Optimization of growth conditions of local leeches for hirudin production.

Leeches (Phylum: Annelida, Class: Hirudinea) are widely distributed all over the world in various habitats, such as freshwater, seas, desert, and oases (Gouda, 2006). In this study, the effect of light intensity, temperature and diet on the reproductive efficiency of local leeches was examined with eight different conditions. After 3 months of culture, the number of cocoons produced was very significantly different among the different conditions (p=0.00). The average number of hatchings per cocoon was significantly different (p ≤ 0.05) where Condition 1 gave the highest number (6.23±0.25), but hatching rate was not (p=0.354). The Condition 5 produced the highest mortality of parent leeches (52±13.86%). The sizes of the cocoons were not significantly different among the treatments, with the Condition 1 having the largest cocoon of 22.19±0.92 mm and 13.26±0.07 mm according to their length and diameter, respectively. The wet weight of cocoons was significantly different (p ≤ 0.05) with the Condition 1 producing the heaviest cocoons of 1.26±0.11g compared to Condition 5 producing the lightest cocoons of 0.22±0.38 g. The effect of diet (FT1: fresh eel blood and FT2: juvenile commercial product ‘A’) on the growth and survivorship of the juvenile leeches was also studied. After 2 months of culture, the final body weight was significantly different among the treatments, with juveniles in the FT1 (fresh eel blood) gave the highest final body weight (0.8893±0.012g). Percentage weight gain (WG) and specific growth rate (SGR) of the juveniles in the treatment FT2 (juvenile commercial product ‘A’) were lowest with mean and standard deviation of 769.41±11.54% and 3.6±0.02%, respectively. Juveniles in the FT2 (fresh eel blood) treatment had the highest survival rate (93.33±5.77%). Hirudin is the generic name for a family of closely related homologous peptides that are all found in the cranial salivary glands of leech (Wallis, 1996). In this study, the leech crude extract was prepared by using conventional method and tested to fresh rabbit blood. The results showed that the rabbit blood treated with leech crude extract gave the longest time of blood to clot and the clotted blood was did not contract up to 77.67±2.52 minutes when compared with treatments 1 (Control) and treatment 2 (Rabbit blood + distilled water) where the blood clot in 0.59±0.01 and 2.83±0.3 minutes and the clotted blood did not contract up to 1.17±0.29 and 3.17±0.29 minutes, respectively. These results revealed that crude extract of leeches contain an anti-coagulant agent to prevent blood from clotting. It has long been known that local leeches contain a substance with anticoagulant properties. The purification of hirudin, protein profiling and protein quantification from different parts of leeches are reported. The result shows the sample from leech head gave the highest result in protein concentration with value of 69.33 µg/ml compared with either the sample from the whole body and headless body which was 36.67 µg/ml and 26.67 µg/ml, respectively. Sample from leech head was purified by gel permeation chromatography on Sephadex G-75. Fractions which contain significant amounts of protein with absorbance value more than 0.2 at 595 nm were tested for their anti-coagulant activity. The result shows that the Peak III (Fraction Number= 23, Optical Density (O.D) = 0.256, protein concentration = 82.00 µg/ml) gave the longest time for blood to clot (123.33±15.28 minutes) and the clotted blood did not contract up to 380.00±17.32 minutes compared with Peak II (Fraction Number= 21, Optical Density (O.D) = 0.293, protein concentration = 94.33 µg/ml) and Peak V (Fraction Number= 36, Optical Density (O.D) = 0.214, protein concentration = 68.00 µg/ml ) where the blood took 13.33±2.89 and 23.33±2.89 minutes to clot and the clotted blood did not contract up to 24.33±0.58 and 41.67±2.89 minutes, respectively. Crude protein and eluted fractions which contain significant amounts of protein with the longest time of blood to clot were subjected to SDS-PAGE analysis. The result shows that the molecular weight of hirudin was about 11.36 kDa. From the purification made the profiles of leech crude extract from different parts of body indicate that anti-coagulant agent (hirudin protein) was only found in leech head.