Effect of sulfur on rumen ecology, blood metabolites, hormones and production in lactating dairy cows supplemented with fresh cassava foliage or cassava hay
 

C Promkot and M Wanapat*

Faculty of Natural resources ,Rajamangala  University of Technology Isan,
Sakon Nakhon Campus ,Sakon Nakhon, 47160, Thailand
promkot@yahoo.com
* Tropical Feed Resources Research and Development Center,
Department of Animal Science, Faculty of Agriculture
 Khon Kaen University , Khon Kaen, 40002, Thailand
 

Abstract

The effect of sulfur (S) supplementation on utilization of fresh cassava foliage and cassava hay was evaluated using sixteen multiparous Holstein-Friesian crossbred dairy cows in mid-lactatation. The experimental design was a 2x2 factorial arrangement using two roughages (FCF: rice straw + fresh cassava foliage, CH: rice straw + cassava hay, CH) and two elemental sulfur levels (0.15 and 0.4% S of DM). The four dietary treatments were offered in the form of a totally mixed ration with concentrate to roughage (chopped rice straw + chopped cassava foliage) at 60:40 ratio and offered ad libitum.

Fresh cassava foliage or cassava hay did not affect DM intake, rumen ecology parameters, total tract digestibility, blood metabolites and hormones. However, HCN intake and blood thiocyanate concentration were significantly higher in cows fed with fresh cassava foliage, although with no sign of toxicity. DM intake, average daily live weight gain, molar percentage of propionate in the rumen, rumen bacteria and fungal zoospores were increased in cows fed the higher level of  sulfur.  The higher S level also appeared to facilitate cassava cyanide detoxification especially when the cassava foliage was given in the fresh state.

It is concluded that supplementation with 0.4% sulfur will be nutritionally beneficial when cassava foliage is used as a portion of the roughage component in diets for milking cows.

Key words: Bacteria, digestibility, feed intake, fungal zoospores, HCN, VFA
 

Introduction

Feeding of dairy cattle in the tropics is often difficult because of deficiencies in feed supply, in both quality and quantity (Wanapat and Devendra 1992). The use of rice straw as a feed in the dry season, in spite of its low nutritive value, has been a common feeding system, generally practiced by dairy farmers in the tropics when green forages are often scarce (Leng and Preston 1983; Wanapat 1994). Chemical treatment of rice straw to improve its quality has been reported (Wanapat et al 1990, 1996). The development and utilization of cassava hay (cassava whole crop at a young growth stage, 3-4 months and being harvested about 30-45 cm above ground, sun-dried for 1-2 days until having a final dry matter of at least 85%, Wanapat 1999; 2003) as an on-farm feed has been recommended as a possible solution to the lack of good-quality roughages during the dry season in the tropics (Wanapat et al 1997). Cassava hay has high protein content, (20 to 27% in DM) and condensed tannins (1.5 to 4%. The use of cassava hay at 0.56 to 1.70 kg/head/day or about 0.1 to 0.5 % BW has proved to be an excellent ruminant protein feed (Wanapat 1999; 2003). The use of cassava hay has been successfully implemented in several ways by either direct feeding or as a protein source in the concentrate mixtures (Wanapat et al 2000a,b,c; Hong et al 2003; Kiyothong and Wanapat 2004; Wanapat et al 2007), as a combination in a pellet of cassava hay, soybean meal and urea (Wanapat et al  2006) or inclusion in a high quality feed block (Wanapat et al 1999; Wanapat and Khampa 2006). 

Fresh cassava foliage supported high growth rates (>800 g/day) when fed to fattening cattle as the sole source of roughage and protein in a liquid-based diet of molasses/urea (Ffoulkes and Preston 1978). However, there is a lack of information of feeding fresh cassava foliage as a supplement for dairy cows. During the rainy season, it is difficult to make cassava hay, therefore feeding fresh cassava foliage to ruminants could be a possible alternative method. However, cassava foliage especially fresh cassava contains cyanogenic glucosides, linamarin and lotaustralin. After tissue damage, these are hydrolyzed by the endogenous enzyme linamarase to cyanohydrins. Further hydrolysis to HCN may result in chronic toxicity. In ruminants, HCN can be rapidly detoxified by rhodanese and b-mercaptopyruvate sulfurtransferase (Martensson and Sorbo 1978; Frankenberg 1980) by rumen microbes (Majak and Cheng 1984) and animal tissue (rumen wall, liver, kidney and red blood cell) (Aminlari and Gilapour 1991; Aminlari et al 1989). Rhodanase is a sulfur transferase that catalyses the formation of thiocyanate from cyanide and thiosulphate or other suitable sulfur donor, then the less toxic thiocyanate is excreted in the urine.

The main sources of sulfur for HCN detoxification are the sulfur amino acids, cysteine and methionine or elemental sulfur (Oke 1978). Detoxification of cyanide into -SCN causes an increased demand for sulfur-containing amino acids (Maner and Gomez 1973). NRC (2001) also mention that the sulfur requirement of dairy cattle consuming cyanogenic plants (such as cassava or sorghum) may be increased because of the need for sulfur in the detoxification of cyanogenic glucosides. Hence, the hypothesis behind this study was that feeding fresh cassava foliage or cassava hay (10% in total ration) combined with adequate sulfur could provide good sources of protein for milk production in dairy cattle with no toxic effects from cyanide.

The objective:

This was to evaluate the effect of sulfur supplementation of fresh cassava foliage and cassava hay in milking dairy cows, on:

• rumen pH, ammonia nitrogen, total volatile fatty acids, cyanide concentration and microflora population and microbial protein synthesis

• nutrient digestibility

• serum concentration of urea nitrogen, thiocyanate, thyriod gland hormones and liver enzymes

• milk production and composition.

Materials and Methods

Experimental design, animals and treatments:

Sixteen , multiparous Holstein Fresian crossbred dairy cows were used in a randomized complete block design (RCBD) to determine the effects of sulfur on utilization of fresh cassava foliage and cassava hay. The experiment period was 30 days and carried out on the dairy farm of the Faculty of Natural Resources, Rajamangala University of Technology Isan, Sakon Nakon Campus.   The cows were from 100 to150 days-in- milk  with a range of 12-15 kg/day milk yield. The treatments in a 2x2 factorial arrangement were: two levels of sulfur (0.15 and 0.4% in diet DM) and either fresh cassava foliage (FCF) or cassava hay (CH) supplying 10 % of the diet DM. Summary of treatment feeds (total mixed rations, TMR) and animals in the experiment are shown in Table 1. 

Feeds and cows management:

Whole fresh cassava foliage crop (variety Rayong 72, bitter variety) was harvested from plots on the university farm. Cutting height was 30-45 cm above ground (at the point where the woody stem charges to the green stem) at a young growth stage (3-4 months). Harvesting was once a day in the morning (07:00 am) throughout the experimental period. The foliage was kept in the cooling room (4⁰ C) until the total mixed rations (TMR) were prepared. The cassava hay was made by chopping the fresh foliage which was then sun-dried for 1-2 days until reaching a final DM of at least 85%. Concentrate and roughage (30% chopped rice straw and 10% chopped FCF or CH, DM basis)  were provided every day (09:00 am and 15:00 pm) in the form of total mixed ration (TMR) with concentrate to roughage at 60:40 using a mixing machine. Water was added to obtain  TMR moisture at 45-50 %.

The TMR diets were formulated for two levels of S and two supplemental roughages (FCF and CH) (Table 3).  The cows were housed individually for each treatment and had ad libitum access to the TMR (fresh quantities offered three times a day), fresh water and mineral block. The cows were milked twice daily by milking machine. The cows were adjusted to the diets for the first two weeks, and actual intakes and other measurements were taken subsequently during the experimental period of 30 days of milk collection.

Feeds, feces, rumen fluid and blood sampling

Feed intakes were recorded daily. Samples of feeds (fresh feed offered and feed residues) were randomly collected (250 g) once a day in the morning before new fresh feed was offered and were kept in a refrigerator until analysis. During the last five days of each period, the cows were in metabolism crates. Feces and urine were collected daily before the morning feeding; a 1% sub-sample of urine and a 3% sub-sample of feces were collected, bulked  by period and stored at –20°C until later analysis.

Rumen fluid and blood samples were collected at 0 and 4 h-post feeding on the last day of each period. About 100 ml of whole rumen fluids were collected by stomach tube from individual cows and pH of rumen contents was determined immediately using a glass electrode pH meter. Ruminal fluid samples were then filtered through four layers of cheesecloth and were centrifuged (3,000´g, 4⁰ C for 15 min) immediately and supernatant of centrifuged rumen fluid was divided into two parts. The first 50 ml of  rumen fluid was kept in a plastic bottle where 5 ml of 1M H₂SO₄ was added and stored at –20 ⁰C and later used for NH₃-N and VFA analysis. The second portion of 1 ml rumen fluid was kept in plastic bottle with 9 ml of 10% formalin solution and stored at 4⁰ C for later measurement of total direct count of bacteria, protozoa and fungal zoospores. Blood samples were taken from a coccygeal vessel into heparinized Vacutainer tubes and centrifuged immediately to separate plasma that was stored at -20°C before analysis. Milk yields of each cow were recorded daily. Milk samples of 60 ml (in ratio of morning milk samples to afternoon milk samples at 60:40) were collected twice daily during milking (05.00 and 17.00 h) on the last five days of each period for later chemical analysis. 

Chemical Analyses

Feed (both fresh feed offered and feed residues) and fecal samples were analyzed for DM, ash, CP (AOAC 1990) and NDF and ADF (Goering and Van Soest 1970). Hydrogen cyanide content of feed and cassava foliage was determined by spectrophotometer (SpectroSC, LaboMed, inc. USA.) with the 2, 4-quinolinediol-pyridine reagent (Lambert et al 1975). Feeds (TMR, CH, FCF) were extracted with 0.1 M phosphoric acid and the acid extract (feed material removed by filtration) hydrolyzed in 2 M H₂SO4 at 100 ⁰C for 50 minutes (Bradbury et al 1991). To 4.0 ml of the acid solution was added 5 ml of 3.6 M NaOH, after which the solution was filtered. After five minutes 1 ml aliquots were taken for colorimetric determination of cyanide by Modified Konig Reaction (Lambert et al 1975).

Rumen fluid samples were analyzed for NH₃-N using the procedure of AOAC (1990). VFA analysis was by High Performance Liquid Chromatography (HPLC; Model Water 600; UV detector, Millipore Corp.) according to the method of  Samuel et al (1997). Total direct count of bacteria, protozoa and fungal zoospores in rumen fluid were done according to the method of Galyean (1989).

Plasma samples were analyzed for urea-nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), triiodothyronine (T₃), and thyroxin (T4) using automated clinical chemistry analyzers (Vitallab Flexor E). Thiocyanate was determined using the method of Lambert et al (1975). Plasma samples  (1 ml) were added to 3 ml 15% w/v trichloroacetic acid, and centrifuged for 15 min at 1,000 g. One ml of supernatant was used for colorimetric procedure with 2, 4-quinolinediol-pyridine as the coupling reagent (Lambert et al 1975).

Milk samples were analyzed for fat, protein, lactose and solids-not-fat using infrared apparatus (Milko-scan 104, Foss Electric, Denmark). A sub-sample of the composite was analyzed for milk urea nitrogen according to the method of Roseler et al. (1993) using a Sigma diagnostics kit #535 reading at 540 nm in a spectrophotometer (Spectonic 20, Milton Roy company, USA) and milk thiocyanate using the method of Lambert et al (1975). One ml of milk sample was added to 3 ml 15% w/v trichloroacetic acid, centrifuged for 15 min at 1,000xg. One ml of supernatant was used for colorimetric procedure with 2, 4-quinolinediol-pyridine as the coupling reagent (Lambert et al 1975).

Statistical analyses

 Data were analyzed using Proc. GLM model in the software of SAS (1996). Differences among means were tested using Duncan’s New Multiple Range Test.
 

Results and Discussion

Chemical composition of feed

All values for chemical composition of cassava hay and fresh cassava foliage (Tables 2 and 3) are in the range of reports in the literature (Wanapat et al., 2000a; Man and Wiktorsson, 2001; Wiktorsson and Man, 2002; Wanapat, 2003; Kiyothong and Wanapat, 2004; Wiktorsson and Khang, 2004; Wanapat et al. 2006).   

Dry matter intake, digestibility and body weight change

 DM intake tended to be higher (P=0.065) when the sulfur level was increased (Table 4). These results could be due to the significant increase in DM digestibility especially in the fresh cassava foliage treatment when the sulfur level was increased. Hydrocyanic acid (HCN) intakes were higher in cows fed fresh cassava foliage than in those fed cassava hay. However, there were no signs of toxicity.

The tendency for apparent digestibility of ADF to be higher in cows fed the higher S level could be attributed to increased numbers of rumen bacteria and fungal zoospores (Table 5). The tendency for a negative effect of the low S diet on live weight change may be due to reduction in muscular development as a result of depletion of the sulfur-containing amino acids necessary for formation of S-amino acids as well as for cyanide detoxification (Onwuka et al 1992). These authors reported that goats on the low sulfur, cassava-based diet had the greatest weight losses as compared to S supplemented groups.   They proposed that a dietary level of 0.5% elemental S ensured adequate cassava cyanide detoxification in sheep and goats. However, if the difference in our experiment was due to the needs of S for cyanide detoxification, there should have been an interaction between LW change and cassava, as the loss of weight should have been much more pronounced with the fresh cassava foliage, which had ten times higher concentration of HCN. In fact, there was no interaction.

Rumen ecology

There were no effects of the form of cassava foliage, level of S or the interaction of the two, on rumen pH, ammonia nitrogen, and total or individual VFA.  Similar findings were reported by de Oliveira et al (1996) that moderately high percentages of sulfur (0.4 to 0.6%) in the diet generally had no effects on these parameters. Qi et al (1993) also indicated that rumen ammonia was not affected by added sulfur. Wora-anu et al (2004) reported that cattle fed cassava hay or fresh cassava foliage showed no differences in terms of effects on rumen pH, VFA and ammonia concentrations.

There was an indication (P=0.081) that the molar percentage of propionate was increased on the higher level of sulfur. This finding is supported by Thompson et al (1972) and Zinn et al (1997) who reported increased rumen propionate in feedlot cattle when dietary sulfur was increased from 0.12 to 0.37 and 0.15 to 0.25 %, respectively. According to Goodrich et al (1978), low rumen propionate concentrations could occur in ruminants fed with sulfur-deficient diets because little lactate would be converted to propionate via the acrylate pathway. It is hypothesized that in cattle fed with cassava foliage, rumen microbes might need more sulfur for hydrogen cyanide detoxification (Onwuka et al 1992; Promkot et al 2007) leading to sulfur-deficiency in cattle fed a low sulfur diet which in turn might result in low rumen propionate concentration.

Rumen microbes

Rumen bacteria and fungal zoospores were in higher concentrations in the cows fed the higher S levels with a tendency for interaction with the form of the cassava (P=0.16 and P=0.085), indicating the effects were more pronounced on fresh cassava foliage than on cassava hay (Table 6).

It was reported by de Paiva (2007) that a dose of 0.31% sulfur could increase the population of  rumen microorganisms (including protozoa). Slyter et al (1986) reported that there were reduced numbers of cellulolytic bacteria in sulfur deficient sheep (0.04% S in the diet) in comparison to sulfur-supplemented sheep (0.34% S). Sulfur is one of many important factors for microbial growth (Leng and Preston 1983). Low levels of sulfur supplementation in the diet containing cassava foliage reduced microbial biomass in the rumen (Promkot et al 2007). Rumen fungi concentrations and activity may be increased by supplementation with a variety of sulfur sources according to Morrison et al  (1990) and Gutierrez et al (1996). In Australia, sulfur-fertilized grass (Akin et al 1983) and a methionine-supplemented diet (Gordon et al, cited by Akin and Borneman 1990) resulted in increased fungal populations. In research conducted by Orpin and Greenwood (1986) it was reported that N. patriciarum required a reduced form of sulfur.     

Blood metabolites and hormones

Thyroid hormones and liver enzymes were in normal physiological ranges (Table 7 which indicates that feeding fresh cassava foliage or cassava hay at 10% of the diet did not damage the liver and the thyroid gland. Similar findings have been reported by Khang and Wiktorsson (2004) that 11 % of total DMI of fresh cassava foliage did not affect liver enzymes and triiodothyronin and thyroxin concentration of the thyroid gland in local yellow cattle. Soto-Blanco et al (2001) also found that the levels of the thyroid hormones were unaffected when lactating goats were orally dosed with 3 mg/kg/day KCN for 90 days.

 The concentrations of SCN in plasma were greater in cows given fresh cassava foliage diets than in cows on the cassava hay diet.  This increase in the levels of thiocyanate in plasma in the cows fed fresh cassava foliage follows from the higher levels of HCN in the fresh foliage, which has to be detoxified to thiocyanate (Martensson and Sorbo 1978; Frankenberg 1980). The higher levels of SCN with increased S in the diet indicate that the S supplementation facilitated the detoxification of the HCN. This effect was more marked on the fresh cassava foliage diet, as indicated by the significant interaction. This finding indicates that sulfur supplementation could facilitate HCN detoxification (thiocyanate is a metabolic product of cyanide detoxification) in dairy cows fed with cassava foliage. Similar results were reported by Promkot et al (2007) who found that sulfur can stimulate the rate of HCN detoxification by rumen microbes. Onwuka et al (1992) also observed that levels of S in the diet were correlated with serum SCN when sheep were fed with a cassava-based diet. The fact that SCN differences in blood were not reflected in SCN levels in milk could be due to passage of SCN from the blood stream into milk being apparently slow. Grün et al (1995) reported that in cows with healthy udders the SCN content of blood plasma was higher than in milk (on average twice). Jose (2004) stressed that the mammary gland barrier reduces the SCN passage from maternal serum to milk.

HCN can be rapidly detoxified by rhodanese and b-mercaptopyruvate sulfurtransferase (Martensson and Sorbo 1978; Frankenberg 1980) in the rumen (Majak and Cheng 1984; Promkot et al 2007) and in animal tissues (rumen wall, liver, kidney and red blood cells) (Aminlari and Gilapour 1991; Aminlari et al 1989). Rhodanase is a sulfur transferase that catalyses the deformation of cyanide and thiosulphate or other suitable sulfur donor to less toxic thiocyanate which is secreted to the blood steam and then excreted in the milk and urine. The main sources of sulfur for HCN detoxification are the sulfur amino acids, cysteine and methionine or elemental sulfur (Oke 1978). Therefore, adding sulfur in feeds containing HCN is essential for HCN detoxification.

Milk yield and milk production

Milk production and composition did not differ among treatments except for protein content, which was higher in the high sulfur treatment (Table 8). Similar results have been reported by Stobbs and Wheeler (1977) that protein content of cow’s milk was increased following S supplementation.

 
Conclusions

• An increase in the level of sulfur supplementation from 0.15 to 0.4% (in diet DM) was beneficial to cows consuming fresh cassava foliage or cassava hay in terms of dry matter intake, live weight change, concentration of propionate in the rumen, population of rumen bacteria and fungi and milk protein content.

• The higher S level also appeared to facilitate cassava cyanide detoxification especially when the cassava foliage was given in the fresh state.

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