Introduction

Se is an essential trace element, and its importance for animal health and productivity has been well confirmed. Se has known to be involved in enzyme activity and preventing oxidative damage to body tissue [1]. Se is also associated with protein fractions in microorganisms and plants [2]. Se has a very narrow margin between its lowest acceptable level of intake and its toxicity. The toxicity of Se depends on the chemical form, species, physiological state, nutrition, and dietary interactions [3, 4].

Diets for ruminants are almost exclusively of plant origin and the Se concentration within plants can be varied remarkably in China and other countries [5, 6]. The deficiency of Se always causes great losses in livestock. Thus, addition of Se to animal diets is required around the low Se region. Inorganic informs, typically sodium selenite (Na2SeO3) or selenate (Na2SeO4) and organic Se such as Se-enriched yeast (YS), are two principal forms used in ruminants [7] and EEC (2006) [8]. Prior studies have confirmed that organic Se is more effective than inorganic Se in ruminants [9–15] and inorganic Se is more toxic than organic Se [16]. Recently, elemental nano-Se caused great interest in the high bioavailability and low toxicity [17–19]. Shi et al. (2011) reported addition of nano-Se to sheep feedstuffs could improve rumen fermentation and feed utilization. However, little is known about the influence of different Se sources at high dose on fermentation characteristic and nutrient digestibility [20].

Thus, the objectives of this research were to investigate the effects of feeding high-dose YS and NS on feed digestibility, rumen fermentation, and excretion of purine derivatives in sheep.

Materials and Methods

Chemicals

The Se-enriched yeast was purchased from Angel Yeast Co., Ltd., China. The concentration was 1,000 mg/kg of Se. Nano-Se was purchased from Shanghai Stone Nano-Technology Port Co., Ltd., China, and concentration was also 1,000 mg/kg of Se.

Animals, Diets, and Experimental Design

Six Dorset sheep × Small Tail Han × Tan sheep, average 43.32 ± 4.8 kg of body weight (BW), were ruminally cannulated and used in a replicated 3 × 3 Latin square experimental design with three 25-day periods (15-day adaptation and 10-day sampling collection each period). The three treatments were a basal diet (control), a basal diet supplemented with 4 g YS (provide 4 mg Se) per kilogram of diet dry matter (DM), and a basal diet supplemented with 4 g NS (provide 4 mg Se) per kilogram of dry matter (DM). The supplement of YS or NS was mixed into concentrate in the diets. Sheep were fed twice daily (07:00 and 19:00 h) at maintenance nutrition requirements with a basal diet consisting of 300 g/kg (dry matter) DM of basal concentrates and 700 g/kg DM of forage. The proximate composition of basal diet is shown in Table 1. Sheep were placed in metabolic cages individually and fresh water was freely available during the experimental period.

Table 1 The ingredients and chemical composition of the basal diet (g/kg dry matter)
Full size table

Digestibility in the Total Tract

A digestion trial was conducted with all sheep on days 11–20 of each period. Sheep was dosed via ruminal cannula with 1 g chromic oxide twice daily at 07:00 and 19:00 h for 10 days. The initial 5 days were used for uniform chromic oxide excretion and next five sampling days were used for collection of feces. Fecal pellets were collected from rectum at 07:00 and 19:00 h, and representative samples of the feces were pooled by sheep for each period. After drying in an oven at 60 °C, the samples were ground to pass a 1-mm sieve for chemical analyses. Dry matter excreted in feces was evaluated by dividing chromium input by chromium concentration in the feces. Excretion of other nutrients in the feces was evaluated by multiplying DM flow by their concentration in fecal DM.

Ruminal pH and Fermentation

Samples of rumen fluid were collected through the cannula at 0 h (i.e., just before feeding in the morning), 3, 6, and 9 h after feeding on days 23 and 24 of each collection period for pH, ammonia-N, and volatile fatty acids (VFAs) determination. Ruminal pH was immediately measured using an electric pH meter (Sartorius Basic pH Meter PB-20; Sartorius AG, Goettingen, Germany).

In Situ Ruminal Degradability

Ruminal degradability of soybean meal and Leymus chinensis was determined using nylon bag technique on days 16–18 of each experimental period. Soybean meal and Leymus chinensis were ground through a 2.5-mm screen, and about 3.5 g of L. chinensis and 4.5 g of soybean meal were weighed into 6 cm × 10 cm nylon bags made of monofilament Pecap polyester (Guangzhou Minyuan Business Co., Ltd., Guangdong, China). The bags were incubated in the rumen of each sheep for 0, 6, 12, 24, 48, and 72 h. As soon as the bags were removed from the rumen, they were rinsed in cold water until they were clean, and then dried in an oven at 65 °C for 12 h, 105 °C for 24 h. Residual DM of bags were ground to pass a 1-mm screen for chemical analysis. Ruminal nutrient degradability was fitted to the exponential model:

$$ y = a + b\left( {{1} - {e^{{ - c}}}^{{(t{ } - { }L)}}} \right), $$

for t > L [21], where a is the soluble fraction, b is the slowly degradable fraction, c is the fractional degradation rate constant at which b is degraded, L is the lag time (h), and t is the time of incubation (h). The non-linear parameters “a”, “b”, and “c” were estimated using the non-linear regression procedure of SAS (1996). Effective degradability (ED) of feeds in the rumen were calculated as \( {\text{ED}} = a + [\left( {b \times c} \right)/\left( {c + k)} \right] \) [22], where k was the passage rate of the digesta from the rumen and was 0.02/h for L. chinensis and 0.052/h for soybean meal according to our measurements.

Urine Collection and PD Measurements

Total collections of urine were conducted for 10 days (from day 16 to 25). Urine volume was measured daily and collected into buckets containing approximately 10 mL of 10 % H2SO4 to reduce pH below 3 for prevention bacteria destruction of purine derivatives (PD). At the end of the collection days, 20-mL urine samples were diluted to 100 mL with distilled water, then sub-sampled and stored at −20 °C for analysis of allantoin, uric acid, xanthine, and hypoxanthine. The relationship between microbial purines absorbed (X, mmol/day) and the purine derivatives (PD) excreted (Y, mmol/day) was estimated using the equation described by Chen and Gomes [23]:

$$ Y = { }0.{84}X + { }\left( {0.{15}0{W^{{0.{75}}}}{e^{{ - 0.{25}X}}}} \right) $$

Laboratory Analysis

All dried samples were ground through a mill to pass a 1-mm screen and later analyzed for dry matter (DM, ID no. 934.01), organic matter (OM, ID no. 942.05), and crude protein (CP, ID no. 984.13) according to methods of AOAC [24]. Neutral detergent fiber (aNDF, with heat-stable alpha amylase and sodium sulfite, and expressed inclusive of residual ash) and acid detergent fiber (ADF) were determined according to Van Soest et al. [25]. Ruminal VFA was determined using gas chromatography (GC102AF; Shanghai Specialties Ltd., China). The concentrations of ammonia N (NH3–N) was determined by the method of AOAC [24]. Allantoin and uric acid in urine was determined according to the procedure of IAEA (1997).

Statistical Analyses

Data on digestibility were analyzed using the mixed model procedure of SAS (Proc Mixed) [26] to test the effects of square, period within square, animal within square, and treatment. The treatment was as a fixed effect; square, period within square, and animal within square were as random effects. Rumen fermentation parameters were also analyzed using the same mixed model but with time included as a repeated measures. Relationships between measured variables were analyzed using linear correlation procedure of SAS. Effects of factors were considered significant at P <0.05.

Results

Digestibility in the Total Tract

As shown in Table 2, digestibility of DM, organic matter (OM), crude protein (CP), ether extract (EE), aNDF, and ADF in the total tract were higher in Se supplemented animals than in control sheep (P < 0.01), and also with significantly (P < 0.01) higher values in NS group compared to SY group.

Table 2 Effects of high-dose nano-selenium (NS) and selenium–yeast (SY) supplementation on nutrient digestibility in the total tract of sheep
Full size table

Ruminal pH and Fermentation

Ruminal fermentation characteristics are shown in Table 3. Mean ruminal pH, ammonia N content, molar proportion of propionate, and the ratio of acetate to propionate were lower for NS supplemented sheep compared to the control and YS treatment (P < 0.01). Total ruminal VFA concentration was higher (P < 0.01) in YS group. Molar proportions of acetate and butyrate were not affected by Se supplementation (P > 0.05).

Table 3 Effects of high-dose nano-selenium (NS) and selenium–yeast (SY) supplementation on ruminal pH and fermentation in sheep
Full size table

Effective Ruminal Degradability

Table 4 showed the in situ ruminal digestion kinetics and effective degradability (ED) of L. chinensis and soybean meal. For L. chinensis, the soluble fraction, slowly degradable fraction, fractional degradation rate, and the ED of L. chinensis DM and aNDF were higher for NS than YS and control treatments (P <0.01), and the YS was higher than control treatments (P < 0.01).

Table 4 In situ ruminal digestion kinetics and effective degradability (ED) of Leymus chinensis and soybean meal
Full size table

For soybean meal, the soluble fraction, slowly degradable fraction, and ED of DM were higher for NS compared to the YS and control treatments (P < 0.01), and YS was higher than control treatments (P < 0.01). However, fractional degradation rate in NS was the lowest among treatments (P < 0.01). Similarly, the soluble fraction, slowly degradable fraction, fractional degradation rate, and the ED of CP were higher in NS compared with other treatments (P < 0.01), and YS was higher than control treatments (P < 0.01).

Urinary Purine Derivatives

Urinary purine derivatives are shown in Table 5. Daily urinary excretion of uric acid, xanthine, and hypoxanthine were not affected by NS and YS addition (P > 0.05). However, urinary excretion of allantoin and PD were higher in NS and YS treatments than control treatment (P < 0.01), and urinary excretion of PD in NS treatment was higher than YS treatment (P < 0.01).

Table 5 Effects of high-dose nano-selenium (NS) and selenium–yeast (SY) supplementation on urinary purine derivatives in sheep
Full size table

Discussion

Ruminal Fermentation

Supplementation of NS or YS in sheep switched rumen fermentation pattern from acetate to propionate as shown by the reduction in ratio of acetate to propionate. The decrease of the ratio of acetate to propionate and the increase of total VFA concentration resulted from the increased propionate concentration. It is suggested that Se supplementation in sheep diets particularly improved ruminal microbial activity in propionate production. In the present study, mean ruminal pH was within the optimum range for cellulolytic bacteria activity [27]. The increase in the total VFA and propionate production and reduction in the mean ruminal pH by high dose NS or YS supplementation in our research agree with the results of Shi et al. (2011) who obtained higher VFA concentration and lower pH in the sheep fed with the basal diet supplemented with 3 g of nano-Se/kg DM (provide 3 mg Se) in sheep [20]. The similar results were also found in cows [28] and in lambs [29]. However, Serra et al. showed concentration of total VFA and propionate decreased slightly by administration 0.2 mg Se (Na2SeO3 or Na2SeO4) per kilogram of DM in sheep [30]. The conflicting results may ascribe the conversion of inorganic Se to insoluble forms in the rumen, thus decreasing its availability. In the present study, the total VFA concentration was higher in NS than YS treatment. Thus, we concluded absorption and availability of Se was closely related to chemical form of Se in ruminant. Zhang et al. (2008) noted nano-Se possessed a higher bioavailability because of its excellent catalytic efficiency, low toxicity, and strong adsorbing ability [19]. All these novel properties of nano-Se and higher bioavailability may explain increased rumen fermentation by NS supplementation compared with YS.

Apparent Digestibility in the Total Tract and In Situ Ruminal Degradability

The increase of in situ ruminal ED of L. chinensis DM and NDF was consistent with higher ruminal total VFA concentration and increased nutrient digestibility in the total tract in NS treatment compared to control and YS. Our results are in accordance with that published by Shi et al. (2011) who found in situ ruminal ED of L. chinensis DM and NDF was increased by supplementation with 3 g nano-selenium (provide 3 mg Se) per kilogram of DM in sheep [20]. However, the results are in contrast with the report by Serra et al. who found that supplementation of sheep feed with 0.2 mg Se (Na2SeO3 and Na2SeO4, per kilogram of DM) had no effect on digestibility of NDF [30]. The results showed addition 4 g/kg dietary DM NS could significantly increase growth and activity of cellulolytic bacteria compared to YS, and thus improved rumen fermentation. This allowed us to conclude that different metabolic ways exist between inorganic Se and organic Se in rumen. Further studies are necessary to clarify the metabolic mechanisms of different Se forms.

The increase of in situ ruminal ED of soybean meal DM and CP by NS or YS supplementation was also in line with the prior study that digestibility of crude protein was significantly increased by supplementation with 3 mg/kg dietary DM Se (nano-Se) in sheep [20]. The similar results were also found in rats [31], in pigs [32], and in dairy cows [28]. In the present study, the digestibility of CP and in situ ruminal ED of soybean meal CP were higher in NS than YS treatment. The results suggest nano-Se supplementation could significantly increase activity of protein-decomposing bacteria and promote proteolytic digestive enzymes activity.

Nitrogen Metabolism and Urinary Purine Derivatives Excretion

Se supplementation could increase rumen microbial population and activity [18, 33, 34]. The reduction of ammonia N concentration by NS or YS supplementation could be due to an enhanced growth of ruminal microbial populations which increased the ammonia N consumption. The increased urinary excretion of PD (Table 5) suggested that Se supplementation may increase the microbial protein production in the rumen. Besides, Russell et al. observed that cellulolytic bacteria obtain their N exclusively from ammonia N [35]. One can suggest that NS or YS supplementation may increase the ruminal microbial protein synthesis as effective ruminal degradability and the nutrient digestion in the total tract was improved.

Conclusions

Supplementation of NS and YS in sheep increased ruminal VFA concentration and switched rumen fermentation pattern from acetate to propionate. Nutrients digestibility in the total tract, in situ ruminal aNDF degradation of L. chinensis and CP of soybean meal, and urinary excretions of PD were also improved by Se supplemented. Feeding 4 g nano-Se per kilogram of dietary DM could significantly improve feed utilization and rumen fermentation compared with yeast–Se.