Animals and ethics

Carcasses of Japanese monkeys were provided by Fukushima City. Monkeys were culled as a measure against crop damage with the permission of the governor of Fukushima Prefecture, according to the Fukushima Japanese Monkey Management Plan which was established based on the Wildlife Protection and Hunting Management Law. Monkeys were captured using box traps and killed with a gun by licensed hunters at the request of Fukushima City. The capture and killing method was in accordance with the guidelines of the management plan stated above and should not be an ethical concern. This killing method was also in accordance with guidelines published by the Primate Research Institute, Kyoto University21. The Japanese monkeys inhabiting this study area were not listed as an endangered species on the Japanese Red List, as revised by the Ministry of the Environment in 201222.

Fetuses and muscle samples

Fetuses were collected from pregnant Japanese monkeys from 2008 to 2016. Carcasses were transported under refrigerated conditions to our laboratory and subjected to autopsy. The body weight of each monkey was measured in grams. During the autopsy after the NPP disaster, 500–1000 g of muscle tissue was collected from the hind limbs to measure radiocesium content. Skeletal muscle was used because organs weighing 500 g or more were required to measure radiocesium concentration. The muscle tissue was stored frozen at −30 °C until it was used for radioactivity measurements.

After the fetuses were removed from the uterus during autopsy, body weight was measured to the nearest gram, and crown rump length (CRL), the length of the fetus from the top of its head to bottom of torso, was measured to the nearest millimeter. CLR was most commonly used as the somatometric measures for age assessment in physical and neurological examination23.

Fetuses were preserved in 10% neutral buffered formalin solution. The fetuses analyzed including specimens whose CRL was 90 mm or greater (fetal age of about 3 months or greater) and whose crania were ossified, allowing external measurement. Fetal head size was defined as the product of the biparietal diameter and occipital frontal diameter. The biparietal diameter was one of the basic biometric parameters used to assess fetal size, and maximum width of the head. The occipital frontal diameter should be measured on maximum length between forehead and occipital region. All specimens were measured by the same person, and to the nearest 0.1 mm using a caliper.

The Japanese monkeys in Fukushima City were seasonal breeders that bear young in March and April17. Accordingly, the fetuses collected in 2011 were almost fully developed at time of the NPP disaster. Thus, the fetuses were divided into those conceived before 2011 (pre-disaster) and those conceived in 2011 or later (post-disaster) and compared. The sample comprised 31 fetuses conceived pre- disaster and 31 fetuses conceived post-disaster.

Fat index

During autopsy, fat indices were calculated to evaluate the monkeys’ nutritional status. In a previous study24, the ratio of mesenteric fat weight to body weight was proportional to the percentage of body fat in Japanese monkeys. The fat index was defined as mesenteric fat weight (g) divided by body weight (g), multiplied by 1000.

Radioactivity measurements

Muscle radiocesium concentration was measured in 31 mothers that had conceived after the disaster. The radioactivity of radiocesium in the muscle samples was analyzed with a germanium semiconductor spectrometer (GC2020-7500SL-2002 CSL, Canberra, Meriden, CT) and a NaI (T1) scintillation detector (AT1320A, Atometex, Minsk, Belarus). Data were corrected to a background radiation dose in the measurement environment on an as-needed basis. 134Cs was detected using 604.70 and 795.85 keV gamma-rays, whereas 137Cs was detected using 661.6 keV gamma-rays. The radioactivity of radiocesium was adjusted to the value on the day of capture based on its physical half-life. The limit of detection was 10 Bq/kg. Muscle cesium concentration was calculated as the combined concentration of 134Cs and 137Cs per kilogram of fresh muscle.

To estimate the external exposure in monkeys, we used measurements taken 1 m above the ground at two air dose monitoring sites maintained by Fukushima Prefecture at Iizuka (N37°49′33.7″, E140°26′52.8″) and Ohzaso (N37°47′11.6″, E140°24′10.8″) near the monkeys’ habitat in Fukushima City. Between April 2011, just after the NPP disaster, and May 2016, Fukushima Prefecture performed nine air dose measurements at these monitoring sites25. Mean accumulated external exposure doses were estimated to multiply the average value of air doses in two monitoring site by the number of days between consecutive measurement dates.

Statistics

All the monkeys who were undergoing fetal growth prior to the disaster were categorized in one “unexposed” group (pre-disaster), and those who underwent fetal growth after the disaster were classified as “exposed” (post-disaster) because there was no misclassification of this measure of exposure. The Shapiro-Wilk test was used to check for normality of each variable. The Student’s t-test was used to compare CRL, biparietal diameter, occipital frontal diameter, and head size to see if there was any difference between pre- and post-disaster groups, and the Wilcoxon-Mann-Whitney test was used to compare body weight between groups. Body weight growth rate was determined by using body weight divided by CRL, and the proportional head size was determined by using head size divided by CRL. The Student’s t-test was performed to compare the body weight growth rate and the Wilcoxon-Mann-Whitney test was performed to compare the proportional head size between pre- and post-disaster groups. Regression lines were generated describing the association of body weight and CRL, and head size and CRL. Multiple linear regressions were performed with dependent variables (body weight and head size) and explanatory variables (CRL and pre/post disaster) with an interaction term (pre/post disaster*CRL). Models were fit for each regression and tested using likelihood ratio test with and without interaction.

Stata/IC 13.1 (Stata Corp LP, College Station, Texas USA) was used for all analyses. For statistical estimation and inferences, two-sided hypothesis tests were used with a 5% significance level.

Significant differences in fat index were evaluated by the Wilcoxon rank-sum test.