Ferric citrate is a safe and digestible source of iron in broilers and piglets

Study location & quality standards

The four studies were performed at the Institute of Animal Nutrition, Department of Veterinary Medicine, Freie University of Berlin (Berlin, Germany). All studies were conducted according to the Animal Welfare Act of Germany approved by the local state office of occupational health and technical safety (Landesamt für Gesundheit und Soziales, LaGeSo, no. A 0439/17).

Experimental design and diets

The test item ferric citrate contains ca. 17% Fe, hence at 200, 2,000, 500 and 5,000 mg/kg delivers 34, 340, 85 and 850 mg Fe/kg feed, respectively.

Haematological and biochemical analyses (Study A, Study B and Study D)

In Study A, blood samples were collected on 35 d (study end), from two birds/pen with body weight (BW) closest to the treatment mean. For Study B and Study D, blood samples were collected on day 42 (Study B) and on day 0 and 42 (Study D) from one piglet/pen with body weight (BW) closest to the treatment mean.

In all studies, samples were collected from the anterior vena cava into different tubes: plain, heparinised for clinical chemistry, and EDTA tubes for blood counts.

Haematology parameters measured were erythrocytes, leukocytes, haemogram: lymphocytes, monocytes, eosinophils, basophils, neutrophils, haemoglobin, haematocrit, mean corpuscular volume–MCV, mean corpuscular haemoglobin–MCH, mean corpuscular haemoglobin concentration–MCHC and electrolyte analyses (sodium, potassium, chloride, calcium, magnesium, phosphate). Biochemistry analyses included total cholesterol, triglycerides, bilirubin, creatine, urea, glucose, albumins, globulins, total protein, enzymes (α-amylase, aspartate-amino-transferase -AST, alanine aminotransferase–ALT, glutamate-dehydrogenase–GLDH, alkaline phosphatase–AP, L-lactate dehydrogenase–LDHA), and serum amyloid A.

For Study D, blood serum was additionally analysed for Fe, superoxide dismutase (SOD) and haptoglobin. SOD is an enzyme that eliminates superoxide radicals and is an important indicator of antioxidation and oxidation levels caused by free radicals created by unbound Fe. Haptoglobin is an acute phase protein that binds to free haem and reduces oxidative activity. Blood cells were measured by flow cytometry, blood concentrations in plasma were determined by using photometry, while sodium, potassium, and chlorides were measured by ionic liquid-polyacrylamide gel electrophoresis.

Zootechnical parameters

Standard zootechnical parameters (body weight, BW; feed intake, FI; weight gain, WG; mortality-corrected feed conversation rate, MFCR) were recorded in all studies for the different study periods.

Fe digestibility (Study D)

On day 42, the apparent total digestibility of crude ash and Fe was determined using titanium (IV) dioxide added at a rate of 3 g/kg feed as an inert indigestible marker. A total of seven replicates from each treatment (pooled faeces collected rectally from 5 piglets per pen, giving a total of seven replicates) were collected.

To calculate the apparent total Fe digestibility, the following formula was used:

$$\text{Total digestibility}(\mathrm{\%})=100-(\frac{\text{\% Marker in feed}}{\text{\% Nutrient in faeces}}\times \frac{\text{\% Nutrient in faeces}}{\text{\% Nutrient in feed}})\times 100$$

Statistical Analyses

Study A and Study B were based on demonstrating non-inferiority, where the tolerance group (T3) does not perform worse than the untreated control (T1). The null hypothesis being that the animals fed the tolerance dose perform worse that the control animals. The alternative hypothesis was that the animals fed the tolerance dose perform the same or not worse than the animals fed the control diet.

Study C and Study D were based on demonstrating superiority, where the animals fed diets supplemented with ferric citrate or another source of Fe, perform better than the untreated control. The null hypothesis being that the animals fed diets containing ferric citrate (or another source of Fe) perform the same as the control animals. The alternative hypothesis being that the animals fed ferric citrate (or another source of Fe) perform better than the animals fed the control diet.

For the different studies, sample size was calculated according to the hypothesis (Sealed Envelope Ltd, 2012; primary outcome measure = BW), and were designed to be optimal or near optimal in the trade-off between power and resources.

For all studies, the basic study design was a random complete block design, with pen as the experimental unit for statistical purposes for zootechnical and faecal parameters. The individual animal was used as the experimental unit for blood parameters. All parameters were analysed using a general lineal model as follows: Y_ij = μ + treatment_i + block_j + ε_ij, where Y_ij was the dependent variable, μ was the overall mean, treatment_i was the effect of treatment (Study A = 3 levels; Study B = 3 levels; Study C = 4 levels and Study D = 5 levels), block_j was the effect of the block, and ε_ij was the residual error. All experimental units were used for statistical analysis.

All parameters were reported as group least squares mean. Standard error of the mean were also reported. Multiple comparisons between treatment groups were made by Tukey test and significant differences were declared at P ≤ 0.05, while near significant trends were considered for 0.05 < P ≤ 0.10.

The analyses were performed using the software packages SPSS (IBM SPSS, Version 21, Study A, Study B, Study D) and Unistat (Unistat Ltd., version 10.05, Study D).

Study A

The results summarising the safety of ferric citrate on male broiler performance are presented in Table 9. For the overall study period (0–35 days), broilers receiving the T2 (200 mg ferric citrate/kg) and T3 (2,000 mg ferric citrate/kg) diets significantly outperformed the T1 Control group in terms of BW (T2 + 4.31%; T3 + 3.32%), ADG (T2 + 4.30%; T3 + 3.34%) and MFCR (T2 - 3.26%; T3 - 3.77%). Overall mortality rate was 2.5%, indicating good bird health. Blood data from samples taken at 35 days (study end) are presented in Table 9. No significant differences were observed between the birds fed ferric citrate and birds fed the Control diet for any blood parameters measured except for erythrocytes, MCV, sodium, potassium, chloride, urea and glucose. Erythrocytes were significantly lower in T3 birds (ferric citrate, 200 mg/kg) compared to T1 Control birds (−5.04%) and MCV was significantly higher in birds fed the T3 diet (ferric citrate 2,000 mg/kg) compared to birds fed the T1 Control (+2.75%). The minerals sodium and chloride were significantly higher in birds fed T2 (ferric citrate, 200 mg/kg) and T3 (ferric citrate, 2,000 mg/kg) compared to birds fed T1 Control diet (sodium = T2 + 3.13%; T3 + 2.24%; chloride = T2 + 2.66%; T3 + 2.15%). Potassium was significantly lower in T2 birds (ferric citrate, 200 mg/kg) compared to T1 Controls (−8.98%), while urea and glucose were significantly higher in T2 birds (ferric citrate, 200 mg/kg) compared to T1 (urea = +7.5%; glucose = +4.13%).

Study B

Results summarising the safety of ferric citrate in weaned piglets are presented in Table 10. For the overall study period (0–42 days) there were no significant differences between treatment groups for BW, ADG or ADFI. However, FCR was significantly improved in piglets fed T2 (ferric citrate, 500 mg/kg) and T3 (ferric citrate, 5,000 mg/kg) diets compared to those fed the T1 Control diet (T2 = −4.02%; T3 = −5.31%). There were no significant differences between treatment groups for any of the blood parameters measured.

Study C

Results summarising the effects of ferric citrate and other ferric Fe sources fed to weaned piglets are presented in Table 11. For the overall study period, significant improvements were observed in groups fed the ferric Fe sources compared to those fed the control diets. On day 42, animals in T3 (ferric citrate) and T4 (ferric tartrate) were 1.41 kg and 1.45 kg heavier, respectively compared to the T1 Control animals. Furthermore, animals fed T3 and T4 diets grew significantly better than T1 Controls (ADG: T3 = +7.09%; T4 = +7.30%). FCR was also significantly improved in animals fed T3 and T4 compared to those fed T2 (ferric lactate) (T3 = −3.84%; T4 = −3.22%) and the T1 Controls (T3 = −8.36%;T4 = −7.77%).

Study D

Results summarising the effects of ferric citrate and other sources of Fe fed to weaned piglets are presented in Table 12. On day 42 piglets fed T4 (ferric citrate) and T5 (ferric citrate + Fe (II) sulphate) weighed significantly more compared to piglets fed T3 (Fe (III) oxide) (+0.81 kg and +0.77 kg, respectively), T2 (Fe (II) sulphate, +0.95 kg and +0.91 kg, respectively) and T1 (Control, +1.53 kg and +1.49 kg, respectively). Piglets in T4 and T5 also grew significantly better and had improved FCR compared to piglets fed T2 and T1 diets (ADG: T4 vs. T2 = +5.63%; T4 vs. T1 = +9.39%; T5 vs. T2 = +5.39%; T5 vs. T1 = +9.13%; FCR: T4 vs. T2 = −4.13%; T4 vs. T1 = −6.93%; T5 vs. T2 = −4.41%; T5 vs. T1 = −7.20%). Faecal ash digestibility was significantly better in all treatment groups compared to the T1 Control (T2 = +15.27%; T3 = +19.43%; T4 = +14.27%; T5 = +16.80%) and Fe digestibility was significantly improved in piglets fed T4 (ferric citrate, +96.71%) and T5 (ferric citrate + Fe (II) sulphate, +70.84%) compared to the T1 Control animals. Fe content in blood serum was not significantly different between treatment groups at either 25 or 66 days of age.

The effects of ferric citrate and other sources of Fe on blood parameters of weaned piglets are presented in Tables 13 and 14. On day 0 there were no significant differences in blood parameters between treatment groups. On day 42, no significant differences were observed for any of the blood parameters except for total bilirubin and SOD. Total bilirubin was significantly higher in T2 compared to the other treatment groups (T1 = +74.73%; T3 = +63.54%; T4 = +78.49%; T5 = +95.29%), while SOD was significantly higher in the animals fed ferric citrate (T4 and T5) compared to the piglets fed the T1 Control diet (T4 = +33.73%; T5 = +29.51%), T2 (T4 = +35.36%; T5 = +31.09%) and T3 (T4 = +30.58%; T5 = +26.47%).

Safety in broilers - Study A

Haemoglobin is an Fe-rich protein involved in cellular metabolism and the transport of oxygen through animal tissues (Snyder & Sheafor, 1999). Thus, haemoglobin and haematocrit-related parameters are key to detecting any possible negative effects of dietary Fe supplementation in broilers. No significant differences between diets were detected for haemoglobin, haematocrit, MCH or MCHC. In the case of erythrocyte and MCV values, despite the statistically significant differences with the Control diet, all results were in line with values reported in previous studies (Milanovic et al., 2008; Saripinar-Aksu, Aksu & Özsoy, 2010; Tako, Rutzke & Glahn, 2010). Likewise, the statistically significant differences detected between treatments for sodium, potassium, chloride, urea and glucose were considered to have no clinical relevance since there were no indications of a dose-dependent effect and results were in agreement with other reported data for chickens (Simaraks, Chinrasri & Aengwanich, 2004; Café et al., 2012; Odunitan-Wayas, Kolanisi & Chimonyo, 2018). Furthermore, the inflammatory marker serum-amyloid A (O’Reilly, Bailey & Eckersall, 2018) was below the detection level, indicating good health status of all birds. Finally, and with the few exceptions described above, blood cells, enzymes, electrolytes and biochemical blood constituents at the end of the 35 days feeding period did not change significantly across the different doses of ferric citrate. Altogether, the analysis of blood data shows that ferric citrate is a safe source of supplementary Fe for broilers, since no negative effects were detected in either of the ferric citrate treatments, where supplementary Fe from ferric citrate supplied an additional 34 mg (T2) and 340 mg (T3) Fe/kg feed, approximately, in comparison with T1 Control (only endogenous dietary Fe). Additionally, it was confirmed that ferric citrate is well tolerated up to 2,000 mg/kg (×10 the recommended inclusion rate). These results are in line with a recent assessment performed by the European Food Safety Authority (EFSA, 2019), who concluded that ferric citrate is a safe feed additive for weaned piglets up to 5,000 mg/kg of feed, (supplying ca. 850 mg Fe/kg feed).

As indicated by the National Research Council (NRC) (1994), some of the response criteria for Fe supplementation in chickens are growth and feed efficiency. The results presented in Study A support this as, after 35 days, animals fed diets supplemented with ferric citrate at either 200 mg/kg or 2,000 mg/kg feed (34 and 340 mg Fe/kg feed, respectively) showed better performance in terms of ADG, MFCR and final weight compared to birds with no Fe supplementation (T1 Control = $\overline{\text{x}}$ 79 mg Fe/kg Fe). The low mortality rate reported (2.5% at 35 days), coupled with the blood analysis and the growth performance, support the safety of ferric citrate in broilers.

The results of Study A are in agreement with an earlier study that evaluated ferric citrate as a source of Fe, using broiler chickens in a specialised model intended as an intermediate test of in vivo Fe bioavailability of human Fe supplements (Tako, Rutzke & Glahn, 2010). Intestinal Fe absorption and haemoglobin maintenance was assessed in broilers using ferric citrate as the Fe source. From one week of age the control group was fed a diet deficient in Fe (51 mg Fe/kg feed), and the other group was fed a diet supplemented with 500 mg ferric citrate/kg (total Fe: 141 mg/kg feed). During the 6-week study period, there was no difference in feed intake between the treatments, but Fe intake was significantly increased in the ferric citrate supplemented group compared to the Fe deficient control group. Blood haemoglobin (Hb) concentrations were also significantly higher in the ferric citrate group compared with the control. The Hb maintenance efficiency values (indicator for dietary Fe availability) were significantly higher in birds fed the Fe-deficient control diet, indicating that the Fe uptake mechanisms in broiler are responding to dietary Fe concentrations. The final total body Hb and the increase in total body haemoglobin Fe during the study period was significantly greater in the ferric citrate supplemented group than in the control group (85 vs. 54 mg, P < 0.05). No mortalities or adverse effects were reported.

Whilst not evaluated in the present study, it has been demonstrated that organic sources of Fe are more bioavailable in broilers than inorganic forms (Pla & Fritz, 1970; Shinde et al., 2011; Arnaudova-Matey et al., 2013). For example, Pla & Fritz (1970) investigated the bioavailability of organic and inorganic sources of Fe in broiler chicks with severe anaemia. Over a two-week period, response to dietary Fe supplementation was measured using blood Hb. The authors categorized the sources of Fe into good, mediocre and poor. They reported that the organic sources of iron, including ferric citrate, were good sources of iron compared to ferrous sulphate. Shinde et al. (2011) looked at the efficiency of organic and inorganic sources of Fe in Fe-depleted broilers and compared ferric sulphate and ferrous methionine with a negative control diet. During 35 days, improved body weight gain, FCR, and greater dry matter, crude protein retention and Fe digestibility in faeces were observed in birds fed on Fe supplemented diets when compared with birds fed the control diet. After 35 days, the red blood cell, Hb, haematocrit, and Fe concentration in plasma, tibia and liver were higher in birds fed on Fe-supplemented diets than birds fed on the control diet. Most importantly, supplementation of Fe in the organic form resulted in greater Fe concentration in the tibia and liver, and less Fe excretion, when compared with birds receiving inorganic Fe.

Safety in weaned piglets

The results obtained in Study B support the safety of ferric citrate. In this study, no adverse effects on growth or blood parameters were noted. Furthermore, no mortality was recorded, and animals were healthy for the duration of the trial. These results support the safety of ferric citrate when added to the diet of weaned piglets at up to 10 times the recommended dose, and are in agreement with the recent assessment performed by the European Food Safety Authority (EFSA, 2019), who concluded that ferric citrate is a safe feed additive for weaned piglets up to 5,000 mg/kg of feed. Results obtained in Studies C and D further support the safety of ferric citrate as a source of dietary Fe. In these studies, no adverse effects were observed in the animals fed ferric citrate at the recommended dose (ferric citrate at 500 mg/kg feed supplying 85 mg Fe/kg feed); animals fed ferric citrate performed better than animals fed the control diet (containing endogenous Fe only), and the blood data were within the normal ranges for healthy piglets.

Nutritional efficacy in weaned piglets

The results from Studies C and D showed that ferric citrate is an effective source of iron in weaned piglets. Study C demonstrated that ferric citrate and other sources of ferric Fe improved growth rates in pigs fed these compounds compared to pigs fed the Fe deficient negative control diet containing only endogenous Fe. The results from Study D showed that pigs fed ferric citrate alone or in combination with ferrous sulphate performed better than the control animals and the animals fed inorganic forms of iron. Moreover, in Study D, Fe digestibility was significantly improved in piglets fed ferric citrate alone or in combination with ferrous sulphate compared to the control animals and the animals fed the inorganic forms of Fe, thus supporting the nutritional efficacy of ferric citrate. In the same study, no significant improvements in blood Fe parameters were observed. At the beginning of the study (25 days of age), all piglets had Hb of 100 g/L to 102 g/L, which indicated normal iron concentrations and that the animals were not iron deficient or anaemic (Wei et al., 2005). At day 42 (66 days of age), piglets fed ferric citrate had numerically higher Hb compared to the negative controls, but all groups were within the expected range. No significant differences were noted for other blood parameters indicative of Fe status (MCV, MCH, MCHC), highlighting that all piglets included in this study were not seriously iron deficient or anaemic. Furthermore, no significant differences in Fe serum concentrations were seen between treatment groups on day 0 or day 42. These results are not surprising given that all piglets received a parenteral iron dextran injection at 3 days old. Administration of iron dextran is standard practice and performed for welfare reasons to reduce severe Fe deficiency and high mortality in pre- and post-weaning piglets (Perri et al., 2016; Svoboda, Vanhara & Berlinska, 2017). As these studies were run for regulatory approval, using Fe-depleted animals was not appropriate as healthy animals must be used to support the safety and efficacy of feed additives (EFSA, 2017; EFSA, 2018). However, earlier studies reported that ferric citrate supplemented at daily use rates of 48 mg and 144 mg/feed increased blood haemoglobin concentration and plasma Fe in pigs (Furugouri & Kawabata, 1975) and interstingly, Furugouri & Kawabata (1975) also cited a study performed by Ullrey et al. (1973), who reported that ferric citrate is a more effective source of supplemental Fe for piglets than ferrous sulfate.

Even though a Fe-depletion model which is often described as the standard for assessing bioavailability of iron (Henry & Miller, 1995) was not used in the present studies, improved growth performance in animals fed ferric citrate compared to the animals fed diets containing endogenous Fe only was observed. This, coupled with the faecal digestibility of Fe, indicates that ferric citrate is a safe and effective source of dietary Fe. Furthermore, piglets receiving ferric citrate had significantly higher levels of SOD compared to the negative control group and the groups fed the inorganic forms of Fe, indicating that oral administration of ferric citrate improved the oxidative status of the animals. These results were in agreement with a recent study that compared oral administration of ferrous glycine and parenteral iron dextrose with a negative control in anaemic pigs (Dong et al., 2020). These authors reported significantly decreased serum SOD levels in the control animals and those that received iron dextran compared to the animals fed the organic form of Fe.

Some animals in studies C and D were treated with antibiotics, which could raise some concern given that there are reports showing an interference of these compounds with iron absorption (Djaldetti et al., 1981). However, the data presented in study D shows that all animals had recommended Fe levels and no significant differences were found in Fe serum concentrations between treatment groups. Additionally, the proportion of animals treated in each study was low (<9% out of 220 and 175 piglets, respectively) and antibiotics were applied roughly equally across all treatment groups.

Iron requirements for piglets are influenced by several factors, the most important being growth intensity during pre-weaning and post-weaning. Modern pig breeds are selected for fast growth and several recent studies have questioned whether current Fe supplementation practices are meeting Fe requirements (Perri et al., 2016; Svoboda, Vanhara & Berlinska, 2017). Hence, dietary supplementation with Fe is required to ensure that adequate stores of Fe are achieved. Inadequate iron leads to health and economic implications as piglets with depleted iron stores are at risk of a suppressed immune system, leading to an impaired ability to resist infection and parasitic diseases, and have slower growth rate and increased morbidity and mortality (Svoboda & Drabek, 2005).