Technical note
Effects of dietary sulfur level on amino acid concentrations
in ruminal bacteria of goats
H. Carneiro
a, R. Puchala
b, F.N. Owens
c, T. Sahlu
b,*, K. Qi
c, A.L. Goetsch
b aEmbrapa, Gado de Leite, Rua EugeÃnio do Nascimento, 610, Alto dos Passos Juiz de Fora, MG 36038-330, BrazilbE (Kika) de la Garza Institute for Goat Research, Langston University, Langston, OK 73050, USA cAnimal Science Department, Oklahoma State University, Stillwater, OK 74078, USA
Accepted 10 December 1999
Abstract
12 Angora (180.6 kg BW) and 20 Alpine (241.0 kg BW) goat wethers consumed diets (14.3% CP and 1.67±1.80 Mcal/ kg ME, DM basis) with 0.11, 0.20, 0.28 or 0.38% S (supplemental S : CaSO4; N:S ratio is 21, 12, 8 and 6, respectively) for 10
weeks to determine effects of dietary S on amino acid concentrations in ruminal ¯uid bacteria. The concentration of cysteine in bacterial DM changed quadratically (P<0.08) as dietary S increased (3.28, 3.77, 3.80 and 3.65% for 0.11, 0.20, 0.28 and 0.38% S, respectively). However, dietary S did not alter methionine concentration in bacterial DM or total amino acids, and for the few amino acids whose concentrations were affected, magnitudes of change were relatively small. In conclusion, with diets moderate to low in ME, levels of S greater than 0.20% and N:S ratios less than 12:1 had very little effect on amino acid concentrations in ruminal ¯uid bacteria of growing goats, which supports the contention that the primary potential in¯uence of inorganic dietary S on absorbed S-containing amino acids is through the quantity of microbial protein synthesized in the rumen.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Sulfur; Goats; Amino acids; Bacteria
1. Introduction
Mohair production by Angora goats can be affected by plane of nutrition, including dietary S concentra-tion (Reis, 1989; Qi et al., 1992; Reis and Sahlu, 1994). For wool production, primary effects of nutri-tional plane are on the rate of division of follicle bulb cells and ®nal size of cells in the follicle bulb and ®ber cortex (Black and Reis, 1979). Cysteine is the primary
S-containing amino acid in animal ®ber protein. The high concentration of cysteine in keratin relative to that in plant material suggests that wool- and mohair-producing ruminants could require greater quantities of S-containing amino acids than other ruminant classes.
NRC (1981) suggested an N:S requirement of 10:1 for goats, similar to that for other ruminants. However, Qi et al. (1992) observed maximum clean mohair production by Angora wethers at 0.26% dietary S and an N:S ratio of 7.2, with diets ranging in CP from 11.8±12.2% DM and ME from 1.51±1.58 Mcal/kg DM. Conversely, with diets slightly higher in ME *Corresponding author. Tel.:1-405-466-3836;
fax:1-405-466-3138.
E-mail address: sahlu@mail.luresext.edu (T. Sahlu)
and CP consumed by young, growing goats, Qi et al. (1993) calculated maximum ADG, DM intake and N retention at 0.22±0.24% dietary S and an N:S ratio of 9.5±10.4.
Most protein and S-containing amino acids avail-able for digestion and absorption by ruminants are derived from dietary protein escaping ruminal fermen-tation and microbial protein synthesized in the rumen. The former is determined by the quantity of protein fed and susceptibility to degradation by ruminal microorganisms. Microbial protein formed in the rumen depends on the quantity of OM fermented and availability of required nutrients such as ammonia (NRC, 1985). Low ruminal S concentration can also depress microbial growth and ®ber digestion (Kandy-lis, 1984). Besides effects on the quantity of microbial protein synthesized, nutrient availability can impact
composition of microbial cells, and Weston et al. (1989) proposed that the level of amino acids contain-ing S (methionine, cysteine and cysteine) in ruminal microbes might be reduced by a de®ciency of S. Therefore, the objective of this experiment was to determine the effect of dietary S on amino acid concentrations in ruminal ¯uid bacterial cells of goats.
2. Materials and methods
2.1. Animals and diets
This report is a companion to that of Qi et al. (1993). The report of Qi et al. (1993) includes a detailed description of performance, acid±base balance and nutrient digestibilities. In the present experiment, 12
Table 1
Diet composition (DM basis)
Item Dietary sulfur (%)
0.11 0.20 0.28 0.38
Ingredient(%)
Ground peanut hulls 50.00 50.00 50.00 50.00
Corn starch 37.40 37.40 37.40 37.40
Soybean meal 6.10 6.10 6.10 6.10
Urea 1.50 1.50 1.50 1.50
Na2HPO4 1.35 1.35 1.35 1.35
CaCO3 0.85 0.53 0.25 0.00
CaSO4 0.00 0.40 0.80 1.15
Trace mineralized salta 1.50 1.50 1.50 1.50
Vitamin premixb 1.00 1.00 1.00 1.00
SiO2 0.30 0.22 0.10 0.00
DCABc(mEq/100 g) 35.62 34.11 36.03 36.52
S (mEq/100 g) 3.35 6.08 8.68 11.91
DCAB:Sd(mEq/100 g) 32.27 29.03 27.35 24.61
aContained 95.5±98.5% NaCl and more than 0.24% Mn, 0.24% Fe, 0.05% Mg, 0.032% Cu, 0.011% Co, 0.007% I, and 0.005% Zn. bContained 2200/g IU Vitamin A, 1200/g IU Vitamin D
Angora wether kids (18.10.6 kg BW) and 20 Alpine wether kids (23.71.0 kg BW) were housed indivi-dually in 2.3 m1.0 m stainless steel cages in an open-front barn with forced-air ventilation during the 10-week experiment. Wethers were randomly assigned within breed to four dietary treatments. Thus, there were three Angoras and ®ve Alpines assigned to each treatment. Diets (Table 1) were isonitrogenous (14.3% CP; DM basis), similar in estimated ME concentration (1.67±1.80 Mcal/kg) and formulated to meet ME, CP, Ca and P requirements of growing goats (NRC, 1981). Diets contained 0.11, 0.20, 0.28, or 0.38% S (DM basis), with supplemental S supplied by CaSO4. Calcium from CaSO4 was balanced via different dietary levels of CaCO3, and SiO2was added to achieve constant ME concentration. The N:S ratio was 21, 12, 8 and 6 for diets with 0.11, 0.20, 0.28 and 0.38% S, respectively. Wethers were fed once daily at approximately 110% of consumption on the preceding
few days. DM intake was 747, 874, 859 and 756 g/day and DM digestibility was 52, 48, 50 and 49% for 0.11, 0.20, 0.28 and 0.38%, respectively.
2.2. Sample collection and analyses
On Day 70 of the experiment, ruminal ¯uid was collected by stomach tube just before feeding (i.e., 0 h) and at 4 h after feeding, with combining of the two 140 ml aliquots to form a 280 ml composite sample for each animal. The ®rst 20±30 ml of ruminal ¯uid sample was discarded to minimize saliva con-tamination. Immediately after the second collection, ruminal ¯uid was centrifuged at 500g for 10 min to remove feed particles and protozoa, then supernatant was centrifuged again at 20,000gfor 20 min to sedi-ment bacteria. The bacterial pellet was washed twice with 0.9% (w/v) saline and once with distilled water. Examined microscopically, pellets were essentially
Table 2
Amino acid concentrations (mg/g DM) in bacterial cells from ruminal ¯uid of goat kids consuming diets differing in sulfur concentration
Amino acid Dietary sulfur (%) SEb Effect (Pvaluea)
0.11 0.20 0.28 0.38 Linear Quadratic
Essential
Arginine 13.28 13.91 13.73 13.57 0.65
Histidine 5.16 5.33 5.37 5.05 0.23
Isoleucine 13.79 13.71 14.06 13.81 0.54
Leucine 21.63 22.16 22.39 22.24 0.76
Lysine 19.36 19.71 20.46 18.97 0.94
Methionine 9.84 9.84 10.02 9.56 0.56
Phenylalanine 13.67 14.06 13.74 13.61 0.46
Threonine 14.28 14.68 14.29 14.11 0.52
Valine 15.39 15.62 15.59 15.30 0.67
Total 126.40 129.02 129.65 126.22 4.82
Nonessential
Alanine 23.83 23.94 23.51 23.41 0.98
Aspartate 32.09 33.30 32.94 32.34 1.14
Cysteine 3.28 3.77 3.80 3.65 0.18 0.08
Glutamate 38.16 40.59 39.55 38.72 1.35
Glycine 16.79 16.62 16.91 16.53 0.64
Proline 7.34 7.75 7.80 7.72 0.39
Serine 12.81 13.14 14.35 13.71 0.46 0.07
Tyrosine 21.01 20.99 23.97 22.27 1.21
Total 155.31 160.10 162.83 158.35 5.40
Cysteinemethionine 13.12 13.61 13.82 13.21 1.42 Phenylalaninetyrosine 34.67 35.06 37.71 35.89 1.50
aListed if0.10.
free of contaminants. Pellets were lyophilized and DM and amino acids were quanti®ed. Complete details for the protein hydrolysis and amino acid analysis are given by Barkholt and Jensen (1989) and Puchala et al. (1995). Other analyses were described by Qi et al. (1993).
2.3. Statistical analyses
Data were analyzed by General Linear Models procedures of SAS (1985), with a 23 factorial arrangement of treatments. Breed and dietary S did not interact; therefore, the mean effect of S level was evaluated by regression to determine linear, quadratic and cubic effects. As the analyzed S concentration were not equally spaced, polynomial values were calculated (Verneque, 1984) to employ in these regres-sions. Also, cysteine concentration in bacterial cell DM was regressed against dietary S. Mean amino acid
concentrations for isolated ruminal bacteria in the present experiment were compared with literature values. The probability that values of the present experiment differed from earlier estimates was assessed using standard errors and t-tests.
3. Results
As dietary S level increased, cysteine concentration in bacterial DM changed quadratically (P<0.08), and cysteine as a percentage of total amino acids increased linearly (P<0.04; Tables 2 and 3, respectively). A regression of cysteine concentration in bacterial cell DM against dietary S (Fig. 1) revealed a peak in cysteine concentration at dietary S of 0.263% DM, which is only slightly greater than S requirements suggested by Qi et al. (1993) for maximal live weight gain (i.e., 0.22%) and DM intake (i.e., 0.24%). Serine
Table 3
Amino acid concentrations (% total amino acids) in bacterial cells from ruminal ¯uid of goat kids consuming diets differing in sulfur concentration
Amino acid Dietary sulfur (%) SE Effect (Pvaluea)
0.11 0.20 0.28 0.38 Linear Quadratic Cubic
Essential
Arginine 4.71 4.81 4.69 4.75 0.10
Histidine 1.83 1.85 1.84 1.78 0.06
Isoleucine 4.90 4.75 4.80 4.87 0.11
Leucine 7.69 7.67 7.67 7.81 0.08
Lysine 6.84 6.80 6.98 6.66 0.15
Methionine 3.49 3.39 3.41 3.35 0.10
Phenylalanine 4.87 4.87 4.70 4.78 0.06
Threonine 5.08 5.08 4.88 4.97 0.08
Valine 5.46 5.40 5.31 5.38 0.08
Total 44.87 44.60 44.29 44.34 0.14 0.07
Nonessential
Alanine 8.47 8.27 8.01 8.22 0.11 0.06 0.07
Aspartate 11.42 11.52 11.26 11.38 0.09 0.06
Cysteine 1.16 1.30 1.30 1.28 0.04 0.04 0.06
Glutamate 13.56 14.06 13.53 13.61 0.20
Glycine 5.97 5.74 5.77 5.81 0.55 0.09 0.04
Proline 2.59 2.68 2.66 2.70 0.09
Serine 4.56 4.55 4.96 4.84 0.16 0.10
Tyrosine 7.39 7.27 8.22 7.82 0.19
Total 55.13 55.40 55.71 55.66 0.29
Cysteinemethionine 4.65 4.69 4.71 4.63 0.12 Phenylalaninetyrosine 12.26 12.14 12.92 12.60 0.29
in bacterial DM and total amino acids increased linearly (P<0.07 and 0.09, respectively) as dietary S increased. Methionine levels in bacterial DM and total amino acids were not affected by dietary S. Increasing dietary S decreased alanine linearly as a percentage of total amino acids (P<0.07). Cubic effects of dietary S were observed for glutamate, aspartate and tyrosine as percentages of total amino acids (P<0.09, 0.06 and 0.11, respectively). Essential amino acids as a percen-tage of total amino acids decreased linearly (P<0.07) as dietary S increased.
4. Discussion
4.1. Comparison with literature values
Mean amino acid concentrations in ruminal bacteria (percentages of total amino acids) in the present experiment were compared with values presented by Clark et al. (1992), Ibrahim and Ingalls (1972) and Hoeller and Harmeyer (1964) as cited by Purser and Buechler (1966) (Table 4). Amino acid
concen-trations in the present experiment were lower (by more than 2 S.D.) than those observed by Clark et al. (1992) for proline, isoleucine and phenylalanine and greater for tyrosine, although our values were within the range of values from individual experiments summarized by Clark et al. (1992). Estimates of Ibrahim and Ingalls (1972) for bacteria harvested from dairy cattle gen-erally were gengen-erally intermediate to values in the present experiment and those of Clark et al. (1992). The level of cysteine observed in the present experi-ment (1.3% of total amino acids) was slightly greater than reported by Purser and Buechler (1966) (1% of total amino acids) and Weller (1957) (0.7±0.8% of total amino acids) for sheep and Hoeller and Harmeyer (1964) (1.1% of total amino acids) for goats, although, our values were less than one-half of estimates of Ibrahim and Ingalls (1972) (3.0±3.2% of total amino acids) with lactating dairy cows.
4.2. Dietary S
Similarity in the predicted dietary S concentration at which concentration of cysteine in bacterial DM was maximal and S levels determined by Qi et al. (1993) that facilitated maximal live weight gain and DM intake re¯ects high degradability of protein in these diets and, hence, relatively large impact of microbial amino acids on those available for metabo-lism. In accordance, levels may not so closely align with diets higher in ruminally undegraded protein, for which the array of amino acids in microbial protein would have less effect on the pro®le of absorbed amino acids and the array in undegraded protein a greater in¯uence.
Overall, results of this experiment do not depict appreciable in¯uence of dietary S on the level of S-containing amino acids in ruminal ¯uid microbial cells. Even for the diet with a N:S ratio of 20 there was no effect on methionine and not a substantial one on cysteine. Hence, there was no evidence for a marked impact of dietary S on S-containing amino acids available for absorption on metabolism via change in microbial cell concentrations of amino acids. The importance of dietary S would thus appear to lie primarily with potential effects on the total quantity of microbial protein synthesized in the rumen, which was not the focus of this experiment. Factors responsible for the determination by Qi et al. Fig. 1. Best ®t curve for the relationship between dietary sulfur
level and cysteine concentration in bacterial cells from ruminal ¯uid of goat kids.YabX2cX2, whereYcysteine concentration in mg/g DM,Xdietary sulfur concentration (% DM),a4.1483,
(1992) of a N:S ratio lower than 10 being optimal for mohair growth by Angora goats were not identi®ed in that report, and relevant insight was not provided by results of the present experiment either. Diets used by Qi et al. (1992) were lower in digestibility than those employed by Qi et al. (1993) and in the present, companion experiment, suggesting lower microbial protein synthesis in goats of Qi et al. (1992). Perhaps differences in animal types used in these experiments were important, as presumably in¯uencing propor-tions of absorbed nutrients used for muscle growth versus mohair production and perhaps modulating S recycling.
5. Conclusions
Dietary S as in¯uenced by level of an inorganic source did not appreciably alter amino acid concen-trations in bacteria from ruminal ¯uid of goats
har-vested immediately before and at 4 h after feeding a diet moderate to low in digestibility. Thus, there was no evidence to suggest effect of dietary S on S-con-taining amino acids available for animal metabolism, apart from possible impact on the quantity of synthe-sized microbial protein.
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