Effect of Different Dietary Levels of Protein on Growth Performance of Penaeus monodon Families

Song Jiang1,2, Xianbin Mo1,2, Falin Zhou2,3, Jianhua Huang2,3, Qibin Yang2,3, Lishi Yang2,3 and Shigui Jiang2,3* 1College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China 2Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture and Rural Affairs, P.R.China, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Guangzhou 510300, Guangdong, China 3Shenzhen Base of South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shenzhen 518108, P.R. China.


INTRODUCTION
I n shrimp culture, feed cost accounts for more than half of the total cost. At present, shrimp feed mainly relies on fish meal as protein source and the resource of fish meal is limited and the demand is increasing rapidly (Zhang et al., 2018). The international market price of fish meal is rising sharply, and price of shrimp feed are increasing accordingly, which affects the economic benefit and sustainable development of shrimp culture industry (Liang et al., 2017;Leelatanawit et al., 2017). Some investigations have been carried out on the replaceable plant protein to reduce the demand of fish meal in shrimp feed (Yu et al., 2016;Riche et al., 2011;Liu et al., 2012), while others have selected new shrimp varieties with low O n l i n e

F i r s t A r t i c l e
content in the feed of P. monodon is about 30% ~ 50%, and the optimal protein demand of P. monodon cultured in seawater is about 40% (Miao et al., 2001;Wang et al., 1998). The rapid growth of new varieties of P. monodon, which are suitable for low fish meal protein feed culture, are selected by the method of family selection, in order to reduce the fish meal protein level in the feed, and save the cost of culture and increase the economic benefit, which will have a better application prospect. In this study, the growth and survival of P. monodon families were tested under different fish meal protein content feed culture, and the differences of growth and survival were evaluated under different feed diet conditions, in order to provide basic data for P. monodon to adapt to low fish meal protein level feed heritage breeding.

Experimental materials
The experiment was carried out in the genetic and breeding center of P. monodon in the Ministry of Agriculture of the People's Republic of China. The P. monodon used in the experiment were selected as the core breeding family of the research group after several generations of continuous breeding. A total of 36 P. monodon families with similar spawning date were selected for the experiment. The average body weight of the experimental families was 1.96-2.06g.

Family construction
According to the mating plan, artificial insemination (Jiang et al., 2013) was used to culture the P. monodon families. In order to reduce the impact of the environment in the process of family construction, standardized operating procedures were used during the cultivation of family seedlings. After artificial insemination, the female shrimps with mature gonads were put in a 500 L spawning bucket. After spawning, the female shrimps were transferred out of the spawning bucket, and the fertilized eggs were hatched at 30 o C. After hatching the nauplii, each family randomly selected 10,000 nauplii and put them into a new 500 L incubation bucket for individual cultivation. During the process of metamorphosis, the larvae were fed costal algae (Guangdong Haid Group), shrimp chips and artemia. After the larvae metamorphosis to the 2nd day, a total of 2,000 shrimps were randomly selected from each family and were transferred into the new cultivation tank for independent cultivation. On the 10 th day, a total of 500 shrimps were randomly selected from each family and transferred to a 3m 3 tank for intermediate cultivation. After a period of time feeding, the uniform individuals were selected from each family with a length of more than 3 cm for fluorescent labeling (Jiang et al., 2013), and then divided into groups according to the experimental design.

Feed formula and preparation
Fishmeal, soybean meal, concentrated dephenolization cottonseed protein and peanut meal were used as the main protein sources, and the formula was designed according to the principle of equal energy and protein. Two kinds of diets (Table I)

F i r s t A r t i c l e
feed is 30%) and Diet B (fishmeal content in feed is 10%, concentrated dephenolized cottonseed protein content is 20%). All feed materials were crushed through 80 mesh sieve, mixed step by step, and then stirred with water to make 1.00 and 1.50 mm diameter pellet feed, baked in 90 o C oven (put some water in the oven) for 2 h, dried in the air-conditioned room, stored in -20 o C refrigerator for standby.

Growth performance test under different feeds
A total of 36 families with similar incubation date were tested. The shrimps of each family were labeled by fluorescence and then mixed cultured in a 500 cm × 400 cm × 150 cm cement tank. Each feed group was set up with three parallel groups. A total of 150 shrimps in each family were randomly selected and each feed group was randomly allocated 50 shrimps.

Feeding management
Shrimps were feed 3 times a day at 8:00, 17:00 and 22:00. The daily feeding amount was 7 -8% of the shrimp weight and the feeding amount was adjusted according to the weather and feeding conditions. The aquaculture water was sand filtered seawater. The water was changed 1/3 every week, and the test period was 56 days. During the experiment, the micro tube aerator was used for 24 h to aerate and increase oxygen. The water temperature was 27-32 o C, the salinity was 31-33 PPT, the pH was 7.8-8.2, and the dissolved oxygen was 6.6-7.0 mg / L during the whole test period.

Data collection
After the test, the body length and weight of each shrimp in each feed group were measured, and the fluorescent color of each shrimp was identified, and the survival number of each family was counted.

Data calculation and processing
Spss19.0 software was used to calculate the phenotypic parameters of growth and survival of P. monodon in different diet groups. ANOVA was used to analyze the weight traits. LSD method was used to compare the weight of different families. The significance test level was set to P < 0.05, and the test data was expressed by mean ± SD.
Calculation formula of AGR: A (g/d) = (W 2 -W 1 )/t In formula, A was the absolute weight gain rate, t was the test days, W 1 was the initial weight; W 2 was the harvested weight.
Survival calculation formula: S(% )= n/N X 100 In formula, S was the survival rate%, n was the survival mantissa and n was the initial mantissa.

Phenotypic parameters of growth traits of P. monodon in different feed groups
The phenotypic parameters of body length and body weight of each P. monodon family harvested in different feed groups are shown in Table II. In different feed groups, the variation coefficient of body length was larger than that of body weight, in which the variation coefficient of body length was 32.45 -37.36%, and the variation coefficient of body weight was 22.71 -22.84%. The variation coefficient of body length and body weight of test group in Diet B group was 22.71 -22.84% and both of them were larger than that in Diet A group. The survival rate of P. monodon families in Diet A group and Diet B group were 76.52 -85.91% and 51.20 -86.34%, respectively, and the average survival rate of the Diet B group was lower than that of the Diet A group and the coefficient of variation of the Diet B group was higher than that of the Diet A group. Comparison of growth performance of P. monodon families in different feed groups The results of one-way ANOVA of body weight of P. monodon families in different feed groups were shown in Table III. The difference of body weight at harvest time among families in different feed groups were significant (P < 0.01).
LSD multiple comparison results of the mean body weight at harvest in different feed groups of P. monodon were shown in Table IV, the AGR range of the Diet A group and the Diet B group were 0.14-0.28 and 0.13-0.27, respectively. The AGR of the families with the fastest weight gain in the Diet A group and the Diet B group was 100.00% and 107.69% higher than that of the families with the slowest weight gain. At the same significant level, the top 10% of the families were selected as fast-growing families, and No. 10 and No. 6 families performed well in two different feed groups.

Survival of P. monodon families
The results of survival rate of P. monodon families in different feed groups are shown in Table V. The average survival rates of P. monodon families in the Diet A group and the Diet B group were 80.59 and 77.88%, respectively. The χ 2 test showed that there were no significant difference between the two diet groups (P > 0.05). The survival rates of the Diet A group and the Diet B group were 72.51 -86.52% and 70.68 -84.65%, respectively. In the same significant level, No. 14 and No. 10 families all performed well in two different feed groups.

Production performance of Penaeus monodon family
The yield of each family calculated by combining the body weight at harvest and the number of surviving tails is quite different. The highest yield of each family in the control group and the test group is 100% and 124.44% higher than the lowest, respectively (Table VI). No. 10 and No. 6 were in the top 10% in two different feed groups, and the two families performed better production performance.

DISCUSSION
As a traditional breeding method, selective breeding technology has been widely used in the study of animal and plant genetic improvement and has become one of the effective means of breeding new varieties and new strain (Lou, 2001). Family selection is an important means of selection and breeding (Li, 2007). Through the directional construction of families and the performance test under the common environment, and then the selection between families and within families, and the breeding according to the pedigree and the performance test results during the subculture, the effect of inbreeding controllable and sustainable breeding can be achieved. The technology of family selection and breeding has been applied in many aquatic animals and cultivated breeding a large number of good aquatic animals (Luo et al., 2015;Chen et al., 2008;Gao et al., 2010;Zhang et al., 2007Zhang et al., , 2008 which has become one of the most important breeding methods.    All aquatic animals have a certain range of adaptation to the protein content in the diet. They can survive and grow normally within the appropriate range of protein content. If the protein content in the feed is too high or too low, it will affect the survival rate and normal growth of aquatic animals (Paripatananont et al., 2015). P. monodon is a kind of omnivorous aquatic organism. Under the condition of artificial culture, the protein content in feed is generally 30%-50% (Jiang et al., 2013). However, the growth rate and survival rate of P. monodon fed with different protein content are quite different (Jiang et al., 2013). In this experiment, there are significant differences in the growth of different P. monodon families when they are fed the same feed, and there are very significant differences among individual families, which is consistent with the previous research results on P. monodon (Jiang et al., 2013) and Penaeus chinensis (Zhang et al., 2007), and there were significant differences in the growth of different families of P. chinensis in the development period. This shows that there are differences in gene level among different families of P. monodon, therefore, the different families are good breeding materials.

O n l i n e F i r s t A r t i c l e
The level of fish meal in feed will affect the growth performance and feed utilization of the shrimp (Zhang et al., 2013;Lim et al., 1990). Zhang et al. (2013) replaced 6% of the fish meal with the mixed plant protein composed of soybean protein concentrate and peanut bran, so that the growth and feed coefficient of P. monodon were not significantly affected after the fish meal level was reduced from 30% to 24%, with the declining of the fish meal level, the growth and feed coefficient of P. monodon were significantly affected. Alvarez et al. (1997) used soybean meal instead of 7% of fish meal to reduce the level of fish meal from 29% to 22%, the growth and feed coefficient of P. vannamei were not affected. Similarly, Lim et al. (1990) used soybean meal instead of 10.7% of fish meal to reduce the fish meal level from 32% to 21.3%, which did not affect the growth, feed coefficient and protein efficiency of P. vannamei, but with the increase of substitution level, the fish meal level decreased and the weight gain rate decreased significantly. In this study, 36 families in the same diet group had different growth and survival performance and the absolute growth rate of the fastest growing families was 97.16% and 95.46% higher than that of the slowest growing families in two diet groups. The difference of growth and survival between families in the same diet group may be caused by the genetic difference of families. By carrying out family selection and breeding according to the feed, families with good growth and survival characteristics can be better obtained. The growth and survival performance of the same P. monodon family fed with different diets were not the same and the growth rate of family No.22 was the 2 nd in Diet A, but the growth rate is 23 rd in Diet B. The growth rate and survival rate of family No.10 were both in the top 10 in two diet groups, which had the characteristics of good adaptability to feed with low protein level. Through the test and selection of large-scale families fed with different feeds, a group of families with wide adaptability to protein level can be obtained, which can lay a good foundation for further breeding.
According to the results of the yield index of each family, the yield of each family was still quite different. The yield order of each family in each feed group was not the same. The yield of family No.10 and family No.6 were in the top three of the two diet groups. Family yield could be used as an index to evaluate the production performance of P. monodon fed with different diets. Families adapted to low fish meal protein level diet performed certain advantages in growth rate and survival rate in the breeding process and showed good production performance.

CONCLUSION
In this study, P. monodon families were established by artificial insemination and seedling standardized cultivation. The growth, survival and production performance of different families in different diet groups were compared and analyzed. The results showed that the growth and survival characteristics of P. monodon families were quite different and had the potential for further genetic improvement. The results of this study could be used for the next step of feeding P. monodon at low fish meal protein level the breeding of new material varieties laid the foundation.