الفهرس 
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Abstract
Enzyme production, chemical and microbiological analyses were carried out at the Laboratories of Applied Microbial Genetics Lab., Genetics and Cytology Department in collaboration with Animal Production Department, National Research Centre, Dokki, Giza, Egypt. Laboratory in-vitro trials were carried out at the Laboratories of Animal Production Department, Faculty of Agriculture, Ain Shams University, Egypt. The farm trials were carried out at the Animal Production Unit, Agricultural Experimental Station of the National Research Centre, Al-Noubareya, Egypt.
This study was carried out to:
1- Screening various fungal genotypes for their ability to produce phytase and improve the produced enzyme by ethyl-methane sulfonate (EMS) mutagenesis.
2- Evaluate the potential use of different levels of laboratory produced phytase enzyme (PE) for improving in-vitro nutrients degradability of lactating animals’ ration compared with different levels of commercial phytase enzyme (Axtra® PHY) to decide the most effective level of phytase supplementation from both sources.
3- Investigate the impact of adding Axtra® PHY and PE enzymes, at the best level that was obtained from the results of the in-vitro study, to the ration of lactating buffaloes on nutrients digestibility, some blood parameters, milk yield and composition.
Three experiments were carried out in this study:
1. The first experiment [Laboratory microbial phytase production trials]
Based on the previous studies in the literature, submerged fermentation (SmF) was used to evaluate the effect of different phytase screening media (PSMG and PSMS) and incubation times (2, 4 and 8 d) on phytase production by various fungal genotypes (Aspergillus niger (syn. A. ficuum) NRRL 3135, AN1; Aspergillus niger NRRL 326, AN26; Aspergillus terrus F2-Kh, AT; and Mucor racemosus NRRL 3639, MI). A complementary experiment was designed to examine the impact of various incubation times (2, 4, 6, 8, 10, 12 and 14 d) on phytase enzyme production by the most efficient fungal strain at the selected medium. The fungal spore suspension was treated with EMS (200 mM) at different time intervals (20, 40 and 60 minutes). Obtained mutants were chosen by spreading EMS-treated spores on Petri plates containing 100 ml of phytase screening medium (PSM) supplemented with 2% agar, 0.1% Triton X 100 and heavy metals (250 ppm cobalt or 500 ppm manganese). The selection was determined by the observation of heavy metal resistance, survival percentage was also estimated.
The obtained results from this experiment could be revealed as follows:
• Aspergillus niger (AN1) showed the highest phytase activity on phytase screening medium (PSMG) after 8 d of incubation (reached 1875.40 IU/mL) therefore, they were selected for further work.
• The phytase activity of AN1 was increased with increasing incubation period and the highest value was achieved at 12 d of incubation (2859.33 IU/mL).
• The exposure of AN1 spore suspension to 200 mM of EMS for different time intervals (20, 40 and 60 minutes) enhanced the phytase activity.
• Increasing the exposure time to EMS mutagenesis from 20 to 40 min. decreased the survival percentages of AN1 from 59.43% to 35.38%. Moreover, the survival percentages continued to be decreased as the exposure time to EMS mutagenesis increased to 60 min. where it reached 22.17 %.
• Mutant 20 Mn exhibited the highest phytase activity (reached
4520.5 IU/mL) therefore it was chosen for the next experiments.
2. The second experiment [in-vitro trials]
In-vitro dry and organic matter degradability (IVDMD and IVOMD), total gas production (GP), short chain fatty acids (SCFA’s) and inorganic phosphorus (Pi) concentration were determined for the experimental ration (ER) that consisted of 40% berseem hay and 60% concentrate feed mixture (CFM). The experimental ration was separately supplemented with the commercial phytase (Axtra® PHY) and the laboratory produced phytase (PE) at six different levels (0, 400, 800, 1200, 1600 and 2000 IU phytase-1Kg dry matter).
The results of this study showed that:
• In-vitro dry and organic matter degradability, GP, SCFA’s and Pi concentration were improved significantly (P<0.05) by phytase addition from the two sources of phytase and the highest significant (P<0.05) values achieved at the level of 1200 IU.
3. The third experiment (in-vivo trials)
Twelve lactating buffaloes after 3 months of parturition were divided into three groups, four animals each, using a complete random design. The experimental groups were: experimental ration (ER) without enzyme (G1), ER plus 1200 IU commercial enzyme (Axtra® PHY) per kilogram of diet (G2) and ER plus 1200 IU produced enzyme (PE) per kilogram of diet (G3).
The results obtained from this experiment could be summarized in the following:
3.1. Digestibility and nutritive values
• The digestion of DM, OM, CP, CF and NFE were improved significantly (P<0.05) in buffaloes fed diets with exogenous phytase (78.53, 80.48, 82.89, 67.02, 87.04% and 79.53, 81.80, 83.80, 67.53, 87.77% for G2 and G3, respectively) compared with G1 (74.20, 77.61, 75.37, 63.47 and 83.69%), with no significant differences (P>0.05) between the two sources of phytase (G2 and G3).
• Phytase supplementation from both sources had significantly (P<0.05) improved the TDN and DCP values (79.64, 12.91% and 80.33, 13.06% for G2 and G3, respectively) compared with G1 (75.79 and 11.74%), without any significant differences (P>0.05) between the two sources of phytase.
3.2. Fecal calcium, phosphorus and magnesium excretion
• The fecal excretion of Ca, P and Mg were significantly (P<0.05) decreased in buffaloes fed diets with exogenous phytase (1.92, 0.30, 0.51% and 1.90, 0.29, 0.50% for G2 and G3, respectively) compared with G1 (2.40, 0.36 and 0.75%), with no significant differences (P>0.05) between the two sources of phytase (G2 and G3).
3.3. Blood serum parameters
• The phytase treated buffaloes (G2 and G3) showed a significant increase (P<0.05) in blood serum total protein, albumin, globulin and glucose concentrations than those of G1 (6.57 and 6.63 vs. 6.02 g/dl; 4.03 and 4.07 vs 3.84 g/dl; 2.54 and 2.56 vs 2.18 g/dl; 73.55 and 74.18 vs 66.73 mg/dl for G2 and G3 vs G1, respectively). While, there were no significant differences (P>0.05) between the two sources of phytase (G2 and G3).
• Blood serum urea, AST and ALT concentrations were not significantly (P>0.05) affected by phytase supplementation among all groups (40.43, 40.16 and 40.25 mg/dl; 50.00, 50.33 and 50.67 U/ml; 30.25, 30.50 and 31.00 U/ml for G1, G2 and G3, respectively).
• Blood serum Ca and Pi values were significantly increased (P<0.05) with phytase supplementation from both sources of phytase (G2 and G3) compared with G1 (10.14 and 10.19 vs 9.64 mg/dl; 5.64 and 5.74 vs 4.86 mg/dl for G2 and G3 vs G1, respectively). There were no significant differences (P>0.05) between the two sources of phytase (G2 and G3) except for Pi values. where the buffaloes of G3 showed the highest Pi values (P<0.05).
3.4. Milk yield and its composition
• The yields of milk (kg/d), 4% fat corrected milk (FCM, kg/d), and other milk components (total protein, fat, lactose, ash, total solids and solids not fat, g/d) were significantly (P<0.05) increased by phytase supplementation (G2 and G3) compared with G1 (7.49 and 7.54 vs 6.70; 10.18 and 10.40 vs 8.94; 294 and 302 vs 228; 479 and 492 vs 417; 421 and 425 vs 324; 65.91 and 67.11 vs 54.27; 1260 and 1286 vs 1023; 781 and 794 vs 606; for G2 and G3 vs G1, respectively). No significant differences (P>0.05) were detected in the milk yields between the two sources of phytase (G2 and G3).
• Compared with buffaloes fed the experimental ration without phytase (G1), buffaloes fed the phytase supplemented ration (G2 and G3) showed a significant increase (P<0.05) in milk total protein, fat, lactose, ash, total solids and solids not fat percentages (3.93, 4.00 and 3.40; 6.40, 6.53 and 6.22; 5.62, 5.64 and 4.83; 0.88, 0.89 and 0.81; 16.83, 17.06 and 15.26; 10.43, 10.53 and 9.04% for G2, G3 and G1, respectively). No significant differences (P>0.05) between the two sources of phytase (G2 and G3) were found.
• The percentage of milk P was significantly (P<0.05) improved by
phytase supplementation (G2 and G3) compared with G1 (0.24 and 0.26 vs 0.18; for G2 and G3 vs G1, respectively), with no significant differences (P>0.05) between the two sources of phytase (G2 and G3). However, the percentages of milk Ca and Mg were not significantly (P>0.05) affected by phytase supplementation among all groups (0.13, 0.15 and 0.15; 0.01, 0.02 and 0.02% for G1, G2 and G3, respectively).
3.5. Economical evaluation (feeding cost/kg of milk)
• The economic values expressed as the profit above feeding cost (LE/Kg of milk/105 day) for both G2 and G3 were greatly increased compared with G1 (798, 892 and 73 LE/Kg of milk/105 day for G2, G3 and G1, respectively).
• Buffaloes fed the experimental ration supplemented with the produced phytase (G3) showed the highest economic.
CONCLUSION
It could be concluded that Aspergillus niger (ficuum) NRRL 3135 (AN1) exhibited the highest phytase activity at PSMG medium, and the best incubation period was 12 days. Phytase activity was influenced by exposure to EMS mutagen compared to the wild type. Supplementation of phytase (Axtra® PHY and PE) at 1200 IU/kg of ration tended to increase nutrients digestibility, improve milk yields and composition and decrease fecal excretion of Ca, P and Mg in lactating buffalos. Buffaloes fed the experimental ration supplemented with the PE showed the highest economic value.
These results should be considered to make the right decision to expand the local production of such an important enzyme (phytase) to achieve the dual benefit of preventing environmental pollution and supporting the Egyptian economy by maintaining the hard currency stock.