comparison of means considering ˛ = 0.05. The GLM procedure of SAS (SAS Institute, 2004) used for analysis of data.
3. Results 3.1. Performance Final body weights, ADG, ADFI, and gain efficiency are shown in Table 1. Final BW, ADG, ADFI, and gain efficiency were improved (p < 0.05) by Co supplementation. Final BW, ADG and gain efficiency were higher (p < 0.05) in the treatment group supplemented with 0.50 mg Co/kg DM compared to other groups. There was no difference in ADFI among Co supplemented groups (p < 0.05). 3.2. Vitamin B12 and glucose There was no significant difference in plasma vitamin B12 concentration on day 0 among treatments (Table 1). Plasma vitamin B12 concentrations on day 68 of the experiment increased (p < 0.05) at all levels of Co supplementation. Plasma glucose concentrations differed only on day 68 of the experiment (p < 0.05) and increased at all levels of Co supplementation.
Table 1 Effects of dietary Co supplementation on plasma metabolites and performance of lambs. Co supplemental levels (mg/kg) 0 Vitamin B12 (pmol/L) d1 d 68
808.73 ± 101.9 213.26c ± 25.0
Glucose (mg/dL) d1 d 34 d 68
49.52d 176.33d 1508.1b 0.117d
± ± ± ±
0.5
789.51 ± 107.2 1404.69b ± 168.5
69.22 ± 3.32 68.28 ± 4.13 65.73b ± 3.40
Performance Final BW (kg) ADG (g) ADFI (g/day) Gain:feed
p-Value
0.25
782.54 ± 103.6 1745a ± 161.9
67.23 ± 3.94 71.07 ± 3.40 73.15a ± 4.21 51.37b 202.45b 1574.55a 0.128b
2.70 4.73 50.7 0.002
± ± ± ±
2.60 4.57 53.9 0.004
1 841.84 ± 98.3 1812.34a ± 105.4
68.33 ± 4.60 72.22 ± 3.05 73.51a ± 4.46 52.29a 215.71a 1618.33a 0.133a
± ± ± ±
2.74 8.12 45.2 0.003
68.78 ± 4.47 71.33 ± 3.66 74.13a ± 3.11 50.92c 196.73c 1580.4a 0.124c
± ± ± ±
2.62 4.92 57.2 0.003
0.7013 0.0001 0.8241 0.2192 0.001 0.3292 0.0001 0.0053 <0.0001
Means within the same row with different letters are significantly different (p < 0.05). Table 2 Effects of dietary Co supplementation on ruminal fluid pH and TVFA. Co supplemental levels (mg/kg)
p-Value
0
0.25
0.5
1
TVFA (mmol/day) d 33 d 67
82.58 ± 10.85 85.85 ± 12.54
86.38 ± 12.85 92.33 ± 15.04
89.47 ± 13.86 94.42 ± 9.82
91.57 ± 12.83 95.20 ± 10.20
0.5793 0.4736
Ruminal fluid pH d 33 d 67
7.153 ± 0.191 7.154 ± 0.190
7.136 ± 0.218 7.075 ± 0.242
7.073 ± 0.273 7.038 ± 0.161
74.05 ± 0.233 7.017 ± 0.268
0.8113 0.6667
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Table 3 Effects of dietary Co supplementation on apparent nutrient digestibility in lambs (DM basis). Item
Co supplemental levels (mg/kg) 0
DM OM CP EE NDF NFC
65.02b 66.47b 61.12b 65.91 45.11b 89.35
p-Value
0.25 ± ± ± ± ± ±
2.26 2.91 2.24 3.26 4.39 3.19
68.97a 70.85a 66.59a 68.52 51.73a 91.62
0.5 ± ± ± ± ± ±
2.25 2.20 3.33 2.17 4.31 2.69
69.71a 71.59a 66.88a 70.05 52.86a 92.71
1 ± ± ± ± ± ±
3.03 2.47 3.02 3.14 3.32 2.21
69.57a 71.33a 66.71a 69.11 53.20a 92.37
± ± ± ± ± ±
1.78 2.31 3.19 1.86 2.56 2.19
0.0494 0.0412 0.0478 0.2093 0.0309 0.3067
Means within the same row with different letters are significantly different (p < 0.05).
3.3. TVFA and ruminal fluid pH As it is shown in Table 2 there were no significant differences in ruminal fluid pH and TVFA production between treatments on days 33 and 67 of the experiment (p > 0.05). 3.4. Digestibility of nutrients Digestibility of DM, OM, CP and NDF increased (p < 0.05) by Co supplementation, but did not differ among Co supplemented treatments (Table 3). Co supplementation had no effect on digestibility of EE and NFC. 4. Discussion
(within 3 days) and dramatic increase in ruminal fluid succinate and a decline in ruminal propionate concentration (Kennedy et al., 1991). Considering propionate as major precursor of glucose in fed ruminants, lower proportions of ruminal propionate might be the reason for lower blood glucose concentration seen in the control group. Also considering importance of propionate as an energy source lowered performance is an expected result. It appears that the basal diet in the present experiment containing 0.088 mg Co/kg DM was no adequate for optimum vitamin B12 synthesis and glucose formation in the Mehraban lambs and supplemental 0.50 mg Co/kg DM seems to be optimum level for enhanced plasma vitamin B12 concentration in Mehraban lambs.
4.1. Performance
4.3. TVFA and ruminal fluid pH
As the obtained results showed a level of 0.088 mg Co/kg DM fed to the lambs was inadequate and Co supplementation increased lambs performance. It was supported by Johnson et al. (2004) who indicated that Omani goats fed with diet containing 0.12 mg Co/kg DM exhibited slower growth rate. When steers were fed a diet moderately Codeficient (0.04–0.05 mg/kg of DM), intake, ADG, and gain efficiency were decreased relative to Co supplemented steers (Tiffany et al., 2002). The present study showed that maximum growth rate in Mehraban lambs was achieved at a level of 0.50 mg/kg DM Co supplementation. Similar to our results, Wang et al. (2007) reported that a 0.50 mg Co/kg DM addition to a basal diet containing 0.08 mg Co/kg DM increased ADG and ADFI by Chinese Poll Dorset × Small Tailed Han lambs. These results suggest a higher Co requirement of lambs than recommended by NRC (1985) (i.e., 0.10–0.20 mg Co/kg DM).
Results of the present study indicate that there were no differences in TVFA production and ruminal fluid pH between treatments (p > 0.05). Similar to our results, Kisidayova et al. (2001) found the supplementation of Co had no effects on TVFA production. Adding large amounts (5.0 or 10.0 mg/kg) of Co to cultures did not affect TVFA in vitro (Hussein et al., 1994).
4.2. Vitamin B12 and glucose Results of the present study showed that control lambs were deficient in Co because the plasma values for vitamin B12 was below normal level (220 pmol/L) on day 68 of the experiment. Plasma vitamin B12 concentrations decreased when sheep (Kennedy et al., 1991) and cattle (Stangl et al., 1999) are fed Co-deficient diets. The results in plasma glucose concentration was in agreement with Tiffany and Spears (2005) who indicated that addition of supplemental Co to the basal diets increased (p < 0.01) plasma glucose concentrations of steers. Feeding diets severely deficient in Co (0.004 mg of Co/kg) to sheep caused an immediate
4.4. Digestibility of nutrients The results in nutrient digestibility were consistent with the findings of Kadim et al. (2003) who reported that low levels of dietary Co in goats resulted in lower apparent nutrient digestibility compared to goats supplemented with parenteral injections of vitamin B12 . Similarly, Wang et al. (2007) observed that a 0.50 mg Co/kg DM addition to a basal diet containing 0.08 mg Co/kg DM increased apparent nutrient digestibility (DM, OM, CP, metabolizable energy (ME) and ADF) in lambs. Divalent cations such as Co may act as bridges between the bacteria and plant cell walls, both of which tend to be negatively charged (Lopez-Guisa and Satter, 1992). When a negatively charged bacterium has difficulty attaching to similarly charged fiber particles, a divalent cation, possessing two positive charges, can serve as a link between the two negatively charged surfaces (Somers, 1983). Moreover, the increased digestibility of NDF indicates the possible role of Co in fiber digestion. This may largely be due to the increase in activity of fiber digesting bacteria (Tomlinson and Socha, 2003). The results of this study showed lambs fed the basal diet containing 0.088 mg Co/kg DM resulted in lowering the
S. Bishehsari et al. / Small Ruminant Research 90 (2010) 170–173
apparent nutrient digestibility compared to lambs supplemented with Co. The lower digestibility of the control group is likely due to several factors. Cobalt deficient diets have been indicated to reduce the number of rumen microorganisms (Marston and Smith, 1952), leading to lower rumen digestibility (Kadim et al., 2003). 5. Conclusion A diet containing 0.088 mg Co/kg DM did not meet the Co requirement of Mehraban lambs during the growing period. Based on growth performance, nutrient digestibility and plasma vitamin B12 concentration an addition of 0.50 mg Co/kg DM to the diet (total of dietary Co 0.58 mg/kg DM) will meet the Co requirement of Mehraban lambs. References AOAC, 1990. Official Methods for Analysis, 15th ed. Association of Official Analytical Chemists, Arlington, VA, pp. 69–90. Johnson, E.H., AL-Habsi, K., Kaplan, E., Srikandakumar, A., Kadim, I.T., Annamalai, K., AL-Busaidy, R., Mahgoub, O., 2004. Caprine hepatic lipidosis induced through the intake of low levels of dietary cobalt. Vet. J. 168, 174–179. Hussein, H.S., Fahey Jr., G.C., Wolf, B.W., Berger, L.L., 1994. Effects of cobalt on in vitro fiber digestion of forages and by-products containing fiber. J. Dairy Sci. 77, 3432–3440. Kadim, I.T., Johnson, E.H., Mahgoub, O., Srikandakumar, A., ALAjmi, D.S., AL-Maqbaly, R.S., AL-Saqri, N.M., 2003. Effect of low levels of dietary cobalt on apparent nutrient digestibility in Omani goats. Anim. Feed Sci. Technol. 109, 209–216. Kennedy, D.G., Young, P.B., McCaughey, W.J., Kennedy, S., Blanchflower, W.J., 1991. Rumen succinate production may ameliorate the effects of cobalt-vitamin B12 deficiency on methylmalonyl CoA mutase in sheep. J. Nutr. 12, 1236–1242.
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Kisidayova, S., Sviatko, P., Siroka, P., Jalc, D., 2001. Effect of elevated cobalt intake on fermentative parameters and protozoan population in RUSITEC. Anim. Feed Sci. Technol. 91, 223–232. Lopez-Guisa, J.M., Satter, L.D., 1992. Effect of copper and cobalt addition on digestion and growth in heifers fed diets containing alfalfa silage and corn crop residues. J. Dairy Sci. 75, 247–256. Marston, H.R., Smith, R.M., 1952. Control of cobalt deficiency in sheep by injection vitamin B12 . Nature (London) 17, 791. McDowell, L.R., 2000. Vitamins in Animal and Human Nutrition, 2nd ed. Iowa State Press, Ames. USA. NRC, 1985. Nutrient Requirements of Sheep, 6th ed. National Academy Press, Washington, DC, USA. SAS Institute, 2004. User’s Guide. Version 9.1: Statistics. SAS Institute, Cary, NC. Singh, K.K., Chhabra, A., 1995. Effect of dietary cobalt on ruminal vitamin B12 synthesis and rumen metabolites. J. Nucl. Agric. Biol. 24, 112–116. Somers, G.F., 1983. The affinity of onion cell walls for calcium ions. Am. J. Bot. 60, 987–990. Stangl, G.I., Schwarz, F.J., Kirchgessner, M., 1999. Moderate longterm cobalt-deficiency affects liver, brain and erythrocyte lipids and lipoproteins of cattle. Nutr. Res. 19, 415–427. Stangl, G.I., Schwarz, F.J., Müller, H., Kirchgessner, M., 2000. Evaluation of the cobalt requirement of beef cattle based on vitamin B12 , folate, homocysteine and methylmalonic acid. Br. J. Nutr. 84, 645–653. Tiffany, M.E., Spears, J.W., 2005. Differential responses to dietary cobalt in finishing steers fed corn vs. barley-base diets. J. Anim. Sci. 83, 2580–2589. Tiffany, M.E., Spears, J.W., Xi, L., Valdez, F.R., 2002. Effects of dietary cobalt source and concentration on performance, vitamin B12 status, and ruminal and plasma metabolites in growing and finishing steers. J. Anim. Sci. 80 (Suppl. 1), 183. Tomlinson, D., Socha, M., 2003. More cobalt for mature cows? Feed Int. 8, 20–22. Van Soest, P.J., Roberston, J.B., Lewis, B.A., 1991. Methods for dietary fibre NDF and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Wang, R.L., Kong, X.H., Zhang, Y.Z., Zhu, X.P., Narenbatu, Z.H. Jia., 2007. Influence of dietary cobalt on performance, nutrient digestibility and plasma metabolites in lambs. Anim. Feed Sci. Technol. 135, 346–352.
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Effect of dietary cobalt supplementation on plasma and rumen metabolites in Mehraban lambs Shahab Bishehsari, Mohamad Mehdi Tabatabaei ∗ , Hassan Aliarabi, Daryoush Alipour, Pouya Zamani, Ahmad Ahmadi Department of animal Science, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran
a r t i c l e
i n f o
Article history: Received 6 October 2009 Received in revised form 18 January 2010 Accepted 16 February 2010 Available online 19 March 2010 Keywords: Mehraban lambs Cobalt Performance Nutrient digestibility
a b s t r a c t Two experiments were conducted to study the effects of different levels of dietary cobalt on performance, plasma and rumen metabolites and nutrient digestibility in Mehraban male lambs. Experiment 1: 28, 8–9-month-old lambs were randomly divided into four groups. Animals were fed a basal diet containing 0.088 mg Co/kg DM and were supplied with 0 (control), 0.25, 0.50, or 1.00 mg Co/kg DM as reagent grade CoSO4 ·7H2 O. The experiment lasted for 70 days. Experiment 2: four lambs from each group in Experiment 1 were randomly allocated to the individual metabolic crates to measure the effects of dietary Co on nutrient digestibility. Final body weight, average daily gain and gain efficiency were higher (p < 0.05) in the group supplemented with 0.50 mg Co/kg DM compared to other groups. Plasma glucose and vitamin B12 concentrations increased (p < 0.05) at all levels of Co supplementation on day 68 of the experiment and for vitamin B12 were higher (p < 0.05) at 0.50 and 1.00 mg Co/kg DM compared to 0.25 mg Co/kg DM. There was no significant difference among treatments for TVFA and ruminal fluid pH. Digestibility of dry matter, organic matter, crude protein and neutral detergent fiber increased (p < 0.05) by Co supplementation, but did not differ among Co supplied treatments. The obtained results showed that lambs fed the control diet containing 0.088 mg Co/kg DM had a reduced appetite and gained less than the supplemented animals, suggesting that the level of 0.088 mg Co/kg DM was inadequate for normal growth of Mehraban male lambs, and a total level of 0.58 mg Co/kg DM might be optimum level for enhancing performance. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Cobalt (Co) has been demonstrated to be an essential nutrient for ruminants and is required by ruminal microorganisms for the synthesis of vitamin B12 (McDowell, 2000). In ruminants, a Co-induced vitamin B12 deficiency results in reduced intake and ADG (Wang et al., 2007), decreased plasma, liver and ruminal vitamin B12 (Tiffany and Spears, 2005), elevated plasma methylmalonic acid
∗ Corresponding author. Tel.: +98 8114424090; fax: +98 8114424012. E-mail addresses: [email protected], [email protected] (M.M. Tabatabaei). 0921-4488/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2010.02.010
(MMA) and homocysteine (Stangl et al., 2000). Recent findings have demonstrated that 0.10 mg/kg DM of Co intake does not meet the rumen microbial requirements (Kisidayova et al., 2001; Johnson et al., 2004) and ADG, ADFI and nutrient digestibility had been increased in growing lambs when a diet with 0.086 mg Co/kg DM was supplemented with 0.50 mg Co/kg DM (Wang et al., 2007). Singh and Chhabra (1995) reported that 0.3–0.5 mg Co/kg DM enhanced ruminal microbial activity, fermentation and vitamin B12 synthesis. The effects of dietary Co concentration on growth, nutrient metabolism and ruminal microbial activity in Mehraban lambs are not well understood. Therefore, objectives of the present study were to determine effects of dietary Co levels on performance, plasma and
S. Bishehsari et al. / Small Ruminant Research 90 (2010) 170–173
rumen metabolites, nutrient digestibility and microbial protein synthesis in Mehraban lambs. 2. Materials and methods To conduct Experiment 1, 28 Mehraban male lambs 8–9-month-old with 37.1 ± 2.3 kg initial body weight were housed in individual pens (1.9 m × 1.1 m) and fed a basal diet containing 0.088 mg Co/kg DM. The basal diet was formulated to meet or exceed all nutrient requirements with the exception of Co (NRC, 1985). Diets were offered twice a day. After a 2-week adjustment period to the experimental feeding system, lambs were weighed and randomly assigned to four treatments with seven each including: control group fed the basal diet, and three supplemental groups fed the basal diet supplied with 0.25, 0.50, or 1.00 mg Co/kg DM. Co was added as CoSO4 ·7H2 O to the premix using wheat bran as a carrier. The experiment lasted for 70 days. Body weights were obtained before the lambs were fed in the morning on 2 consecutive days at the start and end of the experiment. Blood samples were collected on days 0, 34 and 68 via jugular vein in heparinized tubes before lambs were fed in the morning and their plasma were removed and frozen at −80 ◦ C until they were analyzed. On days 37 and 67, ruminal fluid, prior to the morning feeding, was collected by stomach tube and filtered through four layers cheesecloth. At the end of the growing period four lambs from each group were randomly selected and allocated to individual metabolism crate to study the effects of dietary Co on apparent nutrient digestibility for the Experiment 2. The digestibility trial lasted for 12 days with 6-day adaptation period and subsequent 6-day collection period during which daily feed intake, residual of diet and feces were collected and recorded. Lambs were fed the same diet as in Experiment 1. Daily fecal output was weighed and 20% was kept for subsequent analysis. The Co concentrations in feeds and feces were determined using an atomic absorption spectrophotometer (PHILIPS Model PU9100, single beam) according to the procedures of AOAC (1990). Feed and fecal samples were analyzed for dry matter (DM), organic matter (OM), crude protein (CP), ether extract (EE) and non-fiber carbohydrate (NFC) according to AOAC (1990) while the neutral detergent fiber (NDF) was measured according to Van Soest et al. (1991). Plasma concentration of vitamin B12
171
was determined using a competitive binding radio-immunoassay kit (ICN, Costa Mesa, CA, USA) and plasma glucose concentration by a commercial kit (Pars Azmon, Tehran, Iran). Content of volatile fatty acids was estimated in the laboratory by markham apparatus. All traits were analyzed according to a completely randomized design. The model used for analysis was: Yij = + ci + eij ; where , ci and eij are overall mean, level of cobalt and residual effects, respectively. Initial body weight was considered as a covariate for analysis of final body weight and average daily gain. Duncan’s multiple range test was used for comparison of means considering ˛ = 0.05. The GLM procedure of SAS (SAS Institute, 2004) used for analysis of data.
3. Results 3.1. Performance Final body weights, ADG, ADFI, and gain efficiency are shown in Table 1. Final BW, ADG, ADFI, and gain efficiency were improved (p < 0.05) by Co supplementation. Final BW, ADG and gain efficiency were higher (p < 0.05) in the treatment group supplemented with 0.50 mg Co/kg DM compared to other groups. There was no difference in ADFI among Co supplemented groups (p < 0.05). 3.2. Vitamin B12 and glucose There was no significant difference in plasma vitamin B12 concentration on day 0 among treatments (Table 1). Plasma vitamin B12 concentrations on day 68 of the experiment increased (p < 0.05) at all levels of Co supplementation. Plasma glucose concentrations differed only on day 68 of the experiment (p < 0.05) and increased at all levels of Co supplementation.
Table 1 Effects of dietary Co supplementation on plasma metabolites and performance of lambs. Co supplemental levels (mg/kg) 0 Vitamin B12 (pmol/L) d1 d 68
808.73 ± 101.9 213.26c ± 25.0
Glucose (mg/dL) d1 d 34 d 68
49.52d 176.33d 1508.1b 0.117d
± ± ± ±
0.5
789.51 ± 107.2 1404.69b ± 168.5
69.22 ± 3.32 68.28 ± 4.13 65.73b ± 3.40
Performance Final BW (kg) ADG (g) ADFI (g/day) Gain:feed
p-Value
0.25
782.54 ± 103.6 1745a ± 161.9
67.23 ± 3.94 71.07 ± 3.40 73.15a ± 4.21 51.37b 202.45b 1574.55a 0.128b
2.70 4.73 50.7 0.002
± ± ± ±
2.60 4.57 53.9 0.004
1 841.84 ± 98.3 1812.34a ± 105.4
68.33 ± 4.60 72.22 ± 3.05 73.51a ± 4.46 52.29a 215.71a 1618.33a 0.133a
± ± ± ±
2.74 8.12 45.2 0.003
68.78 ± 4.47 71.33 ± 3.66 74.13a ± 3.11 50.92c 196.73c 1580.4a 0.124c
± ± ± ±
2.62 4.92 57.2 0.003
0.7013 0.0001 0.8241 0.2192 0.001 0.3292 0.0001 0.0053 <0.0001
Means within the same row with different letters are significantly different (p < 0.05). Table 2 Effects of dietary Co supplementation on ruminal fluid pH and TVFA. Co supplemental levels (mg/kg)
p-Value
0
0.25
0.5
1
TVFA (mmol/day) d 33 d 67
82.58 ± 10.85 85.85 ± 12.54
86.38 ± 12.85 92.33 ± 15.04
89.47 ± 13.86 94.42 ± 9.82
91.57 ± 12.83 95.20 ± 10.20
0.5793 0.4736
Ruminal fluid pH d 33 d 67
7.153 ± 0.191 7.154 ± 0.190
7.136 ± 0.218 7.075 ± 0.242
7.073 ± 0.273 7.038 ± 0.161
74.05 ± 0.233 7.017 ± 0.268
0.8113 0.6667
172
S. Bishehsari et al. / Small Ruminant Research 90 (2010) 170–173
Table 3 Effects of dietary Co supplementation on apparent nutrient digestibility in lambs (DM basis). Item
Co supplemental levels (mg/kg) 0
DM OM CP EE NDF NFC
65.02b 66.47b 61.12b 65.91 45.11b 89.35
p-Value
0.25 ± ± ± ± ± ±
2.26 2.91 2.24 3.26 4.39 3.19
68.97a 70.85a 66.59a 68.52 51.73a 91.62
0.5 ± ± ± ± ± ±
2.25 2.20 3.33 2.17 4.31 2.69
69.71a 71.59a 66.88a 70.05 52.86a 92.71
1 ± ± ± ± ± ±
3.03 2.47 3.02 3.14 3.32 2.21
69.57a 71.33a 66.71a 69.11 53.20a 92.37
± ± ± ± ± ±
1.78 2.31 3.19 1.86 2.56 2.19
0.0494 0.0412 0.0478 0.2093 0.0309 0.3067
Means within the same row with different letters are significantly different (p < 0.05).
3.3. TVFA and ruminal fluid pH As it is shown in Table 2 there were no significant differences in ruminal fluid pH and TVFA production between treatments on days 33 and 67 of the experiment (p > 0.05). 3.4. Digestibility of nutrients Digestibility of DM, OM, CP and NDF increased (p < 0.05) by Co supplementation, but did not differ among Co supplemented treatments (Table 3). Co supplementation had no effect on digestibility of EE and NFC. 4. Discussion
(within 3 days) and dramatic increase in ruminal fluid succinate and a decline in ruminal propionate concentration (Kennedy et al., 1991). Considering propionate as major precursor of glucose in fed ruminants, lower proportions of ruminal propionate might be the reason for lower blood glucose concentration seen in the control group. Also considering importance of propionate as an energy source lowered performance is an expected result. It appears that the basal diet in the present experiment containing 0.088 mg Co/kg DM was no adequate for optimum vitamin B12 synthesis and glucose formation in the Mehraban lambs and supplemental 0.50 mg Co/kg DM seems to be optimum level for enhanced plasma vitamin B12 concentration in Mehraban lambs.
4.1. Performance
4.3. TVFA and ruminal fluid pH
As the obtained results showed a level of 0.088 mg Co/kg DM fed to the lambs was inadequate and Co supplementation increased lambs performance. It was supported by Johnson et al. (2004) who indicated that Omani goats fed with diet containing 0.12 mg Co/kg DM exhibited slower growth rate. When steers were fed a diet moderately Codeficient (0.04–0.05 mg/kg of DM), intake, ADG, and gain efficiency were decreased relative to Co supplemented steers (Tiffany et al., 2002). The present study showed that maximum growth rate in Mehraban lambs was achieved at a level of 0.50 mg/kg DM Co supplementation. Similar to our results, Wang et al. (2007) reported that a 0.50 mg Co/kg DM addition to a basal diet containing 0.08 mg Co/kg DM increased ADG and ADFI by Chinese Poll Dorset × Small Tailed Han lambs. These results suggest a higher Co requirement of lambs than recommended by NRC (1985) (i.e., 0.10–0.20 mg Co/kg DM).
Results of the present study indicate that there were no differences in TVFA production and ruminal fluid pH between treatments (p > 0.05). Similar to our results, Kisidayova et al. (2001) found the supplementation of Co had no effects on TVFA production. Adding large amounts (5.0 or 10.0 mg/kg) of Co to cultures did not affect TVFA in vitro (Hussein et al., 1994).
4.2. Vitamin B12 and glucose Results of the present study showed that control lambs were deficient in Co because the plasma values for vitamin B12 was below normal level (220 pmol/L) on day 68 of the experiment. Plasma vitamin B12 concentrations decreased when sheep (Kennedy et al., 1991) and cattle (Stangl et al., 1999) are fed Co-deficient diets. The results in plasma glucose concentration was in agreement with Tiffany and Spears (2005) who indicated that addition of supplemental Co to the basal diets increased (p < 0.01) plasma glucose concentrations of steers. Feeding diets severely deficient in Co (0.004 mg of Co/kg) to sheep caused an immediate
4.4. Digestibility of nutrients The results in nutrient digestibility were consistent with the findings of Kadim et al. (2003) who reported that low levels of dietary Co in goats resulted in lower apparent nutrient digestibility compared to goats supplemented with parenteral injections of vitamin B12 . Similarly, Wang et al. (2007) observed that a 0.50 mg Co/kg DM addition to a basal diet containing 0.08 mg Co/kg DM increased apparent nutrient digestibility (DM, OM, CP, metabolizable energy (ME) and ADF) in lambs. Divalent cations such as Co may act as bridges between the bacteria and plant cell walls, both of which tend to be negatively charged (Lopez-Guisa and Satter, 1992). When a negatively charged bacterium has difficulty attaching to similarly charged fiber particles, a divalent cation, possessing two positive charges, can serve as a link between the two negatively charged surfaces (Somers, 1983). Moreover, the increased digestibility of NDF indicates the possible role of Co in fiber digestion. This may largely be due to the increase in activity of fiber digesting bacteria (Tomlinson and Socha, 2003). The results of this study showed lambs fed the basal diet containing 0.088 mg Co/kg DM resulted in lowering the
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apparent nutrient digestibility compared to lambs supplemented with Co. The lower digestibility of the control group is likely due to several factors. Cobalt deficient diets have been indicated to reduce the number of rumen microorganisms (Marston and Smith, 1952), leading to lower rumen digestibility (Kadim et al., 2003). 5. Conclusion A diet containing 0.088 mg Co/kg DM did not meet the Co requirement of Mehraban lambs during the growing period. Based on growth performance, nutrient digestibility and plasma vitamin B12 concentration an addition of 0.50 mg Co/kg DM to the diet (total of dietary Co 0.58 mg/kg DM) will meet the Co requirement of Mehraban lambs. References AOAC, 1990. Official Methods for Analysis, 15th ed. Association of Official Analytical Chemists, Arlington, VA, pp. 69–90. Johnson, E.H., AL-Habsi, K., Kaplan, E., Srikandakumar, A., Kadim, I.T., Annamalai, K., AL-Busaidy, R., Mahgoub, O., 2004. Caprine hepatic lipidosis induced through the intake of low levels of dietary cobalt. Vet. J. 168, 174–179. Hussein, H.S., Fahey Jr., G.C., Wolf, B.W., Berger, L.L., 1994. Effects of cobalt on in vitro fiber digestion of forages and by-products containing fiber. J. Dairy Sci. 77, 3432–3440. Kadim, I.T., Johnson, E.H., Mahgoub, O., Srikandakumar, A., ALAjmi, D.S., AL-Maqbaly, R.S., AL-Saqri, N.M., 2003. Effect of low levels of dietary cobalt on apparent nutrient digestibility in Omani goats. Anim. Feed Sci. Technol. 109, 209–216. Kennedy, D.G., Young, P.B., McCaughey, W.J., Kennedy, S., Blanchflower, W.J., 1991. Rumen succinate production may ameliorate the effects of cobalt-vitamin B12 deficiency on methylmalonyl CoA mutase in sheep. J. Nutr. 12, 1236–1242.
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