Effects of dietary energy intake and cold exposure on kinetics of plasma phenylalanine, tyrosine and protein synthesis in sheep

An isotope dilution method of [²H₅]phenylalanine (Phe) and [²H₂]tyrosine (Tyr) was used to determine the effects of metabolisable energy (ME) intake and cold exposure on plasma Phe and Tyr turnover rates in sheep. Whole body protein synthesis (WBPS) was calculated with the [²H₅]Phe model. Eight adult sheep were assigned to two dietary treatments receiving the same amount of crude protein and either 515 or 828 kJ · kg BW^−0.75 · d⁻¹ of ME (Me-ME diet and Hi-ME diet, respectively) with a crossover design for two 28 d periods. The sheep were exposed from a thermoneutral environment (23 ± 1°C) to a cold environment (2 ± 1 to 4 ± 1°C) for 6 d for each dietary treatment. The primed-continuous infusion method of isotope dilution was conducted in both environmental temperatures. Plasma Phe turnover rate (PheTR) tended to be greater and plasma Tyr turnover rate (TyrTR) was greater (p = 0.03) for the Hi-ME diet compared with the Me-ME diet. Plasma PheTR increased (p = 0.04) and plasma TyrTR tended to increase during cold exposure. Whole body protein synthesis tended to be greater for the Hi-ME diet compared with the Me-ME diet and increased (p = 0.03) during cold exposure compared to the thermoneutral environment, but no interaction was detected. It was concluded that in sheep, plasma PheTR and WBPS (as determined by the [²H₅]Phe model) tended to be influenced by and plasma TyrTR was influenced by ME intake. Further, plasma PheTR and WBPS increased and plasma TyrTR tended to increase during cold exposure.

Keywords: cold stress; energy intake; phenylalanine; protein synthesis; sheep; tyrosine

Under cold environments, livestock animals must generate heat from oxidation of body reserves and ingested diets to maintain homoeothermy. Exposure of livestock to the cold has negative effects on productivity through modified digestive, metabolic and endocrine functions (Kennedy et al. [10]; Young et al. [24]; von Keyserlingk and Mathison [11]). It is important to understand the kinetics of plasma amino acids and whole body protein of livestock during cold exposure because of their economic significance.

Dietary energy intake also influences the intermediary and protein metabolism in ruminants (Harris et al. [8]; Fujita et al. [6]). Fujita et al. ([6]) reported that in goats, whole body protein synthesis (WBPS) increased with increasing energy intake supplemented as corn starch. Therefore, it was hypothesised that metabolisable energy (ME) intake might modulate the stress of a cold environment on animals through modification of intermediary metabolism and endocrine function. Sano et al. ([16]) reported that in sheep, plasma leucine (Leu) turnover rate (LeuTR) and WBPS, determined by a [1–13C]Leu technique, were influenced by diet and cold exposure, whereas the responses of WBPS to cold exposure were not modified by diet. However, in ruminants, the response of plasma amino acid turnover rate to cold exposure differed between amino acids (Tsuda et al. [22]; Early et al. [5]; Scott et al. [20]). Moreover, in sheep, turnover rates of plasma amino acids increased to different extents with increasing dietary intake (Savary-Auzeloux et al. [19]). These results indicate that the effect of ME intake and cold exposure is influenced by amino acids. Therefore, this experiment was designed to confirm the combined effect of ME intake and cold exposure on plasma phenylalanine (Phe) and tyrosine (Tyr) turnover rates (PheTR and TyrTR, respectively) and protein kinetics, determined by a [2H5]Phe model (Thompson et al. [21]), in sheep fed two levels of ME and exposed from a thermoneutral environment to a cold environment.

All experimental procedures were reviewed and approved by the Animal Care Committee of Iwate University. All experiments were carried out without noticeable stress to the animals, except for exposure to a cold environment.

Eight crossbred (Corriedale × Suffolk) shorn sheep (four ewes and four weathers), aged 1–3 years and weighing 31.1 ± 3.7 kg body weight (BW) were used. The basal diet consisted of mixed hay of orchardgrass and reed canarygrass, ground corn, and soybean meal (Table 1). Metabolisable energy and metabolisable protein for each diet was assumed from the Agricultural and Food Research Council (AFRC [1]). Two dietary treatments were designed according to ME intake; medium ME intake (515 kJ · kg BW−0.75 · d−1; Me-ME diet) and high ME intake (828 kJ · kg BW−0.75 · d−1; Hi-ME diet). The Me-ME diet was estimated to be maintenance ME level and 150% of maintenance CP level (National Research Council [NRC] 1985). The crude protein intake was similar for both dietary treatments (Table 1). The animals were fed once daily at 14:00 h. Drinking water was available ad libitum. The experiment utilised a crossover design with two 28 d periods in which either the Me-ME or the Hi-ME diets were fed. The sheep were randomly divided into two groups. Four sheep were fed the Me-ME diet during the first period and then fed the Hi-ME diet during the second period. The other four sheep were subjected to the dietary treatments in reverse order. The sheep were housed in individual pens in an animal room during the first two weeks of the experiment. The sheep were then moved to metabolic cages in a controlled environment chamber at an air temperature of 23 ± 1°C, with lighting present from 07:00 to 21:00 h. After completing the experiment in the thermoneutral environment, the sheep were shorn again and exposed to a cold environment for 6 d. During the cold exposure period (day 23 to day 28), the temperature in the chamber was maintained at 2 ± 1°C from 10:00 to 21:00 h and at 4 ± 1°C from 21:00 to 10:00 h. The sheep were weighed on the day before and days 15 and 21 and after each dietary treatment.

Table 1. Ingredients and contents of metabolisable energy (ME), crude protein (CP) and metabolisable protein (MP) of experimental diets, and nutrient intake.*

On days 20 and 28 of each dietary treatment, an isotope dilution method of [2H5]Phe and [2H2]Tyr was conducted to determine the kinetics of plasma Phe, Tyr and WBPS in the thermoneutral environment and cold exposure, respectively. Catheters were inserted into both jugular veins for infusion and blood sampling on the morning of each determination of the isotope dilution method. Catheters were filled with a sterile solution of 0.13 mol/l trisodium citrate. At 12:00 h, 2.5 μmol/kg BW0.75 of [2H5]Phe (L-phenylalanine, ring-D5, 98%; Cambridge Isotope Laboratories, Inc., USA), 1.6 μmol/kg BW0.75 of [2H4]Tyr (L-4-hydroxyphenyl-2,3,5,6-D4-alanine, 98 atom %; Isotec Inc., A Matheson, USA Co., USA) and 1.6 μmol/kg BW0.75 of [2H2]Tyr (L-tyrosine, ring-3,5-D2, 98%; Cambridge Isotope Laboratories, Inc., USA) dissolved in 25 ml of saline solution (0.15 mol/l sodium chloride) were injected into the jugular catheter as priming doses. Then, [2H5]Phe and [2H2]Tyr (1.7 and 1.0 mmol/l saline, respectively) were continuously infused by a multichannel peristaltic pump (AC-2120, Atto Co., Ltd, Japan) at rates of 2.6 and 1.5 μmol · kg BW−0.75 · h−1, respectively, through the same catheter for 4 h. Blood samples were taken through another jugular vein catheter immediately before and at 30 min intervals during the last 120 min of the isotope infusion. Samples were transferred into centrifuge tubes containing heparin sodium and were chilled until centrifugation. Blood samples were centrifuged in the cold (10,000 _I_g_i_, 10 min, 2°C), and the plasma was stored at −30°C until further analysis.

The concentrations of plasma Phe and Tyr and enrichments of plasma [2H5]Phe, [2H4]Tyr and [2H2]Tyr were determined with a gas chromatography mass spectrometry (QP-2010, Shimadzu, Japan) by the procedures of Rocchiccioli et al. ([13]) and Calder and Smith ([3]). Plasma free amino acid and urea concentrations at the preinfusion periods were determined with an automated amino acid analyser (JLC-500/V, JEOL, Japan). Plasma glucose concentrations were enzymatically determined (Huggett and Nixon [9]). The concentrations of plasma non-esterified fatty acids (NEFA) were determined using a diagnostic kit (NEFA C Test, Wako, Japan). Nitrogen content in the diets were determined by the Kjeldahl method (Tecator Digestor System and Kjeltec 2300, Foss Tecator, Sweden).

Mean values with standard errors of the mean are given. Metabolisable protein (MP) was calculated as described in AFRC ([1]):

Graph

where MCP [g · kg BW−0.75 · d−1] is microbial crude protein supply and UDP [g · kg BW−0.75 · d−1] is undegradable dietary protein. Turnover rate (TR) of plasma Phe (PheTR) and Tyr (TyrTR) were calculated using equations by Wolfe et al. ([23]).

Graph

where I is the infusion rate of each stable isotope and E is the corresponding plasma isotope enrichment during the steady states. The rate of Phe hydroxylation to Tyr (PheOX) was calculated as described by Thompson et al. ([21]):

Graph

where Etyr and Ephe are the plasma isotope enrichments of [2H5]Phe and [2H4]Tyr, respectively, and Iphe is the infusion rate of [2H5]Phe. Whole body protein synthesis (WBPS) was calculated from the following equation:

Graph

The phenylalanine concentration in carcass protein was estimated to be 3.5% (Harris et al. [8]).

Data were analysed by a split-plot design with the MIXED procedure (SAS [18]). The main-plots were period and diet, and sub-plots were environment and diet and environment interaction. The gender effect was also tested. Results were considered significant at the p < 0.05 level. The tendency was defined as 0.05 ≤ p < 0.10. Repeated measures were used for the time course of changes during the isotope dilution method and the difference in least square means with Tukey adjustment was used (p < 0.05).

Because one weather of eight sheep could not eat all of the Hi-ME diet, the experiment was not performed for that sheep. Therefore, the number of sheep was seven for the Hi-ME diet. The other seven sheep ate all the diets offered (Table 1). Body weight was greater (p < 0.01) for the Hi-ME diet compared with the Me-ME diet, but was not influenced by cold exposure, as shown in Table 2. The weathers were heavier (p < 0.01) compared with the ewes. The concentrations of plasma Phe and Tyr and the enrichments of plasma [2H5]Phe, [2H4]Tyr and [2H2]Tyr were constant during the latter period of the isotope dilution (data not shown). The mean coefficients of variation during the latter period of infusion were 5.3 and 5.5% for concentrations of plasma Phe and Tyr, and 8.4, 11.4 and 11.3% for enrichments of plasma [2H5]Phe, [2H4]Tyr and [2H2]Tyr, respectively. Plasma PheTR tended to be greater (p = 0.07) and plasma TyrTR was greater (p = 0.03) for the Hi-ME diet compared with the Me-ME diet (Table 2). Plasma PheTR increased (p = 0.04) and plasma TyrTR tended to increase (p = 0.07) during cold exposure. The PheOX tended to be greater (p = 0.09) for the Hi-ME diet compared with the Me-ME diet and did not change during cold exposure (p = 0.51). Whole body protein synthesis tended to be greater (p = 0.08) for the Hi-ME diet compared with the Me-ME diet. Whole body protein synthesis increased (p = 0.03) during cold exposure, but a diet × environment interaction was not detected. No gender difference was detected in the parameters of plasma Phe, Tyr and protein metabolism.

Table 2. Effects of metabolisable energy (ME) intake and cold exposure on plasma phenylalanine (Phe) and tyrosine (Tyr) concentrations, rates of Phe and Tyr turnover, Phe hydroxylation to Tyr (PheOX), and whole body protein synthesis (WBPS) in sheep.

Plasma free amino acids and urea concentrations were determined in four sheep (two ewes and two weathers) (Table 3). The concentration of plasma Tyr was greater (p = 0.01) and those of plasma Leu, glycine and serine tended to be greater for the Hi-ME diet compared with the Me-ME diet. The concentrations of plasma Leu and valine increased (p < 0.05) during cold exposure, whereas those of plasma serine and glutamine decreased (p < 0.05) and plasma glycine concentration tended to decrease. Plasma urea concentration was lower (p = 0.01) for the Hi-ME diet compared with the Me-ME diet, and remained unchanged during cold exposure. No significant interaction of diet and environment was detected in plasma amino acids and urea, except for plasma Tyr. Concentrations of plasma threonine, glutamine, valine, Leu and Tyr were higher (p < 0.05), but that of plasma glutamic acid was lower (p < 0.01) for weathers compared with ewes. Plasma glucose concentrations remained unchanged with the dietary treatment and increased (p < 0.01) during cold exposure. Plasma NEFA concentration was lower (p = 0.01) for the Hi-ME diet compared with the Me-ME diet and increased (p < 0.01) during cold exposure, and a diet × environment interaction was detected (p < 0.01). No gender effect was detected in the concentrations of plasma urea, glucose and NEFA.

Table 3. Effects of metabolisable energy (ME) intake and cold exposure on concentrations of plasma amino acids, urea, glucose and non-esterified fatty acids (NEFA) in sheep (n = 4, two ewes and two weathers).

The current experiment demonstrated that in sheep, plasma PheTR and WBPS (as determined by the [2H5]Phe model) (Thompson et al. [21]), tended to be influenced by and plasma TyrTR was influenced by dietary ME intake. Further, plasma PheTR and WBPS increased, whereas plasma TyrTR tended to increase during cold exposure. Turnover rates of plasma amino acids increased with increasing dietary intake level in ruminants (Harris et al. [8]; Savary-Auzeloux et al. [19]; Fujita et al. [6]). Savary-Auzeloux et al. ([19]) reported that in sheep fed at 0.5–2.5 times energy maintenance, irreversible loss rates of plasma Phe and Tyr increased from 1.36 and 0.99 mmol/h to 3.31 and 2.65 mmol/h, respectively. Harris et al. ([8]) reported that in young growing sheep, turnover rates of Leu and Phe increased as the dietary intake level increased. Fujita et al. ([6]) reported that in goats, supplemental corn starch (0, 0.5 and 1.0 times of maintenance energy) increased plasma PheTR (0.923, 0.971 and 1.194 μmol · kg BW−0.75 · min−1, respectively) and TyrTR (0.763, 0.772 and 1.058 μmol · kg BW−0.75 · min−1, respectively). Moreover, Dawson et al. ([4]) reported that in young steers, plasma Leu flux and Leu concentration increased with intake.

The response of WBPS to diet and cold exposure showed similar trends to that obtained by the [1–13C]Leu technique in sheep (Sano et al. [16]). Of the isotope dilution methods for the estimation of WBPS, the [1–13C]Leu technique is the most widely applied, but the methodology needs to determine CO2 production derived from Leu oxidation (Lobley [12]). The [2H5]Phe model allows the determination of WBPS using a blood sample. Moreover, Al-Mamun et al. ([2]) reported that in sheep fed at two levels of intake (431 and 632 kJ ME · kg BW−0.75 · d−1), WBPS determined by the [2H5]Phe model was comparable to the [1–13C]Leu technique, and intake level influenced WBPS in a similar direction for both methods (10.3 and 12.8 g · kg BW−0.75 · d−1 for the [2H5]Phe model, and 14.8 and 16.1 g · kg BW−0.75 · d−1 for the [1–13C]Leu technique, respectively). They concluded that the [2H5]Phe model could be used as an alternative to the [1–13C]Leu technique for the determination of WBPS in sheep. The rate of WBPS was comparable to those reported previously in sheep (Lobley [12]; Savary-Auzeloux et al. [19]; Sano et al. [14]). Therefore, it is confirmed that the Hi-ME diet enhances microbial protein synthesis and intestinal amino acid absorption, even though CP intakes were similar in both dietary treatments, as reported previously (Fujita et al. [6]).

Increased plasma NEFA and glucose concentrations suggested that the sheep were stressed by the cold exposure, as observed previously (Sano et al. [15]). In the present experiment, cold exposure (2–4°C for 6 d) increased plasma PheTR and tended to increase plasma TyrTR, resulting in enhanced WBPS of the sheep. However, the effect of cold exposure on plasma free amino acid kinetics has not been well investigated in ruminants and available data are not consistent. Sano et al. ([16]) reported that in sheep fed similar diets to this experiment, Leu turnover and oxidation rates as well as WBPS increased during cold exposure (2–4°C for 5 d). In humans, short-term mild cold exposure (10°C for 105 min) increased the appearance rate of Leu, but not significantly, and catabolic rates of protein increased significantly (Goodenough et al. [7]). On the contrary, turnover rates of plasma glutamic acid and alanine, glucogenic amino acids, were not influenced by acute and chronic cold exposure in sheep (Tsuda et al. [22]; Early et al. [5]). Although protein metabolism in response to cold exposure differed between the amino acids used as isotope tracers, the responses of plasma PheTR and TyrTR to cold exposure were of a similar direction to plasma LeuTR in sheep (Sano et al. [16]).

Greater ME intake was expected to moderate the stress of cold exposure on protein metabolism in sheep. However, WBPS in response to cold exposure was not modified by ME intake in the present experiment, because no significant diet × environment interaction was detected in protein metabolism, as observed previously (Sano et al. [16]). Therefore, no clear evidence was obtained that large amounts of ME intake moderated the effect of cold exposure. Although increases in ME intake were expected to increase intestinal amino acid absorption and to reduce mobilisation of body reserves, energy expenditure was enhanced to maintain homoeothermy during cold exposure (Sano et al. [15]). These factors may partly explain why no combined effect was detected in protein metabolism in the present experiment.

In the present experiment, the gender effect was significant in six plasma free amino acid concentrations, but was not detected in other parameters, including amino acid and protein metabolism. In our previous experiment using ewes and rams, significant differences were observed in eight plasma free amino acid concentrations and plasma LeuTR (Sano et al. [17]). Therefore, it may be possible that some plasma amino acid concentrations are influenced by gender, and the effect is more remarkable between ewes and rams compared to between ewes and weathers.

Metabolisable energy intake tended to influence, and cold exposure influenced, WBPS in sheep. However, ME intake did not modify the impact of cold exposure on the kinetics of protein metabolism. The [2H5]Phe model was comparable to the [1–13C]Leu technique for determining WBPS, and responses to diet and cold exposure were similar for both of the techniques.

This work was in part supported by a Grant-in-Aid (17580244) for Scientific Research from the Japan Society for the Promotion of Science (JSPS). The authors are also grateful to Kim Taylor, University of Guelph, Canada, for his kind comments on the manuscript.