Effect of Rubus coreanus Miquel Byproducts on Performance and Hormone Secretion of Crossbred Chicks
Kyeong Seon Ryu
The present study was undertaken to investigate the feeding Rubus coreanus Miquel byproducts (RCMB) on the performance, blood composition and hormone concentrations of crossbred chicks. Day-old male crossbred chicks (384) were allocated to four RCMB (0, 0.25, 0.5, 1.0%) level in the diets with six replication having 64 chicks in each treatment. Four dietary energy (3,000, 3,100, 3,100 and 3,200 kcal kg-1 ME) and CP (22, 21, 19 and 17%) levels were provided to the starting (0-2 weeks), growing (3-5 weeks), growing-finishing (6-8 weeks) and finishing (9-10 weeks) period. No significant performances were observed during the starting and growing periods but weight gain and feed intake were increased in the growing-finishing and finishing period. Thus the total weight gain and feed intake were significantly higher in RCMB treatment group as compared to those of the control (p<0.05), except feed conversion. Triglycerides were significantly lower in birds fed 1.0% RCMB compared to those in the controls and in the 0.25% RCMB fed birds (p<0.05). Total protein concentrations were not different among the treatments, but albumin concentration was increased in the 1.0% RCMB treatment compared to those of the other treatments (p<0.05). The melatonin, growth hormone and testosterone concentration was increased significantly (p<0.05) in RCMB treatment than that of the control. Therefore, the inoculation of 1% level of RCMB in the diet of crossbred chicks appeared to enhance the performance and hormone secretion. Meanwhile, further follow-up studies should be conducted to investigate RCMB additions of more than 1% in chicken diets.
Received: February 17, 2012;
Accepted: March 29, 2012;
Published: July 09, 2012
Rubus coreanus Miquel is a perennial wild berry type of plants that
belongs to the Rosaceae family and its cultivation is limited to the southern
parts of the Korean peninsula, China and Japan. During 2001 to 2005, Rubus
coreanus Miquel production have increased from 1510 to 2733 tons (Ku
and Mun, 2008) and which influenced the development of various Bokbunja
products (Jin et al., 2008). It is a rich source
of flavonoids, tannins, triterpenoids and phenolic compounds (Ju
et al., 2009). It composed mainly of triterpenoids that include coreanoside
F1 suavissimoside and nigaichigoside F1 and F2 (Kim
and Kang, 1993). The presence of antioxidative agents such as phenolic acid
and organic acids has been reported (Lee, 1995; Yoon
et al., 2002) along with the presence of Quercetin (Yoon
et al., 2003). In the berries of Rubus coreanus Miquel, hydrolyzable
tannins of gallotannin and ellagitannin were found along with gallic acid, 2,
3-(S)-HHDP-D-glucopyranose, sanguiin H-4 and sanguiin H-6 (Lee
and Lee, 1995; Pang et al., 1996).
Recently, it was reported that weight gain and feed conversion ratio in crossbred
chicks had increased by inoculation of Rubus coreanus Miquel extracts
with the diet (Kim et al., 2009). Several studies
have been conducted to create high value products by agri-byproducts such as
Rubus coreanus Miquel byproducts (Isci and Demirer,
2007). From 2001 to 2005, the growth area in South Korea has increased about
13 times and the harvest quantity increased nearly two times (Ku
and Mun, 2008). Most Bokbunja seeds are byproducts from wine processing
and thus, the search for other applications may contribute to minimize waste
disposal problems, improving the value of Bokbunja fruit and enhancing the agricultural
economy (Ku and Mun, 2008). Therefore, the potentialities
of the RCM byproducts started to draw researchers attention; the usefulness
and feasibility of using RCM byproducts for various applications becomes the
subject of studies. Researchers generally focused on the application of RCM
in human and experimental rodents, but there have not been studied on the subject
to the inoculation of RCM byproducts to the poultry yet.
Thus, the present study investigated the effects of adding different levels of Rubus coreanus Miquel byproducts to the crossbred chicks diet and reviewed overall performance, hematology and the differentiation of hormone secretion levels of the chicks to confirm the potential value of Rubus coreanus Miquel byproduct as a feed for crossbred chicks.
MATERIALS AND METHODS
Preparation and analysis of RCMB: The fruit flesh and residues containing
seeds of the Rubus coreanus Miquel, which were acquired after manufacturing
Rubus coreanus Miquel wine (Ku and Mun, 2008).
The RCM byproduct were separated and dried at room temperature and then crushed
and mixed with the basal diet.
The Rubus coreanus Miquel byproducts (RCMB) was dried in the oven at
60°C for 24 h and powdered by a roller mill. Thereafter, moisture, crude
protein and crude ash were analyzed by the AOAC, (1995)
methods. The dried RCMB was contained 4.98% moisture, 12.99% CP, 1.48% crude
ash and 13.61% crude fat. Oil content was extracted by Soxhlet extraction and
amino acids were analyzed as follows. One gram RCMB was added into 10 mL of
conical flask and mixed with 5 mL of 1% boric acid solution and kept at 4°C
for 24 h. It was centrifuged at 5,000 rpm for 30 min and 0.5 mL of supernatant
were separated from the solution and eluted through the column (Sephadex G-50)
and then 200 μm eluted sample added with 200 μm chloroform and mixed
by vortex. The mixture was again centrifuged at 15,000 rpm for 5 min. Supernatant
liquid was filtered by 0.22 μm syringe filter and reacted derivatization
by kit (AccQ-Flour Reagent, Water). Amino acid compositions were determined
by amino acid analyzer (water 2690, Waters Co., USA) equipped with a Waters
747 scanning fluorescence detector (Water Co., USA) and a AccQ-Tag column (3.9x150
mm, Waters) where the sample injection volume was 10 μm with flow rate
was 1.0 mL min-1. Fatty acids in RCMB were analyzed as described
by Ku and Mun (2008).
Feeding trials: In a 70 days (March-May, 2007) feeding trial, 384, one-day-old
male crossbred chicks (Hanhyup 3 Ho) was placed into floor pens for ten weeks.
A basal diet was prepared by mixing corn and soybean meals and the prepared
feeds were provided in the starting (0-2 weeks), growing (3-5 weeks), growing-finishing
(6-8 weeks) and finishing (9-10 weeks) periods to meet the metabolic energy
levels of 3,000, 3,100, 3,100 and 3,200 kcal kg-1, respectively.
The crude protein levels of the feeds were prepared at 22, 21, 19 and 17%, respectively,
as shown in Table 1.
|| Basal diet composition
|Provide per kilogram of diet: vit. A: 12,000 IU: vit D3: 5,000
IU, vit E: 50 mg, vit K3: 3.0 mg, vit B1: 2.0 mg, vit B2: 6.0 mg, vit B6:
4.0 mg, vit B12: 0.025 mg, Biotin: 0.15 mg, Pantothenic acid: 20.0 mg, Folic
acid: 2.0 mg, Nicotinic acid: 7.0 mg, 2Contents per kg; Fe: 66.72
mg, Cu: 41.7 mg, Mn: 83.4 mg, Zn: 66.72 mg, I: 0.834 mg, Se: 0.25 mg
Rubus coreanus Miquel byproducts (RCMB) were added at the rate of 0,
0.25, 0.5 and 1.0% with the basal diets and each treatment was replicated six
times, with 96 chicks assigned to each treatment and a total of 384 chicks allocated
with similar body weight. Chicks were provided feed and water without restriction
and 24 h continuous lighting was available during the entire trial period.
Weight gain, feed intake and feed conversion ratio: The body weights of the chicks were measured per week and the feed intake was calculated by deducting the residual amount of feed from the total amount of feed provided. The feed conversion ratio was calculated by dividing the feed intake amount by the weight gain.
Blood test and hormone assay: At the end of the feeding trial, the blood tests were conducted by selecting ten chicks from each treatment group. Blood was collected from the brachial vein into a vacuum tube using a 5 mL syringe. After blood collecting, serum was separated and stored at -70°C until analysis. Blood total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol concentrations were measured using a blood analysis kit that used an enzymatic colorimetric method (AM 202-K; Asan Pharm Co., LTD; Korea) and total protein, albumin and glucose levels were measured using an automatic blood analyzer (Minos BAT; France).
The blood growth hormone level was measured using the RIA test kits manufactured by Linco Research (USA) to measure the blood IGF-1 level. Two hundred microlitres of blood plasma was mixed with 800 μL acid-ethanol (2 M HCl: ethanol in the ratio of 1:7) and centrifuged at 4°C for 30 min at a speed of 3,000 rpm. Then, 500 μL of the supernatant fraction containing the free IGF-1 was mixed with 200 μL 0.855 M trizma base and the 100 μL of the reactant was added to 100 μL of the [125I]-IGFs (20000 cpm/100 μL) prepared using the Chloramin-T method. To this, 50 μL of 1000 times diluted polyclonal anti-IGFs (Gro-pep, Australia) was added to react for 18 h at 4°C. After the reaction, 50 μL serum and 1 mL 12% polyethylene glycol # 8000 (PEG) were added and the mixture was centrifuged for 30 min at a speed of 3,000 rpm in order to separate the fraction into the bound and free forms of IGF. The radioactivity of the bound form was measured using a gamma counter (Packard, ILL, USA). The measurement of blood testosterone level was performed by applying the radioimmunoassay method using the Immuchem testosterone kit (ICN Biomedicals, INC. Diagnostics, U.S.A.). Specifically, 100 μL serums and the same amount of testosterone standard were contained within anti-testosterone coated tables and 1.0 mL 125I-testosterone was placed onto the total tube and gently shaken and incubated at 37°C for 90 min. After incubation, the liquid fractions within the tube were discarded and radioactivity was measured for 60 sec using the gamma counter. The blood melatonin concentration was measured using the RIA test kits manufactured from Buhlmann Laboratories AG (Switzerland). The RIA test kits utilize two types of G280 anti-melatonin antibodies (primary antibody and secondary antibody) to measure the melatonin concentration. The analysis procedure of the RIA includes the initial pre treatment procedure with a column preparation step, the control procedure in which 1 mL of methanol and the same amount of distilled water are injected and a washing step in which 1 mL of 10% methanol was injected two times along with 1 mL of hexane. Next, the sample was diluted five-fold and 1 mL methanol was added for the final extraction step. After the pretreatment procedure, samples were reacted with 100 μL primary antibodies and 100 μL I-125 for 20±4 h and 100 μL secondary antibodies was injected before measuring the radioactivity using the gamma counter.
Statistical analysis: Statistical analysis of all data was performed
using analysis of variance on the GLM program of SAS (version 9.0). The post-hoc
tests were performed using Duncans multiple range tests (Steel
and Torrie, 1980) and the statistical significance between the treatment
intervals was determined at the 5% level.
Chemical and nutritional properties of RCMB: Moisture, Crude protein, ash and oil contents of RCMB were 4.98, 12.99, 1.48 and 13.61%, respectively. The amino acids composition of RCMB is presented in Table 2 where proline, leucine and lysine were showed highest concentration (up to 30 μg g-1) and aspartic acid, glutamic acid, alanine and phenyl alanine were shown to be 20 μg g-1. Furthermore, Serine, threonine, arginine, tyrosine, valine, methionine and lsoleucine concentration ranged from 10 to 20 μg g-1. The values of glycine and histidine were 4.28 and 4.78 μg g-1, respectively. The concentration of fatty acids, palmitic, stearic, oleic, linoleic and linolenic acid composition of RCMB are presented in Table 3. The linoleic acid content was much higher than that of others fatty acids. Out of total fatty acid, saturated and unsaturated fatty acids content in RCMB was approximately 5 and 95%, respectively.
Weight gain, feed intake and feed conversion ratio: The productivities
of the crossbred chicks fed with RCMB are shown in Table 4.
In the starting period, weight gains, feed intake and feed conversion ratios
did not show any statistical differences among the treatments, but weight gain
of the treatment groups (194, 193 and 195 g in 0.25, 0.5 and 1.0% RCMB, respectively)
were increased than that of control group (187 g). During the growing period,
the performance trends of the crossbred chicks were similar to those of starting
period. Though, the productivities of the growing-finishing and finishing periods
were non-significant but higher feed intake and weight gain was obtained in
|| Amino acid composition in Rubus coreanus Miquel byproducts
|Values are presented as means of tri-replicated analysis
|| Fatty acid compositions in Rubus coreanus Miquel byproducts
|Values are presented as means of tri-replicated analysis,
SFA: Saturated fatty acids, UFA: Unsaturated fatty acids
Meanwhile, weight gain and feed intake pattern of the 1.0% RCMB fed group (2203
and 5251 g) was significantly (p<0.05) higher in the whole experimental period
as compared to the control (2093 and 4951 g) but the feed conversion ratios
were not influenced by the treatments.
Blood cholesterol and hormone: Blood total cholesterol and LDL-cholesterol concentrations was decreased in RCMB feeding chicks compared to those of the control, but the HDL-cholesterol concentration increased with the RCMB level in the diets (Table 5). The triglyceride concentration was decreased significantly (p<0.05) with the increasing level of RCMB in the diets (13.6, 10.9 and 8.6 mg dL-1 in 0.25, 0.5 and 1.0% RCMB treatments, respectively). Thus, the triglyceride concentrations in chicks blood were significantly higher in 1% RCMB treatment group than that of control and 0.25% RCMB feeding group. Though, the glucose level and total protein content was non-significant, but blood albumin concentration in 1.0% RCMB treatment (1.3 g dL-1) group was significantly higher (p<0.05) than those of other treatments.
|| Effects of Rubus coreanus Miquel byproducts on performance
in crossbred chicks
|Values are Mean±SD, a,b Values with the
same letters in the column are not significantly different at the 5% level,
1 Feed conversion ratio, 2Rubus coreanus Miquel
byproduct levels in the diets
|| Effects of feeding Rubus coreanus Miquel byproducts
on blood composition in crossbred chicks
|Values are means±standard error, a,b Values
with the same letters in the column are not significantly different at the
5% level, 1Rubus coreanus Miquel byproduct levels in the
In the present experiment, growth hormone level in the blood (28.80, 62.60, 66.20 and 77.00 ng mL-1 for 0, 0.25, 0.5 and 1.0% RCMB, respectively) was increased (p<0.05) with dietary inoculation of RCMB in the diet (Table 6). Consequently, the blood IGF-1 concentration also tended to increase with increasing level of RCMB in the diet, but the differences was non-significant (Table 6). The blood testosterone concentration rapidly increased with increased RCMB feeding levels, with concentrations of 68.60±8.30, 231.80±19.02, 664.20±15.03 and 753.60±35.97 ng mL-1 for 0, 0.25, 0.5 and 1.0% RCMB, respectively. On the other hand, the blood melatonin concentration of the control group was 65.20±3.31 pg mL-1 and the 0.25, 0.5 and 1.0% RCMB treatments were 92.00±4.91, 131.60±14.43 and 209.80±19.00 pg mL-1, respectively (Table 6). Therefore, these results demonstrated that blood melatonin concentration was sharply responsive (p<0.05) to the RCMB treatment (0.5 and 1.0% level in the diet).
|| Effect of feeding Rubus coreanus Miquel byproducts
on blood hormone levels in crossbred chicks
|Value are means±Standard error, a,b,c,d Value
with the same letters in the column are not significantly different at 5%
level, 1Rubus coreanus Miquel by-products feeding levels
Fruits of Rubus coreanus have been used in oriental traditional medicine
as the remedies for impotence, pollution, premature ejaculation and frequency
of urination (Pang et al., 1996). The physiological
activity and antioxidative effects of Rubus coreanus Miquel have been
mainly performed on human and experimental animals. No other studies have ever
been conducted to know the physiological effects of feeding Rubus coreanus
Miquel byproducts on poultry. Therefore, the present study was aimed to investigate
the effect of Rubus coreanus Miquel byproduct (RCMB) on the growth-related
hormonal changes in crossbred chicks.
In composition of the RCMB, fat, crude protein content was higher than that
of moisture and crude ash content. But, in the previous observation Cha
et al. (2007) determined that Rubus coreanus Miquel fruit
with seed and fresh pulp contained lower level of crude protein, fat and ash
and higher level of moisture which contradicts with the present findings. Likewise
amino acid compositions also have differences. This disparity attributed due
to the sample state between the present study and previous findings. The major
fatty acid were palmitic, stearic, oleic, linoleic and linolenic acid in RCMB
where linoleic acid was the most general, accounting for up to 50% of the total
fat in RCMB and which agreed with the previous findings (Ku
and Mun, 2008). Therefore, the linolenic acid may become an important indicator
of adulteration of RCMB. The present results were indicated that unsaturated
fatty acid contained up to 95% of the total fatty acids in RCMB which is higher
than the previous results (Ku and Mun, 2008). However,
chemical and nutritional values in Rubus coreanus Miquel fruit are changed,
according to maturating level or collecting time (Cha et
al., 2007). Thus, the author suggested that the RCMB fruit residues
needed to analyze before starting of the experiment.
During the entire period of the experiment, weight gain and feed intake were
increased significantly with 1.0% RCMB treatment (p<0.05). Due to the lack
of previous studies regarding the effect of RCMB treatment in poultry productivity,
a direct comparison was not possible with the present results; however, our
results were some what similar to those of a previous report (Kim
et al., 2009) who showed that the improvements of body weight gain
and feed conversion ratio of birds fed RCMB. In the berries of Rubus coreanus
Miquel, contain 52 types of volatile flavor components that produce its unique
flavor (Lee and Do, 2000) and which increase the favorability
of feeds and correspondingly stimulates the feed intake and which reflect on
weight gain of chicks. Since the body weight gain of the birds showed a linear
increase up to the 1% RCMB treatment level in this experiment, follow-up studies
should be conducted to investigate RCMB additions of more than 1% in chicken
diets. In the peel of Rubus coreanus Miquel, a polyphenolic antioxidative
agent of anthocyanin has been reported as a preventive effect on the occurrence
of cardiovascular diseases (Toufektsian et al., 2008).
Although, no statistically significant difference was observed between the RCMB
treatments and the control, the HDL-cholesterol concentrations tended to increase
with RCMB treatment, while the LDL-cholesterol concentration tended to decrease.
Similar results also obtained by Seo et al. (2011).
Several studies have shown that an RCM extract has higher electron donation
ability and prevents low-density lipoprotein (LDL) oxidation in in vitro
studies (Yoon et al., 2003; Cho
et al., 2005; Lee and Do, 2000). In the case
of triglyceride concentration, a significant reduction was observed in the 1.0%
RCMB treatment compared to that of the control (p<0.05). Given the reduction
in triglyceride concentration in the RCMB treatments, the present study suggests
that Rubus coreanus Miquel byproducts may play a role in internal lipid
metabolism. Synthesized in the liver, blood albumin has been widely used as
an important indicator of the synthesis activities and abnormality of the liver.
The albumin concentration in blood was highest with 1% RCMB treatment, showing
the same pattern as the weight gains of the chicks. In the present study, the
growth hormone level was increased (p<0.05) with the inoculation of RCMB
to the diet. Brooks et al. (2006) reported that
growth hormones act on most growing cells to affect cell size, as well as to
increase the cell population through mitosis, which will have a potentially
positive effect on growth. In the present study, the levels of IGF-1 were numerically
increased with the inoculation of RCMB in the crossbred chicks diet. Thereafter,
Rubus coreanus Miquel byproducts was increased the blood testosterone
concentrations of male crossbred chicks (p<0.05). This result is thought
to be attributable to the flavonoids present in Rubus coreanus Miquel,
which enhance the red coloring and male sexual performance. In addition, Chen
et al. (1996) reported that the administration of Rubus coreanus
Miquel increased the level of testosterone secretion and sperm count. It also
activate the sexual performances of males (Lee, 1966;
Kim et al., 2005; Oh et
al., 2007), by increasing in testis weight, sperm count, activity and
expression rate of the cAMP-responsive element modulator (CREM) gene, along
with a significant increase in CREM protein expression rate. In the present
study, the melatonin concentration was increased significantly (p<0.05) with
increasing level of RCMB in the diet. On the other hand, melatonin is a hormone
secreted from the pineal gland to control reproductive function and is known
to act effectively on life span extension through the prevention of aging (Tan
et al., 1993). Although Rubus coreanus Miquel has been reported
to have both stimulatory and inhibitory effects on the reproduction capacities
of animals (Herbert, 1981). The present study revealed
that the feeding of RCMB increased the testosterone levels and melatonin concentrations
in the crossbred chicks, suggesting mutual dependence.
The present study indicated that the addition of 1% RCMB into the diet of crossbred chicks resulted in increases of feed intake, weight gain and blood albumin concentration, but decrease in triglyceride level in the blood. The inoculation of RCMB also increased the levels of growth hormone, testosterone and melatonin hormones in crossbred chicks blood. Therefore, the inoculation of 1% level of RCMB in the diet of crossbred chicks appeared to enhance performance and hormone secretion. Meanwhile, further follow-up studies should be conducted to investigate RCMB additions of more than 1% in chicken diets.
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