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Effects of dietary supplementation with different sulfate sources on

nitrogen and sulfur metabolism and rumen fermentation in
cashmere goats
H W Deng, Y Y Cong, Y L Feng and J Chen
Shenyang Agricultural University, China P R
yuyancong@syau.edu.cn

Abstract

Sulfur is an essential nutrient for cashmere goats. In order to investigate the effects of dietary supplementation with different
sulfate on nitrogen and sulfur metabolism and rumen fermentation in cashmere goats, we adopted a 4×4 Latin-square experiment
design using four 1-year old castrated Liaoning cashmere goats with permanent ruminal cannulas. All goats were placed in four
equally sized metabolism cages with bag excrement collectors. There were four experiment periods, in each of which the goats
were respectively fed with the control diet (no additional sulfate supplemented), Na2SO4 diet (sodium sulfate supplemented),
ZnSO4 diet (zinc sulfate supplemented) and MnSO4 diet (manganese sulfate supplemented). Feces, urinary and rumen fluid were
collected, and nitrogen (N) intake, fecal N content, urinary N content, sulfur (S) intake, fecal S content, urinary S content, rumen
pH, NH3-N concentration and volatile fatty acids (VFA) concentration were determined.

The results showed that: there were no significant differences in N intake, fecal N content, urinary N content, retained N amount
and N apparent digestibility among four diets (P>0.05); S intake and fecal S content were significantly increased with dietary
sulfate supplementation (P<0.05), but there were no significant differences in urinary S content, retained S amount and S apparent
digestibility among the diets (P>0.05); dietary sulfate supplementation has no effect on rumen pH (P>0.05), but can decrease
rumen NH3-N concentration (P<0.01) and increase VFA concentration (P<0.05) effectively. We conclude that dietary
supplementation with sulfate has a significant effect on sulfur metabolism and rumen fermentation in cashmere goats, and there
are significant differences among diets with different sulfates supplementation, of which MnSO4 supplementation could decrease
S digestion of cashmere goats than other two sulfates according to this study.

Key words: manganese sulfate, nitrogen digestibility, rumen NH3-N, rumen pH, rumen VFA, sodium sulfate, sulfur digestibility,
zinc sulfate

Introduction

Sulfur is an essential nutrient for animals, and it can only be obtained from diets. Sulfur in rumen microbe
protein is from sulfides which decompose from sulfate and sulfur amino acid (Bray and Till 1975). Many
studies indicated that synthesis of rumen microbe protein, digestion of cellulose, wool growth and quality
were promoted by supplementation with sulfur (Bray and Hemsley 1969, Reis and Schinckel, 1963, Qi et al
1992). In addition, some studies showed that sulfur supplementation can promote the digestion and
metabolism in cashmere goats (Wang and Jia 1999, Peng et al 2001), and our previous researches also
indicated that the higher level of dietary sulfur can improve the metabolism of nitrogen and sulfur and
rumen fermentation and 0.23% was the optimal level of dietary sulfur in Liaoning cashmere goats (Cong et
al 2010). Whereas, there are several types of sulfur sources that can serve as sulfur supplement in diets.
Peng et al (2001) found that there were significant differences among the effects of dietary supplementation
with different sulfur sources (pure sulfur, Na2S and Na2SO4) on nutrient digestion and metabolism in Inner
Mongolia cashmere goats. However, few data are available about different sulfates on rumen fermentation
in cashmere goats. Based on the previous studies, this study was conducted to investigate the effects of
dietary supplementation with different sulfate on nitrogen and sulfur metabolism and rumen fermentation in
cashmere goats, which could provide preliminary analysis on the optimal sulfate source and lay the
groundwork for further studying on cashmere production in cashmere goats.
 Material and Methods

Experimental design
We used a 4×4 Latin-square experimental design with four healthy, 1-year old castrated Liaoning cashmere
goats weighing about 30 kg each. The four goats were installed permanent ruminal cannulas and placed in
four equally sized metabolism cages with urinary and fecal collectors. There were four experiment periods,
each of which consisted of 14 d—7 d for adjusting and 7 d for testing. Four experimental diets were fed to
different goat respectively during the same period and the same goat was fed with different diet during the
different period. All goats were fed twice a day (07:00 and 19:00) and were offered water ad libitum under
the same environmental conditions.

Experimental diets
The experimental diets included the control diet (no additional sulfate supplemented) at 0.12% sulfur level
and three diets respectively supplemented with sodium sulfate (Na2SO4 diet), zinc sulfate (ZnSO4 diet),
manganese sulfate (MnSO4 diet) at 0.23% sulfur level (Table 1). The diets were formulated by referring to
NRC (1985).
Table 1: Dietary composition and nutrient levels (% air dry basis)
Control Na2SO4 ZnSO4 MnSO4
Ingredient
diet diet diet diet
Hay 75 75 75 75

Corn 15 15 15 15

Wheat bran 3 3 3 3
Cottonseed
3 3 3 3
meal
Soybean meal 1.62 1.62 1.62 1.62

CaHPO4 0.15 0.15 0.15 0.15

Urea 0.75 0.75 0.75 0.75
NaCl 0.5 0.5 0.5 0.5

MgO 0.1 0.1 0.1 0.1

Na2SO4 — 0.50 — —

ZnSO4.H2O — — 0.63 —

MnSO4.H2O — — — 0.60

Zeolite powder 0.63 0.13 — 0.03

Premix 1) 0.25 0.25 0.25 0.25

Total 100 100 100 100

Nutrient levels 2)
ME, MJ/kg 8.96 8.96 8.96 8.96

CP 10.6 10.6 10.6 10.6

Ca 0.431 0.431 0.431 0.431

P 0.252 0.252 0.252 0.252

N 1.69 1.69 1.69 1.69

S 0.121 0.229 0.229 0.229

1) Provided per kg of premix: FeSO ·H O 120 g, Cu (OH) Cl 30 g,
4 2 2 3
ZnSO4·H2O 160 g, Ca(IO3)2 25 g, Na2SeO3 14 g, MnSO4·H2O 90 g,
CoCl2·6H2O 1 g, Vitamin A 50 g and Vitamin D3 10 g.
2) ME is a calculated value and others are measured values.
 Sample collection and Measurement
The remaining hay was collected every morning, and feces and urine were collected at 6:00 and 18:00 each
day. The feces and hay were weighed, dried at 65℃ till constant weight, and grinded for analysis. Urine was
added to concentrated hydrochloric acid to prevent NH3-N from volatilizing, and stored at -4℃ for analysis.
Rumen fluid was collected four times per day during the 4th to 6th days of the formal period. Sampling time
was different each day during the 3 d so that it was evenly distributed into different time frames.

N content of diet, feces and urinary was analyzed with by Kjeldahl determination. S content was determined
by magnesium nitrate determination (GB/T17776-1999). Rumen pH was determined by digital pH meter
immediately following sample collection. The sample was then filtered with a four-layers wool fabric mesh,
centrifuged at 3500 rpm for 10 min, and the supernatant was collected and mixed with 50% sulfuric acid to
obtain a ratio of 50:1 (supernatant to sulfuric acid, v/v). Samples were kept at -200C until NH3-N and
volatile fatty acids (VFA) analysis. Rumen NH3-N concentration was determined using colorimetric method
(Feng and Gao 1993). Rumen VFA concentration was determined by gas chromatography after pretreatment
using the method of Erwin et al (1961).

Statistical analysis
We used SPSS 16.0 for Windows for statistical analysis. Variance analysis was performed with One-way
analysis of variance and significant differences were determined by Duncan's multiple range test.

Results and Discussion

Effects of dietary supplementation with sulfate on nitrogen metabolism in cashmere goats

There were no significant differences in N intake among four diets (P>0.05) in this study (Table 2).
However, Wang (1999) found that dietary supplementation of inorganic sulfur could increase N intake,
which agreed with the result of Kennedy and Siebert (1972). This is probably because their experimental
animals were different from our study. We also found that there were no significant differences in fecal N
and urinary N among different sulfate supplementation, which agreed with Peng et al (2001).

In this study there was no effect on digestible N amount when the sulfur levels increased to 0.23% by
dietary supplementation with sulfate. Similarly, we didn’t find that there were significant differences in
retained N or N apparent digestibility among Na2SO4 diet, ZnSO4 diet and MnSO4 diet at the sulfur level of
0.23%. Thus, the nitrogen metabolism was not affected by dietary supplementation with sulfate, and there
were no significant differences in nitrogen metabolism among the above three diets with the same sulfur
level.
Table 2: Nitrogen metabolism in Liaoning cashmere goats with different sulfate diets

Control Na2SO4
Items ZnSO4 diet MnSO4 diet SEM P
diet diet
N intake, g/d 12.2 11.9 12.1 12.0 0.279 0.863
Fecal N, g/d 1.10 1.05 1.09 1.15 0.0338 0.785
Urinary N, g/d 1.89 1.78 2.22 1.95 0.0112 0.351
Digestible N, g/d 11.1 10.9 10.95 10.8 0.327 0.564
Retained N, g/d 9.18 9.07 8.73 8.87 0.308 0.495
N apparent
90.7 91.2 90.9 90.2 0.724 0.387
digestibility, %

Effects of dietary supplementation with sulfate on sulfur metabolism in cashmere goats

The study found that S intake significantly increased at the dietary sulfur levels of 0.23% by supplementing
with sulfate compared with the dietary sulfur level of 0.12% (Table 3). The fecal S content of the diets with
Na2SO4 and ZnSO4 was significantly greater than that of the control diet but lower than that of MnSO4 diet,
which indicated that the sulfate supplementations can increase S loss in feces accordingly, especially with
MnSO4. The results also showed that there were no significant differences in urinary S content among the
four diets, which agreed with Qi et al (1992).
 Both digestible S amount and retained S amount of Na2SO4 diet and ZnSO4 diet were respectively greater
than that of the control diet. The digestible S amount of MnSO4 diet was significantly greater than the
control diet but lower than Na2SO4 diet and ZnSO4 diet. There was no significant difference in retained S
amount between MnSO4 diet and the control diet, but the retained S amount of MnSO4 diet was lower than
that of Na2SO4 diet and ZnSO4 diet. There was no significant difference in S apparent digestibility among
the four diets.

The above results indicated that dietary supplementation with sulfate can affect sulfur metabolism in
cashmere goats, but there were significant differences among different sulfates, of which dietary MnSO4
supplementation probably could not benefit S utilization in cashmere goats, compared with other two
sulfates. The differences were made possibly because that MnSO4 supplementation could inhibit S digestion
than Na2SO4 and ZnSO4
Table 3: Sulfur metabolism in Liaoning cashmere goats with different sulfate diets

Control Na2SO4
Items ZnSO4 diet MnSO4 diet SEM P
diet diet
S intake, g/d 1.17b 1.59a 1.59a 1.58a 0.0193 0.024
Fecal S, g/d 0.41 c 0.51b 0.48 b 0.63a 0.0218 <0.001
Urinary S, g/d 0.28 0.36 0.32 0.42 0.0229 0.125
Digestible S, g/d 0.76 c 1.08a 1.11 a 0.95b 0.0413 0.001
Retained S, g/d 0.48 b 0.72a 0.79 a 0.53b 0.0212 0.039
S apparent
64.9 67.8 68.7 60.5 0.258 0.165
digestibility/%
ab Means in the same row without common letter are different at P<0.05.

Effects of dietary supplementation with sulfate on rumen pH in cashmere goats

Rumen pH is a comprehensive parameter which can reflect fermentation status in the rumen. The variation
of rumen pH is determined by dietary composition and nutrient levels (Russell and Rychlik 2001). Too high
or too low pH has an adverse impact on the growth and fermentation of rumen microbe.

This study showed that the variation ranges of rumen pH with different sulfate diets (Table 4) were
consistently in its optimal pH range (pH 6.0 to 7.0) for the synthesis of rumen microbial protein (Satter and
Slyter 1974). Rumen pH gradually decreased after feeding with each diet and the lowest pH occurred about
2 h post-feeding, after which it gradually increased. Similar variation tendency of rumen pH appeared after
the next feeding. Thus, our study demonstrated that the variation of rumen pH is determined by time interval
after feeding.

Moreover, we found that the average values of rumen pH with four diets were all within the optimal rumen
pH range for cellulose digestibility ranged from 6.4 to 6.8 (Edman 1988), and there were no significant
differences in the average values of rumen pH among four diets. These results indicated that dietary
supplementation with sulfate had no significant effects on rumen pH in Liaoning cashmere goats, which is
similar to the results of Elliott and Armstrong (1982) and Qi et al (1992). However, our results do not
support the results of Weston et al (1988) who found that dietary sulfur supplementation decreased rumen
pH in sheep. It is clear that there are inconsistent results of previous studies, which probably is due to the
differences among species of experimental animals.
Table 4: Rumen pH after feeding with diets containing different sulfates

Hours after Control Na2SO4

ZnSO4 diet MnSO4 diet SEM P
feeding diet diet
0 6.73 6.76 6.73 6.71 0.0186 0.317
2 6.43 6.50 6.46 6.35 0.0365 0.435
4 6.28 6.45 6.34 6.23 0.0540 0.621
6 6.36 6.46 6.55 6.54 0.0301 0.416
8 6.50 6.56 6.50 6.45 0.0449 0.572
10 6.62 6.63 6.62 6.50 0.0497 0.538
Average 6.49 6.56 6.53 6.46 0.0239 0.513
 Effects of dietary supplementation with sulfate on rumen NH3-N utilization in cashmere goats
The concentration of NH3-N can reflect the microbial decomposition of nitrogenous substances and the
utilization of NH3 in rumen. Rumen microbes can reutilize the degraded NH3-N to synthesize microbial
proteins (Or-Rashid et al 2001), and the optimal NH3-N concentration for microbial protein synthesis in
rumen is more than 5mg/100 mL (Satter and Slyter 1974).

Our study showed that the ranges of rumen NH3-N concentration with four diets (Table 5) were all within
the optimal range of rumen NH3-N concentration for microbial protein synthesis as proposed in previous
studies (0.35~29 mg/ml, Owens and Bergen 1983; 6~30 mg/100 ml, Preston and Leng 2009). The
concentration of rumen NH3-N all gradually increased after feeding with each diet and reached the highest
level at 2 h after feeding, after which it decreased till next feeding.

The results showed that the concentrations of rumen NH3-N of the diets supplemented with Na2SO4,
ZnSO4, and MnSO4 was lower than that of the control diet during 2 h to 6 h after feeding, respectively. In
addition, we found that dietary supplementation with sulfate can significantly decrease the average
concentration of rumen NH3-N in Liaoning cashmere goats, similarly to Kennedy and Siebert (1972). The
above results indicate that dietary sulfate supplementation can promote the absorption of NH3 by the rumen
of cashmere goats, and there are no significant differences in this promoting role among different sulfates. It
is speculated that sulfate supplementation could increase synthesis of microbial protein and then promote
utilization of NH3 in rumen of cashmere goats.
Table 5: Rumen NH3-N concentration (mg/100 mL) after feeding with diets containing different sulfates

Hours after Control Na2SO4

ZnSO4 diet MnSO4 diet SEM P
feeding diet diet
0 18.1 19.0 20.9 18.3 0.410 0.214
2 27.7 a 25.3 b 23.1 b 23.9b 0.345 0.043
4 20.2 a 17.9 b 17.4 b 18.5b 0.253 0.031
6 15.1a 12.3b 13.1b 12.5b 0.158 0.039
8 12.1 10.9 11.2 11.7 0.179 0.088
10 11.7 10.3 10.7 10.1 0.143 0.175
Average 17.5a 16.0b 16.1b 15.8b 0.175 <0.001
ab Means in the same row without common letter are different at P<0.05.

Effects of dietary supplementation with sulfate on rumen VFA production in cashmere goats
VFA is a key parameter of measuring the status of rumen fermentation in ruminants, and its content,
composition and proportion depend on dietary composition, rumen environment, and microbial population.
The fatty acids are readily absorbed and assimilated as a nutrient source by ruminants (Bergman 1990).

Our study showed that rumen VFA concentration gradually increased after feeding and peaked at about hour
4 post-feeding, and then it decreased and reached the lowest level just before the next feeding (Table 6). The
results also showed that the concentrations of rumen VFA of the diets supplemented with Na2SO4, ZnSO4,
and MnSO4 was higher than that of the control diet at 2 h and 4 h after feeding, respectively. Additionally,
we found that the average concentration of rumen VFA of the diets supplemented with sulfate were
significantly greater than that of the control diet between the two adjacent feeding in Liaoning cashmere
goats. The results indicated that dietary sulfate supplementation can effectively increase rumen VFA
concentration in cashmere goats, and there was no significant difference among three diets supplemented
with different type of sulfate.

Dietary nutritional balance can improve the ability of microbe to digest feedstuffs in ruminants. Therefore,
sulfate supplementation of the basal diet may enhance the microbe growth, which then can promote rumen
VFA production in cashmere goats. However, no significant difference was found in rumen VFA
concentration among the diets supplemented three types of sulfate in our study, which presumably suggested
that there is no significant difference in the ability of rumen microbe to produce VFA among the diets
containing different sulfates.
Table 6: Rumen VFA concentration (mmol/L) after feeding with diets containing different sulfates
Hours after Control Na2SO4
ZnSO4 diet MnSO4 diet SEM P
feeding diet diet
0 26. 1 28.9 27.9 29.3 0.566 0.184
2 35.3 b 40.7 a 41.2 a 42.8a 0.714 0.041
4 46.7 b 52.5 a 53.4 a 52.3a 0.676 0.036
6 41.1 46.9 43.2 45.5 0.743 0.133
8 36.7 38.8 38.7 40.6 0.745 0.083
10 31.1 33.8 35.1 34.5 0.658 0.159
Average 36.2b 40.3a 39.9a 40.8a 0.629 0.042
ab Means in the same row without common letter are different at P<0.05.

Conclusions

Based on the results of this research it is conclude that dietary supplementation with sulfate doesn’t
affect nitrogen metabolism but has a significant effect on sulfur metabolism in cashmere goats. This
treatment has no significant effect on rumen pH but can decrease rumen NH3-N concentration and
increase rumen VFA concentration significantly. It is demonstrated that dietary sulfate
supplementation can promote sulfur metabolism and improve rumen fermentation in cashmere goats,
and there are significant differences among the diets supplemented respectively with Na2SO4, ZnSO4,
and MnSO4, of which MnSO4 supplementation could decrease S digestion of cashmere goats than
other two sulfates according to this study.

Acknowledgements

The work was supported by the Nature Science Foundation of China (NO. 30700577) and National Key
Technology R&D Program (No. 2008BADB2B05 and 2009BADA5B02). We thank Prof. Yujun Zhao and
Dr. Lin Li for helping us install ruminal cannulas for the experimental goats, and we also thank Shizheng
Yu, Xinxin Wang and Wenxiao Qin who participated actively in this study.

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Received 12 December 2012; Accepted 3 February 2013; Published 1 March 2013

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