I.INTRODUCTION
In current feeding systems, the energy requirement is indicated with accuracy based on in vivo metabolism studies to access energy requirement for maintenance and production. Moreover, ruminal microbial nitrogen supply to the small intestine has been also evaluated quantitatively to estimate metabolizable protein (MP) supply to ruminants (ARC 1984; AFRC 1993).
Recently Kim et al. (2015) reported that low-quality forage, such as Italian ryegrass straw (IR), can be used for a long-term feeding system for growing ruminants. The growing wethers were fed on two types of forage based iso-energetic diet; and both of nitrogen (N) intake and digestible crude protein intake were greater for timothy hay (TH) based diet (THD) feeding as compared to IR based diet (IRD) feeding without significant difference in N retention (% of N intake). Additionally, dressing rate, lean meat weight per empty body weight (EBW), weight proportion of subcutaneous fat and abdominal fat were numerically greater for THD as compared to IRD in hogget production performance of Suffolk wethers which were slaughtered at 18 months of age (Kim et al., 2014). The lack of differences in N retention and carcass traits between the diets may be due to the differences in metabolizable energy intake related digestible organic matter intake (Yan and Agnew, 2004), differences in MP supply originating rumen microbes (Clark et al., 1992), amino acid sparing (Neale, 1971; Neale and Waterlow, 1974), gluconeogenesis from the ruminal propionic acid, and urea recycling into the rumen (Sands and Layton, 2009). However, the effect of factors above mentioned was not cleared in the previous studies (Kim et al., 2014; 2015). The plausible reason for the difference in carcass traits might be due to the difference in amount of nutrients absorbed though the gastrointestinal tract (GIT) or delivered to the liver, rate and extant of nutrients processing in the hepatic tissue, which in turn, resulted in the difference in amount of available nutrients for the development and growth of adipocytes or muscle cells.
To elucidate our hypothesis, there is a need to determine the difference of nutrients flux in sheep fed with different type of forage diets. The objective of this experiment was to determine the effect of feeding forage based diet on blood plasma nutrients flow to muscle tissue and adipose tissue in relevant to N balance determination in growing wethers.
II.MATERIALS AND METHODS
The animal experiment of this study was conducted at Shimane University from May to October 2014. Use and animal care procedures were approved by Animal Care Committee of Shimane University (Permit Number: MA26- 23).
1.Animals and diets
Three spring born (January to February 2014) Suffolk wethers, initial mean body weight (BW) of 27.3 ± 2.3 kg, were used in this study. The animals were allocated to three dietary treatments at the age of 6 to 7 months. First cut TH, IR and rice straw (RS) were used as basal forages with commercial concentrate in this study (Table 1). The animals were kept individually in metabolism crates throughout the experimental period, and fed THD, IRD or RS with commercial concentrate RSD in a one-way layout design. The forages were allowed at 2% BW(dry matter basis) and amount of concentrate allowance was set at 40% of forage intake (as fed basis) of each animal. The diets were designed to satisfy metabolizable energy requirements for 100 g daily gain with a provision of 5% safety margin based on the estimation equations by the AFRC (1993). The animals were fed two equal sized meals at 09:00 and 21:00 hours throughout the experiment.
The animals were surgically prepared under general anesthesia for forming a skin loop enclosing the right carotid artery and for locating indwelling catheters in the mesenteric vein and the hepatic portal vein at 3 months before the start of the experiment. Surgical preparation, post-surgical care and management were conducted in accordance with the guideline of the animal use regulation of Shimane University and also with the ‘Guide for the Care and Use of Agricultural Animals in Agricultural research and Teaching’ (Federation of Animal Science Societies, 1999).
2.Experimental procedures
The animals were housed in an environment controlled room with air temperature being 23 ± 1 ℃ with free access to water throughout the experimental period. The experiment was carried out in three periods of 11 days, first 7 days for adaptation to the diet, 3 days for metabolism trials and the last one day for blood collections.
During the metabolism trials, daily excretion of feces and urine were recorded for 3 consecutive days. Urine was collected into the container containing 50 mL of 10% (v/v) H2SO4 to keep urine pH below 3. Representative samples of feeds, orts, feces and urine were obtained daily during the total collection period. On d-11 of the experiment, continuous infusion of para-aminohippuric acid (PAH) solution (1% w/v) was conducted via the mesenteric vein as described by Ortigues et al. (1994). The blood samples at portal vein and artery were simultaneously collected into 10 mL heparinized syringes at every 60 min after morning feeding. Total 12 sets of blood samples were immediately analyzed for hematocrit value. Whole blood samples were centrifuged for 15 min at 4 ℃, and the blood plasma samples were stored at -21 ℃ until further analysis.
3.Chemical analysis
The samples of feeds, orts and feces were dried in a forced air oven at 60 ℃ for more than 48 h. Air dried samples were ground to pass through a 1 mm screen. The N in feeds and feces samples was determined by AOAC method. Blood plasma concentrations of glucose, nonesterified fatty acids (NEFA) and triglyceride (TG) was determined by commercial kits (Glucose C-test Wako, NEFA C-test, TG E-test, Wako Pure Chemical Industries, Osaka, Japan) and PAH concentration of blood plasma samples were determined by the method of Huntington (1982).
4.Calculations
Blood plasma flow rate was calculated using an indicator-dilution technique previously described by Katz and Bergman (1969). One-compartmental pharmaco kinetics model (Oriuchi et al., 1995) was introduced to estimate the rate constant of nutrients absorption from the GIT and first stage nutrients elimination in the hepatic tissue. According to the graphical shape of the change in nutrients concentration of arterial blood plasma (glucose, NEFA and TG) postprandial time, analytical equation was generated based on the pharmacokinetics model. In this model, change in specific nutrients concentrations of arterial blood plasma post feeding (t, hr) can be described as:
where Cnutr, nutrient concentration of arterial blood plasma; C, scale parameter; ka, rate constant of nutrients absorption though the small intestine (per h) and kd, rate constant of nutrients disappearance at the hepatic tissue by the metabolism of first passage effect (per h). In this model, assumptions, ka ≠ kd and ka > kd, were introduced for estimating the parameter relating nutrients absorption, hepatic tissue metabolism and supply to the body tissue. All curves fitted to estimate the parameters were performed using commercial software generated by Daniel G. (Curve Expert 1.4, http://curveexpert.net). The amount of bioavailable nutrients delivered to the tissues the weight of muscle and adipose tissue were calculated using allometric equations listed in by ARC (1980).
5.Statistical analysis
Effects were deemed to be statistically significant when P<0.05, and tendencies were considered to exist when 0.05<P<0.15. Data were analyzed by one-way ANOVA and differences between the means were separated using Duncan’s multiple range test. All statistical procedures were performed using SPSS 12.0 (SPSS, 2006).
III.RESULTS AND DISCUSSION
1.Blood metabolites
Blood metabolites were shown in Table 2. Blood plasma concentrations of all items investigated were higher for arterial blood than for portal vein blood except that of TG in IRD. Blood plasma concentration of glucose at arterial and portal vein did not differ significantly between the diets. Sletmoen et al. (2000) has suggested that plasma glucose concentration was increased by supplementing with ruminal degradable protein. However, other studies have shown no effect of protein supplementation on blood glucose concentration in ruminants (Krysl et al., 1987; Cheema et al., 1991), and our result agreed with their results. The concentrations of NEFA and TG at arterial of portal blood were not different among the diets.
2.Plasma flows and net flux of nutrients
The EBW, estimated portal plasma flow rate, absorption rate constant of nutrients from the GIT and available nutrient circulations were shown in Table 3. The EBW and portal plasma flow rate (L/min) did not differ among the diets. The ka (fraction/h) variable of glucose and NEFA did not differ among the diets, and the ka for TG was significantly lower for THD than for IRD or RSD (P=0.01). Additionally, available amount of glucose and NEFA were estimated to be higher for THD than for IRD or RSD, but statistical differences were not detected between the diets. The available amount of TG was significantly higher for IRD than for THD (P=0.04). The difference of basal forages might have resulted the changes in nutrients absorption rate constant and portal plasma flow rate.
3.The proration of nutrition in the body
The daily amount of bioavailable nutrients supply to muscle tissue and adipose tissue in growing wethers were shown in Table 4. Bioavailable glucose and NEFA supplied to both of tissues were higher for THD than for IRD or RSD, however, they did not differ among the diets. In addition, the amount of TG showed no significant differences among the diets. As shown in our results, supplement of bio-available nutrients in forage diets showed no significantly different in muscle and adipose tissue of growing wether lambs. However, little is known that the roles of bioavailable nutrients in the concentration of blood plasma were investigated in the in-vivo model because of its very complex process.
4.Effect of forage based diets on N balance
The N balance in growing wethers fed with three forage based diets was shown in Table 5. The N intake was not significant differences among the diets. However, fecal N excretion tended to be higher for IRD than the THD or RSD. The N digestibility was higher for THD than for IRD and RSD, it accounted for 71.4, 50.5 and 57.3%, respectively. However, urinary N excretion was higher for THD than IRD and RSD. The N retention and proration of N intake were higher for THD than IRD and RSD. However, there were no significant differences among the diets. N retention increased with an increase in apparent digestible N intake, which was also reported in other sheep breeds (Sarraseca et al., 1998; Lobley et al., 2000; Marini et al., 2004; Kamalzadeh and Shabani, 2007). In addition, the ratio of N retention to apparent digestible N intake with an increase in apparent digestible N intake increased in 40~50 kg Suffolk wether sheep (Sarraseca et al., 1998).
IV.CONCLUSION
In conclusion, amount of bioavailable glucose supplement was exhibited no significant differences among the diets. Our results showed that bio-available glucose supplement was higher for muscle tissue than adipose tissue in THD. This reason was thought to be closely related to N retention (g/d or % of N intake) which was higher for THD than for IRD or RSD.
