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Effects of hainanmycin or monensin supplementation on ruminal protein metabolism and populations of proteolytic bacteria in Holstein heifers
Animal Feed Science and Technology 201 (2015) 99–103
Contents lists available at ScienceDirect
Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci
Short communication
Effects of hainanmycin or monensin supplementation on ruminal protein metabolism and populations of proteolytic bacteria in Holstein heifers Z.B. Wang a , H.S Xin b , J. Bao b , C.Y. Duan c , Y. Chen a , Y.L. Qu a,∗ a b c
College of Animal Science & Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China Xijie Feed Co., Ltd. Shenyang 110164, China
a r t i c l e
i n f o
Article history: Received 22 July 2014 Received in revised form 1 January 2015 Accepted 2 January 2015 Keywords: Heifer Hainanmycin Monensin Proteolytic bacteria Protein metabolism
a b s t r a c t Three ruminally cannulated cows were used in a 3 × 3 Latin square design (21-d periods) to evaluate effects of hainanmycin (HAI) or monensin (MON) supplementation on ruminal protein metabolism and populations of proteolytic bacteria in Holstein heifers. The three dietary treatments were: (1) basal diet (control); (2) basal plus 20 mg/heifer per day HAI and (3) basal plus 350 mg/heifer per day MON. The supplementation with HAI and MON had minor effects on ruminal pH and total VFA. In general, HAI and MON decreased acetate, butyrate concentrations, and acetate to propionate ratio, and increased propionate concentration. Supplementation of HAI and Mon resulted in higher relative population sizes of the genus Prevotella and Prevotella ruminicola, and fewer Butyrivibrio fibrisolvens and hyperammonia-producing bacteria (P<0.05). HAI supplementation increased concentrations of peptide nitrogen and amino acid nitrogen, and reduced ammonia concentration (P<0.05), and decreased peptidase and deaminase activities (P<0.05). Overall, HAI was as effective as monensin as ionophore in regulation of ruminal protein metabolism and populations of proteolytic bacteria for dairy cows. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The breakdown of protein to ammonia in the rumen may lead to an appreciable loss of nitrogen to the animal (Wallace, 1996). Bacteria are thought to be responsible for the majority of the breakdown in the rumen (Wallace et al., 1987). Many of ruminal bacteria have peptidase and deaminase activities, for instance Prevotella sp. and hyper-ammonia-producing (HAP) bacteria (Wallace et al., 1997). Monensin caused an accumulation in amino acids N and peptide N by mixed ruminal microorganisms in vitro, and decrease rumen ammonia production (Chen and Russell, 1991; Ghorbani et al., 2008). Much of the decrease in ruminal ammonia production caused by monensin addition can be specifically attributed to the inhibitory effects on HAP bacteria (Eschenlauer et al., 2002). Hainanmycin is a polyether monocarboxylic acid ionophore from one of fermentation products of a
Abbreviations: ADF, acid detergent fiber; A:P, acetate to propionate ratio; BW, body weight; CP, crude protein; DM, dry matter; HAI, hainanmycin; HAP, hyper-ammonia-producing; ME, metabolisable energy; MNA, methoxynapthylamide; MON, monensin; N, nitrogen; NDF, neutral detergent fiber; RPS, relative population sizes; VFA, volatile fatty acid. ∗ Corresponding author. Tel.: +86 0459 6819195; fax: +86 0459 6819195. E-mail address: [email protected] (Y.L. Qu). http://dx.doi.org/10.1016/j.anifeedsci.2015.01.001 0377-8401/© 2015 Elsevier B.V. All rights reserved.
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rare streptomycete, Streptomyces padanus var dangfangeus, which was isolated from soil sample collected in Hainan province of China. The chemical formula of hainanmycin is C47 H79 O15 N, which capable of forming stable complexes to monovalent cations with a specific affinity for Na+ . Hainanmycin lowers ruminal ammonia production in the sheep, and increase ruminal concentrations of peptide N and amino acid N, and affects growth of ammonia-producing bacteria in vitro (Wang et al., 2013). However, there is little information on the effects of hainanmycin on ruminal protein metabolism and populations of proteolytic bacteria in cattle. The objective of the present study was to evaluate the effects of the supplementation of hainanmycin on ruminal protein metabolism and populations of proteolytic bacteria in Holstein heifers. 2. Material and methods The protocol used in this experiment was approved by the Northeast Agricultural University Animal Science and Technology College Animal Care and Use Committee. 2.1. Feed additives The hainanmycin was obtained from Sheng Li Inc. Shandong province, China. 2.2. Animals and treatments Three Holstein heifers fitted with ruminal cannulas were used in a 3 × 3 Latin square design to evaluate effects of hainanmycin (HAI) or monensin (MON) supplementation on ruminal protein metabolism and populations of proteolytic bacteria in Holstein heifers. The heifers were fed a diet which consisted of dry-rolled corn (99.7 g/kg), soybean meal (109.7 g/kg), Chinese wild rye grass (378.9 g/kg), and corn silage (358.9 g/kg). The CP, NDF, ADF, and ME contents of the diet (DM basis) were 128.8 g/kg, 454.2 g/kg, 319.4 g/kg, and 9.17 MJ/kg, respectively. Treatments consisted of: (1) basal diet (no ionophore); (2) basal plus 20 mg/heifer per day HAI; and (3) basal plus 350 mg/heifer per day MON. The HAI dose was calculated according to the heifer’s body weight (0.05 mg/kg BW per day) (Ren et al., 1998). Each period lasted 21 with 19 d of adaptation and 2 d of sample collection. Diet was offered twice daily at 08:00 and 18:00 h and 10.5 kg (DM basis)/day per heifer. 2.3. Sampling and measurements On d 20 and 21 of each period, ruminal fluid was collected immediately at 0, 2, 4, 6, 8, and 10 h for ruminal pH, VFA concentration, protein fractions (peptide N, amino acid N, and NH3 ), and total DNA extraction. Strained ruminal fluid at 4 h after feeding was used to determine in vitro protease and deaminase activities (Siddons and Paradine 1981). The peptidase activity was assayed using the substrate Glycine-Arginine methoxynapthylamide (MNA) (Wallace and McKain 1989). Concentrations of peptide N plus amino acid N in ruminal fluid were determined as described by Winter et al. (1964). In this method, the tungstic acid precipitates small peptides only partially (Raacke 1957), therefore the values for peptides N as determined by tungstic acid would be low in present experiment. PCR assays for enumeration of selected bacterial species were performed according to the methods described by Stevenson and Weimer (2007). Enumeration for HAP were determined according to the methods described by Russell et al. (1988). 2.4. Statistical analysis Data were statistically analyzed as a 3 × 3 Latin square design using the PROC MIXED procedure of SAS (SAS Institute, version 9.1) according to the following model. Yijk = + Ti + Pj + Ck + Eijk . Where Yijk is observation, is overall mean, Ti is treatment (i = 1–3), Pj is period (j = 1–3), Ck is heifer (k = 1–3), and Eijk is residual error. Differences were declared significant at P<0.05. Results are reported as least squares means ± standard error of the means. 3. Results and discussion 3.1. Ruminal fermentation characteristics Ruminal fermentation characteristics are shown in Fig. 1 and Table 1. In general, the supplementation with HAI and MON had minor effects on ruminal pH and total VFA. Similar to the results in the present trial with HAI, HAI also had no changed pH and total VFA in our previous study in vitro, (Wang et al., 2013). Two ionophores caused lower (P<0.05) acetate concentrations at 6 h after feeding, and higher (P<0.05) concentrates of propionate at 0, 2, 4, and 8 h after feeding. Supplementation of HAI or MON decreased (P<0.05) acetate: propionate ratio at 0, 2, 4, and 6 h after feeding. Changes in acetate, propionate and their ratio in the present study were in agreement with the observations of Ren et al. (1998). HAI and MON caused lower (P<0.05) concentrations of butyrate at 0 and 2 h after feeding, respectively. Concentrations of valerate or isobutyrate were not affected by HAI and MON (data not shown). MON decreased (P<0.05) isovalerate concentrations at 2 and 6 h after feeding compare with control, and HAI also caused lower concentrations of isovalerate (P<0.05) at 6 h after feeding (data not shown).
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Fig. 1. Effects of hainanmycin (HAI) or monensin (MON) on ruminal pH in Holstein heifers. a−b Means within 2 h with different superscripts differ (P<0.05). Control = no ionophore; HAI = hainanmycin (20 mg/heifer per day); MON = monensin (350 mg/heifer per day).
The branched-chain VFA are derived from amino acid catabolism in the rumen (Mackie and White, 1990). The reduction in isovalerate concentrations implied that amino acid deamination were inhibited. 3.2. The number of proteolytic bacteria Supplementation of HAI and MON increased (P<0.05) relative population sizes (RPS) of the genus Prevotella and P. ruminicola (Table 2). It was similar to the results of Weimer et al. (2008), who observed that MON increased RPS of ruminal the genus Prevotella and P. ruminicola in lactating dairy cows. Supplementation of HAI and MON reduced RPS of B. fibrisolvens. In the present study, measured Butyrivibrio fibrisolvens is a highly atypical strain, but the true populations of B. fibrisolvens are much larger (Tajima et al., 1999). HAI and MON had no effect (P>0.05) on RPS of other bacteria compared with control. Supplementation of HAI and MON caused a large decrease in numbers of hyper-ammonia-producing (HAP) bacteria, which was associated with a decrease in HAP bacteria in our previous study (Wang et al., 2013). Chen and Russell (1989) demonstrated that MON reduced NH3 –N concentration through the inhibition of the HAP bacteria that are responsible for the production of most of the ammonia. Table 1 Effects of hainanmycin (HAI) or monensin (MON) on ruminal VFA in Holstein heifers. Acids, mol/L
Total VFA
Acetate
Propionate
Butyrate
A:PA
a−c
Hour after feeding
0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10
TreatmentB Control
HAI
MON
80.5 99.4 90.7 87.2 80.9 73.9 61.0 70.9 65.8 64.5a 60.1 54.9 13.0b 19.8b 16.8b 15.3 13.8c 12.8 5.28a 6.97 6.68 6.17 5.89 5.18 4.71a 3.40a 3.94a 4.23a 4.36 4.28
77.5 94.4 87.7 84.3 77.0 72.5 53.3 64.1 59.7 58.7b 51.7 51.8 16.0a 21.2a 19.5a 17.1 14.7b 13.7 5.16a 6.40 6.33 6.11 5.52 5.06 3.29b 3.03b 3.14b 3.38b 3.54 3.49
74.8 93.5 86.9 83.1 73.0 71.6 54.5 65.3 60.7 59.4b 54.8 51.6 16.6a 20.9a 19.4a 17.6 15.5a 14.8 4.42b 6.50 6.19 6.04 5.63 5.05 3.33b 3.13b 3.08b 3.44b 3.53 3.80
Means within a row with different superscripts differ (P<0.05). A:P represents acetate to propionate ratio. B Control = no ionophore; HAI = hainanmycin (20 mg/heifer per day); MON = monensin (350 mg/heifer per day).
A
SEM
P-value
2.19 1.45 1.09 0.83 2.21 3.17 2.03 1.39 1.20 0.57 2.47 3.33 0.23 0.20 0.17 0.43 0.16 0.32 0.13 0.14 0.14 0.03 0.09 0.11 0.05 0.09 0.02 0.04 0.21 0.31
0.23 0.09 0.13 0.07 0.13 0.78 0.11 0.07 0.06 0.02 0.15 0.62 0.01 0.04 0.01 0.06 0.02 0.08 0.04 0.09 0.14 0.08 0.11 0.53 0.002 0.04 0.001 0.004 0.09 0.24
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Table 2 Effects of hainanmycin (HAI) or monensin (MON) on the number of proteolytic bacteria and enzyme activities in Holstein heifers. TreatmentF
Item
Target bacterium, RPSA , % Genus Prevotella Prevotella ruminicola Prevotella brevis Prevotella bryantii Butyrivibrio fibrisolvens Selenomonas ruminantium Streptococcus bovis HAP bacteriaB Enzyme activities Proteinase activityC Peptidase activityD Deaminase activityE a−b A B C D E F
Control
HAI
MON
42.33b 1.66b 0.17 0.68 0.034a 0.54 0.00172 3.70a
45.76a 1.95a 0.16 0.64 0.021b 0.60 0.00167 0.69b
47.21a 1.96a 0.16 0.63 0.021b 0.62 0.00167 0.69b
0.64 7.39a 0.15a
0.65 4.54b 0.10b
0.69 4.33b 0.10b
SEM
P-value
1.33 0.1 0.01 0.07 0.001 0.07 0.0001 0.16
0.01 0.03 0.78 0.49 <0.01 0.31 0.70 <0.001
0.05 0.77 0.01
0.60 0.03 <0.01
Means within a row with different superscripts differ (P < 0.05). Relative population sizes: percentages of the 16 S rRNA gene copy number of the total bacterial domain. HAP represents hyper-ammonia-producing, most probable numbers (106 /mL). mg of 14 C-casein hydrolyzed/h per mg of protein. Assayed using the substrate Glycine-Arginine methoxynapthylamide, nmols/min per mg protein. nmol of ammonia produced/h per mg of protein. Control = no ionophore; HAI = hainanmycin (20 mg/heifer per day); MON = monensin (350 mg/heifer per day).
Table 3 Effects of hainanmycin (HAI) or monensin (MON) on contentrations of ruminal peptide N, Amino acid N, and NH3 –N in Holstein heifers. Item
Hour after feeding
Peptide N, mmol/L
0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10
Amino acid N, mmol/L
NH3 –N, mg/dL
a−b A
TreatmentA Control
HAI
MON
1.99 5.65b 4.42b 3.47b 2.82 2.21 2.62 5.99b 3.50b 2.73b 2.58 2.55 14.11a 29.78 17.59 8.14 8.13 9.88
2.30 7.42a 6.01a 4.52a 3.20 2.45 2.88 7.06a 5.50a 4.47a 3.83 3.23 11.74b 28.79 15.48 6.77 7.02 8.07
2.18 7.30a 6.10a 4.44a 3.35 2.47 2.42 6.96a 5.68a 4.62a 3.59 3.14 11.00b 28.41 15.73 6.52 6.89 8.39
SEM
P-value
0.25 0.24 0.15 0.14 0.19 0.15 0.25 0.17 0.14 0.15 0.44 0.27 0.27 0.44 0.62 0.30 0.27 0.48
0.57 0.03 0.01 0.03 0.20 0.35 0.37 0.04 0.01 0.01 0.18 0.21 0.01 0.16 0.13 0.06 0.07 0.11
Means within a row with different superscripts differ (P<0.05). Control = no ionophore; HAI = hainanmycin (20 mg/heifer per day); MON = monensin (350 mg/heifer per day).
3.3. Protein fractions and enzyme activities Concentrations of ruminal peptide N, amino acid N, and NH3 –N in heifers are presented in Table 3. Supplementation of HAI and MON increased (P<0.05) peptide N and amino acid N concentrations at 2, 4, and 6 h after feeding, and decreased (P<0.05) NH3 –N concentrations at 0 h after feeding. The accumulation of amino acid N and the decrease in NH3 –N implied that amino acid deamination was inhibited. The inhibition of amino acid deamination has practical implications because it may increase ruminal escape of dietary protein and improve the efficiency of N use in the rumen (Nagaraja et al., 1997). HAI and MON decreased (P<0.05) ruminal peptidase activities, which indicated that hainanmycin inhibited degradation of ruminal peptides to amino acids. HAI and MON also decreased ruminal deaminase activities, which corresponded with the increase in amino acid N concentration and the decrease in NH3 –N. 4. Conclusions Similarly to monensin, hainanmycin was shown to be an effective ionophore in terms of regulation of ruminal protein metabolism and populations of proteolytic bacteria for dairy cows.
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Conflict of interest The authors declare that there are no conflict of interests. Acknowledgments The authors are grateful to National Natural Science Foundation of China (Grant No. 31340031), Synergetic Innovation Center of Food Safety and Nutrition, and National Key Technologies R&D Program (No. 2012BAD12B05-1) for providing financial support for this project. References Chen, G., Russell, J., 1989. More monensin-sensitive: ammonia-producing bacteria from the rumen. Appl. Environ. Microbiol. 55, 1052–1057. Chen, G., Russell, J.B., 1991. Effect of monensin and a protonophore on protein degradation, peptide accumulation, and deamination by mixed ruminal microorganisms in vitro. J. Anim. Sci. 69, 2196–2203. Eschenlauer, S.C., McKain, N., Walker, N.D., McEwan, N.R., Newbold, C.J., Wallace, R.J., 2002. Ammonia production by ruminal microorganisms and enumeration, isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen. Appl.Environ. Microbiol. 68, 4925–4931 http://www.ncbi.nlm.nih.gov/pubmed/12324340 Ghorbani, B., Ghoorchi, T., Amanlou, H., Zerehdaran, S., 2008. Effects of monensin and increasing crude protein in early lactation on performance of dairy cows. Pak. J. Biol. Sci. 11, 1669–1675, http://dx.doi.org/10.3923/pjbs.2008.1669.1675. Mackie, R.I., White, B.A., 1990. Recent advances in rumen microbial ecology and metabolism: potential impact on nutrient output. J. Dairy Sci. 73, 2971–2995, http://dx.doi.org/10.3168/jds.S0022-0302(90)78986-2. Nagaraja, T.G., Newbold, C.J., van Nevel, C.J., Demeyer, D.I., 1997. Manipulation of rumen fermentation. In: Hobson, P.N., Stewart, C.S. (Eds.), The Rumen Microbial Ecosystem. Springer, Netherlands, London, pp. 523–632, http://dx.doi.org/10.1007/978-94-009-1453-7 13. Raacke, I.D., 1957. Protein synthesis in ripening pea seeds. I. Analysis of whole seeds. Biochem. J. 66, 101–110 http://www.ncbi.nlm.nih. gov/pubmed/13426114 Ren, M.Q., Shen, Z.M., Zhao, R.Q., Lu, T.S., Chen, J., 1998. Effects of novel polyether ionophore hainanmycin on nutrient digestion, metabolism and ruminal characteristics of goats. J. Anim. Feed Sci. 25, 21–28. Russell, J., Strobel, H., Chen, G., 1988. Enrichment and isolation of a ruminal bacterium with a very high specific activity of ammonia production. Appl. Environ. Microbiol. 54, 872–877. Siddons, R.C., Paradine, J., 1981. Effect of diet on protein degrading activity in the sheep rumen. J. Sci. Food Agric. 32, 973–981, http://dx.doi.org/10.1002/jsfa.2740321005. Stevenson, D.M., Weimer, P.J., 2007. Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR. Appl. Microbiol. Biotechnol. 75, 165–174, http://dx.doi.org/10.1007/s00253-006-0802-y. Tajima, K., Aminov, R.I., Nagamine, T., Ogata, K., Nakamura, M., Matsui, H., Benno, Y., 1999. Rumen bacterial diversity as determined by sequence analysis of 16 S rDNA libraries. FEMS Microbiol. Ecol. 29, 159–169, http://dx.doi.org/10.1111/j.1574-6941.1999.tb00607.x. Wallace, R., Broderick, G., Brammall, G., 1987. Microbial protein and peptide metabolism in rumen fluid from faunated and ciliate-free sheep. Br. J. Nutr. 58, 87–93 http://www.ncbi.nlm.nih.gov/pubmed/2802611 Wallace, R.J., McKain, N., 1989. Analysis of peptide metabolism by ruminal microorganisms. Appl. Environ. Microbiol. 55, 2372–2376 http://www.ncbi.nlm.nih.gov/pubmed/2802611 Wallace, R.J., 1996. Ruminal microbial metabolism of peptides and amino acids. J. Nutr. 126, 1326S–1334S http://www.ncbi.nlm.nih.gov/pubmed/8642480 Wallace, R.J., Onodera, R., Cotta, M.A., 1997. Metabolism of nitrogen-containing compounds. In: Hobson, P.N., Stewart, C.S. (Eds.), The Rumen Microbial Ecosystem. Springer, Netherlands, London, pp. 283–328, http://dx.doi.org/10.1007/978-94-009-1453-7 7. Wang, Z., Xin, H., Wang, M., Li, Z., Qu, Y., Miao, S., Zhang, Y., 2013. Effects of dietary supplementation with hainanmycin on protein degradation and populations of ammonia-producing bacteria in vitro. Asian-Australas. J. Anim. Sci. 26, 668–674, http://dx.doi.org/10.5713/ajas.2012.12589. Weimer, P.J., Stevenson, D.M., Mertens, D.R., Thomas, E.E., 2008. Effect of monensin feeding and withdrawal on populations of individual bacterial species in the rumen of lactating dairy cows fed high-starch rations. Appl. Microbiol. Biotechnol. 80, 135–145, http://dx.doi.org/10.1007/s00253-008-1528-9. Winter, K.A., Johnson, R., Dehority, B., 1964. Metabolism of urea nitrogen by mixed cultures of rumen bacteria grown on cellulose. J. Dairy Sci. 47, 793–797, http://dx.doi.org/10.3168/jds.S0022-0302(64)88766-X.
Contents lists available at ScienceDirect
Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci
Short communication
Effects of hainanmycin or monensin supplementation on ruminal protein metabolism and populations of proteolytic bacteria in Holstein heifers Z.B. Wang a , H.S Xin b , J. Bao b , C.Y. Duan c , Y. Chen a , Y.L. Qu a,∗ a b c
College of Animal Science & Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China Xijie Feed Co., Ltd. Shenyang 110164, China
a r t i c l e
i n f o
Article history: Received 22 July 2014 Received in revised form 1 January 2015 Accepted 2 January 2015 Keywords: Heifer Hainanmycin Monensin Proteolytic bacteria Protein metabolism
a b s t r a c t Three ruminally cannulated cows were used in a 3 × 3 Latin square design (21-d periods) to evaluate effects of hainanmycin (HAI) or monensin (MON) supplementation on ruminal protein metabolism and populations of proteolytic bacteria in Holstein heifers. The three dietary treatments were: (1) basal diet (control); (2) basal plus 20 mg/heifer per day HAI and (3) basal plus 350 mg/heifer per day MON. The supplementation with HAI and MON had minor effects on ruminal pH and total VFA. In general, HAI and MON decreased acetate, butyrate concentrations, and acetate to propionate ratio, and increased propionate concentration. Supplementation of HAI and Mon resulted in higher relative population sizes of the genus Prevotella and Prevotella ruminicola, and fewer Butyrivibrio fibrisolvens and hyperammonia-producing bacteria (P<0.05). HAI supplementation increased concentrations of peptide nitrogen and amino acid nitrogen, and reduced ammonia concentration (P<0.05), and decreased peptidase and deaminase activities (P<0.05). Overall, HAI was as effective as monensin as ionophore in regulation of ruminal protein metabolism and populations of proteolytic bacteria for dairy cows. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The breakdown of protein to ammonia in the rumen may lead to an appreciable loss of nitrogen to the animal (Wallace, 1996). Bacteria are thought to be responsible for the majority of the breakdown in the rumen (Wallace et al., 1987). Many of ruminal bacteria have peptidase and deaminase activities, for instance Prevotella sp. and hyper-ammonia-producing (HAP) bacteria (Wallace et al., 1997). Monensin caused an accumulation in amino acids N and peptide N by mixed ruminal microorganisms in vitro, and decrease rumen ammonia production (Chen and Russell, 1991; Ghorbani et al., 2008). Much of the decrease in ruminal ammonia production caused by monensin addition can be specifically attributed to the inhibitory effects on HAP bacteria (Eschenlauer et al., 2002). Hainanmycin is a polyether monocarboxylic acid ionophore from one of fermentation products of a
Abbreviations: ADF, acid detergent fiber; A:P, acetate to propionate ratio; BW, body weight; CP, crude protein; DM, dry matter; HAI, hainanmycin; HAP, hyper-ammonia-producing; ME, metabolisable energy; MNA, methoxynapthylamide; MON, monensin; N, nitrogen; NDF, neutral detergent fiber; RPS, relative population sizes; VFA, volatile fatty acid. ∗ Corresponding author. Tel.: +86 0459 6819195; fax: +86 0459 6819195. E-mail address: [email protected] (Y.L. Qu). http://dx.doi.org/10.1016/j.anifeedsci.2015.01.001 0377-8401/© 2015 Elsevier B.V. All rights reserved.
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rare streptomycete, Streptomyces padanus var dangfangeus, which was isolated from soil sample collected in Hainan province of China. The chemical formula of hainanmycin is C47 H79 O15 N, which capable of forming stable complexes to monovalent cations with a specific affinity for Na+ . Hainanmycin lowers ruminal ammonia production in the sheep, and increase ruminal concentrations of peptide N and amino acid N, and affects growth of ammonia-producing bacteria in vitro (Wang et al., 2013). However, there is little information on the effects of hainanmycin on ruminal protein metabolism and populations of proteolytic bacteria in cattle. The objective of the present study was to evaluate the effects of the supplementation of hainanmycin on ruminal protein metabolism and populations of proteolytic bacteria in Holstein heifers. 2. Material and methods The protocol used in this experiment was approved by the Northeast Agricultural University Animal Science and Technology College Animal Care and Use Committee. 2.1. Feed additives The hainanmycin was obtained from Sheng Li Inc. Shandong province, China. 2.2. Animals and treatments Three Holstein heifers fitted with ruminal cannulas were used in a 3 × 3 Latin square design to evaluate effects of hainanmycin (HAI) or monensin (MON) supplementation on ruminal protein metabolism and populations of proteolytic bacteria in Holstein heifers. The heifers were fed a diet which consisted of dry-rolled corn (99.7 g/kg), soybean meal (109.7 g/kg), Chinese wild rye grass (378.9 g/kg), and corn silage (358.9 g/kg). The CP, NDF, ADF, and ME contents of the diet (DM basis) were 128.8 g/kg, 454.2 g/kg, 319.4 g/kg, and 9.17 MJ/kg, respectively. Treatments consisted of: (1) basal diet (no ionophore); (2) basal plus 20 mg/heifer per day HAI; and (3) basal plus 350 mg/heifer per day MON. The HAI dose was calculated according to the heifer’s body weight (0.05 mg/kg BW per day) (Ren et al., 1998). Each period lasted 21 with 19 d of adaptation and 2 d of sample collection. Diet was offered twice daily at 08:00 and 18:00 h and 10.5 kg (DM basis)/day per heifer. 2.3. Sampling and measurements On d 20 and 21 of each period, ruminal fluid was collected immediately at 0, 2, 4, 6, 8, and 10 h for ruminal pH, VFA concentration, protein fractions (peptide N, amino acid N, and NH3 ), and total DNA extraction. Strained ruminal fluid at 4 h after feeding was used to determine in vitro protease and deaminase activities (Siddons and Paradine 1981). The peptidase activity was assayed using the substrate Glycine-Arginine methoxynapthylamide (MNA) (Wallace and McKain 1989). Concentrations of peptide N plus amino acid N in ruminal fluid were determined as described by Winter et al. (1964). In this method, the tungstic acid precipitates small peptides only partially (Raacke 1957), therefore the values for peptides N as determined by tungstic acid would be low in present experiment. PCR assays for enumeration of selected bacterial species were performed according to the methods described by Stevenson and Weimer (2007). Enumeration for HAP were determined according to the methods described by Russell et al. (1988). 2.4. Statistical analysis Data were statistically analyzed as a 3 × 3 Latin square design using the PROC MIXED procedure of SAS (SAS Institute, version 9.1) according to the following model. Yijk = + Ti + Pj + Ck + Eijk . Where Yijk is observation, is overall mean, Ti is treatment (i = 1–3), Pj is period (j = 1–3), Ck is heifer (k = 1–3), and Eijk is residual error. Differences were declared significant at P<0.05. Results are reported as least squares means ± standard error of the means. 3. Results and discussion 3.1. Ruminal fermentation characteristics Ruminal fermentation characteristics are shown in Fig. 1 and Table 1. In general, the supplementation with HAI and MON had minor effects on ruminal pH and total VFA. Similar to the results in the present trial with HAI, HAI also had no changed pH and total VFA in our previous study in vitro, (Wang et al., 2013). Two ionophores caused lower (P<0.05) acetate concentrations at 6 h after feeding, and higher (P<0.05) concentrates of propionate at 0, 2, 4, and 8 h after feeding. Supplementation of HAI or MON decreased (P<0.05) acetate: propionate ratio at 0, 2, 4, and 6 h after feeding. Changes in acetate, propionate and their ratio in the present study were in agreement with the observations of Ren et al. (1998). HAI and MON caused lower (P<0.05) concentrations of butyrate at 0 and 2 h after feeding, respectively. Concentrations of valerate or isobutyrate were not affected by HAI and MON (data not shown). MON decreased (P<0.05) isovalerate concentrations at 2 and 6 h after feeding compare with control, and HAI also caused lower concentrations of isovalerate (P<0.05) at 6 h after feeding (data not shown).
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Fig. 1. Effects of hainanmycin (HAI) or monensin (MON) on ruminal pH in Holstein heifers. a−b Means within 2 h with different superscripts differ (P<0.05). Control = no ionophore; HAI = hainanmycin (20 mg/heifer per day); MON = monensin (350 mg/heifer per day).
The branched-chain VFA are derived from amino acid catabolism in the rumen (Mackie and White, 1990). The reduction in isovalerate concentrations implied that amino acid deamination were inhibited. 3.2. The number of proteolytic bacteria Supplementation of HAI and MON increased (P<0.05) relative population sizes (RPS) of the genus Prevotella and P. ruminicola (Table 2). It was similar to the results of Weimer et al. (2008), who observed that MON increased RPS of ruminal the genus Prevotella and P. ruminicola in lactating dairy cows. Supplementation of HAI and MON reduced RPS of B. fibrisolvens. In the present study, measured Butyrivibrio fibrisolvens is a highly atypical strain, but the true populations of B. fibrisolvens are much larger (Tajima et al., 1999). HAI and MON had no effect (P>0.05) on RPS of other bacteria compared with control. Supplementation of HAI and MON caused a large decrease in numbers of hyper-ammonia-producing (HAP) bacteria, which was associated with a decrease in HAP bacteria in our previous study (Wang et al., 2013). Chen and Russell (1989) demonstrated that MON reduced NH3 –N concentration through the inhibition of the HAP bacteria that are responsible for the production of most of the ammonia. Table 1 Effects of hainanmycin (HAI) or monensin (MON) on ruminal VFA in Holstein heifers. Acids, mol/L
Total VFA
Acetate
Propionate
Butyrate
A:PA
a−c
Hour after feeding
0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10
TreatmentB Control
HAI
MON
80.5 99.4 90.7 87.2 80.9 73.9 61.0 70.9 65.8 64.5a 60.1 54.9 13.0b 19.8b 16.8b 15.3 13.8c 12.8 5.28a 6.97 6.68 6.17 5.89 5.18 4.71a 3.40a 3.94a 4.23a 4.36 4.28
77.5 94.4 87.7 84.3 77.0 72.5 53.3 64.1 59.7 58.7b 51.7 51.8 16.0a 21.2a 19.5a 17.1 14.7b 13.7 5.16a 6.40 6.33 6.11 5.52 5.06 3.29b 3.03b 3.14b 3.38b 3.54 3.49
74.8 93.5 86.9 83.1 73.0 71.6 54.5 65.3 60.7 59.4b 54.8 51.6 16.6a 20.9a 19.4a 17.6 15.5a 14.8 4.42b 6.50 6.19 6.04 5.63 5.05 3.33b 3.13b 3.08b 3.44b 3.53 3.80
Means within a row with different superscripts differ (P<0.05). A:P represents acetate to propionate ratio. B Control = no ionophore; HAI = hainanmycin (20 mg/heifer per day); MON = monensin (350 mg/heifer per day).
A
SEM
P-value
2.19 1.45 1.09 0.83 2.21 3.17 2.03 1.39 1.20 0.57 2.47 3.33 0.23 0.20 0.17 0.43 0.16 0.32 0.13 0.14 0.14 0.03 0.09 0.11 0.05 0.09 0.02 0.04 0.21 0.31
0.23 0.09 0.13 0.07 0.13 0.78 0.11 0.07 0.06 0.02 0.15 0.62 0.01 0.04 0.01 0.06 0.02 0.08 0.04 0.09 0.14 0.08 0.11 0.53 0.002 0.04 0.001 0.004 0.09 0.24
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Table 2 Effects of hainanmycin (HAI) or monensin (MON) on the number of proteolytic bacteria and enzyme activities in Holstein heifers. TreatmentF
Item
Target bacterium, RPSA , % Genus Prevotella Prevotella ruminicola Prevotella brevis Prevotella bryantii Butyrivibrio fibrisolvens Selenomonas ruminantium Streptococcus bovis HAP bacteriaB Enzyme activities Proteinase activityC Peptidase activityD Deaminase activityE a−b A B C D E F
Control
HAI
MON
42.33b 1.66b 0.17 0.68 0.034a 0.54 0.00172 3.70a
45.76a 1.95a 0.16 0.64 0.021b 0.60 0.00167 0.69b
47.21a 1.96a 0.16 0.63 0.021b 0.62 0.00167 0.69b
0.64 7.39a 0.15a
0.65 4.54b 0.10b
0.69 4.33b 0.10b
SEM
P-value
1.33 0.1 0.01 0.07 0.001 0.07 0.0001 0.16
0.01 0.03 0.78 0.49 <0.01 0.31 0.70 <0.001
0.05 0.77 0.01
0.60 0.03 <0.01
Means within a row with different superscripts differ (P < 0.05). Relative population sizes: percentages of the 16 S rRNA gene copy number of the total bacterial domain. HAP represents hyper-ammonia-producing, most probable numbers (106 /mL). mg of 14 C-casein hydrolyzed/h per mg of protein. Assayed using the substrate Glycine-Arginine methoxynapthylamide, nmols/min per mg protein. nmol of ammonia produced/h per mg of protein. Control = no ionophore; HAI = hainanmycin (20 mg/heifer per day); MON = monensin (350 mg/heifer per day).
Table 3 Effects of hainanmycin (HAI) or monensin (MON) on contentrations of ruminal peptide N, Amino acid N, and NH3 –N in Holstein heifers. Item
Hour after feeding
Peptide N, mmol/L
0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10
Amino acid N, mmol/L
NH3 –N, mg/dL
a−b A
TreatmentA Control
HAI
MON
1.99 5.65b 4.42b 3.47b 2.82 2.21 2.62 5.99b 3.50b 2.73b 2.58 2.55 14.11a 29.78 17.59 8.14 8.13 9.88
2.30 7.42a 6.01a 4.52a 3.20 2.45 2.88 7.06a 5.50a 4.47a 3.83 3.23 11.74b 28.79 15.48 6.77 7.02 8.07
2.18 7.30a 6.10a 4.44a 3.35 2.47 2.42 6.96a 5.68a 4.62a 3.59 3.14 11.00b 28.41 15.73 6.52 6.89 8.39
SEM
P-value
0.25 0.24 0.15 0.14 0.19 0.15 0.25 0.17 0.14 0.15 0.44 0.27 0.27 0.44 0.62 0.30 0.27 0.48
0.57 0.03 0.01 0.03 0.20 0.35 0.37 0.04 0.01 0.01 0.18 0.21 0.01 0.16 0.13 0.06 0.07 0.11
Means within a row with different superscripts differ (P<0.05). Control = no ionophore; HAI = hainanmycin (20 mg/heifer per day); MON = monensin (350 mg/heifer per day).
3.3. Protein fractions and enzyme activities Concentrations of ruminal peptide N, amino acid N, and NH3 –N in heifers are presented in Table 3. Supplementation of HAI and MON increased (P<0.05) peptide N and amino acid N concentrations at 2, 4, and 6 h after feeding, and decreased (P<0.05) NH3 –N concentrations at 0 h after feeding. The accumulation of amino acid N and the decrease in NH3 –N implied that amino acid deamination was inhibited. The inhibition of amino acid deamination has practical implications because it may increase ruminal escape of dietary protein and improve the efficiency of N use in the rumen (Nagaraja et al., 1997). HAI and MON decreased (P<0.05) ruminal peptidase activities, which indicated that hainanmycin inhibited degradation of ruminal peptides to amino acids. HAI and MON also decreased ruminal deaminase activities, which corresponded with the increase in amino acid N concentration and the decrease in NH3 –N. 4. Conclusions Similarly to monensin, hainanmycin was shown to be an effective ionophore in terms of regulation of ruminal protein metabolism and populations of proteolytic bacteria for dairy cows.
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Conflict of interest The authors declare that there are no conflict of interests. Acknowledgments The authors are grateful to National Natural Science Foundation of China (Grant No. 31340031), Synergetic Innovation Center of Food Safety and Nutrition, and National Key Technologies R&D Program (No. 2012BAD12B05-1) for providing financial support for this project. References Chen, G., Russell, J., 1989. More monensin-sensitive: ammonia-producing bacteria from the rumen. Appl. Environ. Microbiol. 55, 1052–1057. Chen, G., Russell, J.B., 1991. Effect of monensin and a protonophore on protein degradation, peptide accumulation, and deamination by mixed ruminal microorganisms in vitro. J. Anim. Sci. 69, 2196–2203. Eschenlauer, S.C., McKain, N., Walker, N.D., McEwan, N.R., Newbold, C.J., Wallace, R.J., 2002. Ammonia production by ruminal microorganisms and enumeration, isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen. Appl.Environ. Microbiol. 68, 4925–4931 http://www.ncbi.nlm.nih.gov/pubmed/12324340 Ghorbani, B., Ghoorchi, T., Amanlou, H., Zerehdaran, S., 2008. Effects of monensin and increasing crude protein in early lactation on performance of dairy cows. Pak. J. Biol. Sci. 11, 1669–1675, http://dx.doi.org/10.3923/pjbs.2008.1669.1675. Mackie, R.I., White, B.A., 1990. Recent advances in rumen microbial ecology and metabolism: potential impact on nutrient output. J. Dairy Sci. 73, 2971–2995, http://dx.doi.org/10.3168/jds.S0022-0302(90)78986-2. Nagaraja, T.G., Newbold, C.J., van Nevel, C.J., Demeyer, D.I., 1997. Manipulation of rumen fermentation. In: Hobson, P.N., Stewart, C.S. (Eds.), The Rumen Microbial Ecosystem. Springer, Netherlands, London, pp. 523–632, http://dx.doi.org/10.1007/978-94-009-1453-7 13. Raacke, I.D., 1957. Protein synthesis in ripening pea seeds. I. Analysis of whole seeds. Biochem. J. 66, 101–110 http://www.ncbi.nlm.nih. gov/pubmed/13426114 Ren, M.Q., Shen, Z.M., Zhao, R.Q., Lu, T.S., Chen, J., 1998. Effects of novel polyether ionophore hainanmycin on nutrient digestion, metabolism and ruminal characteristics of goats. J. Anim. Feed Sci. 25, 21–28. Russell, J., Strobel, H., Chen, G., 1988. Enrichment and isolation of a ruminal bacterium with a very high specific activity of ammonia production. Appl. Environ. Microbiol. 54, 872–877. Siddons, R.C., Paradine, J., 1981. Effect of diet on protein degrading activity in the sheep rumen. J. Sci. Food Agric. 32, 973–981, http://dx.doi.org/10.1002/jsfa.2740321005. Stevenson, D.M., Weimer, P.J., 2007. Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR. Appl. Microbiol. Biotechnol. 75, 165–174, http://dx.doi.org/10.1007/s00253-006-0802-y. Tajima, K., Aminov, R.I., Nagamine, T., Ogata, K., Nakamura, M., Matsui, H., Benno, Y., 1999. Rumen bacterial diversity as determined by sequence analysis of 16 S rDNA libraries. FEMS Microbiol. Ecol. 29, 159–169, http://dx.doi.org/10.1111/j.1574-6941.1999.tb00607.x. Wallace, R., Broderick, G., Brammall, G., 1987. Microbial protein and peptide metabolism in rumen fluid from faunated and ciliate-free sheep. Br. J. Nutr. 58, 87–93 http://www.ncbi.nlm.nih.gov/pubmed/2802611 Wallace, R.J., McKain, N., 1989. Analysis of peptide metabolism by ruminal microorganisms. Appl. Environ. Microbiol. 55, 2372–2376 http://www.ncbi.nlm.nih.gov/pubmed/2802611 Wallace, R.J., 1996. Ruminal microbial metabolism of peptides and amino acids. J. Nutr. 126, 1326S–1334S http://www.ncbi.nlm.nih.gov/pubmed/8642480 Wallace, R.J., Onodera, R., Cotta, M.A., 1997. Metabolism of nitrogen-containing compounds. In: Hobson, P.N., Stewart, C.S. (Eds.), The Rumen Microbial Ecosystem. Springer, Netherlands, London, pp. 283–328, http://dx.doi.org/10.1007/978-94-009-1453-7 7. Wang, Z., Xin, H., Wang, M., Li, Z., Qu, Y., Miao, S., Zhang, Y., 2013. Effects of dietary supplementation with hainanmycin on protein degradation and populations of ammonia-producing bacteria in vitro. Asian-Australas. J. Anim. Sci. 26, 668–674, http://dx.doi.org/10.5713/ajas.2012.12589. Weimer, P.J., Stevenson, D.M., Mertens, D.R., Thomas, E.E., 2008. Effect of monensin feeding and withdrawal on populations of individual bacterial species in the rumen of lactating dairy cows fed high-starch rations. Appl. Microbiol. Biotechnol. 80, 135–145, http://dx.doi.org/10.1007/s00253-008-1528-9. Winter, K.A., Johnson, R., Dehority, B., 1964. Metabolism of urea nitrogen by mixed cultures of rumen bacteria grown on cellulose. J. Dairy Sci. 47, 793–797, http://dx.doi.org/10.3168/jds.S0022-0302(64)88766-X.