Programming effects of intrauterine hyperthermia on adrenal gland development

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Animals and Adrenal Gland Collection Procedures. Multiparous pregnant Holstein cows, in their dry period (preceding lactation), were exposed to environmental heat stress (Temperature-Humidity Index > 68) with access to shade and natural ventilation (HS, n = 41), or provided active cooling (CL) via access to shade plus fans, and water soakers (n = 41) to maintain thermoneutrality. The treatments of heifers born to these cows were reflected by their treatment during the dry period. Thus, female calves (heifers) born to HS dams were considered HS, and heifers born to CL dams were considered CL. Unless assigned to euthanasia at birth (d 0), heifers were fed 3.78 L if high-quality colostrum (protein concentration > 21%) within 2 h of birth (0.8 ± 0.5 h). Prophylactic interventions and vaccinations were performed according to the farm standard operating protocols and are available at Dado-Senn et al. (2021). Six heifers from each treatment were euthanized at birth (day 0). The remaining heifers were raised as a cohort, receiving the same postnatal management and nutrition, to isolate the in utero effects. A second subset of six heifers per group was euthanized one week after weaning (day 63). Adrenal glands were located above the kidneys and carefully dissected and washed in sterile PBS. The right adrenal was fixed in 10% NBF, and the left adrenal was bisected and snap-frozen in liquid nitrogen for RNA-sequencing.

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Adrenal glands were harvested and dissected form dairy calves euthanized day 0 (at birth) and on day 63 of age and srtored in 10% NBF overight or in RNA later at -80 degree celcius. Supplemental Figure S1. Adrenal gland tissue was paraffin embeded and section via microtome in 5um slices and stained for hematoxylin and eosin following standard procedures. Cell proliferation was assessed via immunohistochemistry (Ki67 mouse anti-human Ki67, DAKO #M7240, clone MIB-1) and quantified according to Field et al. (2021). Supplemnetary Tables S1-S4. RNA extraction, library preparation, gene expression and pathway analysis. Total RNA was extracted from approximately 5 g of adrenal gland tissue using the TRIzol® (Invitrogen Corp, Carlsbad, CA.). . Libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit for Illumina (#E7775; New England BioLabs, Ipswich, MA) following the manufacturer’s protocol. The libraries were sequenced using the Illumina NovaSeq 6000 platform (Illumina Inc., CA), generating 150 base-pair paired-end reads. The RNA-Seq data can be accessed by NCBI GEO with accession number GSE235160. Differentially expressed genes were detected using the R package edgeR Differentially expressed genes (FDR ≤ 0.20) were uploaded into the IPA software (QIAGEN Inc., http://www.qiagenbioinformatics.com34) to determine the association of DEGs with Canonical Pathways and Upstream Regulators.

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