Effect of dietary cotton stalk on nitrogen and free gossypol metabolism in sheep

Diets, animals, and sampling

Three dietary treatments were used in this study. The control consisted of 50% wheat straw, 10% corn silage (DM based), and 40% formula concentrate feed. In Treatment (Treat) 1, half the wheat straw was replaced by an equal amount of crushed cotton stalk. For Treat 2, crushed cotton stalk substituted for all the wheat straw. The chemical compositions and components of the experimental diets are shown in Table 1. Six Xinjiang daolang wethers (35.6±4.21 kg) were divided into three groups after being fitted with ruminal and duodenal T-type cannulae. They were assigned to one of three dietary treatments using a 3×3 Latin square design (Exp 1). The sheep were fed twice daily at 0800 h and 1700 h. Chromic dioxide (Cr₂O₃) at a concentration of 2 g/kg DM was injected into the rumen via cannula at each feeding time. It was used to estimate duodenal digest flow and fecal output. Fresh water and mineral block (Kang Te Er Feed Technology Co. Ltd., Yue Yang, China) were available ad libitum throughout the study.

Table 1

Ingredient and chemical composition of basal diet1) (dry matter based)

Each experimental period lasted 21 d with 13 d adaptation and 8 d sampling. Feeds were sampled and recorded daily during the first 4 d of the sampling period. Fecal samples were collected from each sheep. Combined feed, orts, and feces samples were dried at 60°C for 48 h in a forced-air oven, ground to pass through a 1-mm screen, and stored for subsequent analyses. Duodenal digest was sampled over a 2-d period. On the first day, samples were drawn from the duodenal cannulae starting at 0800 h then collected again every 6 h. On the next day, sampling began at 1100 h. One half of the material collected was lyophilized and milled through a 1-mm screen for use in later analyses. The other half was acidified with 50% H₂SO₄, frozen at −20°C, and stored for later free gossypol measurements. On the seventh day of the sampling period, rumen fluid was extracted at 0800 h and again at 2000 h. Sample pH was determined immediately upon collection. The rumen fluid was then strained through four layers of gauze, acidified with 50% H₂SO₄, frozen at −20°C, and stored until analysis. Blood samples were collected from the jugular vein at the end of experiment and stored in vacutainer tubes. No anticoagulant was added so that the serum could be separated out and its free gossypol content measured.

In the second experiment, 15 healthy Xinjiang daolang wethers were randomly divided into three groups (five sheep per group) and fed for 60 consecutive days ad libitum feed containing 10% residue. At the end of the experimental period, three sheep from each group were killed. Muscle, liver and kidney samples were taken for histological observation and determination of tissue free gossypol levels.

Immediately after slaughter, about 2 cm×3 cm of liver and kidney samples, respectively were fixed in 2.5% glutaraldehyde and dehydrated with ethanol, washed with xylene and paraffin embedded. The paraffin embedded tissue blocks were sectioned with a microtome, and the sections placed on a glass slide and stained with stained with hematoxylin and eosin (HE). The slides were sealed with neutral rubber and examined with an Olympus BH-2 optical microscope (Olympus Corp., Shinjuku, Tokyo, Japan).

Another 1 mm×1 mm×1 mm of liver and kidney samples were fixed at 4°C in 2.5% glutaraldehyde. Then the tissue blocks were cropped and fixed in 1% osmium tetroxide, dehydrated in an acetone gradient, embedded in epoxy resin (#812) and electronic dyed with uranyl acetate. Ultrathin sections were made with a DIATOME ultra 45° and examined in a Nissan JEOL TEM 1230 transmission electron microscope (JEOL, Tokyo, Japan).

Chemical analyses

Samples of feed, duodenal digest, and feces were analyzed for crude protein [9]. Natural detergent fiber and acid detergent fiber were analyzed according to the procedures described by Van Soest et al [9]. Calcium and phosphorus were determined by the methods described by AOAC [10]. The chromium concentrations of the duodenal digest and the feces were measured by colorimetry [11]. The free gossypol content in the rumen fluid, duodenal digest, feces, blood, and tissue were determined according to the method of Mena et al [12].

Volatile fatty acid (VFA) composition of the rumen fluid was determined by high-performance liquid chromatography fitted with flame ionization detector detection and a capillary column (C18, 150×4.6 mm, 1 mL/min, 210 nm).

At the start of the trial and every 15 d thereafter, the lambs were weighed before- and after feeding, and the weight gain was calculated.

Tissue samples from all major organ systems were fixed in 10% v/v neutral buffered formalin. They were prepared for histological examination by routine paraffin embedding. Sections (6-μm thick) were made and stained with HE. They were examined under an Olympus BH-2 optical microscope (Olympus Corp., Shinjuku, Tokyo, Japan) and an EM-400 electron microscope (Koninklijke Philips N.V., Amsterdam, The Netherlands).

Calculations and statistical analysis

The data for DM, free gossypol, and nitrogen intake, duodenal flow, and apparent digestibility were all reported as least square means±standard error of the mean. They were analyzed as a Latin square with a factorial treatment arrangement using SPSS v. 17.0 (IBM Corp., Armonk, NY, USA). Overall differences in treatment means were considered significant when p<0.05.

Dry matter intake and digestion

Feed intake increased with compositional percentage of cotton stalk supplementation (p<0.01) (Table 2). The DM digestibility in the rumen and post-rumen significantly differed among treatments. The DM excretion into the feces decreased with increasing proportion of cotton stalk. There were no significant differences in total tract DM digestibility among treatments.

Table 2

Dry matter intake and digestibility based on dry metter content by sheep fed dietary cotton stalk

Rumen fermentation quality

For all animals tested, the rumen fluid pH was in the normal range of 5.93 to 6.13. After various proportions of cotton stalk were added to the diet, the ammonium nitrogen content in the rumen fluid changed to varying degrees. The rumen ammonium level in Treat 1 was significantly lower than those for the control and Treat 2 (p<0.01). The acetic acid content in the experimental groups was significantly lower than that of the control group (p<0.05). There were significant differences in total VFAs or propionic acid concentrations among the groups (p>0.05). The ratio of acetic acid to propionic acid decreased with increasing cotton stalk content in the diet (Table 3).

Table 3

Rumen fermentation quality

Nitrogen intake and balance

Nitrogen intake, digestion, and retention are shown in Table 4. Nitrogen intake was significantly higher in Treat 2 than the other treatments (p<0.05). Fecal nitrogen excretion did not significantly differ among treatments (p>0.05). Nitrogen retention increased with the proportion of cotton stalk in the diet (p<0.05).

Table 4

N intake and digestibility based on dry matter

Free gossypol intake and digestion

Dietary free gossypol intake and digestion, including free gossypol its disappearance and digestibility in the rumen and post-rumen did not significantly differ among treatments (Table 5). Free gossypol intake increased with the proportion of cotton stalk in the diet. Free gossypol excretion in the feces and urine increase with gossypol intake (p<0.05). The almost of intake free gossypol disappeared in the rumen. There were no significant differences among treatments in terms of the amount of free gossypol disappearing from the post-rumen (p>0.05). Free gossypol retention was significantly higher in Treat 2 than in either the control or Treat 1.

Table 5

Free gossypol intake and digestibility (based on dry matter)

Growth performance

The mean body weight and daily body weight gains for the 60-d study are presented in Table 6. There were significant differences in body weight gain and average daily gain among treatments.

Table 6

Sheep growth performance

Blood composition

Relative to the control group, the platelet counts, hemoglobin levels, and Fe content were significantly lower in Treat 1 and Treat 2 (p<0.05). For the other blood components, however, no significant differences among treatments were detected (p>0.05). Serum aspartate aminotransferase and γ-glutamyltransferase were significantly higher in Treat 2 than the others (p<0.05) (Table 7).

Table 7

Sheep blood chemistry

Tissue free gossypol levels

Plasma gossypol concentrations significantly increased (p<0.05) with supplemental cotton stalk levels. Free gossypol residue levels in the liver, kidney, and muscle significantly differed among treatments. Free gossypol tissue retention was higher in Treat 2 than in either the control or Treat 1. Free gossypol residues very significantly increased in the liver with free gossypol intake (Table 8).

Table 8

Free gossypol retention in fattening sheep body tissues (dry matter; mg/kg)

Microstructural changes in the liver and kidney

Hepatocyte swelling and nuclear dissolution, central venous- and portal vein congestion, local tissue necrosis, and inflammatory cell infiltration in necrotic areas were observed in the livers of the sheep ingesting cotton stalk (Figures 1A–1C).

Figure 1

Hematoxylin and eosin staining of the liver from sheep fed control (A), 25% cotton stalk (B), and 50% cotton stalk (C) (10×40 objective view).

Control kidneys (Figure 2A) have normal glomerular morphology and no swelling in the glomerular capillary network. Diseased kidneys have relatively smaller glomerular volumes (Figures 2B–2C), capillary network atrophy, thickening of the glomerular basement membrane, necrosis of the splenic cells in the renal tubules, and enlargement of the renal interstitium. Kidney ultrastructure (Figures 3D–3F) shows changes in the podocytes, vacuolar necrosis, swelling of the internal structures of the glomeruli and secondary structures, shrinkage of the endothelial cells, and mitochondrial swelling.

Figure 2

Hematoxylin and eosin staining of the kidney from sheep fed control (A), 25% cotton stalk (B), and 50% cotton stalk (C) (10×40 objective view).

Figure 3

Ultrastructure of the liver from sheep fed control (A), 25% cotton stalk (B), and 50% cotton stalk (C).