**5. Feed development and nutrient requirement**

## **5.1 Development of formulated feed**

We [18] began our studies in this area with several preliminary tests on biofilm because *C. sandwicensis* ate biofilm well which should be close to their natural diet. We also tested several dry diets, gelatin, and agar diets. We discovered that several were preferred and some were not. Several chemical attractants were tested including betaine, gamma aminobutyric acid (GABA), and dimethyl propiothetin (DMPT), but these did not enhance feeding. Among the feeds tested in a preliminary way, fish meal and soybean meal as well as feeds incorporating biofilm were preferred. Eventually, we found that *Porphyra* preparations could replace biofilm as a feeding stimulant in formulated feed.

Nutrient requirement was our next step to develop the commercial feed available for limpet, and the authors assumed that the nutrient requirement of limpet and abalone is the same as they are marine gastropod [18]. For abalone, a series number of researches had been done, and the optimum nutrient requirement as protein, carbohydrate, and lipid was focused. However, the results still varied among researchers. For example, the protein requirements of abalone found by previous studies [33, 34] were higher than those reported in the previous studies [35–37]. Poor growth was found for abalone when the animal was fed with formulated diet containing amino acid profile that does not match the animals' tissue [38]. Moreover, other studies [39, 40] found that a significant lower growth rates when abalone fed with dried kelp *Ecklonia maxima* and *Laminaria*. Therefore, these studies raised the hypothesis that the growth rate of abalone is related to the degree of the amino acid profile of feed and the amino acid profile of tissue.

**95**

*1*

*2*

*3*

**Table 5.**

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*Corn oil and menhaden oil (1:1; v/v).*

*Composition of formulated diet (% dry matter).*

*Reproductive Biology, Seed Production, and Culture of the Hawaiian Limpet* Cellana*…*

Based on the results of previous study [18], further studied on the determination of protein and carbohydrate requirement for Hawaiian limpet *C. sandwicensis* [41]. Experimental animals. Adult *C. sandwicensis* limpets (shell length above 3.0 cm) were collected from a remote area in Oahu, Hawaii, used for this study. After collection they were immediately placed into a 14 L ice plastic insulation box with plastic liner and then transported to the laboratory at the University of Hawaii in Monoa. The limpet was held in a plastic aquaria 150 L with water flow for a week; during this period, the animal were fed with the experimental diet and the commercial algae *Porphyra tenera* or *yezoensis*, known as Nori (Nishimoto

Experimental diets. Formulations of dietary protein and carbohydrate levels are shown in **Table 5**. The first trial was done for dietary protein level, following by dietary carbohydrate. For carbohydrate trial, four different dietary carbohydrate levels of 18, 27, 32, and 37% were tested. The amino acid profiles of *C. sandwicensis* tissue and of the dietary protein in trial 1 were analyzed at the Aquatic Feed and Nutrition Laboratory, Oceanic Institute, Hawaii, USA, according to the described method [42]. The results are presented as A/E ratio (**Table 6**). Most of the essential amino acids of diets were identical and/or close to the amino acid profile of *C.* 

**Ingredient Dietary protein trial 1 Dietary protein trial 2 Carbohydrate trial**

Fishmeal 16.5 19.5 22.5 25.5 28.5 13.4 17.0 21.0 30.4 16.5 16.5 16.5 16.5

Krill meal 4.5 7.5 10.5 13.5 16.5 7.1 8.0 11.0 16.1 4.5 4.5 4.5 4.5 Porphyra1 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 Wheat flour 15.4 14.3 13.3 12.2 11.1 8.98 5.3 4.3 0.8 15.4 26.9 33.7 40.5

Alginate 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Corn/fish oil2 2.5 1.9 1.2 0.6 0.0 2.32 1.4 0.9 0.6 1.0 1.0 1.0 1.0 Vit. mix3 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Cholesterol 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 Total 100 100 100 100 100 100 100 100 100 100 100 100 100 Water 100 100 100 100 100 133 133 133 133 100 100 100 100

Crude protein 26.5 31.7 37.0 42.4 47.7 21.2 30.5 35.8 49.2 26.5 26.5 26.5 26.5 Crude lipid 4.97 4.97 4.97 4.97 4.97 5.13 5.13 5.13 5.13 3.47 3.47 3.47 3.4.7 Carbohydrate 17.5 17.8 18.1 18.3 18.0 11.0 11.0 11.2 11.4 18.0 27.0 32.0 37.0

*This is commercial seasoned seaweed known as nori or the red algae Porphyra tenera or yezoensis. Nishimoto* 

*Commercial vitamin mix (NRC 1981) was kindly provided from Dr. Warren Dominy (Oceanic Institute).*

**270 320 370 420 470 210 300 350 500 180 270 320 370**

11.5 14.5 17.5 20.5 23.5 11.0 12.7 16.6 24.4 11.5 11.5 11.5 11.5

29.2 21.9 14.6 73.0 0.0 36.8 35.2 25.8 7.3 30.9 19.4 12.6 5.8

*DOI: http://dx.doi.org/10.5772/intechopen.87128*

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Defatted soybean

Diatomaceous earth

Analyzed and calculated nutrient "as fed"

**5.2 Protein and carbohydrate requirement**
