Study on Reduction of Fish Meal Dosage – Case 2.Applied Research on Extreme Low Fishmeal Application Solution for Largemouth Bass Feed

[Review of the Previous Issue] Study on Reduction of Fish Meal Dosage - Case 1. Study on the Application of Fishmeal Reduction Solutions in High-Fishmeal Bass Diets

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• I Application Background and Solution

In recent years, with the gradual maturity of largemouth bass feed technology, the fishmeal inclusion in the formulas has continuously decreased. The inclusion is concentrated at 35-40% (decreasing by 10-15% compared with 3-4 years ago). This solution evaluates the effects of extreme fishmeal reduction on the growth and health of largemouth bass. It also studies if enzymatically hydrolyzed cottonseed protein could guarantee the growth, health and disease resistance of largemouth bass under low fishmeal formulations. Meanwhile, it provides references for the application of enzymatically hydrolyzed cottonseed protein in low-fishmeal formulations. The largemouth bass feed application solutions are as follows:

Control group: 35% Peruvian Super Steam Fishmeal dosage

Low-fishmeal group: 25% Peruvian Super Steam Fishmeal dosage (On the basis of the control group, 10% fishmeal is reduced, and the formulated plant protein is increased in equal proportions to maintain the consistent crude protein level.)

Low-fishmeal + Enzymatically Hydrolyzed Cottonseed Protein group: On the basis of the low-fishmeal group, 2-6% Cottonseed Protein Concentrate is replaced with Enzymatically Hydrolyzed Cottonseed Protein at equal crude protein levels.

Group	Daily Diet Design				Evaluation Indicators
	Nutritional Level	Fishmeal	Cottonseed Protein Concentrate	Enzymatically Hydrolyzed Cottonseed Protein
Control Group	CP 50% EE 11% 3 Replicate/Group 65 fish/replicate 24 g/fish	35.0%	10.0%	0%	Growth Performance Intestinal Digestive and Absorptive Capacity Hepatic Metabolic and Deposition Capacity   Disease Resistance: Dextran sulfate sodium (DSS)-induced colitis Formulation Cost: Reduce by 1100-1400 CNY/t (Based on a fish meal price of 17,700–18,500 CNY/t)
Low-fishmeal Group		25.0%	13.0%	0%
Low-fishmeal + 2% Enzymatically Hydrolyzed Cottonseed Protein		25.0%	11.0%	2.0%
Low-fishmeal + 4% Enzymatically Hydrolyzed Cottonseed Protein		25.0%	9.0%	4.0%
Low-fishmeal + 6% Enzymatically Hydrolyzed Cottonseed Protein		25.0%	7.0%	6.0%
Note: For the dosage of other plant proteins, the control group contained 6% SPC and 8% soybean meal; the low-fishmeal group and enzymatically hydrolyzed cottonseed protein group contained 12% SPC and 10% soybean meal.

•  II Application Effect

(1) Enzymatically hydrolyzed cottonseed protein compensates for the impaired growth performance under low-fishmeal diets, bringing it up to the level of regular fishmeal diets

Compared with the control group, growth performance in the low-fishmeal group was reduced significantly, whereas the 4% enzymatically hydrolyzed cottonseed protein group showed comparable.

Compared with the low-fishmeal group, supplementation with 2% and 4% enzymatically hydrolyzed cottonseed protein improved growth performance significantly, whereas the 4% inclusion level yielding the optimal results.

	Control Group	Low-fishmeal Group	Low-fishmeal+2% EHCP Group	Low-fishmeal+4% EHCP Group	Low-fishmeal+6% EHCP Group	Improvement range of 4% EHCP vs low-fishmeal group
WGR %	506.80a	448.46c	476.94b	494.29ab	437.80c	↑ 45.8 pts
SGR %/d	2.73a	2.58c	2.66b	2.70ab	2.55c	↑ 0.12%/d
FI g/fish	104.43	108.24	106.79	109.86	105.75	——
FE %	116.40a	99.69b	107.39ab	108.26ab	100.06b	↑ 8.57 pts
FCR	0.86	1.00	0.93	0.92	1.00	↓ 8%

(2) Enzymatically hydrolyzed cottonseed protein repairs intestinal structural and functional injuries under low-fishmeal diets to reach/exceed the performance of normal fishmeal feeds

① Intestinal development and digestive capacity

Compared with the control group, the intestinal development index of the low-fishmeal group and the digestibility were decreased significantly, and were comparable with the 4% EHCP group.

Compared with the low-fishmeal group, 4% EHCP significantly increased the intestinal development and digestibility.

		Control Group	Low-fishmeal Group	Low-fishmeal+ 4% EHCP	Improvement range compared with the low-fishmeal group
Intestinal Development	Intestinal length index %	73.62ab	70.86c	74.44a	↑ 3.6 pts
	Intestinal fold height μm	423a	384b	421a	↑ 9.6 %
	Intestinal fold thickness μm	97.16a	83.76c	96.63ab	↑ 15.4 %
	Intestinal structure				——
Intestinal digestion	Trypsin U/mg	1425.06a	1127.40c	1401.08a	↑ 24.3 %
	Chymotrypsin U/mg	2.44a	2.14b	2.49a	↑ 16.4 %
	Lipase U/mg	2.09a	1.84c	2.10a	↑ 14.1 %
	Apparent protein digestibility %	89.27a	87.35b	89.85a	↑ 2.5 pts

② Intraoral gavage with 3% DSS for 90 h to induce enteritis

Compared with the control group, the low-fishmeal group exhibited obvious impairment of intestinal structure, significantly reduced immunity and antioxidant capacity, aggravated oxidative damage and marked enteritis, while the 4% EHCP group showed equivalent/superior indicators.

Compared with the low-fishmeal group, 4% EHCP was beneficial to alleviating DSS-induced intestinal structural injury, and elevated immunity and antioxidant capacity to suppress enteritis progression.

		Control Group	Low-fishmeal Group	Low-fishmeal+ 4% EHCP	Improvement range compared with the low-fishmeal group
Macroscopic appearance of enteritis					Unremarkable inflammation
Intestinal structural integrity					Better structural integrity
Immunity	Anti-inflammatory capacity				Greater anti-inflammatory capacity
	LZM μg/mg	0.26ab	0.24c	0.27a	↑ 12.5%
	AKP U/L	292.62a	273.71c	290.46a	↑ 6.1%
Antioxidant capacity	T-AOC U/mg	0.14a	0.12b	0.15a	↑ 25%
	GSH mg/g	6.79b	5.61c	7.98a	↑ 42.2%
	MDA nmol/g	9.66c	10.91a	9.80c	↓ 10.2%

(3) Enzymatically hydrolyzed cottonseed protein repairs hepatic structural and functional injuries under low-fishmeal diets to reach/exceed the performance of normal fishmeal feeds

① Hepatic Architecture

Compared with the control group, the hepatic contour of the low-fishmeal group was less distinct and more enlarged and the nuclei gradually dissolved with vacuolization appearing. In the 4% EHCP group, hepatocytes were arranged in an orderly manner. The nuclei were round and located in the center of the cytoplasm. Moreover, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were comparable to those of the control group.

		Control Group	Low-fishmeal Group	Low-fishmeal+ 4% EHCP	Improvement range compared with the low-fishmeal group
Hepatic development	Hepatic Architecture				The hepatic architecture was more intact
Hepatic metabolism	Serum ALT U/L	18.64bc	19.83a	18.42c	↓ 7%
	Serum AST U/L	27.80c	30.64a	27.69c	↓ 9.6%

② Hepatic Metabolic Capacity

Compared with the control group, the capacities of protein anabolism and lipid catabolism in the low-fishmeal group were significantly decreased, whereas the 4% EHCP group was comparable to the control group.

Compared with the low-fishmeal group, the 4% EHCP group was beneficial for promoting lipid catabolism and reducing fat deposition, thereby directing more protein toward anabolism and improving protein deposition efficiency.

		Control Group	Low-fishmeal Group	Low-fishmeal+ 4% EHCP	Improvement range compared with the low-fishmeal group
Protein Anabolism	Serum total protein mg/ml	37.35a	35.85b	37.66a	↑ 5.0%
	Serum albumin g/L	11.71ab	9.89c	11.74a	↑ 18.7%
Lipid Catabolism	Carnitine palmitoyltransferase I U/g	2.36b	1.98c	3.06a	↓ 54.5%
全鱼体成分Whole-body composition
CP %		16.68a	16.16b	16.55ab	——
Crude fat %		8.99ab	8.85c	9.01a	——
Protein deposition efficiency %		0.39a	0.32c	0.38a	↑ 18.75 pts
Fat deposition efficiency %		1.15a	0.99c	1.15a	↑ 16.2 pts

 Based on the above results, when 4% EHCP was used to replace an equivalent amount of protein from cottonseed protein concentrate in a diet with an extremely low fishmeal level (25% Peruvian super prime steam fishmeal) for largemouth bass, the fish showed the best growth performance, health status, and disease resistance. Moreover, they achieved growth and health performance comparable to those of the group fed 35% fishmeal, and the feed cost was reduced by ¥1200–¥1300 per tonne.

 [Please look forward to the next issue]

Study on Reduction of Fish Meal Dosage – Case 2.

Study on the Application of Fishmeal Reduction Solutions in Low-Protein Diets for Yellow Catfish