Our investigations focused on serum thyroid hormone levels at 11 DPI in piglets, as T3 hormone levels were most extremely suppressed at this timepoint post PRRSV challenge (Pasternak et al., 2021 (link)). Fetal T3 and T4 levels were measured on sera collected on day of termination at 12 or 21 DPMI, depending on the animal experiment the fetuses were derived from (Pasternak et al., 2020b (link)). There are two primary reasons we have not used the T3:T4 ratio in our analysis. The first is that this value is typically evaluated in the human clinical setting, particularly when evaluating the response to levothyroxine treatment. The value in this measure is derived from an established ratio of production (1:13) in the healthy thyroid, however such a ratio has not been effectively established in a healthy fetal pig throughout gestation. The second stems from our past investigations into fetal thyroid hormone response to PRRSV (Pasternak et al., 2020b (link); Ko et al., 2022 (link)) which indicates that the relative response of these two hormones is dependent on fetal phenotype. In short fetuses classified as viable show a decrease in both T3 and T4 while those classified as meconium stained primarily have a decrease in T4 while maintaining near normal levels of T3. A genome-wide association study (GWAS) analysis on the ratio is therefore likely to identify SNPs associated with the phenotype rather than those directly associated with thyroid hormone. All sera was stored at −20°C or −80 °C, respectively, until used to test total T3 (piglet_T3, or fetal_T3, ng/dL) or T4 (fetal_T4, µg/dL) levels using commercial RIA kits (MP Biomedical, Irvine, CA) as previously described (Pasternak et al., 2020b (link)).
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