Radiocesium transfer rates among pigs fed haylage contaminated with low levels of cesium at two differentiation stages

The objective of this study was to determine the radiocesium transfer rates of pigs fed haylage contaminated with low levels of cesium at different growth stages. We measured the body weight of juvenile and adult pigs during the treatment period to confirm their health status. We also performed pig blood hematologic and biochemical analyses at both growth stages. To our knowledge, this is the first study to report pig radiocesium transfer coefficient rates after 1 month of chronic oral treatment, which is the period assumed to be required for body equilibrium under a diet of radiocesium-contaminated food. The results showed higher radiocesium retention rates in the kidneys, liver, spleen, genitals, psoas major, bladder, thyroid, and urine than in the blood and bone (tibia and femur) of pigs at both growth stages. The radiocesium retention levels were generally higher in juvenile pigs than in adult pigs, with the highest transfer coefficient ratio in the kidneys (16.2%).


Introduction
On March 11, 2011, a magnitude 9.1 earthquake occurred off the Pacific coast of Tohoku, Japan. This earthquake triggered a powerful tsunami that destroyed local villages and took a devastating toll on human life. The earthquake and subsequent tsunami also led to the Fukushima Daiichi nuclear power plant disaster, in which a nuclear reactor meltdown resulted in the release of large amounts of radioactive material, contaminating plants and animals in a wide surrounding area. The Animal Resource Science Center (ARSC) of the University of Tokyo (Tokyo, Japan) is located 140 km southwest of the Fukushima Daiichi nuclear power plant. Contamination of the ARSC pastures has been confirmed [1]. However, Italian ryegrass that had been seeded in September 2010 was harvested in May 2011 as usual. The raw grass was dried for several days and then packed in plastic film. The grass was allowed to ferment for 1 month for conversion into animal feed as haylage. The haylage was confirmed on June 3, 2011, to be contaminated with radioactive nuclides at 1.91-2.18 kBq/kg 134 C and 2.03-2.20 kBq/kg 137 C; however, 131 I was not detected [2]). Within 2 months after the Fukushima Daiichi nuclear power plant accident, this contaminated haylage was used to study radiocesium transfer ratios from feed to cow milk. Cesium (Cs) is a naturally occurring element found at low concentrations in rocks, soil, and dust. Natural Cs is present in the environment in only one stable form, as the isotope 133 Cs. Radioactive forms of Cs are unstable and are eventually transformed into other, more stable elements through radioactive decay. The two most important radioactive Cs isotopes are 134 Cs and 137 Cs. Each atom of 134 Cs changes into either xenon-134 ( 134 Xe) or barium-134 ( 134 Ba), neither of which is radioactive, whereas 137 Cs atoms decay to barium-137 ( 137 Ba), which is also not radioactive. As 134 Cs and 137 Cs decay, beta particles and gamma radiation are given off. The half-life, or time required for half of the isotope to give off radiation and transform into a different element, of 134 Cs is about 2 years, whereas that of 137 Cs is about 30 years [3]. In the present study, we examined the health status of 26 pigs that had been raised 17 km northwest of the Fukushima Daiichi nuclear power plant. These pigs were rescued about 110 days after the disaster, and subsequently maintained for over 3 months on an uncontaminated feedlot at the ARSC, after which radiocesium contamination was detected in multiple tissue samples (ovary, testis, spleen, liver, kidney, psoas major, urine, and blood) [1]. The pig farm owners confirmed that the pigs were not fed radiation-contaminated concentrate and that they had access to uncontaminated groundwater while they lived within the radiation alert area. However, radiocesium was detected in pig organs 9 months after the nuclear disaster. Thus, removal of radiocesium from the pig body requires a relatively long period. A previous study reported that the decrease in whole-body activity among voles fed regularly with 134 Cs dropped to below the minimum detectable level after 21 days [4]; however, our own previous findings [1] did not confirm those of this early report.
Our previous hematological and biochemical index analyses showed different plasma serum chemistry values between pigs rescued from the nuclear disaster area and normal pigs fed at the ARSC [1]. We also detected reproductive dysfunction among rescued sows fed uncontaminated concentrate and groundwater at the ARSC. In the present study, we examined radiocesium transfer rates in organ tissues of pigs fed with haylage contaminated with low levels of Cs for 1 month at different growth stages. We measured radiocesium levels in all organ tissue samples and calculated hematological and biochemical indices to confirm the health status of the pigs. Because pigs share many physiological similarities with humans and offer breeding and handling advantages compared to non-human primates, they are model organisms for preclinical experimentation in the fields of reproductive physiology, xenotransplantation, and neurotransplantation. Therefore, the results of the present study may have broader research applications for humans and other animal species.

Materials and methods
All experiments were approved by the Animal Care and Use Committee of the Graduate School of Agricultural and Life Sciences at the University of Tokyo (Approval ID: P15-061).
In this study, we used 10 Middle Yorkshire × Duroc juvenile pigs, including castrated males and females. Each treatment group contained five pigs (mean body weight: 57.9 ± 10.9 kg). The control group also consisted of five pigs (mean body weight: 54.8 ± 12.9 kg); this group was used only for comparing body weight and hematological and biochemical indices.
Grant-in-Aid for Scientific Research (C) (no. JP25517004) and by the Livestock Promotion Agency of Japan Racing Association. The funding of "Japan Racing Association", the project leader is our colleague, same laboratory. I am a member of this project. The present research used pocH-100iV Diff hematology analyzer (Sysmex, Hyogo, Japan) for hematological parameters analyze, and using a DRI-CHEM 3500s (Fujifilm, Japan) did blood biochemistry analyze. Both of which was purchased by above grant. But present research mainly done by the grant of "Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Scientific Research (C) (no. JP25517004)". I am the leader of this project. Both of the funders above had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests:
The funding of "Japan Racing Association", the project leader is our colleague, same laboratory. I am a member of this project. The present research used pocH-100iV Diff hematology analyzer (Sysmex, Hyogo, Japan) for hematological parameters analyze, and using a DRI-CHEM 3500s (Fujifilm, Japan) did blood biochemistry analyze. Both of which was purchased by above grant. But present research mainly done by the grant of "Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Scientific Research (C) (no. JP25517004)". I am the leader of this project. The corresponding author, on behalf of all authors declare no competing interests associated with this manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
We also used four adult Middle Yorkshire pigs (all male; mean body weight: 244.3 ± 11.0 kg). There was no adult pig control group; therefore, the hematological and biochemical indices were compared among pigs of the same age.

Radiocesium treatment
The pigs were fed radiocesium-contaminated haylage (620-709 Bq/kg Cs) consisting of Italian ryegrass grown from September 2010 to May 2011 in an ARSC field. This study was conducted 2.5 years after the 2011 Fukushima Daiichi nuclear power plant disaster; therefore, radioactivity levels have declined to some extent. The haylage was cut to a length of 1.3 cm using a power feed cutter (Cowa Cutter Inc., Mishima, Shizuoka, Japan) and mixed with concentrate (0.7 kg contaminated haylage + 0.7-1.5 kg concentrate). The control group feed consisted of uncontaminated Italian ryegrass (harvested 2013) haylage cut to a length of 1.3 cm and mixed with concentrate (0.7 kg uncontaminated haylage + 0.7-1.5 kg concentrate).

Feed and treatment period
Contaminated haylage (0.7 kg) was mixed with feed concentrate daily for 1 month, at which point the highest cumulative radiocesium level was expected to be reached. After 1 month of treatment, all pigs were provided only uncontaminated concentrate (1.5-2.2 kg/day) until the end of the study period.

Radiocesium transfer coefficients
The general formula for transfer coefficients of nuclide transfer from feed to animal products is defined as [5,6]: Transfer coefficient (%) = Concentration in meat or organ (Bq/kg)/Daily nuclide intake (Bq/animal/day)×100 The daily nuclide intake (Bq/day) is calculated as radiocesium concentration in feed (Bq/ kg) × feed intake (kg/day). A longer period is required for pig meat or organs to reach equilibrium. In the present study, pigs were fed contaminated feed for 1 month to reach equilibrium (or the highest cumulative radiocesium level).

Blood sample collection
Blood samples were collected from the jugular vein using an 18-gauge needle (Venoject II; Terumo, Tokyo, Japan) into 5-mL collection tubes containing EDTA-Na. The hematological parameters were analyzed using a pocH-100iV Diff hematology analyzer (Sysmex, Hyogo, Japan), and the blood tubes were then centrifuged for 20 min at 3,000 rpm and 4˚C to obtain plasma. To determine blood biochemistry parameters, the plasma was analyzed using DRI-CHEM slides (Fujifilm, Tokyo, Japan) and a DRI-CHEM 3500s blood biochemistry analyzer (Fujifilm).
Blood samples were collected from juvenile pigs six times, at 1 day before treatment (Day 0), 2 weeks after the start of treatment, at the end of treatment (1 month after the start of treatment), and then at 1, 3, and 5 months after the end of treatment. Blood samples were collected from adult pigs on the same schedule.

Tissue and organ sample selection
Tissue and organ samples were collected from the liver, spleen, kidneys, genitals, psoas major, bladder, thyroid, urine, thymus, tibia (left hind leg), and femur (left hind leg) of pigs following euthanization with Ravonal (Nipro ES Pharma Co. Ltd., Osaka, Japan) and exsanguination as described previously [7]. These methods were in accordance with the approved guidelines.
Organ and tissue samples were collected from juvenile pigs five times, at 1 month after the start of treatment, and then at 1, 3, 7, and 8 months after the end of treatment. Organ and tissue samples were collected from adult pigs four times, at 1 month after the start of treatment, and then at 1, 4, and 6 months after the end of treatment.
Radiocesium levels were determined as 134 Cs and 137 Cs counts at the Isotope Facility for Agricultural Education and Research at the University of Tokyo (Tokyo, Japan).
In the present study, we fed haylage contaminated with low levels of radiocesium to pigs over a 1-month period. This diet met the nutrition requirements for swine growth and maintenance according to the National Research Council. Juvenile pigs showed good growth and body weight gain, and adult pigs maintained good body condition throughout the experimental period. No health problems or illnesses were detected.

Body weight
In juvenile pigs, body weight gain was normal in both the treatment and control groups, with no difference between groups (Table 1). Adult pigs were found to be in good health, with normal body weights (Table 2).

Hematology and biochemistry
Hematological and biochemical indices for juvenile pigs in the treatment and control groups are shown in Tables 3 and 4, while those for adult pigs are shown in Tables 5 and 6. The WBC counts increased during the first month and then decreased. No similar trends were observed for RBCs or other hematological indices during the experimental period.
Few significant changes in the biochemical indices were detected throughout the study period in juvenile and adult pigs (Tables 4 and 6, respectively). ALP-P III differed greatly between the two growth stages; however, no medical condition was recognized. In adult pigs, the WBC counts were lower at 4 months after the end of treatment, but recovered at 6 months (Table 5) and no clinical symptoms associated with low WBCs were observed. After the first week of treatment, TP-P III and ALB-P decreased and NH3-P II increased in adult pigs.

Radiocesium levels in tissues and organs
In this study, radiocesium radioactivity was determined as total concentrations of 134 Cs and 137 Cs. Radiocesium levels in tissue and organ samples from juvenile pigs are shown in Fig 1. After 1 month of treatment, most samples showed high radiocesium levels, except for blood and bone samples. No radioactivity data were collected for the genitals of juvenile pigs, since the males were castrated at nursing. The highest radiocesium levels in the samples from juvenile pigs were found in the kidneys (105 Bq/kg), whereas the lowest were found in bone (tibia and femur) and were undetectable until 8 months after the end of treatment.
The radiocesium levels in the samples from adult pigs are shown in Fig 2. After 1 month of treatment, radiocesium contamination was detected in most organs, with the highest levels in the liver (44.8 Bq/kg) and kidneys (43.9 Bq/kg), and no detectable contamination in the tibia and femur. These results are consistent with those for the tissue and organ samples from the juvenile pigs. However, a comparison of Figs 1 and 2 shows greater radiocesium accumulation in juvenile pigs than in adult pigs.

Radiocesium transfer coefficients
In juvenile pigs, the radiocesium transfer coefficient ratios were highest in the kidneys (16.2%) ( Table 7). The transfer coefficient ratios were lower in blood and bone than in all other organs. The transfer coefficient ratios of juvenile pigs were roughly double those of adult pigs.

Discussion
Among juvenile pigs, ALP-P III was significantly different between the control and treatment groups; however, the highest ALP-P III value was a normal level [8] and no medical conditions

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were detected. The type of pasture has been shown to have a significant effect on cattle ALP-P III values, but not on other biochemical indices [9]. In the present study, pigs were fed haylage, although not within a pasture. ALP levels have been shown to vary widely with age and sex in children and adolescents [10]; therefore, the large variation observed in the control and treatment ALP levels in the present study may have been caused by factors other than radiocesium administration, especially considering the good health condition of the pigs. Adult pigs had lower WBC counts in the treatment group than in the control group; however, these differences were not accompanied by differences in RBC count and PLT level, and no clinical symptoms were detected. Our biochemical data showed that the TP-P III and ALB-P levels decreased and the NH3-P II levels increased after the first week of treatment, perhaps due to mixing haylage with concentrated feed, which was confirmed to affect the amino acid balance. Amino acid imbalances can negatively affect protein synthesis and increase protein metabolism, resulting in a high blood ammonia concentration. However, no symptoms of these processes were observed in the juvenile pigs.
After 1 month of treatment, when radiocesium accumulation was expected to be highest, the radiocesium levels were high in all organ samples from juvenile pigs, but lower in blood and bone. Tibia and femur samples require longer extraction times [1]. We obtained no genital radioactivity data for juvenile pigs in the present study because the males were castrated at

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nursing and the female ovaries were too small. Radiocesium retention in all organs of the adult pigs was lower than that in the juvenile pigs. Stable equilibrium was reached quickly in similar-sized voles [4] and after 1 month of continuous radiocesium administration in wild and captive hispid cotton rats [11]. Therefore, we expected that 1 month of radiocesium treatment would be sufficient for stable equilibrium in the present study. We also found that juvenile pigs had higher transfer coefficient rates than adult pigs, which suggests that age is an important factor in radiocesium retention. The radiocesium elimination time for both growth stages was 5-6 months or longer. Transfer coefficient rates have been found to be higher in lambs than in than adult sheep [12,13]. These results are consistent with those of the present study; however, effective Cs retention was found to be lower in younger mice, and progressively higher in older mice [14,15]. These inconsistent results may be due to differences in treatment duration since both older studies administered single-dose treatments.
The blood and bone samples had lower radiocesium levels than other organs in both adult and juvenile pigs. Radiocesium was not detected in adult bone samples 1 month after the end of treatment, when radiocesium accumulation was expected to be highest, but increased at 4 and 6 months post-treatment. Few studies have reported similar results, although Lei [4] found that the lowest radiocesium levels in Microtus canicaudus organ samples were detected in bone.
Get together the radiocesium transfer coefficient rates after 1 month of chronic oral treatment, showed higher radiocesium retention rates in the kidneys, liver, spleen, genitals, psoas major, bladder, thyroid, and urine than in the blood and bone (tibia and femur) of pigs at both growth stages. And the radiocesium retention levels were generally higher in juvenile pigs than in adult pigs, with the highest transfer coefficient ratio in the kidneys.