Dietary Supplementation Of Rice Bran Crude Lecithin Affect Feed Digestion, Blood And Rumen Prole In Crossbred Calves

The present study was conducted to evaluate the effect of rice bran crude lecithin (RBCL) on nutrient digestion and balance, performance, methanogenesis, blood and rumen prole. Eighteen crossbred calves were randomly divided into three groups; RBCL-0, RBCL-8 and RBCL-12 and fed wheat straw based diet with concentrate mixture containing 0, 8 and 12 % RBCL respectively. The dry matter, organic matter and crude protein intake were comparable but tended to decrease with the RBCL levels. The digestibility of dry matter, organic matter, total carbohydrate and gross energy tended to decrease while crude protein and ether extract digestibility tended to increase were with RBCL levels. The bre fractions (NDF and ADF) digestibility was signicantly (P < 0.01) lower in RBCL supplemented groups in comparison to control group. The body weight gain and average daily gain tended to decrease with increasing the level of rice bran crude lecithin. The percent of nitrogen and calcium retention tended to decrease, while phosphorus retention was signicantly (P < 0.01) lower with inclusion level of RBCL. The methane production (L/d, L/kgW 0.75 ) were signicantly (P < 0.05) lower in RBCL-12 followed by RBCL-8 as compared to RBCL-0 group. Serum biochemical did not show signicant difference among dietary treatment groups. The cholesterol and blood urea concentration was signicantly (P < 0.01) higher in RBCL-12 group as compared to control group. The rumen metabolites and microbiota showed reduction in RBCL-8 group as compare to RBCL-0 group without reach to signicant (P < 0.05) level. It can be concluded, that detrimental effect of present levels (8 and 12%) of RBCL was seen in the performance of crossbred calves, which was associated with decreased bre digestibility and fermentation in rumen. Beside this, RBCL is helpful in methane mitigation for cleaner production and can be a cheap source of energy in place of corn for ruminant. Further studies in large number of livestock are warranted to explore the potential of RBCL in the ruminant ration. body weight gain in the morning at 9:30 AM. The wheat straw was offered after the concentrate mixture was completely consumed by the animals. Fresh and clean drinking water was made available ad libitum twice a day. The body weight of calves was recorded at fortnightly intervals of experiment on the two consecutive days.


Introduction
Although India ranks rst in milk production but the productivity of milk per animal is very less in rural areas. The main problem is the chronic shortage of protein (Digestible crude protein (DCP), 58%) and energy (Total digestible nutrients (TDN), 31%) rich animals feed. It is predictable that India will require 550 million tonnes of dry fodder, 105 million tonnes of concentrate and 1000 million tonnes of green fodder in year 2025 (Ravi Kiran et al., 2012). Due to high feedstuff prices and lacks of dietary grains, it is needed to explore the nonconventional agro-industrial byproducts in improving the availability of dietary energy sources for dairy animals. During re ning of rice bran oil in the enzymatic degumming process, rice bran crude lecithin (RBCL) is produced as by product, roughly 1.5-2.0% of oil weight depending on the drying condition (Jala and Prasad, 2015). The current rice bran crude lecithin (RBCL) production in India is expected to uctuate between 9000 and 12000 MT. In a number of countries, the gummy materials containing phospholipids are lost during degumming of crude rice bran oil, due to scarce literature (Senger et al., 2014). When using raw, standardized, liquid, non-fat and / or modi ed lecithin of various origins, fat feeding inhibition of rumen fermentation does not occur. Using lecithin instead of oil as the only lipid supplement was found to cause a lower ruminal degradation of crude protein due to their high a nity to protein as a result of their amphiphatic properties which are not present in triglycerides and free fatty acids (Jenkins et al., 1989). Because they are amphiphatic (having hydrophobic and hydrophilic moieties), phospholipids are readily dispersed in aqueous solutions as micelles, giving surface active properties that enhance their wettability, fat dispersion and their a nity for protein.Therefore, when lecithin are used, attachment to feed particles or rumen microbes might be less pronounced and the release of the fatty acids could be delayed resulting in less adverse effects on rumen fermentation (Nagaraja et al., 1997). Sontakke  Therefore present study is planned to investigate the effect of rice bran crude lecithin replacing corn on nutrient utilization, performance, methanogenesis and metabolic pro le in crossbred calves.

Experimental animals and feeding
Eighteen crossbred male calves (Bos taurus x Bos indicus) (about 12 months old) were randomly divided into three groups (RBCL-0, RBCL-8 and RBCL-12) six animals each by completely randomized design. Prior to initiation of the experimental trial, animals were treated for ecto and endo-parasites as per the standard protocol. RBCL-0, RBCL-8 and RBCL-12 group calves were fed wheat straw based diet with three concentrate mixtures i.e. CM1, CM2 and CM3 for 120 days, respectively (Table 1). All the experimental calves were offered daily a weighed amount of concentrate mixture and wheat straw as per Kearl (1985) feeding standards for 500 g daily body weight gain in the morning at 9:30 AM. The wheat straw was offered after the concentrate mixture was completely consumed by the animals. Fresh and clean drinking water was made available ad libitum twice a day. The body weight of calves was recorded at fortnightly intervals of experiment on the two consecutive days. digestibility and balance after 60 days experimental feeding.
Indirect respiration chamber study Following 90d of experimental feeding, animals were kept in respiration chamber for proper adaptation (2-3 days). The concentration of CH 4 , CO 2 and O 2 in the chamber air was measured for three consecutive days using an infrared gas analyzer. The methane energy and heat production was calculated with the help of Brouwer's equation.

Metabolic pro le
The blood from all calves' jugular vein was collected at morning during pre (0 th d) and post (120 th d) experiment. The serum was separated within 2 h after blood collection and analyzed promptly.
Biochemical analysis was performed to determine the glucose, non-esteri ed free fatty acids (NEFA), total cholesterol, 3-hydroxy butyric acid (βHBA) levels to evaluate energy metabolism; total protein, serum albumin, globulin, A:G ratio and blood urea levels to evaluate protein metabolism; calcium and phosphorus level to evaluate mineral metabolism using commercial diagnostic kits as per manufacturer's recommendations. The growth related hormones viz. Insulin, IGF-1 and Leptin were analyzed by using commercial available ELISA diagnostic kits.

Rumen pro le
Rumen liquor samples were collected at the 120 th d of experimental feeding from RBCL-0 (nonsupplemented) and RBCL-8 (supplemented) group animal, on the basis of growth trial results, by stomach tube method to observe the effect of RBCL on rumen fermentation and microbiota. Rumen metabolite mainly NH 3 -N and VFA and its fractions were assessed. The NH 3 -N concentration was analysed by using colorimetric assay as described in Weatherburn, (1967). Estimation of VFA concentration was done using a gas chromatograph equipped with a Flame Ionization Detector (Agarwal et al. 2008). Total bacteria, Fibrobacter succinogenes, Ruminococcus albus, R. avifaciens, methanogens and fungi populations were assessed by qPCR (Real time PCR). In brief, the plasmid was extracted and serially diluted to make a standard curve and copy number was calculated (Ritalahti et al., 2006). The ampli cation reactions were performed in a total volume of 20 µl, containing 2 ng of template DNA, 10 µl of 2X Kappa SYBR master mix , 0.6 µl of each primer (10 µM) and nuclease free water ( to make up the volume 20 µl). The number of protozoa was counted as per the procedure described by Kamra et al. (1991).

Chemical and statistical analysis
Analysis of offered feed, residual feed and faeces was done by standard procedure (AOAC, 2000). The gross energy of feed offered, faeces and urine was measured by ballistic bomb calorimeter (Gallenkamp), taking benzoic acid as standard. The data were statistical analyzed by statistical package SPSS version 20.0 in which data were subjected to ANOVA and Duncan's multiple range test (DMRT) was used to determine the signi cant differences between the means. Comparisons were made at 5% probability level.

Nutrient Intake and digestibility
The chemical composition of feeds, daily nutrient intake and digestibility in experimental calves under dietary treatments (RBCL-0, RBCL-8 and RBCL-12) is presented in the Tables 1 & 2. The concentrate DM intake was tended to increase, while wheat straw DM tended to decrease with the inclusion level of RBCL.
The DMI and OMI (g/KgW 0.75 ) was 1.5 and 8.5% lower in RBCL-8 and RBCL-12 groups compared to control group. The CPI (g/KgW 0.75 ) was comparable, while EEI (g/KgW 0.75 ) was signi cantly (P < 0.001) higher in RBCL-12 followed by RBCL-8 compared to RBCL-0 group. The TCHO intake (g/KgW 0.75 ) was signi cantly (P < 0.01) lower in RBCL-12 followed by RBCL-8 as compare to RBCL-0. The NDF and ADF intake was signi cantly (P < 0.05) lower in RBCL-12 group as compared to RBCL-0, while RBCL-8 group had intermediate position between RBCL-0 and RBCL-12 groups. The digestibility of DM, OM, TCHO and GE tended to decrease, while CP digestibility tended to increase with inclusion of RBCL levels. The EE digestibility was signi cantly (P < 0.05) higher in RBCL-12 group as compare to RBCL-0, however RBCL-8 had intermediate position between RBCL-0 and 12 groups. The NDF and ADF digestibility in RBCL-12 group were signi cantly (P < 0.05) lower than RBCL-0 followed by RBCL-8 group. The calcium and phosphorus balance in calves is shown in Table 4. The Ca intake (g/d) was comparable, while faecal calcium (g/d, %) tended to increase with inclusion level of RBCL. The urinary Ca and Ca retention (g/d, %) tended to decrease with the level of RBCL. The P intake (g/d) was signi cantly (P < 0.01) higher in RBCL-12 and RBCL-8 as compare to RBCL-0. The faecal P (g/d, %) was comparable among the RBCL-0, RBCL-8 and RBCL-12, however urinary P (%) was tended to decrease with RBCL levels.
The phosphorus retention was signi cantly (P > 0.010) lower in RBCL-12 and RBCL-8 as compared to RBCL-0 group. Effect of various dietary levels of RBCL on growth performance and methane production and heat production is presented in Table 5. Initial and nal body weights were comparable among the groups. The total BW gain and ADG tended to decrease with the level of RBCL in the ration of calves. The nal body weight of RBCL-8 and RBCL-12 group calves was 3 and 6% lower than RBCL-0 group calves. During respiration chamber study, the metabolic body size was comparable among dietary treatments. The DMI (kg) depression was 13

Blood pro le
The data of serum biochemical related to protein, energy and mineral metabolism and serum hormones is presented in Table 6. The treatment mean of serum total protein, albumin, globulin (g/dl) and A:G ratio was comparable, while period mean of serum total protein, albumin (g/dl) and A:G ratio was signi cantly (P<0.01) higher at 120 d compare to 0 d.. The treatment mean of blood urea (mg/dl) was signi cantly (P<0.01) higher in RBCL-12 group than RBCL-8 and RBCL-0 groups while period mean signi cantly higher at 120 d than 0 d. The treatment mean of serum glucose (mg/dl) was comparable among groups, while period mean tended to increase with time. The treatment mean of NEFA (µmol/L) was numerically lower in supplemented groups as compare to control group, while period mean was also quantitatively lower at 120 d as compare to 0 d. The treatment mean of cholesterol (mg/dl) was signi cantly (P<0.001) lower in RBCL-12 group as compare to RBCL-0 and 8 groups, while period mean was signi cantly (P<0.01) lower in 120 d than 0 d. The treatment mean of BHBA (nmol/ml) was numerically lower in lecithin groups than control while period mean was signi cantly (P<0.001) lower in 120 d than 0 d. The treatment and period mean of serum Ca was comparable among groups and period, while treatment mean of serum i-P was comparable between groups and period mean was signi cantly higher at 120 d than 0 d. The treatment mean of serum insulin (μIU/ml) were comparable among groups and tended to decrease with the higher level of RBCL, while period mean were signi cantly (P<0.05) higher at 120d as compared to 0d. The treatment mean of IGF-1 (ng/ml) of RBCL-0, RBCL-8 and RBCL-12 groups was analogous to each other but tended to decrease. The period mean of IGF-1 was numerically higher but not reached to signi cant (P<0.05) level. There was no signi cant difference in Insulin and IGF-1 among dietary groups due to period treatment interaction. The treatment mean of serum leptin (ng/ml) were comparable among groups, while period mean were signi cantly (P<0.05) higher at 120d as compared to 0d.

Rumen pro le
Among three dietary groups, rumen metabolites and microbial pro le were studied in non-supplemented (RBCL-0) and supplemented (RBCL-8) groups on the performance basis. The supplementation of RBCL in RBCL-8 group did not cause signi cant (P<0.05) change in the concentration of NH 3 -N, total VFA and its fraction i.e. acetate, propionate, butyrate, iso-butyrate, valerate and iso-valerate, A:P and (A+B):P ratio in comparison to RBCL-0 calves. The concentration of all above rumen metabolites was lower in RBCL supplemented group. The population density of rumen microbes including total bacteria, fungi, methanogens, ciliate protozoa, brolytic bacteria as Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus avifaciens of crossbred calves was comparable in both groups (Table 7). In RBCL-8 group, numerically lower count of total bacteria, brolytic bacteria, fungi, archaea and protozoa was seen as compare to control group. balance. The lower methane energy loss in rice bran supplemented groups could be related to lower methane production due to either bio-hydrogenation of unsaturated fatty acids or low acetate production in the rumen. The low HI is associated with lower DMI along with the higher level of RBCL in ration of experimental calves. The net energy available for growth and maintenance is only 5 % higher in RBCL-4 group and equal in RBCL-6 group in comparison to RBCL-0 group.

Ca and P balance
Because of limited information about lecithin in ruminants, studies with nonruminant animals fed lecithin is discussed. In the support, Overland et al. (1994) noticed that lecithin had no impact on the retention of total P and Ca in pigs while contrary Huang et al. (2007) observed that lecithin at 2% level signi cantly improved the Ca and P utilization in broilers. In current study, the higher faecal Ca might be attributed to the formation of insoluble calcium soap from by reaction of Ca with free long chain fatty acids (LCFAs) present in RBCL. While RBCL is a good source of phosphorus but the phosphorus absorption is inversely proportional to the intake, might be possible explanation behind less phosphorus retention. The lower Ca and P retention is also correlated with the reduction in ADG in experimental calves.

Growth performance and methanogenesis
In the conformity of present ndings, Shain et al. (1993)  1996) which provide hydrogen as a substrate for the methanogens. The lower reduction of methane release with lecithin than with canola oil indicates that phospholipids were either not hydrolysed to the same extent or slower as triglycerides (Jenkins et al., 1989). In current study the lower methane production may be attributed to lower DMI and presence of PUFAs.

Metabolic pro le
In present study, the higher serum urea and cholesterol level in RBCL-12 group may be attributed to the insu cient utilization of ammonia by ruminal microbes due to lower soluble carbohydrate and supplementation of higher amount of fat in the diet. Plasma glucose, NEFA and BHBA are considered as principle circulating blood metabolite to assess the energy status of the animals (Muwel, 2016 (1989) reported decreased acetate and increased propionate proportions but the proportion of butyrate was not in uenced by phospholipids in sheep. Jenkins (1990) found a decrease in acetate proportion and increased butyrate when lecithin was added in combination with hydrogenated fat to steer diet. Paul (1994) observed a decrease in butyrate production during in-vitro study when pure phospholipids were used in comparison to free fatty acid and triglycerides.

Conclusion
In last it can be concluded that excessive levels of RBCL (8 and 12 %) did not adversely affect the nutrients intake, digestibility, metabolic pro le and growth performance of crossbred calves, except the bre digestibility and its intake. RBCL signi cantly reduce the methane production so it is helpful in methane mitigation for cleaner production and can be a cheap source of energy in place of corn for ruminant. Further studies in large number of livestock are warranted to explore the potential of RBCL at appropriate level in the ruminant ration. Mechanism studies are needed to explore the effect of RBCL on rumen fermentation and body metabolism.