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. 2023 Jan 6;102(3):102482. doi: 10.1016/j.psj.2023.102482

Sodium dehydroacetate-induced disorder of coagulation function in broiler chickens and the protective effect afforded by vitamin K

Jinge Xu *,†,‡,1, Fuxing Hao $,#,1, Cunkai Wang *,†, Zeting Zhao *,†, Meng Zhang *,†, Xin Chen *,†,$, Yumei Zhang *,†,$,2
PMCID: PMC10014351  PMID: 36706663

Abstract

Sodium dehydroacetate (S-DHA) is used widely as a preservative in several products, including poultry feed. The anticoagulation effect of 200 mg/kg S-DHA in rats has been reported to accompany a reduction in hepatic expression of vitamin K epoxide reductase complex 1 (VKORC1). Poultry and mammals have different physiology and coagulation systems, and species differences in VKORC1 expression have been found. The effect of S-DHA on blood clotting of poultry has not been studies deeply. S-DHA was given to yellow-plumage broilers (YBs) as single and multiple administrations. Vitamin K3 (VK3) was injected into YBs 2 wk after S-DHA administration. Then, the prothrombin time (PT), partial activated prothrombin time (APTT), plasma levels of vitamin K (VK), factor IX (FIX), and S-DHA, and hepatic expression of VKORC1 were obtained. Chicken hepatocellular carcinoma (LMH) cells were also exposed to S-DHA, and the cell activity, VK level, and FIX level were measured. S-DHA prolonged the PT or APTT significantly, decreased levels of VK and FIX in blood, and inhibited hepatic expression of VKORC1. The maximum changes were 1.15-fold in the PT, 1.42-fold in the APTT, 0.8-fold in the VK level, 0.7-fold in the FIX level, and 0.35-fold in VKORC1 expression compared with controls. The cell activity, VK level, FIX level, and VKORC1/VKORC1L1 expression of LMH cells were reduced significantly at S-DHA doses of 2.0 to 10.0 mM. Prolongation of the PT/APTT and lower levels of VK/FIX in YBs or the lower cell activity and VK/FIX levels in LMH cells induced by S-DHA therapy were resisted significantly by VK3 treatment. We demonstrated that S-DHA could induce a disorder in coagulation function in YBs or in LMH cells via reduction of VKORC1/VKORC1L1 expression, and that VK could resist this anticoagulation effect.

Key words: coagulation, sodium dehydroacetate, fungicide, chicken, LMH cell

INTRODUCTION

Sodium dehydroacetate (S-DHA) and dehydroacetate acid have antimicrobial effects (Li et al., 2004; Li et al., 2008; Tang et al., 2018) and are used as antiseptic agents. They are employed widely in the production of food (Scordino et al., 2018; Iwakoshi et al., 2019; Yangbo et al., 2021), cosmetics (Foti et al., 2016; Canavez et al., 2021), medicines (Sulser et al., 1958; Park et al., 2017), and animal feed (Tang et al., 2018; Li et al., 2020). They have some adverse effects: lipid peroxidation (Sugihara et al., 1997), allergic contact dermatitis (Milpied et al., 2011; Foti et al., 2012, 2016; Valois et al., 2015), and cellular damage (Izawa et al., 2018). Importantly, S-DHA has been found to induce a coagulation disorder or severe hemorrhage in animals. For example: stomach hemorrhage was documented in a subchronic toxicity test in dogs using S-DHA at 200 mg/kg (Spencer et al., 1950); severe hemorrhage in Sprague-Dawley rats was noted with multiple administration of S-DHA at 200 mg/kg or 400 mg/kg (Sakaguchi et al., 2008); a coagulation disorder in Wistar rats was described in our previous study (Chen et al., 2019, Chen et al., 2021). The coagulation dysfunction induced by S-DHA in rats was inhibition of vitamin K epoxide reductase complex subunit 1 (VKORC1) expression in the liver, followed by decreases in levels of vitamin K (VK) and factor IX (FIX) (Sakaguchi et al., 2008; Chen et al., 2019, 2021).

VK is indispensable for animal growth. It can be synthesized by gastrointestinal microorganisms in domestic animals. VK deficiency is much more serious in poultry than that in other monogastric animals due to their short gut. Hydroquinone VK is involved in γ-carboxylation of the residues of glutamic acid of VK-dependent proteins to their active forms, its hydroquinone converted into vitamin K epoxide (VKO). Reduced vitamin K serves as a cofactor in the γ-glutamyl carboxylase (GGCX)-mediated carboxylation reaction.

Finally, VKO is transformed into VK hydroquinone through vitamin K epoxide reductase (VKOR) in VK cycles (Furie and Furie, 1988; Oldenburg et al., 2008). VKOR is a rate-limited enzyme in the VK cycle, and is essential for posttranslational modification of VK-dependent proteins. VKOR complex 1 (VKORC1) and VKORC1-like 1 (VKORC1L1) are isoenzymes of VKOR. VKORC1 is located mainly in the liver, whereas VKORC1L1 is found in extrahepatic tissues. VKORC1 appears to have higher activity than VKO to VK in vitro. VKORC1L1 supports VK-dependent protein carboxylation in vivo, but might have a different physiological function other than “recycling” VK (Oldenburg et al., 2015; Lacombe and Ferron, 2018; Lacombe et al., 2018). There are differences between species and genera with regard to the expression and activity of VKORC1 and VKORC1L1 (Wilson et al., 2003; Caspers et al., 2015; Nakayama et al., 2020). The distribution of VKORC1 and VKORC1L1 between mice and rats is considerably different. The activity of VKOR in chickens is 6 times that in rats. The inhibition constant of warfarin for VKOR in rats is 0.28 ± 0.09 mM and for chickens is 11.3 ± 2.5 mM. Warfarin metabolism is more rapid in chickens than that in rats (Nakayama et al., 2020).

Several coagulation factors, such as prothrombin (factor II), FIX, VII, X, and XII, are VK-dependent proteins. Lower vertebrates have coagulation mechanisms that are different to those of mammals: they have only FIX, and lack the other proteases in the intrinsic pathway (Doolittle, 2009; Ponczek et al., 2012). In our previous study, the abnormal effect upon coagulation induced by S-DHA in rats was inhibited mainly by VKORC1. The latter reduced γ-carboxylation of VK-dependent coagulation factors (e.g., FIX) and their active forms, then prolonged the prothrombin time (PT) and partial activated prothrombin time (APTT) (Chen et al., 2019, 2021). The physiology of poultry varies greatly from that of mammals, and their coagulation mechanisms are different. Studies on the effect of S-DHA on the coagulation function of poultry when S-DHA is used as a feed fungicide in domesticated animals have not been reported.

We undertook single or multiple administration of S-DHA to broilers to investigate the effect of coagulation function based on coagulation parameters, liver function, growth increase, and hepatic VKORC1 expression. A chicken hepatic carcinoma cell line (LMH) was also used in vitro to evaluate the effect of VK and FIX induced by S-DHA. We aimed to provide information on the effect of S-DHA on blood-clotting function and the risk of VK-deficiency in chickens.

MATERIALS AND METHODS

Single Administration of S-DHA to Chickens

Male and female yellow-plumage broilers (YBs; 1.5 ± 0.25 kg) were purchased from a farmer's market in Yangzhou City in China. YBs were housed in controlled conditions in a room at 24°C and allowed to adapt to their environment for 5 d before experimentation. S-DHA (50, 100, and 200 mg/kg) was administered to YBs once. This single administration of S-DHA was by lavage based on feed consumption. Blood samples were collected from a wing vein at 0, 4, 8, 16, 24, 72, 80, and 96 h after administration, with the blood of 6 chickens being sampled at each time point. After killing, samples of liver tissue were also harvested. The fresh-blood samples were used for measurement of the PT and APTT. Serum samples were used for determination of levels of VK and FIX using enzyme-linked immunosorbent assay (ELISA) kits. The S-DHA concentration in liver samples was measured by a high-performance liquid chromatography method described previously (Zhang et al., 2017).

Multiple Administration of S-DHA to Chickens

A total of 240 1-day-old YBs of similar birthweight (35.0 ± 2.0 g) were divided randomly into 4 groups: control and S-DHA (100, 200, and 400 mg/kg). Each treatment contained 6 replicates, with 12 YBs in each replicate. S-DHA was supplemented to the diet based on our pretreatment. YBs were fed for 14 wk, which was divided into 2 phases: first phase was 0 to 21 d; second phase was >21 d. The diets we used were the commercial broiler products 510S (for baby YBs) and 511S (for larger YBs) from Yangzhou Zhengda (Yangzhou, China) in the first phase and second phase, respectively. The room temperature was maintained at 35°C for the first week and then reduced by 2°C each week until it reached 24°C. The lighting schedule during the experimental period was 23 h of light and 1 h of darkness. Chicken feed and water were provided ad libitum throughout the study. Blood samples were collected from a wing vein each week from 2 wk to 14 wk after administration, and the blood samples of 6 YBs reported at each time point. Some blood samples were used to measure levels of aspartate transaminase (AST), alanine transaminase (ALT), total protein (TP), total bilirubin (TBIL), direct bilirubin (DBIL), and indirect bilirubin (IDBL) using a clinical chemistry analyzer (AU680; Beckman Coulter, San Jose, CA) to evaluate liver function. The “organ index” was defined as the ratio of organ weight:body weight.

Effect of S-DHA on LMH Cells In Vitro

Chicken hepatocellular carcinoma (LMH) cells were cultured in DMEM/F12 medium at 37°C in an atmosphere of 5% CO2. A cell suspension (1 × 106 cells/mL) was adjusted after digestion with 0.25% trypsin. Cells were added to a 96-well plate (2 × 104 cells per well). After cells had become adherent, they were exposed to S-DHA (0.1–10.0 mM) for 24 h. Then, cell activity was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data were derived from the mean of 5 replicate experiments.

Cells were placed in a 6-well plate (1 × 106 cells per well) followed by exposure to S-DHA (2.0–10.0 mmol/L) for 24 h. Culture supernatants and cells were obtained by centrifugation (3,000 rpm, 5 min, room temperature). Digestion was carried out with 0.25% trypsin, and VKORC1/L1 expression was measured by Western blotting. Cells (1 × 106) were resuspended in phosphate buffer (pH 7.2). After centrifugation (3,000 rpm, 5 min, room temperature), the supernatant was obtained. After repeated freezing and thawing of cells, they were used to measure levels of VK and FIX within them by ELISAs. Data were derived the mean of 3 replicate experiments.

Protection Proffered by VK Against the Coagulation Disorders Induced by S-DHA

After administration of S-DHA (200 mg/kg) to YBs for 2 wk, VK3 (2 mg; Shanghai Qunyu Animal Pharmaceuticals, Shanghai, China) was injected (i.m.) into the thigh muscle of each YB. Blood samples were collected 24 h or 48 h after injection for determination of the PT, APTT, VK level, and FIX level. YBs were divided into 4 groups: S-DHA (administration of S-DHA only for 2 wk); S-DHA+VK (administration of S-DHA (200 mg/kg) plus VK3 injection); VK (VK3 injection only); negative control.

LMH cells were cotreated with VK3 (2.0–8.0 μM) and S-DHA (5.0 mmol) for 24 h. Then, cell activity and FIX level were measured.

Assay for PT and APTT

Fresh blood was mixed with trisodium citrate (0.109 M) immediately at 9:1 (v/v). Plasma was collected by centrifugation (950 × g, 15 min, room temperature). The PT and APTT were measured with a PT assay kit or APTT (ellagic acid-type) assay kit, respectively, from Nanjing Jiancheng Biotechnology (Nanjing, China) using a blood coagulation analyzer (Coatron M4; TECO, Nrufahm, Germany) according to manufacturer instructions.

Determination of Levels of VK and FIX

Plasma or cell-culture samples were used to measure levels of VK and FIX using the respective ELISA kits (Shanghai Tongwei Biotechnology, Shanghai, China) according to manufacturer instructions.

Determination of Plasma Recalcification Time

The plasma recalcification time (PRT) is the time required for formation of a fibrin clot when calcium is replenished in anticoagulated plasma. The PRT is also employed to assess coagulation function.

Fresh blood was mixed with sodium citrate solution (0.109 M) at a ratio of 9:1 in tubes immediately. Then, the anticoagulated whole blood was centrifuged (950 × g, 15 min, room temperature). The upper part of the plasma had few platelets. Each test tube containing 0.1 mL of plasma was incubated for 3 min at 37°C, followed by addition of CaCl2 solution (0.1 mL) to start PRT determination. The PRT was recorded when “silky” fibrin appeared in the mixture, which denoted clot formation (Zhou et al., 2019). The experiment was repeated 6 times and an average value obtained.

Western Blotting and Hematoxylin and Eosin Staining

Liver tissues or LMH cells were used to measure VKORC1/L1 expression by Western blotting and hematoxylin and eosin (H&E) staining. The method for Western blotting was as described by Chen and colleagues (Nakayama et al., 2020) using primary antibodies against rat VKORC1 (GTX47837; Gene Tex, San Francisco, CA), VKORC1L1 (PA548618; Thermo Fisher Scientific, Waltham, MA), or β-actin (MilliporeSigma, Burlington, MA). Histopathology using H&E staining was carried out using paraffin sections as described by Zhang and colleagues (Zhang et al., 2017).

Ethical Approval of the Study Protocol

The experimental protocol was approved (protocol number: 202103225) by the Ethics Committee of Experimental Animal Welfare of Yangzhou University (Yangzhou, China). All procedures involving YBs were carried out according to the Guiding Principles for the Use of Animals in Toxicology published by the International Society of Toxicology (www.toxicology.org/) and the Regulations of the People's Republic of China on Laboratory Animals.

Statistical Analysis

Values of coagulation-time parameters were monitored in seconds and are expressed as the mean ± SD of 6 independent experiments. Comparisons were made using one-way analysis of variance, followed by Newman-Keuls post hoc analysis.

RESULTS

Single Administration of S-DHA to YBs

Determination of the PT and APTT

Single administration of S-DHA (150 or 200 mg/kg) to YBs was undertaken by lavage. The PT and APTT were ascertained using the respective detection kits (Figure 1A). Compared with before administration, the PT was increased 1.03- to 1.22-fold at 150 mg/kg S-DHA and 1.03- to 1.27-fold at 200 mg/kg S-DHA. Compared with before administration, the APTT was increased 1.06- to 1.35-fold at 150 mg/kg S-DHA and 1.19- to 1.53-fold at 200 mg/kg S-DHA. The relative increase in the APTT was greater than the relative increase in the PT after S-DHA administration. There was no obvious difference between male and female YBs with respect to changes in the PT or APTT.

Figure 1.

Figure 1

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The PT, APTT, and S-DHA concentration in YBs after single administration of S-DHA. S-DHA (150 or 200 mg/kg) was given as a single dose to YBs by lavage. The PT and APTT were obtained using the respective detection kits. The PT or APTT was compared with that before S-DHA administration in A. The data of 6 YBs at each time point were used. *P < 0.05; **P < 0.01, compared with the PT or APTT before administration by ANOVA. (B) The serum S-DHA concentration was measured by HPLC method (detection at 293 nm).

S-DHA Concentration and Levels of VK and FIX in Blood

The serum S-DHA concentration of YBs after S-DHA administration is shown in Figure 1B. The maximum serum concentration was ∼100 μg/mL and ∼118 μg/mL for S-DHA at 150 mg/kg and 200 mg/kg, respectively, at 4 h or 8 h postadministration. The serum concentration decreased by >50% at 24 h after administration in males and by ∼65% in females. The serum level of VK and FIX of YBs after single administration of S-DHA was measured by ELISAs (Figure 2A and B). The serum level of VK and FIX in YBs decreased 4 d after administration of S-DHA at 100 mg/kg or 200 mg/kg. The serum level of VK of male YBs was reduced significantly 4 h and 8 h after administration of S-DHA at 100 mg/kg, whereas the serum level of VK in female YBs was reduced significantly 8 to 24 h after administration. The FIX level was depressed significantly in the 150 mg/kg group (11–29%) and 200 mg/kg group (25–50%) compared with that in the control group at 0 h. For S-DHA at 200 mg/kg, the serum levels of VK and FIX were reduced significantly compared with those before dosing. The extent of the reduction in VK level at identical time points was more than that for the FIX level after administration of S-DHA at 100 mg/kg or 200 mg/kg.

Figure 2.

Figure 2

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Serum levels of VK and FIX and hepatic expression of VKORC1 in YBs treated by single administration of S-DHA. The serum levels of VK and FIX in YBs after single administration of S-DHA were measured by ELISAs. The left denotes the 150-mg/kg S-DHA group and the right denotes the 200-mg/kg group in A and B; C shows hepatic VKORC1 expression by Western blotting. The experiment was repeated 6 times. *P < 0.05; **P < 0.01, compared with the VK level before S-DHA administration.

Hepatic Expression of VKORC1/L1

Hepatic VKORC1/L1 expression was measured by Western blotting (Figure 2C). After administration of S-DHA (100 or 200 mg/kg), hepatic expression of VKORC1 and VKORC1L1 in YBs was reduced significantly. The maximum decrease in VKORC1 expression was ∼50% at a S-DHA dose of 150 mg/kg and ∼80% at a S-DHA dose of 200 mg/kg. The maximum decrease in VKORC1L1 expression was ∼40% at a S-DHA dose of 150 mg/kg and ∼70% at a S-DHA dose of 200 mg/kg. The decrease in hepatic VKORC1/L1 expression of YBs treated with S-DHA was not restored 3 d after administration.

Multiple Administration of S-DHA to Chickens

YBs administered different doses S-DHA did not have unusual traits in behavior, activity, excretion, eating or drinking throughout experimentation.

General Observations

Increases in growth and certain biochemical parameters in blood related to liver function were measured (Figure 3). The growth increase of YBs in the 3 S-DHA groups was not significantly different compared with that in the control group (P > 0.05). There was no significant difference in blood levels of AST, ALT, TP, TBIL, or DBIL of YBs (P > 0.05). Hence, there was no obvious adverse effect of S-DHA administration on the growth or liver function of YBs.

Figure 3.

Figure 3

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Growth increase and some blood parameters of YBs treated by S-DHA. Average increase every day of 12 YBs. Blood levels of AST, ALT, TP, TBIL, and DBIL of YBs were measured by a blood biochemical analyzer. The experiment was repeated 6 times.

The organ indices of YBs 7, 11, and 14 wk after S-DHA administration (all doses) were not significantly different (P > 0.05) (Figure 4). There were no obvious histological changes in the main organs of YBs 14 wk after administration of S-DHA (200 mg/kg) compared with those in the control group according to H&E staining.

Figure 4.

Figure 4

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Organ indices of YBs by histology. One-day-old chickens were given different doses of S-DHA. The organ index is the organ weight:body weight ratio. A is the organ index using the data from 6 repeated experiments. B is the H&E staining of the main organs of YBs 14 wk after S-DHA administration. The top line is the control; the bottom line is the 400-mg/kg S-DHA group. *P < 0.05, compared with the control.

Parameters of Coagulation Function

The parameters related to coagulation function of YBs upon multiple administration of S-DHA are shown in Figure 5. The PT in S-DHA groups (100 and 200 mg/kg) was increased significantly 5 wk after administration. The PT increased 3 wk after S-DHA (400 mg/kg) administration, and the degree of APTT increase was more than that observed for the groups of 100 mg/kg and 200 mg/kg. The PT did not increase with an increase in the administration time in the 3 S-DHA groups. After S-DHA administration, the increase in the APTT was obviously greater than the changes seen in the PT for all treatment groups; compared with the control, the ATPT increased by 1.08- to 1.39-fold and PT increased 1.01- to 1.04-fold after S-DHA (200 mg/kg) was given. The APTT was increased significantly from 2-wk postadministration, with a rapid increase at 2 to 4 wk, a decline at 5 to 11 wk, and an obvious increase at 12 to 14 wk. When S-DHA (400 mg/kg) was given, the PT increased by a maximum of 1.15-fold, and the APTT increased by a maximum of 1.42-fold.

Figure 5.

Figure 5

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The parameters related to the coagulation function of YBs after multiple administration of S-DHA. One-day-old YBs were given different doses of S-DHA in their feed. The PT and APTT were obtained with the respective assay kits. Levels of VK and FIX were measured by the respective ELISA kits. The plasma recalcification time (PRT) time was also measured. Each experiment was repeated 6 times. *P < 0.05; **P < 0.01, compared with the control.

The PRT is also used to evaluate coagulation function. In general, increases of 1.4-fold in the PRT indicate disturbance of blood coagulation. The PRT was increased by more than 1.4-fold at 12 d after administration of S-DHA (200 mg/kg), whereas it was increased by more than 1.4-fold at 7 d after = administration of S-DHA (400 mg/kg) (Figure 3). Hence, obvious effects on blood clotting were noted for a longer administration time and higher dose of S-DHA.

Plasma levels of VK and FIX of YBs were decreased significantly at all test times in the 3 S-DHA groups. The lowest VK concentration was ∼0.8-fold compared with that in the control group 7 to 8 wk after administration, and the FIX level decreased by 30% at later administration, in the 400-mg/kg group.

Hepatic VKORC1/L1 Expression in YBs

Hepatic expression of VKORC1/L1 in YBs after S-DHA administration was measured by Western blotting (Figure 6). Two weeks after administration, hepatic VKORC1/L1 expression at all times was reduced significantly compared with that in the negative control group (P < 0.01), and there was no obvious difference between male YBs and female YBs. The minimum expression was about 0.35-fold for VKORC1 and VKORC1L1 compared with that in the control group 5 wk after administration. VKORC1/L1 expression increased again slowly by a small amount with increasing time, but VKORC1/L1 expression 14 wk after administration was about 0.6-fold/0.45-fold in male YBs and 0.5-fold/0.42-fold in female YBs compared with that in the control group. Hence, a similar reduction in expression of VKORC1 and VKORC1L1 caused by S-DHA administration was found in YBs.

Figure 6.

Figure 6

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Hepatic VKORC1 expression of YBs at different administration times of S-DHA. One-day-old YBs were given S-DHA (200 mg/kg) added to their feed. Liver samples were used to measure VKORC1 expression by Western blotting, with experiments repeated 3 times for each time point. The upper part shows Western blots, and the part the gray values of VKORC1/β-actin compared with the control, *P < 0.05; **P < 0.01, compared with the control (ANOVA).

Effect of S-DHA on LMH Cells

LMH cells were exposed to different concentrations of S-DHA for 24 h and the effect on cell activity, VK content, FIX content, and VKORC1/L1 expression was documented (Figure 7). The activity of LMH cells was not changed at a S-DHA concentration of 0.2 to 2.0 mM, but was reduced significantly at a dose of 5.0 to 10.0 mM (∼30% reduction at 10.0 mM) (P < 0.05). Levels of VK and FIX in cells or culture medium were reduced significantly at S-DHA doses of 5.0 and 10.0 mM (P < 0.05). Expression of VKORC1 or VKORC1L1 in LMH cells decreased with an increasing dose of S-DHA, with a significant decrease noted at 2.0 to 10.0 mM (P < 0.05).

Figure 7.

Figure 7

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Effect of S-DHA on the activity, VK level, FIX level, and VKORC1/L1 expression of LMH cells. LMH cells were treated by different concentrations of S-DHA for 24 h. The cell activity was measured by the MTT assay. Levels of VK and FIX in cells or culture supernatant were measured by ELISAs. Expression of VKORC1 and VKORC1L1 was measured by Western blotting. Data are the mean of 5 replicates for cell activity, VK level, and FIX level, and of 3 replicates for VKORC1/L1 expression. *P < 0.05, **P < 0.01 compared with control.

Resistance Proffered by VK3 to Coagulation Disorders Induced by S-DHA

The resistance proffered by VK3 to coagulation disorders induced by S-DHA in LMH cells or in YBs is shown in Figure 8. The reduced activity of LMH cells induced by S-DHA (5.0 mM) increased significantly with an increase in VK3 dose (P < 0.01). The activity of LMH cells was not significantly different at a VK3 dose of 8.0 μM compared with that of the control group (P > 0.05). The lower FIX level induced by S-DHA administration increased at a VK3 dose of 4.0 or 8.0 μM in cells or in the culture medium, and a significant difference was noted at a VK3 dose of 8.0 μM (P < 0.01). VK3 could resist the coagulation disorders in LMH cells induced by S-DHA administration.

Figure 8.

Figure 8

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Protective effect of VK against the coagulation disorders induced by S-DHA administration in LMH cells or in YBs. LMH cells were cotreated by VK3 (2.0–8.0 μM) and S-DHA (5.0 mM) for 24 h. Then, the cell activity and FIX level were measured. In the YB experiment, VK3 (2 mg) was injected (i.m.) in the thigh muscle of each YB after 2 wk of administration of S-DHA (200 mg/kg) to YBs. YBs were divided into 4 groups: S-DHA (administration of S-DHA only for 2 wk); S-DHA+VK (administration of S-DHA (200 mg/kg) plus VK3 injection); VK (VK3 injection only); negative control. Then, blood samples were collected 24 h or 48 h after VK3 injection to determine the PT, APTT, VK level, and FIX level. *P < 0.05, **P < 0.01, compared with control.

Prolongation of the PT or APTT induced by S-DHA (200 mg/kg) was reduced in the S-DHA+VK group 24 h or 48 h after injection, with a significant difference noted after 48 h (P < 0.05). There were no significant difference in the PT between the S-DHA+VK group and control group 24 h or 48 h after injection. The APTT of the S-DHA+VK group after 48 h was significantly lower than that of the S-DHA group (P < 0.05), but was clearly higher than that of the control group (P < 0.01). The reduced blood levels of VK and FIX induced by S-DHA therapy were increased significantly in the S-DHA+VK group 48 h after injection, and there was no significant difference compared with the control group (P > 0.05). The disordered coagulation parameters of the PT, VK level, and FIX level induced by S-DHA therapy could return to normal 48 h after VK3 injection apart from the APTT, which was clearly higher than that in the control group. Hence, VK3 seemed to proffer protection against coagulation disorders in YBs induced by S-DHA administration.

DISCUSSION

We discovered that S-DHA (100–400 mg/kg) administration induced a coagulation disorder in YBs, increased the PT and APTT, and reduced the levels of FIX and VK. These effects are similar to the abnormal coagulation effects observed in rats (Sakaguchi et al., 2008; Zhang et al., 2017; Chen et al., 2019, 2021). There was no obvious difference in the effect of S-DHA on coagulation in male and female YBs. Upon multiple administration, compared with the control, the increase in the PT was about 1.01- to 1.04-fold and the increase in the APTT was about 1.08- to 1.39-fold at 200 mg/kg of S-DHA. The increase in the PT and APTT in S-DHA-treated males was 1.27- to 1.48-fold and 1.17- to 1.37-fold, respectively, whereas the corresponding values in S-DHA-treated females was 1.36- to 2.02-fold and 1.20- to 1.70-fold, respectively (Chen et al., 2021). The maximum VKORC1 expression in the rat liver has been reported to be 0.15- to 0.44-fold, and 0.3-fold in the chicken liver treated by S-DHA compared with the control. The effect of S-DHA on coagulation function in rats may be greater than that in chickens based on changes in the PT and APTT and decrease in hepatic VKORC1 expression.

Several VK-dependent coagulation factors (e.g., prothrombin, FIX, FVII, FX) in mammals, lower vertebrates (e.g., birds, fish) only have FIX, and lack other proteases in the intrinsic pathway of their blood-clotting system (Doolittle, 2009; Ponczek et al., 2012). In rats, hepatic VKORC1 expression is inhibited by S-DHA, resulting in the decreased γ-carboxylation and biological activity of several VK-dependent coagulation factors. Only a few VK-dependent coagulation factors (e.g., FIX) in chickens are inhibited, which may be one of reasons for the coagulation disorders caused by S-DHA in rats and chickens. Different metabolism of S-DHA, VKORC1 sensitivity, and other VK sources (Oldenburg et al., 2008) in chickens may be other reasons.

Interestingly, levels of VK and FIX in blood and hepatic VKORC1/L1 expression in YBs decreased to a minimum in the first 5 to 6 wk after administration, and then increased gradually with prolonged duration of medication (Figures 5 and 6). Hence, there may be compensatory coagulation mechanism for free VKORC1/L1-VK cycle-γ-carboxylation of VK-dependent coagulation factors in the liver. In contrast with mammals, avian VKOR activity is supported by VKORC1 and VKORC1L1, and mRNA expression could cause differences in VKOR activity between avian species. VKORC1 has been reported predominantly in the mammalian liver, but VKORC1 and VKORC1L1 are present in the chicken liver. In the chicken liver, VKORC1 has been reported to have 500 and VKORC1L1 to have 700 copy numbers/ng total RNA (Nakayama et al., 2020). Different activity and sensitivity of VKORC1 and VKORC1L1 have been documented (Oldenburg et al., 2015; Lacombe et al., 2018). VKORC1L1 has been reported to be 50-fold more resistant to VK antagonists than VKORC1 (Hammed et al., 2013). Expression of VKORC1 and VKORC1L1 in YBs was reduced by S-DHA administration to a similar extent according to our data, thereby indicating that reduced expression of VKORC1/L1 was involved in the coagulation disorders induced by S-DHA. Whether S-DHA administration alters the activity of VKORC1 or VKORC1L1 in YBs will be investigated in our next work.

The biological effects of VKORC1L1 in extrahepatic tissues have been documented. A reduction in VKORC1L1 expression increases the intracellular vitamin-K2 level and impedes preadipocyte differentiation, VKORC1L1 can promote adipogenesis and possibly obesity (Ding et al., 2018), as well as VK-dependent release of intracellular antioxidants (Westhofen et al., 2011). VKORC1L1 and VK have been reported to mediate protection of the vasculature because VKORC1L1 inhibition induces a proliferative and proinflammatory phenotype of vascular smooth muscle cells (Aksoy et al., 2021). In addition, poultry are sensitive to VK, and diseases due to VK deficiency are not uncommon. The decrease in the blood level of VK caused by sprinkling of S-DHA in chicken feed could increase the risk of VK deficiency, which we will study in the near future.

CONCLUSIONS

We demonstrated that S-DHA could induce a disorder in coagulation function in YBs. S-DHA administration increased the PT and APTT, reduced blood levels of VK and FIX, and inhibited VKORC1/L1 expression in the YB liver. The abnormal coagulation induced by S-DHA administration was associated with a hepatic reduction in VKORC1/L1 expression, and VK could help to resist this phenomenon.

ACKNOWLEDGMENTS

Thank for your help to the staff of the Comparative Medical Center of Yangzhou University.

This work was supported financially by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the 111 Project D18007.

Availability of Data and Materials: The datasets used during the current study are available from the corresponding author on reasonable request.

Authors’ Contributions: J.X. and Y.Z.: methodology, investigation, validation, data curation, visualization, writing (original draft). F.H., C.W., and Z.Z.: investigation, validation, data curation, visualization. M.Z., X.C., and Y.Z.: resources, supervision, review and editing, data analyses. All authors approved the final version of the manuscript.

DISCLOSURES

The authors declare that they have no competing interests.

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