MATERIALS AND METHODS

Plant materials: Samples were collected from two sites of subtropical and tropical regions, namely (i) Southern part of Japan (Okinawa at 26°N127°E) and (ii) Bangladesh (Savar at 23°N 90°E and Shahzadpur at 24°N89°E). In Okinawa, a total of 31 plant samples were taken from 13 fodder plant species in February 2010. In Savar and Shahzadpur, a total of 63 plant samples were taken from 27 fodder plant species in September 2010. Stage of plant samples was fixed at the time of maturity when the farmers usually offer to ruminant livestock for feeding in these sites.

In Savar and Shahzadpur, it has been achieved significant success in increasing the animal production (especially milk) by providing training and extension services. Annual minimum temperature of the country (Bangladesh) varies from 8.0-13.4°C and maximum temperature 25.5-36.8°C. The average annual rainfall varies from 1429 to 4338 mm. The 80% of the rainfall occur in Monsoon which covers July to October of the year (BBS, 2002). Okinawa is a subtropical climate, with a relatively high annual average temperature of 23.0°C and precipitation is about 2100 mm.

Sample preparation and chemical analyses: All samples were cut and divided into plant fraction if necessary before drying. The dried samples were milled to pass through a 1 mm screen using a Wiley mill. Samples were analyzed for total and soluble oxalates following the method of Rahman et al. (2007). Insoluble oxalate content was estimated as the difference between the total and soluble oxalate contents. The calcium (Ca), magnesium (Mg), potassium (K) and sodium (Na) contents in the samples were determined by the flame atomic spectroscopy method after wet digestion with nitric acid and hydrogen peroxide (Laboratory of Agricultural Chemistry, The University of Tokyo, 1978). The correlation coefficient between oxalate and mineral contents in plants was analyzed using SPSS (version 12.0, SPSS Inc., Chicago, IL, USA).

RESULTS

The fodder plants tested were divided into 3 groups based on their soluble oxalate content, namely plants containing over 20 g kg^-1 DM (group 1, probably toxic as reported by McKenzie et al. (1988), 10-20 g kg^-1 (group 2, probably safe) and less than 10 g kg^-1 (group 3, safe).

The oxalate content of the plant samples at Okinawa site is given in Table 1. Samples belonging to group 1 included Setaria sphacelata; group 2 included Pennisetum purpureum, Cenchrus ciliaris and Digitaria decumbens and group 3 included other 9 plants. Chloris gayana, Saccharum officinarum and Leucaena leucocephala did not show any detectable soluble oxalate. The soluble oxalate content in plant samples ranged from 0 to 29.67 g kg^-1 DM and total oxalate content ranged from 0 to 32.12 g kg^-1 DM.

The oxalate content of the plant samples at two sites in Bangladesh is given in Table 2. Samples belonging to group 1 included Pennisetum purpureum and Brachiaria mutica; group 2 included Setaria sphacelata, Panicum maximum, Digitaria decumbens, Vetiveria zizanioides, Oryza sativa, Sesbania sesban and Saccharum officinarum and group 3 included other 18 plants.

Table 1:	Oxalate content (g kg-1 DM) of fodder plants in Okinawa, Japan
nd: not detected, Groups 1, 2 and 3 were classified with soluble oxalate content at >20, 10-20 and <10 g kg-1 DM, respectively, Mean values are in parenthesis

Table 2:	Oxalate content (g kg-1 DM) of fodder plants in Savar and Shahzadpur, Bangladesh
nd: not detected, Groups 1, 2 and 3 were classified with soluble oxalate content at >20, 10-20 and <10 g kg-1 DM, respectively, Mean values are in parenthesis

Leucaena leucocephala did not show any detectable soluble oxalate content. The soluble oxalate content in plant samples ranged from 0 to 31.61 g kg^-1 DM and total oxalate content ranged from 0-49.15 g kg^-1 DM.

The mineral concentration of the plant samples at Okinawa site is given in Table 3. The highest contents of K (16.36 g kg^-1 DM), Na (8.03), Ca (50.99) and Mg (8.92) were observed in Pennisetum purpureum, Setaria sphacelata, Leucaena leucocephala and Brachiaria spp. (Marandu), respectively. The mineral content of the plant samples at 2 sites in Bangladesh is given in Table 4. The highest contents of K (65.48 g kg^-1 DM), Na (7.69), Ca (30.11) and Mg (9.89) were observed in Eichhornia spp., Vetiveria zizanioides, Ficus hispida and Aletris farinosa, respectively. Table 5 represents the correlation coefficient between oxalate and mineral contents in the plants. In most of the cases, no relationship was observed between the oxalate and mineral contents in the plants, except for positive correlation between soluble oxalate and K contents in plants growing in Bangladesh.

DISCUSSION

Oxalate binds and forms insoluble compounds with some essential minerals including Ca, iron, zinc and Mg. High oxalate feeds inhibit mineral absorption. For example, Ca can combine with oxalate to form insoluble Ca-oxalate in the intestine, making Ca unavailable for absorption; Ca-oxalate is then excreted in feces. The decrease in serum Ca impairs normal cell membrane function, causing animals to develop muscle tremors and weakness, leading to collapse and eventually death. Acute oxalate poisoning has been reported with the ingestion of Setaria sphacelata, a high oxalate-containing plant, by Jones et al. (1970). Without adequate knowledge of oxalate content in fodder plants, dietary guidelines could not be established.

The data of present study revealed that the majority of fodder plants commonly consumed by livestock accumulate oxalate much lower content than 20 g kg^-1 DM. For example, Oryza sativa contained up to 14.93 g kg^-1 DM soluble oxalate.

Table 3:	Mineral content (g kg-1 DM) of fodder plants in Okinawa, Japan
Groups 1, 2 and 3 were classified with soluble oxalate content at >20, 10-20 and <10 g kg-1 DM, respectively

Table 4:	Mineral content (g kg-1 DM) of fodder plants in Savar and Shahzadpur, Bangladesh
Groups 1, 2 and 3 were classified with soluble oxalate content at >20, 10-20 and <10 g kg-1 DM, respectively

Table 5:	Correlation coefficient between oxalate and mineral contents in plants
ns: Not significant at p>0.05, significant at *p<0.05

However, some fodder plants (Pennisetum purpureum, Setaria sphacelata and Brachiaria mutica) showed oxalate contents exceeding 20 g kg^-1 DM. Fodder plants having a lower tendency to accumulate oxalate might be selected for cultivation; otherwise, consumption of high oxalate-containing plants by ruminants should be carefully monitored.

A previous study demonstrated that sheep fed by forage containing high oxalate (13.4 g kg^-1 DM) had lower blood Ca than those feeding low oxalate (4.7 g kg^-1 DM) of forage (Rahman et al., 2011). The present results revealed that the formulation of a recommendable ration shall be based not only on the nutrient content of feedstuffs but also on antinutrients as oxalate. Pennisetum purpureum and Brachiaria mutica of Bangladesh contained high soluble oxalate and low Ca which may cause Ca deficiency in animal. Setaria in Okinawa contained plenty of Ca (7.54 g kg^-1 DM) may be safe for animal feeding despite high soluble oxalate content (29.67 g kg^-1 DM). The legume plants contained relatively low contents of soluble oxalate, ranging from 0 in Leucaena leucocephala to 15.70 g kg^-1 DM in Sesbania sesban. In some regions of Bangladesh, Leucaena leucocephala and Sesbania sesban are well recognized in wasteland and are used by most livestock farmers as a feed supplement.

Feeding browse plants has become an essential practice in many parts of the tropics, especially in the dry season, when a scarcity of grass and herbaceous legume plants always occurs. Ficus hispida is a commonly available fodder tree in Bangladesh and this plant is often used as a feed for ruminants. Ficus hispida contained very low content of soluble oxalate (ranging from 2.75-4.24 g kg^-1 DM) and high content of Ca (30.11 g kg^-1 DM).

In the previous study, we observed a positive relationship between oxalate and mineral contents in napier grass (Rahman et al., 2008). However, no significant correlations between these 2 factors were observed in most of plants tested in this study, suggesting that the relationship between oxalate and mineral contents may vary among plant species. Smith (1972) reported that the amounts of inorganic ions in plants can be varied by altering the nutritional conditions and that the ions maintaining ionic balance in plants may alter the amounts of carboxylic acids (including oxalate).

Several factors are concerned in the accumulation of oxalate in fodder plants. For example, seasons can influence on oxalate accumulation in plants (Singh, 2002; Rahman et al., 2006). Singh (2002) reported that the oxalate content in napier grass was found to be 1.5 times higher in June and July than in April, whereas in August the level decreased from 36.0 to 25.6 g kg^-1 DM. In this study, however, samples were not collected during the peak growth of plants, i.e. summer and rainy seasons, but collected in late winter (for the samples in Okinawa, Japan) and late summer (for the samples in 2 sites of Bangladesh). Therefore, unfavorable season might have caused lowering the oxalate content in this study. Samples were also not harvested at the same stage of maturity that might be affected the oxalate accumulation to some extent, because oxalate levels declined as the harvest interval increased (Rahman et al., 2009b).

The oxalate levels of the same grass species we examined in two subtropical and tropical regions were varied and this might be associated with area of origin, although environmental conditions of Bangladesh are more or less similar with southern part of Japan (Okinawa). Moreover, this variation might also be associated with cation uptake, because both N and K application have pronounced effect on oxalate (Rahman and Kawamura, 2011). The order in soluble and insoluble oxalate contents was reversed for Setaria sphacelata in Okinawa and Bangladesh and this variation was not clear. However, this result might be partially explained with the findings of Rahman et al. (2009a) who reported that Ca supply can affect the soluble/insoluble oxalate ratio in plants. Hence, oxalate level in fodder plants grown in two subtropical and tropical regions could differ due to different fertilizer management.

The results of the present study demonstrate that the oxalate content in fodder plants may vary over a wide range, mainly depending upon the plant species. The majority of fodder plants in both subtropical and tropical regions accumulated low contents of oxalate, while a few fodder plants in some cases could accumulate oxalate up to the potentially toxic level. Dietary Ca should be ingested with the rations to maximize the binding of oxalate in the gut. We hope the data obtained herein can help livestock farmer to take necessary step on the prevention from oxalate toxicity.

ACKNOWLEDGMENT

The authors would like to thank the staff of Okinawa Livestock Research Centre and Bangladesh Livestock Research Institute for the facilities to carry out the experiment.