Methionine and Threonine Requirements of Dutch Rabbits Fed under a Cecotrophy Prevention Program

It is suggested that the difference in body size between domestic-type rabbits and small pet-type rabbits results in different nutrient requirements. The nutritional requirements of pet rabbits have been assessed, although such assessments require evaluation throughout the rabbit life span. The present study was conducted under a cecotrophy prevention program with young and adult rabbits. Six male Dutch rabbits were housed individually in a dormitory-type cage, and they were randomly fed graded levels of dietary methionine and threonine, at ratios of 4:0, 3:1, 2:2, 1:3, and 0:4 mixed with two types of feed, 4:0 and 0:4. Four days after switching diets and 4 hrs after starting morning feeding, approximately one milliliter of blood was collected from the vein of the ear. Free amino acid concentrations in the plasma were determined with a high-speed amino acid analyzer. Plasma concentrations of methionine and threonine compared to dietary methionine and threonine levels are shown in young rabbits. The plasma concentration of methionine remained constant at a low level and then increased linearly. The intersection was estimated as 0.16 g/d. In the same manner, the intersection of the plasma threonine value was estimated as 0.27 g/d. These values were calculated as 0.27% and 0.47% of the diets, respectively. In the case of adult rabbits, the baseline was not obtained for dietary methionine and threonine levels. The methionine requirement was estimated as 0.11 g/d. The threonine requirement was estimated as 0.22 g/d. These values were calculated as 0.15% and 0.30% of the diets, respectively. In comparison with young and adult rabbits, both methionine and threonine showed a low baseline level in young rabbits, while their variations in plasma levels of adult rabbits were not determined. The requirement of both amino acids in young rabbits is higher than that in adult rabbits. The dietary values of both amino acids in young rabbits were also higher than those in adult rabbits. Small pet-type adult rabbits required lower amino acid levels than domestic-type rabbits. How to cite this paper: Abe, Y., Muramatsu, H. and Sukemori, S. (2019) Methionine and Threonine Requirements of Dutch Rabbits Fed under a Cecotrophy Prevention Program. Open Journal of Animal Sciences, 9, 163-172. https://doi.org/10.4236/ojas.2019.92014 Received: January 10, 2019 Accepted: March 17, 2019 Published: March 20, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
Detailed information on the nutritional requirements of rabbits has been derived from studies with strains that produce meat and/or fur. Currently, there are more than 50 breeds of rabbits in the world, and feeding objectives do not consider rabbits for production of meat and fur and/or for research. Rabbits, as a pet animal have a very important status in the life of humans. It has been suggested that the difference in body size between the domestic-type rabbits and the small pet-type rabbits causes different nutrient requirements for maintenance [1].
Therefore, assessment of the nutritional requirements of pet rabbits has increased their life span. However, nutritional requirement data for pet rabbits are not sufficient. In this study, the free amino acid levels in the plasma of rabbits fed various dietary amino acid levels were used to determine amino acid requirements. This method has been reported for several animal strains and the authors previously reported the lysine, valine, methionine and threonine requirements of domestic rabbits [2] [3]. Hoover and Heitmann [4] suggested that free amino acids were synthesized with non-protein nitrogen in the digestive tract and were supplied by cecotrophy. Our previous study [5] also showed that rabbits can use amino acids synthesized by microorganisms in their digestive tracts. Moughan et al. [6] proposed that the whole-body amino acid pattern can be used to approximate an ideal balance of dietary amino acids, and their data indicate discrepancies in the recommended essential amino acid requirements for growth in rabbits. Furthermore, in our earlier study [3], it was suggested that methionine and threonine were supplied by cecotrophy of the soft feces produced by cecum microorganisms; therefore, the present study was conducted under a cecotrophy prevention practice with young and adult Dutch rabbits as pet rabbits. In this study, methionine and threonine were chosen, because no report has indicated metabolic interaction between these amino acids.

Materials and Methods
The present study was conducted with consideration for animal welfare established in the guidelines for animal experiments at Tokyo University of Agriculture.

Animals and Feeding Environmental Conditions
Six male Dutch rabbits weighing 0.71 ± 0.1 kg at 5 weeks of age were purchased from Kitayama Rabes Co., Ltd. in Nagano. In this study we could not prepare the female rabbits. They were housed individually in a dormitory-type cage (32 × Open Journal of Animal Sciences trolled at 24˚C ± 1˚C and 60%, respectively.

Diets and Time Schedule of the Study
After the preliminary feeding of the rabbits with cecotrophy prevention methods utilizing color, the first study with the young stage was conducted at 7 weeks of age for 20 days to observe the amino acid requirements.
Each experimental diet was provided for 4 days in the following order: 1, 4, 2, 5, 3. It was recognized that the plasma free amino acid concentration responded to the new amino acid dietary levels by 3 days after the dietary changes. In addition, the changes in dietary amino acid levels were reflected in the plasma amino acid levels within 2 hr after eating began. Therefore, in the fourth day after switching diets and 4 hr after starting morning feeding, approximately one mL of blood was collected from the vein of the ear. Body weight was determined before switching diets, and diet intake was recorded everyday.

Sample Preparation
Collected blood was centrifuged at 6.93 g for 5 min, and plasma was obtained.
This plasma was thoroughly mixed with the same volume of 8% sulfosalicylic acid solution. After 10 min of decantation, the mixture was centrifuged at 6.93 g for 5 min under 5˚C. Supernatant fluid was filtered with a 0.45 μm filter (Advantec, Dismic-25) as reported previously [2] [3] and kept in a −40˚C freezer until the determination of free amino acids.
After the first study, the rabbits were fed a commercial diet until they grew up to the adult stage at 35 weeks of age. The second study was conducted with 35-weeks-old rabbits in the same manner as the first study.
Free amino acid concentrations in the plasma kept at −40˚C were determined with a high-speed amino acid analyzer (Hitachi L-8500).

Statistical Treatment
Statistical treatment was carried out by one-way analysis of variance. When a significant difference (P < 0.05) was recognized, the means were analyzed by Tukey's multiple range test. The requirements of methionine and threonine were estimated by the broken line model.

Results and Discussion
Body weight gain and feed efficiency decreased in proportion to a decrease in methionine intake, and there was no change induced by a decrease in threonine intake, as shown in both stages in Table 3. Diet intake showed the maximum values in the methionine:threonine 2:2 groups and decreased in accordance with both amino acid levels. Spreadbury [7] reported that the decrease in methionine and cystine levels in the diet reduced body weight gain and feed intake in a study with New Zealand White rabbits, and the present data agreed with these findings. Taboada et al. [8] reported that there were no significant effects of methionine and cysteine levels on body weight gain and feed intake in their study with cross-bred rabbits. Colin and Ghezal-Triki [9] reported that the dietary threonine level slightly affects the feed intake (P = 0.056 -0.096) and daily weight gain  Each value is means for 6 rabbits. a-c means with different superscripts in the same row are significantly different (P < 0.05).
(P = 0.047 -0.032) with a quadratic effect among the tested threonine levels; the present data showed the same trend.
In the statistical treatment of plasma amino acid concentration (Table 4), serine, alanine, cysteine, and tyrosine without threonine and methionine showed a significant difference under the unstable tendency in young rabbits. Serine, glutamate, glycine, valine without threonine and methionine also showed significant differences under the unstable tendency in adult rabbits ( Table 5). The plasma concentrations of methionine and threonine clearly changed in accordance with the dietary supply levels, except for the stable level of methionine in young rabbits fed a low-methionine diet. Cystine and cysteine can replace 50% of the methionine requirement, but their increase in accordance with the deletion of dietary methionine was not recognized in the previous study [3], and the same trend was also obtained in this study. Therefore, the present results showed the simple methionine requirement. On the other hand, all amino acids except the abovementioned amino acids were not affected by dietary methionine and/or threonine levels, although they showed fractional variation in their levels. The abovementioned amino acids showed significant differences and were also not affected directly by the change in the level of threonine and/or methionine, because their variation was not parallel to it of threonine and/or methionine. It has already been reported that when a given amino acid level is less than required, its plasma concentration remains constant at a relatively low level (baseline), as shown in the variation of methionine of young rabbits [2] [3]. When the dietary level of tested amino acids exceeds the requirement, the plasma concentration increases linearly with increasing dietary levels (linear line). The intersection of both lines predicts the requirement of tested amino acid. In Figure 1 and Figure  2, plasma concentrations of methionine and threonine against dietary methionine and threonine levels are shown in young rabbits. The plasma concentration of methionine remained constant at a low level (under 0.15 g/day intake) and then increased linearly. In our previous study, dietary essential amino acid level is less than the requirement, its level remains constant at relatively low level and Open Journal of Animal Sciences    In a previous study [3] of adult Japanese white rabbits, the methionine and threonine requirement values were 0.23 g/d and 0.42 g/d, and their dietary ingredients were 0.28% and 0.52% of the diets, respectively. The requirements of both amino acids by young rabbits in this study were approximate values. With   i.e., the active growth stage of small pet-type rabbits, does not differ from the requirement level of domestic-type rabbits. The present data of both amino acids from adult Dutch rabbits are half the values of previous data [3]. In this study, the plasma concentration of methionine showed a constant low level as a baseline, but the plasma concentration of threonine did not show a constant low level, i.e., the minimum supply from the present diet was sufficient for the requirement of adult Dutch rabbits. The diet used was the same as that used in the previous study. In a previous study [3], the plasma concentration of both amino acids showed a constant low level, i.e., the minimum supply from the diet was insufficient for the requirement of adult Dutch rabbits. Therefore, small pet-type rabbits required lower amino acid values than do domestic-type rabbits.
In comparison with young and adult rabbits in the present study, both methionine and threonine showed a low baseline level in young rabbits, while their variations in plasma levels of adult rabbits was not determined. Furthermore, the requirement of both amino acids in young rabbits is higher than that in adult rabbits. The dietary values of both amino acids in young rabbits were also higher than those in adult rabbits. Maertens et al. [15] proposed that protein and amino acid requirements decrease with increasing age, and our data agree with their proposal.