Relationships between Plasma L-Carnitine Levels and Genetic Characteristics of Japanese Black Steers

The calving transition period, which lasts several days before and after calving, is a time of high stress for cows, and this stress affects not only cows but also the possibility of calves receiving milk with damaged composition. Some treatments, such as glucose precursors, minerals used as an oral drench solution, L-carnitine or administration of rumen-protected amino acids, have been examined as methods to minimize the negative effects caused by calving [1-6]. In studies of the nutritional role of L-carnitine supplementation on growth performance and meat characteristics, early postnatal skeletal myo-fiber formation in lightweight piglets from large litters was reported by Lösel D et al. [6,7] and Madsen JG et al [8] with L-carnitine supplementation occurring during suckling. These authors reported increases in myo-fiber number, but growth performance was not influenced. Meat production performance of Japanese Black cattle requires large carcass size, i.e., beef cattle strains that show high daily gains preferred by farmers. In contrast, beef with fine marbling texture generally is preferred by consumers [8,9] but preference for beef with coarse marbling has recently increased, as this type of beef is considered a healthier option by older consumers.


Introduction
The calving transition period, which lasts several days before and after calving, is a time of high stress for cows, and this stress affects not only cows but also the possibility of calves receiving milk with damaged composition. Some treatments, such as glucose precursors, minerals used as an oral drench solution, L-carnitine or administration of rumen-protected amino acids, have been examined as methods to minimize the negative effects caused by calving [1][2][3][4][5][6]. In studies of the nutritional role of L-carnitine supplementation on growth performance and meat characteristics, early postnatal skeletal myo-fiber formation in lightweight piglets from large litters was reported by Lösel D et al. [6,7] and Madsen JG et al [8] with L-carnitine supplementation occurring during suckling. These authors reported increases in myo-fiber number, but growth performance was not influenced. Meat production performance of Japanese Black cattle requires large carcass size, i.e., beef cattle strains that show high daily gains preferred by farmers. In contrast, beef with fine marbling texture generally is preferred by consumers [8,9] but preference for beef with coarse marbling has recently increased, as this type of beef is considered a healthier option by older consumers.
Based on the genetic modification of meat characteristics, strains with fine marbling and beef strains with coarse marbling have been developed. Production of beef with fine marbling is an original technique developed in Japan, and beef steers that provide fine marbling show lower weight gain tendencies than do steers that provide beef with coarse marbling [10]. This difference in daily gain between the steers may be induced by other factors, such as nutrient metabolism. Additionally, there are few studies on the endogenous synthesis of nutrients that do not consider ingestion supply. Rumen microbes undergo reproductive functions that produce amino acids using protein ingested from feed, therefore the requirements of amino acids have never been of concern in cattle farming. Recently, it was revealed that a spot supply of specific amino acids for the regulated lactation period, is produced as a result of a rumenbypass characteristic found in high-performance dairy cattle [5,11]. Colostrums has high levels of lactose and milk fat, both of which can be used as an energy source, and L-carnitine, which accelerates the utilization of long-chain fatty acids in β-oxidation, is also present at high levels in colostrums. In a study of humans, it was estimated that 25% of L-carnitine was produced by endogenous synthesis from lysine and methionine in the liver and/or kidneys of mature individuals, and the remaining 75% of total body carnitine levels came from the diet [12].
Therefore, a young animal such as a calf must take in L-carnitine in the form of milk or a starter; additionally domestic steers recognized to have bovine spongiform encephalopathy and physiologically inactive organs require a regulated intake of animal products rich in L-carnitine resources. Artificial milking practices caused by the early separation of mother and child such as in dairy cattle, have increased in the feeding of beef cattle. In this system, artificial milk is produced from skimmed milk and acts as a resource that supplies L-carnitine. The L-carnitine content in milk changes in the lactation phase and varies in colostrums and regular milk, and the content decreases with heat stress [2]. In previous studies [13,5], although the supplementation of L-carnitine into artificial milk tended to improve daily gains, significant differences could not be recognized, and it was deduced that the statistical results were induced by the endogenous synthesis of L-carnitine, which was dominant in the strain.
In this study, changes of plasma L-carnitine levels in accordance with growth of steers from two strains, one that was representative of fine marbling and one that was representative of high gains, were observed to provide further clarification of our previous hypotheses.

Materials and Methods
In this study, the steers used in the experiments were fed under custom conditions which included feed-stuffs from two collaborating farms, and were cared for in accordance with the guidelines of the Animal Welfare Act of the Tokyo University of Agriculture. Blood collection was carried out by veterinarian in each farm.

Steers and artificial milk
The two collaborating farms were located in Kumamoto Prefecture (K farm) and Shimane Prefecture (S farm). Steers were classified into two groups, one of which was a fine marbling group composed of 11 steers (4 in the K farm and 7 in the S farm) and the other was a high-gain group composed of 7 steers (2 in the K farm and 5 in the S farm). They were given their mother's milk until 9 days old, and steer separation occurred at 10 days, after which the calves were fed artificial milk. The makers of commercial artificial milk were different.

Blood collection and treatment
Blood was collected when the calves were 10 days old, which was the starting point of artificial nursing, and 30 days old at which time early weaning was carried out. Blood plasma was separated from whole blood by moderate centrifugation (1500xg) and stored at -20 °C for determination of L-carnitine levels.

Analysis
The same volume of 2% sulfo-salicylic acid solution was added to the stored blood plasma and artificial milk that was prepared at typical concentrations for the removal of protein sediments. The L-carnitine levels in the supernatant of the blood plasma and in the artificial milk samples were determined by UV 21 nm by liquid chromatography (LC-20AD, Shimadzu, Co. Ltd., Kyoto, Japan) and under the following conditions: a Unison UK-C18 column, 20nM phosphate buffer including a 5nM octane sulfonic acid sodium carrier, and a flow rate of 0.8mL/min.

Statistics
The results as shown with real values were statistically analyzed with one-way analysis of variance with a significance level of P<0.05. The differences between the values of 10 days and 30days in each calf, as shown Δ20 values, were treated using the Student t-test with significance level of P<0.05, between the farms in the strain with fine marbling and the strains in the S farm. Furthermore, a linear regression equation for chronological variation was calculated for each strain.

Results and Discussion
The obtained real values and Δ20 values of plasma L-carnitine level and the difference between day 30 value and day 10 values in each calf are shown in Table 1.

L-carnitine contents in artificial milk
The L-carnitine content in the artificial milk used at the K farm was 47.9mg/L and at the S farm 87.4mg/L. The L-carnitine content from the S farm was approximately 1.8 times higher than that of the K farm. In our previous studies, we reported values ranging from 16-64mg/L in colostrums and a value of 47.5mg/L in artificial milk [3][4][5][6]. The high values in these studies were closer to the K farm value, and the lowest value of 16mg/L was calculated from the colostrums of Japanese Black cows (unpublished), as shown in Table 2. In the groups representing fine marbling of both two farms, there was no significant difference in the Δ20 values (Figure 1). Therefore the difference of supplied L-carnitine amounts through artificial nursing did not affect the plasma L-carnitine level. It is recognized that the contents in milk change with environmental conditions, especially atmospheric temperature [4], and this change is reflected in the L-carnitine content of the skimmed milk that was used to produce artificial milk. The existence of plasma proteins that provide immunological substances can have an effect on the L-carnitine content in artificial milk. The difference in L-carnitine contents in the current result might be caused by one or a combination of the above mentioned reasons.  Δ20: Changed plasma L-carnitine level in the calf with the fine marbling strain in both farms during supply with artificial milk for 20 days. There were no significant differences. In the procedure of L-carnitine synthesis, ascorbic acid, riboflavin and iron are required as co-factors. Vaz et al. proposed that ε-N trimethyllysine hydroxylase mediates the first step in the endogenous synthesis of L-carnitine [14]. The activity of γ-butyrobetaine dioxygenase is also necessary to form L-carnitine, and if this enzyme has very low activity, animals become highly dependent on active uptake from blood [15]. Activities of both enzymes are essential factors for L-carnitine biosynthesis, but little research has been conducted on the relationship between the endogenous synthesis of L-carnitine and the growth performance of nursing calves; however many studies have been conducted with infants [13]. In the present study, there was no significant difference between the different strains at the same time after birth. After the separation of mother and child at the age of 10 days, the amount of L-carnitine ingested by steers was different at each farm. It changed in direction, meaning an increase or decrease in plasma L-carnitine level, reflected the endogenous synthesis activities, i.e., an increase reflecting high synthesis and low consumption of L-carnitine, and a decrease showed the opposite relationship. While the obtained linear regression equations were not statistically significant, there was a decreasing trend in the beef strain with fine marbling and a slight increasing trend in the plasma L-carnitine levels in the high-gain beef strain. This present trend was emphasized in the fine marbling strain with significant level of P=0.1 (Figure 2), and the variation of Δ20 values during different strains in the S farm showed opposite direction with decreasing trend (P=0.095) (Figure 3). It was recognized that the present difference in the supplied L-carnitine did not affect the plasma level, as in the abovementioned. In a previous study [16], a positive relationship

Journal of Dairy and Veterinary Sciences
between L-carnitine levels of the blood and daily gain of bulls was recognized but was insignificant. The effect of L-carnitine supplementation of feed on the daily gain of steers was also unable to reach full potential, i.e., it was obviously at a low plasma level, as in the previous result [4], and we deduced that the influence of supplemental L-carnitine on the growth of steers may be related to different levels of endogenous synthesis and supplement levels. The current results support the results of our previous studies, although further verification with more heads of steer is necessary. Δ20: Changed plasma L-carnitine level in the calf with the fine marbling strain in both farms during supply with artificial milk for 20 days. There were no significant differences between the different strains.
In studies on the nutritional roles of administered L-carnitine, it has been reported that dietary L-carnitine prevents fat accumulation in dairy cows [17] and young bulls [18], and there is no improvement in the growth performance of young bulls. This result might be, because the steers are too large; we recorded body weights of 310±24 kg, for steers used for L-carnitine administration tests in our analysis.

Conclusion
Plasma L-carnitine levels in immature steers varied with growth under different conditions of energy production including de novo synthesis and consumption for the energy production as β-oxidation, and this balance may be controlled by the genetic characteristics of beef strain. Current results relate to a particular type of beef production used in Japan, but the supplementation of L-carnitine in the starter feed proves to be useful and therefore is suggested.