Aquaculture and Fisheries xxx (xxxx) xxx

Contents lists available at ScienceDirect

Aquaculture and Fisheries

journal homepage: www.keaipublishing.com/en/journals/aquaculture-and-fisheries

Effects of dietary vitamin K3 levels on growth, coagulation, calcium

content, and antioxidant capacity in largemouth bass, Micropterus salmoides
Xiang Wei a, b, c, 1, Ying Hang a, b, c, 1, Xiang Li a, b, c, Xueming Hua a, b, c, *, Xiangming Cong a, b, c,
Wanting Yi a, b, c, Xu Guo a, b, c
a
Centre for Research on Environmental Ecology and Fish Nutrition (CREEFN) of the Ministry of Agriculture, Shanghai Ocean University, Shanghai, 201306, China
b
Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai, 201306, China
c
National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, 201306, China

A R T I C L E I N F O A B S T R A C T

Keywords: An 8-week feeding trial investigated the optimum dietary vitamin K3 requirements of largemouth bass. A total of
Growth 600 healthy fish (12.96 ± 0.07 g) were fed diets containing varying levels of vitamin K3 at 0.78 (K0), 5.80 (K5),
Blood coagulation 10.82 (K10), 15.84 (K15), and 20.85 (K20) mg/kg with four replicates per level. Results showed that dietary
Antioxidant capacity
vitamin K3 enhanced growth, with weight gain rate and specific growth rate in K15 trials significantly higher
Largemouth bass
than K0 trials (P < 0.05). No significant difference was observed in the content of crude protein, crude lipid,
Vitamin K3
moisture, ash, and phosphorus in muscle tissue (P > 0.05). However, the supplementation of vitamin K3
significantly contributed to calcium accumulation in muscle tissues and vertebrae (P < 0.05) and increased red
blood cell counts (P < 0.05). The blood coagulation time decreased significantly with increasing dietary vitamin
K3 from 0.78 to 10.82 mg/kg (P < 0.05) before stabilizing (P > 0.05). The antioxidant capacity was improved
with the addition of vitamin K3, which was inconsistent with the changes in malondialdehyde and MK-4 (a form
of menaquinone) within liver and serum, suggesting that the toxicity of excessive artificial vitamin K3 may ac­
count for this difference. Using regression analysis, the appropriate dietary vitamin K3 levels within a largemouth
bass diet was determined to be in the range of 9.93–15.22 mg/kg, which will provide a reference for the
preparation of vitamin premix and artificial diet for largemouth bass.

1. Introduction
Vitamin K is a fat-soluble vitamin that acts as a co-factor in the
carboxylation process of vitamin K-dependent enzyme glutamyl
carboxylase (GGCX). GGCX converts glutamic acid residues (Glu) to
γ-carboxy glutamic acid (Gla) in vitamin K-dependent proteins (Booth,
2009). Osteocalcin (BGP), matrix Gla protein (MGP), and protein S are
vitamin K-dependent proteins found in bones (Ferland & Ph.D., 1998;
Vermeer, Jie, & Knapen, 1995). Vitamin K acts as a positive regulator to
facilitate normal calcium content and development (Ferland & Ph.D.,
1998). Vitamin K also promotes the synthesis of prothrombin (coagu­
lation factor II) and regulates the synthesis of three other coagulation
factors (Ⅶ, Ⅸ and Ⅹ), promoting clotting (NRC, 2011).
Vitamin K is classified into three forms, vitamin K1 (phylloquinone,
PK), vitamin K2 (menaquinone, MK), and vitamin K3 (menadione)
(Udagawa, Nakazoe, & Murai, 1993). PK and MK are natural com­
pounds, whereas vitamin K3 is an artificial water-soluble compound. PK
is synthesized predominantly by green plants and algae, MK is synthe­
sized mainly by microorganisms (Shearer, 1995; Stafford, 2005). MK
includes a series of forms, with multiple repeating prenyl units in the
side chain of the molecule. The range of chemicals is thus called MK-n,
where the “n” represents the number of prenyl units (Bügel, 2003;
Vermeer & Braam, 2001). Synthetic water-soluble salts are commonly
used in fish feed, most often menadione sodium bisulphite (MSB) or
menadione nicotinamide bisulphite (MNB) (Krossøy et al., 2009). Di­
etary menadione plays an important biological role after converting to
an active compound (MK-4) in tissues, mainly presenting in fish livers
(Grahl-Madsen & Lie, 1997; Udagawa et al., 1993).
Studies on Vitamin K have mostly focused on its physiological
function within body and its required levels in aquatic species. Vitamin

* Corresponding author. Centre for Research on Environmental Ecology and Fish Nutrition (CREEFN) of the Ministry of Agriculture, Shanghai Ocean University,
Shanghai, 201306, China.
E-mail address: xmhua@shou.edu.cn (X. Hua).
1
These two authors contributed equally to this work.

https://doi.org/10.1016/j.aaf.2021.08.004
Received 16 November 2020; Received in revised form 3 August 2021; Accepted 7 August 2021
2468-550X/© 2021 Shanghai Ocean University. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: Xiang Wei, Aquaculture and Fisheries, https://doi.org/10.1016/j.aaf.2021.08.004
X. Wei et al. Aquaculture and Fisheries xxx (xxxx) xxx

K deficiency might result in such symptoms as bleeding, anemia, Table 1

decreased hematocrit, and prolonged blood clotting times in fish Basal feed formula and nutrition level (g/kg dry basis).
(Halver, 1989; Poston, 1976; Taveekijakarn, Miyazaki, Matsumoto, & Ingredients (g/kg) K0
Arai, 1996). A study conducted in mummichog (Fundulus heteroclitus)
Fish meal 450
reported that diets without supplemented or excessive dosage of MSB Soybean meal 110
caused a higher incidence of vertebral abnormality (Udagawa, 2001). Corn gluten meal 110
The optimal dietary vitamin K requirements of lake trout (Salvelinus Blood meal 50
namaycush) (Poston, 1976), grass carp (Ctenopharyngodon idellus) (Jiang α-starch 100
Fish oil 40
et al., 2007), cod (Gadus morhua) (Grahl-Madsen & Lie, 1997), gibel carp Rapeseed oil 20
(Carassiusauratus gibelio) (Duan et al., 2013), hybrid tilapia (Oreochromis Soy lecithin meal 30
niloticus × O. aureus) (Li, 2003), grouper (Epinephelus malabaricus) (Li, Vitamin premix1 (without vitamin K3) 5
2003), and large yellow croaker (Pseudosciaena crocea) (Cheng, Zhang, Mineral premix2 5
Squid paste 10
Lin, Xu, & Mai, 2015) have been determined, which were 0.5–1 mg/kg,
Choline chloride 5
1.9 mg/kg, 0.2 mg/kg, 3.73–6.72 mg/kg, 5.2 mg/kg, 10.6 mg/kg, and Ca(H2PO4)2⋅H2O 10
10.42 mg/kg per diet, respectively. No additional vitamin K was Taurine 3
required in channel catfish (Ietalurus Punetaus) (Murai & Andrews, Zeolite powder 52
1977), yellowtail (Seriola quinqueradiata) (Shimeno, 1991), or Atlantic Total 1000
Nutrient levels
salmon (Salmo salar L.) (Krossøy et al., 2009) diets. The vitamin K re­ Crude protein (%) 47.14
quirements of fish vary under respective experimental conditions and Crude lipid (%) 12.16
differences may depend on the type of fish, life stage, source of vitamin Gross energy3 (MJ/kg) 18.66
K, and evaluation indicators. Vitamin K3 (mg/kg) 0.78
Largemouth bass (Micropterus salmoides) is a freshwater carnivorous 1, 2
Vitamin premix (without vitamin K) and mineral premix were ob­
species of fish. In recent years, it has become widely popular in China tained from Hangzhou Haihuang Feed Development Co. Ltd.
because of its fast growth, low temperature affinity, strong disease 3
Gross energy was calculated with the following formula.
resistance, and delicious taste. The total aquaculture production of Gross energy (MJ/kg) = [protein (g) × 23640 J + lipid (g) × 39539 J
largemouth bass in China reached 478 thousand tons in 2019 (Yearbook, + NFE (g) × 17154 J] × 10-6.
2020, p. 25). Numerous studies have investigated the nutritional re­
quirements of largemouth bass (Anderson, Kienholz, & Flickinger, 1981; g, purchased from Huzhou, Zhejiang. The feeding experiment was con­
Bright, Coyle, & Tidwell, 2005; Chen et al., 2010, 2015; Dairiki, Dias, & ducted after a two-week acclimation period at the Shanghai Ocean
Cyrino, 2007; Li, Lian, Chen, Wang, & Sang, 2018; Lian, Chen, Wang, University Binhai Breeding Base, located in Huinan Town, Pudong New
Yan, & Ding, 2017; Zhou, Chen, Qiu, Zhao, & Jin, 2012; Zhu et al., District, Shanghai, China. Six-hundred healthy largemouth bass were
2012), but none of these studies have provided information about the randomly distributed within 20 indoor net cages (5 groups x 4 repli­
vitamin K requirements of this species. This study aims to fill this cates/group) at a density of 30 individuals per cage. The fish were fed to
knowledge gap by investigating the effects of dietary vitamin K3 levels apparent satiation twice per day (08:00 and 16:00) for 8 weeks. The
on growth, blood clotting, and antioxidant capacity, and evaluating the feeding of largemouth bass was observed every day. In case of large­
dietary vitamin K3 demands of largemouth bass. mouth bass mortality during the experiment, the fish were removed,
counted, and weighed. During the experimental period, the cages were
2. Materials and methods supplied with tap water and continuous aeration. Feces were siphoned
and water was changed irregularly. The concentration of dissolved ox­
2.1. Experimental diets ygen was more than 5 mg/L and ammonium was less than 0.5 mg/L.
Water temperature varied from 28 to 31 ◦ C and pH was maintained at
Five types of fish diets were prepared, among which fish meal, soy­ 7.5–8.5.
bean meal, corn gluten meal, and blood meal were the main protein
sources, while fish oil and rapeseed oil were the main lipid sources. In 2.3. Sample collection
order to create different levels of vitamin K3, graded levels of menadione
nicotinamide bisulphite (MNB, purity: 96.33 %, Hangzhou Haihuang At the end of the trial, the largemouth bass in each cage were counted
Feed Development Co. Ltd, Zhejiang province, China) were added and and weighed after a 24 h starvation period. Eleven fish were randomly
the amount of zeolite powder was reduced (Table 1). The levels of taken from each cage and anaesthetized with eugenol solution (100
vitamin K3 in the experimental diets were: 0.78 mg (K0), 5.80 mg (K5), ppm). From these specimens (four fish per cage), blood was taken from
10.82 mg (K10), 15.84 mg (K15), and 20.85 mg (K20) kg-1 feed. The the caudal vasculature using 1 ml heparinized syringes and stored in
level of vitamin K1 and K3 in the control group (K0) was 53.2 μg/kg and anticoagulant tubes for hematological parameter analyses. The weights
0.78 mg/kg, respectively, and was measured by HPLC according to the and lengths of the other seven fish per cage were measured individually,
standard method GB 5009.158–2016 and GB/T 18872–2017. Vitamin before additional blood samples were collected without anticoagulation
K2 was not detected by RP-HPLC (Mu et al., 2009) in K0 group. These and stored in ordinary centrifuge tubes, before they were centrifuged at
vitamin K contents were determined by Henan Hai Ruizheng Testing 4000 rpm for 10 min at 4 ◦ C to obtain the serum. Visceral mass and liver
Technology Co. Ltd, Henan province, China. samples were weighed for visceral somatic index and hepatic indices,
All raw materials were weighed and passed through 60-μm mesh muscle samples above the lateral line were collected for approximate
according to the formula specifications and then mixed thoroughly. This component analysis, and liver samples were obtained to analyze
was then thoroughly mixed with fish oil, rapeseed oil, and squid paste, biochemical parameters and content of MK-4. After the dissection of
before water was added and then pelleted with a pellet-making machine, muscle, vertebras were collected for the content of ash, calcium (Ca),
dried for about 12 h in a ventilated oven at 40 ◦ C. Afterwards, the diets and phosphorus (P) analysis. All samples were stored at − 20 ◦ C until
were stored at a cool, dry, and well-ventilated location. analysis. Following the collection, the remaining fish in each net cage
were continually fed experimental diets for 3 days. Then, three fish per
2.2. Experimental fish and feeding trial replicate were used to determine blood coagulation time.

The initial body mass of selected largemouth bass was 12.96 ± 0.07

2
X. Wei et al. Aquaculture and Fisheries xxx (xxxx) xxx

2.4. Sample analysis 3. Results

After freeze-drying, muscle tissues were ground into powder and 3.1. The effect of dietary vitamin K3 on growth
stored in the refrigerator at − 20 ◦ C. Crude protein was determined using
a Kjeldahl apparatus (GB/T6432-94). Moisture content was determined WGR and SGR were highest in the K15 group, which were markedly
by oven to constant weight at 105 ◦ C, and crude ash content was higher than the K0 group (P < 0.05), but had no significant difference
measured according to GB/T64438-92. Crude fat was determined by the with other groups (P > 0.05) (Table 2). VSI in the five groups displayed a
chloroform-methanol method (Folch, Lees, & Sloane-Stanley, 1957). tendency of increasing first and then decreasing, with the K10 group
MK-4 content was measured using an enzyme-linked immunoassay by exhibiting the highest VSI of all groups (Table 3). No significant differ­
Qingdao Sci-tech Innovation Inspection Co. Ltd, Shandong province, ence was observed in SR, FCR, CF, HI, and FI among all groups (P >
China. 0.05).
Liver samples were weighed and homogenized in ice-cold 0.85 %
saline solution and then centrifuged (4000 rpm, 10 min, 4 ◦ C). Super­
3.2. The effect of dietary vitamin K3 on composition of largemouth bass
natants were used to analyze biochemical parameters in 12 h, the same
for serum. Activity of total superoxide dismutase (T-SOD), total anti­
Compared with the K0 and K5 groups, the K10, K15, and K20 groups
oxidant capacity (T-AOC), content of malondialdehyde (MDA), activity
had significantly higher Ca content in muscle tissue (P < 0.05) (Table 4).
of aspartate aminotransferase (AST), and alanine aminotransferase
Dietary vitamin K3 had no significant effect on the content of crude
(ALT) were measured using kits provided by Nanjing Jiancheng Bioen­
protein, crude lipid, moisture, ash, and P in muscle tissue (P > 0.05). The
gineering Institute.
Ca content in the vertebrae of the K0 group was significantly lower other
Blood coagulation time was measured with slides. Time was recor­
groups (P < 0.05) and no significant difference was observed in ash and
ded using stopwatch immediately when blood was taken from the caudal
P among the groups (P > 0.05). The content of MK-4 increased at first
vasculature, then dropped naturally on the glass slide. The blood was
and then tended to decrease in liver, showing the highest level in the
picked up with a clean pin through the bottom of the blood drop.
K10 group, though the K10, K15, and K20 groups were all significantly
Stopwatch was stopped when fibrin filament was observed (Shen, 2000).
higher than the K0 group (P < 0.05).
Red blood cell, hematocrit, and hemoglobin densities were measured
using automatic blood cell analyzer within 48 h after the sampling
(RT-7300, Rayto Instrument Company, China). Red blood cells were 3.3. The effect of dietary vitamin K3 on biochemical parameters in liver
counted using electric impedance method. Hemoglobin was detected and serum of largemouth bass
using the colorimetric method.
The content of calcium in muscle and vertebras were determined The hepatopancreas and serum biochemical parameters are pre­
based on potassium permanganate titration (GB/T6436-2002). The sented in Table 5. In the hepatopancreas, the activity of T-SOD showed a
content of phosphorus in muscle and vertebras were determined based general increasing trend with vitamin K3 content, and the activity of the
on vanadium molybdate yellow colorimetric method (GB/T6437-2002). K0 group was significantly lower than other groups (P < 0.05). The
value of T-AOC in the K15 group was significantly higher than the K0,
2.5. Calculation and statistical analysis K5, and K10 groups (P < 0.05). MDA displayed a tendency of decreasing
first and then increasing and the values of the K0 and K20 groups were
The following equations were used to determine the core indices of significantly higher than the other three groups. No significant differ­
this study: ence was observed in the activity of AST and ALT among groups (P >
0.05).
Weight gain rate (WGR, %) = (final body weight − initial body weight) / initial In serum, the T-SOD activity and T-AOC of the K10 group were
body weight × 100; significantly higher than the K0 group. The trend of MDA content was
Special growth rate (SGR, %/day) = [ln (final body weight) − ln (initial body similar to that in the hepatopancreas, with the lowest found in the K10
weight)] / time (days) × 100; groups (P < 0.05). The activity of ALT in the K20 groups was signifi­
cantly higher than that of other groups (P < 0.05). Although no signif­
Survival rate (SR, %) = (final fish number / initial fish number) × 100; icant difference was observed in the activity of AST among groups (P >
Feed coefficient rate (FCR) = dry diet intake / (final body weight − initial body
weight); Table 2
Effects of dietary vitamin K3 on growth performance of largemouth bass (means
Condition factor (CF, g/cm3) = (weight / body length3) × 100; ± SD, n = 4).
Group K0 K5 K10 K15 K20
Visceral somatic index (VSI, %) = visceral weight / body mass × 100;
Initial 12.91 ± 12.94 ± 13.00 ± 12.96 ± 12.97 ±
Hepatic index (HI, %) = liver weight / body mass × 100; weight 0.07 0.05 0.08 0.04 0.13
(g)
Feed intake (FI, g/fish/day) = dry feed intake / [(final body weight + initial Final body 43.37 ± 46.22 ± 44.38 ± 47.46 ± 44.01 ±
body weight) / 2] / days. weight 3.00a 2.03ab 1.38ab 0.82b 1.61ab
(g)
All figures were created using GraphPad Prism version 8.0. The WGR (%) 235.89 ± 257.11 ± 241.38 ± 266.12 ± 239.51 ±
22.61a 14.39ab 11.04ab 6.43b 14.63ab
optimal dietary vitamin K3 requirements of juvenile largemouth bass
SR (%) 95.00 ± 95.71 ± 98.57 ± 95.71 ± 95.71 ±
were calculated by broken line regression analysis (Robbins, Saxton, & 8.21 2.86 1.65 5.47 2.86
Southern, 2006). SGR (%/d) 2.20 ± 2.31 ± 2.23 ± 2.36 ± 2.22 ±
All data were analyzed via one-way analysis of variance (ANOVA) in 0.12a 0.07ab 0.06ab 0.03b 0.08ab
SPSS version 17.0 and presented as means ± SD. Differences in mean FCR 0.83 ± 0.84 ± 0.85 ± 0.83 ± 0.86 ±
0.03 0.02 0.03 0.03 0.02
values were analyzed via Duncan’s multiple range test when ANOVA
FI (g/fish/ 0.58 ± 0.61 ± 0.59 ± 0.60 ± 0.60 ±
identified differences among groups. The level of significance was set to d) 0.01 0.02 0.03 0.02 0.03
P < 0.05.
In the same line, different superscript letters indicate significant differences (P <
0.05); values with no letter mean no significant difference (P > 0.05).

3
X. Wei et al. Aquaculture and Fisheries xxx (xxxx) xxx

Table 3 Table 5
Effects of dietary vitamin K3 on the morphologic indices of largemouth bass Effects of dietary vitamin K3 on biochemical parameters of largemouth bass
(means ± SD, n = 4). (means ± SD, n = 4).
Group K0 K5 K10 K15 K20 Group K0 K5 K10 K15 K20

CF (g/ 7.09 ± 6.98 ± 6.96 ± 6.85 ± 0.27 6.99 ± Liver

cm3) 0.36 0.26 0.48 0.34 T-SOD (U/ 133.21 ± 148.62 ± 169.00 ± 162.36 ± 179.59 ±
VSI (%) 2.37 ± 2.49 ± 2.52 ± 2.41 ± 2.34 ± mgprot) 6.91a 4.78b 1.07cd 5.97bc 8.57d
0.23ab 0.19bc 0.21c 0.14abc 0.23a T-AOC (U/ 1.17 ± 1.18 ± 1.18 ± 1.40 ± 1.21 ±
HI (%) 2.07 ± 2.02 ± 2.18 ± 2.16 ± 0.26 2.10 ± mgprot) 0.01a 0.08a 0.09a 0.11b 0.01ab
0.20 0.17 0.21 0.20 MDA 3.05 ± 1.76 ± 1.58 ± 2.16 ± 9.06 ±
(nmol/ 0.15b 0.15a 0.13a 0.13a 0.67c
In the same line, different superscript letters indicate significant differences (P < mgprot)
0.05); values with no letter mean no significant difference (P > 0.05). AST (U/ 7.54 ± 7.49 ± 7.24 ± 7.72 ± 7.81 ±
gprot) 0.37 0.43 0.18 0.42 0.60
ALT (U/ 5.37 ± 5.56 ± 5.58 ± 5.52 ± 5.41 ±
Table 4 gprot) 0.09 0.44 0.18 0.40 0.21
Effects of dietary vitamin K3 on approximate composition of largemouth bass Serum
(means ± SD, n = 4). T-SOD (U/ 72.61 ± 77.74 ± 82.58 ± 78.36 ± 78.71 ±
ml) 4.42a 1.12ab 1.17b 3.21ab 2.76ab
Group K0 K5 K10 K15 K20 T-AOC (U/ 17.98 ± 18.09 ± 20.94 ± 18.32 ± 18.69 ±
ml) 0.04a 1.45a 0.31b 1.66ab 0.78ab
Muscle
MDA 53.76 ± 52.74 ± 29.21 ± 45.81 ± 67.46 ±
Crude 191.6 ± 189.5 ± 193.3 ± 191.3 ± 192.4 ±
(nmol/ 0.70c 2.17c 1.12a 1.90b 4.30d
protein (g/ 1.9 1.6 0.9 3.7 5.1
ml)
kg)
AST (U/L) 32.78 ± 32.79 ± 31.98 ± 32.77 ± 34.83 ±
Crude lipid 11.2 ± 0.3 11.3 ± 1.0 11.4 ± 0.6 11.1 ± 0.6 11.2 ± 1.0
0.63 1.81 1.85 1.04 1.80
(g/kg)
ALT (U/L) 7.00 ± 7.09 ± 6.93 ± 7.16 ± 8.08 ±
Moisture(g/ 779.4 ± 780.7 ± 775.8 ± 778.2 ± 775.2 ±
0.22a 0.10a 0.09a 0.22a 0.21b
kg) 2.3 0.2 3.1 4.3 2.2
Ash (g/kg) 12.2 ± 1.0 11.8 ± 0.3 12.5 ± 1.0 11.6 ± 0.9 12.9 ± 0.9 In the same line, different superscript letters indicate significant differences (P <
Ca (g/kg) 7.95 ± 7.81 ± 8.47 ± 8.58 ± 8.62 ± 0.05); values with no letter mean no significant difference (P > 0.05).
0.04a 0.07a 0.13b 0.13b 0.11b
P (g/kg) 13.01 ± 13.53 ± 12.19 ± 12.87 ± 12.92 ±
1.12 1.19 0.72 0.84 0.81
Vertebrae Table 6
Ash (g/kg) 357.7 ± 370.4 ± 371.3 ± 368.3 ± 367.6 ± Effects of dietary vitamin K3 on blood coagulation time and hematological pa­
4.0 8.4 17.2 7.2 6.7 rameters of largemouth bass (means ± SD, n = 4).
Ca (g/kg) 117.89 ± 127.12 ± 133.04 ± 131.16 ± 133.18 ±
Group K0 K5 K10 K15 K20
3.52a 5.57b 6.04b 4.02b 5.38b
P (g/kg) 28.30 ± 28.03 ± 28.82 ± 28.29 ± 29.74 ± Blood coagulation 102.25 ± 66.25 ± 54.17 ± 53.50 ± 55.67 ±
0.77 1.88 1.51 2.09 2.85 time (s) 7.27c 3.84b 9.40a 3.65a 3.88a
Liver Red blood cell 1.51 ± 1.98 ± 1.90 ± 2.08 ± 2.01 ±
MK-4 (ng/g) 53.72 ± 56.27 ± 76.47 ± 62.97 ± 57.62 ± (1012 cells/L) 0.04a 0.06b 0.16b 0.05b 0.08b
2.99a 1.85ab 2.08d 0.99c 1.66b Hemoglobin (g/L) 93.00 ± 93.67 ± 94.25 ± 96.75 ± 97.00 ±
5.00 3.21 4.72 3.18 5.66
In the same line, different superscript letters indicate significant differences (P < Hematocrit (%) 31.66 ± 36.87 ± 34.40 ± 36.33 ± 33.27 ±
0.05); values with no letter mean no significant difference (P > 0.05). 0.79 0.64 2.86 0.18 2.63

0.05), the value in the K20 group was slightly higher than other groups.
3.4. The effect of dietary vitamin K3 on blood coagulation and
hematological parameters of largemouth bass
In the same line, different superscript letters indicate significant differences (P <
0.05); values with no letter mean no significant difference (P > 0.05). Blood
coagulation time: n = 12.

The blood coagulation time shortened significantly with the increase

of dietary vitamin K3 from 0.78 to 10.82 mg/kg (P < 0.05) and then
tended to be stable at higher levels (P > 0.05). The erythrocytic number
of K5, K10, K15 and K20 groups was significantly higher than the K0
group (P < 0.05). However, dietary vitamin K3 content had no signifi­
cant influence on hemoglobin content and hematocrit (P > 0.05)
(Table 6).

3.5. Regression analysis

The optimal dietary vitamin K3 requirement was 11.79 mg/kg and

11.20 mg/kg according to the regression analysis of liver MDA content,
liver MK-4 content with dietary vitamin K3 levels (Figs. 1 and 5). The
dietary vitamin K3 requirement should be not more than 15.22 mg/kg
Fig. 1. Relationship between dietary vitamin K3 levels and liver MDA content is
according to serum ALT (Fig. 2), not less than 9.93 mg/kg according to
fitted by broken line regression analysis. The two straight lines sharing the
blood coagulation time (Fig. 3), not less than 5.93 mg/kg according to
third point.
red blood cell count (Fig. 4).

4
X. Wei et al.
Fig. 2. Relationship between dietary vitamin K3 levels and serum ALT content
is fitted by broken line regression analysis. The two straight lines sharing the
fourth point.
Fig. 3. Relationship between dietary vitamin K3 levels and blood coagulation
time is fitted by broken line regression analysis. The two straight lines sharing
the third point.
Fig. 4. Relationship between dietary vitamin K3 levels and erythrocytic num­
ber is fitted by broken line regression analysis. The two straight lines sharing
the second point.
4. Discussion
Vitamin K was required within the diet of reared Jian carp (Cyprinus
carpio var. Jian) (Yuan et al., 2016), lake trout (Poston, 1976), amago
salmon (Oncorhynchus rhodurus) (Taveekijakarn et al., 1996), Chinese
shrimp (Penaeus chinensis) (Shiau & Liu, 1994) to increase WGR,
otherwise the growth would be impeded. Previous studies found that
vitamin K deficiency could lead to lower HI of rainbow trout (Onco­
rhynchus mykiss)(Kitamura, Suwa, & Ohara, 1967), lower feed efficiency
in hybrid tilapia (Li, 2003) and gibel carp (Duan et al., 2013).
5
Aquaculture and Fisheries xxx (xxxx) xxx
Fig. 5. Relationship between dietary vitamin K3 levels and liver MK-4 content
is fitted by broken line regression analysis. The two straight lines sharing the
third point.
Meanwhile, excess vitamin K3 had negative influence on growth (Shiau
& Liu, 1994). When the concentrations of dietary MSB were 30 mg/kg
and 20 mg/kg, respectively, the growth of Atlantic salmon and cod were
impeded (Grisdale-Helland, Helland, & Åsgård, 1991; Grahl-Madsen &
Lie, 1997). A negative effect on growth performance was also found due
to the lack of vitamin K3 in diets in our present study, and according to
WGR and SGR metrics, the optimal dietary vitamin K3 was 15.84 mg/kg.
That the WGR and SGR decreased when vitamin K3 content was 20.85
mg/kg in the K20 group may be due to the toxicity of artificial mena­
dione, despite the lower toxicity of MNB than MSB (Marchetti, Tassinari,
& Bauce, 1995; Rebhun, Tennant, Dill, & King, 1984; Smith, Ivy, &
Froster, 1943). Although it is difficult for us to understand why VSI
changed significantly among groups, while no significant effects of SR,
FCR, CF, HI, or FI were observed in this experiment, it is necessary to add
optimal vitamin K3 in largemouth bass diet according to WGR and SGR.
Osteocalcin, a vitamin K-dependent protein, is rich within the dentin
and mineralized tissue of animal bones. The special calcium bond
structure in osteocalcin combines with calcium ions and is deposited in
bone (Binkley & Suttie, 1995), so the content of calcium in vertebrae
was a valued indicator of vertebrae quality. Previous studies indicated
that both vitamin K deficiency and the toxicity of the excessive MSB
could impair bone mineralization and increase the occurrence of bone
deformities (Rebhun et al., 1984; Smith et al., 1943; Roy & Lall, 2007).
We saw contents of calcium in muscle and vertebrae increase with
increasing dietary vitamin K3 before reaching a plateau, suggesting that
the addition of vitamin K3 contributes to calcium accumulation in the
body. In animal bodies, vitamin K3 must be converted to MK-4 within
tissues to become biologically active and stored mainly in the liver
(Udagawa, 2001), and further promoted osteoblast differentiation
through pregnane X receptor-mediated transcriptional control of the
Msx2 gene (Igarashi et al., 2007). We found that dietary vitamin K3 was
beneficial to the MK-4 accumulation in the liver, however, the content of
MK-4 existed a decreasing trend in K15 and K20 groups. This phenom­
enon may relate to the hepatocyte health of the K20 group. As two
important transaminases in the cytoplasm of liver cells, AST and ALT are
released from the liver cells into blood resulting from increasing mem­
brane permeability when liver cells become diseased and damaged, with
the liver generally regarded as more sensitive to changes in the ALT level
(Guo et al., 2014). Therefore, the low content of MK-4 in the K20 group
may have resulted from the damaged liver cells.
Antioxidant capacity is another indicator to evaluate the health of
fish. Vitamin K has antioxidant properties, but too much or too little
dietary intake could inhibit the antioxidant reaction (Canfield, Davy, &
Thomas, 1985). Total superoxide dismutase (T-SOD) is an important
reductase, existing in a wide range of organisms. T-SOD has the function
to transform harmful superoxide anion radicals into hydrogen peroxide
to protect body health (Cheng, Zhang, Dong, Lu, & Wang, 2017). MDA is
X. Wei et al.
a metabolite of free radical and lipid peroxidation and widely used as an
indicator of tissue damage caused by free oxygen radicals (Sahin et al.,
2011). T-AOC is a main indicator for assessing the level of the antioxi­
dant enzyme system and non-enzyme system in body and the size can
reflect the ability of both the antioxidant systems to compensate for
external stimuli and the state of free radical metabolism in the body
(Burton & Traber, 1990; Mukai, Morimoto, Okauchi, & Nagaoka, 1993).
Our study showed that appropriate amount of dietary vitamin K3 could
effectively enhance the activity of various antioxidant enzymes and the
antioxidant function of the liver, similar to results of Jian carp (Yuan
et al., 2016). However, in the present study, the activity of T-SOD and
the value of T-AOC were at a high level, coupled with the highest MDA
content in liver and serum in the K20 group. Vitamin K3 has been shown
to react with hemoglobin to yield methemoglobin (Winterbourn,
French, & Claridge, 1979), which was extremely prone to oxidation
accompanied by the generation of superoxide anion radicals or super­
oxide radicals (Grunwald & Richards, 2015; Mi, Guo, Li, & Li, 2016). A
likely scenario was that excessive MNB oxidized with hemoglobin
accompanied the generation of superoxide anion radical or superoxide
radical in the K20 group. On the other hand, the potential toxicity of
excessive supplemental MNB might also suppress the efficiency of
antioxidant system removing reactive oxygen free radicals, which led to
produce more end product of lipid oxidation. The indexes of AST and
ALT in liver of fish were often used to evaluate the state of protein
synthesis and catabolism (Yan, Xie, Zhu, Lei, & Yang, 2007). In this
study, no significant differences in AST and ALT activity in liver was
observed among groups, which was consistent with the trend of crude
protein content in muscle tissue. Therefore, the dietary vitamin K3 had
no effect on protein retention, but the high-dose dietary vitamin K3 may
cause potential damage to fish health.
Vitamin K participates in synthesis of several proteins that mediate
both coagulation and anticoagulation (Ferland & Ph.D., 1998). Our re­
sults show that blood clotting time shortened as a whole with the in­
crease of vitamin K3 in the diet. This indicated that vitamin K3 could
promote blood clotting for largemouth bass and may help fish recover
faster in the case of hemorrhages. The vitamin K-free diet was also found
to prolong the blood clotting time of brook trout (Salvelinus fontinalis)
(Poston, 1964) and large yellow croaker (Cheng et al., 2015). Dietary
vitamin K deficiency could also prolong blood coagulation time and
tissue bleeding in amago salmon (Taveekijakarn et al., 1996) and even
obvious hemorrhages in tissue with 23-week and 12-week feeding trial
on vitamin K demand of cod (Grahl-Madsen & Lie, 1997) and Chinese
shrimp (Shiau & Liu, 1994). To discover vitamin K deficiency in Atlantic
salmon (Salmo salar), the study was conducted over a 16-week period
(Halver, 1989). However, no similar hemorrhagic symptom in tissues of
largemouth bass was observed in this rearing process, possibly due to the
shorter feeding period. In this study, the experimental period was 8
weeks, so it may take a longer time to observe this deficiency.
Red blood cells have functions such as gas transportation and im­
mune defense in the body. That the amount of red blood cell and the
content of hemoglobin in the K0 group were the lowest among all
treatment groups, meant the vitamin K3 additive played an important
role in enhancing oxygen transportation, similar to gibel carp (Duan
et al., 2013), amago salmon (Taveekijakarn et al., 1996), and lake trout
(Poston, 1976).
The optimal range of dietary vitamin K3 was 9.93–15.22 mg/kg
based on the growth and broken-line regression of liver MDA content,
serum ALT content, erythrocytic number, blood clotting time, and liver
MK-4 content, which is higher than that of lake trout (0.5–1 mg/kg diet)
(Poston, 1976), grass carp (1.9 mg/kg diet) (Jiang et al., 2007), cod (0.2
mg/kg diet) (Grahl-Madsen & Lie, 1997), gibel carp (3.73–6.72 mg/kg
diet) (Duan et al., 2013), hybrid tilapia (5.2 mg/kg diet) (Li, 2003), and
Jian carp (3.13 mg/kg diet) (Yuan et al., 2016), but lower than grouper
songpu mirror carp (16.89 mg/kg diet) (Wang, Xu, Xu, Wang, & Shi,
2011). We hypothesize that factors such as fish species, life stage,
breeding condition, and the sources of vitamin K in diet resulted in the
6
Aquaculture and Fisheries xxx (xxxx) xxx
variation of vitamin K requirements. For this reason, the development of
compound feeds for specific species of aquatic animals should be based
on precise nutritional requirements to contribute to the healthy and
sustainable development of aquaculture.
5. Conclusion
In summary, adding proper amount of vitamin K3 could promote the
growth, blood coagulation, calcium content, and antioxidant capacity.
Based on the growth and broken-line regression analyses, the optimal
range of dietary vitamin K3 was estimated to be 9.93–15.22 mg/kg diet.
Further studies should be carried out to clear about the damage of liver
and the deformity inside skeleton, and reveal the mechanisms under­
lying the effects of dietary vitamin K3 on antioxidant status in the
hepatopancreas and serum.
Credit authorship contribution statement
Xiang Wei: conceived the study and designed the experiments,
contributed to the feed production, contributed to feeding, contributed
to the experiment, did the data Formal analysis and wrote the paper.
Ying Hang: contributed to feeding. Xiang Li: contributed to the feed
production. Xueming Hua: conceived the study and designed the ex­
periments, revised the manuscript. Xiangming Cong: contributed to
feeding. Wanting Yi: contributed to feeding. Xu Guo: contributed to the
feed production.
Acknowledgements
This study was supported by Guangdong province key field R&D
program project (No. 2020B0202010001) and Shanghai collaborative
innovation for aquatic animal genetics and breeding.
References
Anderson, R. J., Kienholz, E. W., & Flickinger, S. A. (1981). Protein requirements of
smallmouth bass and largemouth bass. Journal of Nutrition, 111(6), 1085–1097.
Binkley, N. C., & Suttie, J. W. (1995). Vitamin K nutrition and osteoporosis. Journal of
Nutrition, 125(7), 1812–1821.
Booth, S. L. (2009). Roles for vitamin K beyond coagulation. Annual Review of Nutrition,
29, 89–110.
Bright, L. A., Coyle, S. D., & Tidwell, J. H. (2005). Effect of dietary lipid level and protein
energy ratio on growth and body composition of largemouth bass Micropterus
salmoides. Journal of the World Aquaculture Society, 36(1), 129–134.
Bügel, S. (2003). Vitamin K and bone health. Proceedings of the Nutrition Society, 62(4),
839–843.
Burton, G. W., & Traber, M. G. (1990). Vitamin E:antioxi-dant activity biokinetics and
bioavailability. Annual Review of Nutrition, 10, 357–382.
Canfield, L. M., Davy, L. A., & Thomas, G. L. (1985). Anti-oxidant/pro-oxidant reactions
of vitamin K. Biochemical and Biophysical Research Communications, 128(1), 211–219.
Cheng, Y. B., Zhang, Y. X., Dong, Z. Y., Lu, B. Y., & Wang, Y. C. (2017). Effects of dietary
replacement of fish meal and soy protein by wheat gluten on plasma biochemical
indices and liver antioxidative indices of Nibea japonica and Sparus macrocephalus.
Progress in Fishery Sciences of China, 38(3), 106–114.
Cheng, L., Zhang, W. B., Lin, S. Q., Xu, W., & Mai, K. S. (2015). Effects of dietary vitamin
K on growth performances, blood coagulation time and menaquinone-4 (MK-4)
concentration in tissues of juvenile large yellow croaker Pseudosciaena crocea.
Aquaculture Research, 46(5), 1269–1275.
Chen, N. S., Ma, J. Z., Zhou, H. Y., Zhou, J., Qiu, X. J., Jin, L.i. N., et al. (2010).
Assessment of dietary methionine requirement in largemouth bass, Micropterus
salmoides. Journal of Fisheries of China, 34(8), 1244–1253.
Chen, Y. J., Yuan, R. M., Liu, Y. J., Yang, H. J., Liang, G. Y., & Tian, L. X. (2015). Dietary
vitamin C requirement and its effects on tissue antioxidant capacity of juvenile
largemouth bass, Micropterus salmoides. Aquaculture, 435, 431–436.
Dairiki, J. K., Dias, C. T.d. S., & Cyrino, J. E. P. (2007). Lysine requirements of
largemouth bass, Micropterus salmoides: A comparison of methods of analysis of dose-
response trials data. Journal of Applied Aquaculture, 19(4), 1–27.
Duan, Y. H., Zhu, X. M., Han, D., Yang, Y. X., Jin, J. Y., & Xie, S. Q. (2013). Dietary
vitamin K requirement of juvenile gibel carp (Carassius Auratus Gibelio). Acta
Hydrobiologica Sinica of China, 37(1), 8–15.
Ferland, G. (1998). The vitamin K-dependent proteins: An update. Nutrition Reviews, 56
(8), 223–230.
Folch, J., Lees, M., & Sloane-Stanley, G. H. (1957). A simple method for the isolation and
purification of total lipids from animal tissues. Journal of Biological Chemistry, 226
(1), 497–509.
X. Wei et al.
Grahl-Madsen, E., & Lie, Ø. (1997). Effects of different levels of vitamin K in diets for cod
(Gadus morhua). Aquaculture, 151(1), 269–274.
Grisdale-Helland, B., Helland, S. J., & Åsgård, T. (1991). Problems associated with the
present use of menadione sodium bisulfite and vitamin A in diets for Atlantic salmon.
Aquaculture, 92, 351–358.
Grunwald, E. W., & Richards, M. P. (2015). Studies with the heme binding protein
apoShp suggest hemoglobin is the primary promoter of lipid oxidation in pork
muscle mince. Meat Science, 101, 155–156.
Guo, X.-Z., Liang, X.-F., Fang, L., Yuan, X.-C., Zhou, Y., & Li, B. (2014). Effects of non-
protein energy sources on serum biochemical indices and histology of liver in grass
carp (Ctenopharyngodon idella). Acta Hydrobiologica Sinica of China, 38(3), 582–587.
Halver, J. E. (1989). The vitamins. Fish Nutrition. New York: Academic Press.
Igarashi, M., Yogiashi, Y., Mihara, M., Takada, I., Kitagawa, H., & Kato, S. (2007).
Vitamin K induces osteoblast differentiation through pregnane X receptor-mediated
transcriptional control of the Msx2 gene. Molecular and Cellular Biology, 27(22),
7947–7954.
Jiang, M., Wang, W. M., Wen, W., Wu, F., Zhao, Z. Y., Liu, A. L., et al. (2007). Effects of
dietary vitamin K3 on growth, carcass composition and blood coagulation tmie for
grass carp fingerling (Ctenopharyngodon idellus). Freshwater Fisheries of China, 37(2),
61–64.
Kitamura, S., Suwa, T., & Ohara, S. (1967). Studies on vitamin requirements of rainbow
troutII. The defficiency symptoms fourteen kinds of vitamin. Bulletin of the Japanese
Society of Scientific Flsheries, 33(1), 1120–1125.
Krossøy, C., Waagbo, R., Fjelldal, P. G., Wargelius, A., Lock, E., Graff, I. E., et al. (2009).
Dietary menadione nicotinamide bisulphite (vitamin K3) does not affect growth or
bone health in first-feeding fry of Atlantic salmon (Salmo salar L.). Aquaculture
Nutrition, 15(6), 638–649.
Li, Z. Y. (2003). Vitamin K requirements of juvenile hybrid tilapia (Oreochromis niloticus × O.
aureus) and grouper (Epinephelus malabaricus). Thesis for Master of Science. Taiwan,
China: Taiwan Ocean University.
Lian, X. Y., Chen, N. S., Wang, M. L., Yan, C. W., & Ding, G. T. (2017). Dietary vitamin A
requirement of largemouth bass (Micropterus salmoides). Chinese Journal of Animal
Nutrition, 29(10), 3819–3830.
Li, S. L., Lian, X. Y., Chen, N. S., Wang, M. L., & Sang, C. Y. (2018). Effects of dietary
vitamin E level on growth performance, feed utilization, antioxidant capacity and
nonspecific immunity of largemouth bass, Micropterus salmoides. Aquaculture
Nutrition, 24(6), 1–10.
Marchetti, M., Tassinari, M., & Bauce, G. (1995). Tolerance of high dietary levels of
menadione bisulphite-nicotinamide by rainbow trout, Oncorhynchus mykiss.
Aquaculture, 134, 137–142.
Mi, H. B., Guo, X., Li, X. P., & Li, J. R. (2016). Advance in fish hemoglobin-induced lipid
oxidation and the corresponding control approach. Modern Food Science and
Technology, 32(4), 297–304.
Mu, X. Y., Dong, Y. W., Wen, X. J., Fu, Z. Q., Lei, Z., Fang, R. B., et al. (2009). The content
of vitamin K2(20) in vitamin K2 soft capsule was determined by RP-HPLC. Chinese
Journal of Analysis Laboratory, 28(Suppl), 33–35.
Mukai, K., Morimoto, H., Okauchi, Y., & Nagaoka, S.-i. (1993). Kinetic study of reactions
between tocopheroxyl radicals and fatty acids. Lipids, 28(8), 753–756.
Murai, T., & Andrews, J. W. (1977). Vitamin K and anticoagulant relationships in catfish
diets. Bulletin of the Japanese Society of Scientific Flsheries, 43(1), 785–794.
Poston, H. A. (1964). Effect of vitamin K and sulfaguanidine on blood coagulation time,
microhematocrit, and growth of immature brook trout. The Progressive Fish-Culturist,
26(2), 59–64.
Poston, H. A. (1976). Relative effect of two dietary water-soluble analoguesos
menaquinone on coagulation and packed cell volume of blood of lake trout
(Salvelinus namaycush). Journal of the Fisheries Research Board of Canada, 33(8),
1791–1793.
Aquaculture and Fisheries xxx (xxxx) xxx
Rebhun, W. C., Tennant, B. C., Dill, S. G., & King, J. M. (1984). Vitamin K3 induced renal
toxicosis in the horse. Journal of the American Veterinary Medical Association, 184,
1237–1239.
Robbins, K. R., Saxton, A. M., & Southern, L. L. (2006). Estimation of nutrient
requirements using broken-line regression analysis. Journal of Animal Science, 84
(suppl), 155–165.
Roy, P. K., & Lall, S. P. (2007). Vitamin K deficiency inhibits mineralization and enhances
deformity in vertebrae of haddock (Melanogrammus aeglefinus L.). Comparative
Biochemistry & Physiology, 148(2), 174–183.
Sahin, M. A., Yucel, O., Guler, A., Doganci, S., Jahollari, A., Cingoz, F., et al. (2011). Is
there any cardioprotective role of Taurine during cold ischemic period following
global myocardial ischemia? Journal of Cardiothoracic Surgery, 6(1), 31–37.
Shearer, M. J. (1995). Vitamin K. The Lancet, 345, 229–234.
Shen, M. H. (2000). Determination of clotting time in several animals. Qinghai Journal of
Animal Husbandry and Veterinary medicine of China, 30(5), 18.
Shiau, S. Y., & Liu, J. S. (1994). Estimation of the dietary vitamin K requirement of
juvenile Penaeus chinensis using menadione. Aquaculture, 126, 129–135.
Shimeno, S. (1991). Yellowtail, Seriola quinqueradiata. Handbook of Nutrient Requirements
of Finfish, 181–191.
Smith, J. J., Ivy, A. C., & Froster, R. H. K. (1943). The pharmacology of two water-soluble
vitamin K-like substances. The Journal of Laboratory and Clinical Medicine, 28,
1667–1680.
Stafford, D. W. (2005). The vitamin K cycle. Journal of Thrombosis and Haemostasis : JTH,
3(8), 1873–1878.
Taveekijakarn, P., Miyazaki, T., Matsumoto, M., & Arai, S. (1996). Studies on vitamin K
deficiency in amago salmon, Oncorhynchus rhodurus (Jordan & McGregor). Journal of
Fish Diseases, 19(3), 209–214.
Udagawa, M. (2001). The effect of dietary vitamin K (phylloquinone and menadione)
levels on the vertebral formation in mummichog Fundulus heteroclitus. Fisheries
Science, 67(1), 104–109.
Udagawa, M., Nakazoe, J., & Murai, T. (1993). Tissue distribution of phylloquinone and
menaquinone-4 in sardine, Sardinops Melanostictus. Comparative Biochemistry and
Physiology B, 106(2), 297–301.
Vermeer, C., & Braam, L. (2001). Role of K vitamins in the regulation of tissue
calcification. Journal of Bone and Mineral Metabolism, 19(4), 201–206.
Vermeer, C., Jie, K.-S. G., & Knapen, M. H. J. (1995). Role of vitamin K in bone
metabolism. Annual Review of Nutrition, 15, 1–21.
Wang, Y., Xu, Q. Y., Xu, H., Wang, C. A., & Shi, L. Y. (2011). Effects of vitamin K3 on body
composition, meat quality and antioxidant capacity of songpu mirror carp. Chinese
Journal of Animal Nutrition, 23(6), 1058–1064.
Winterbourn, C. C., French, J. K., & Claridge, R. F. C. (1979). The reaction of menadione
with haemoglobin. Biochemical Journal, 179, 665–673.
Yan, Q., Xie, S., Zhu, X., Lei, W., & Yang, Y. (2007). Dietary methionine requirement for
juvenile rockfish, Sebastes schlegeli. Aquaculture Nutrition, 13(3), 163–169.
Yearbook. (2020). China fishery statistics yearbook. Beijing, China: Bureau of Fisheries,
Ministry of Agriculture, China Agriculture Press.
Yuan, J., Feng, L., Jiang, W. D., Liu, Y., Jiang, J., Li, S. H., et al. (2016). Effects of dietary
vitamin K levels on growth performance, enzyme activities and antioxidant status in
the hepatopancreas and intestine of juvenile Jian carp (Cyprinus carpio var. Jian).
Aquaculture Nutrition, 22(2), 352–366.
Zhou, H., Chen, N., Qiu, X., Zhao, M., & Jin, L. (2012). Arginine requirement and effect of
arginine intake on immunity in largemouth bass, Micropterus salmoides. Aquaculture
Nutrition, 18(1), 107–116.
Zhu, Y., Chen, Y., Liu, Y., Yang, H., Liang, G., & Tian, L. (2012). Effect of dietary selenium
level on growth performance, body composition and hepatic glutathione peroxidase
activities of largemouth bass Micropterus salmoide. Aquaculture Research, 43(11), 1–9.