Oral gavage (to mimic ingestion) of low-dose THC

This study was reviewed and approved by the Duke Institutional Animal Care and Use Committee and conducted in accordance with federal guidelines. Duke University maintains an animal program that is registered with the USDA, assured through the NIH/PHS, and accredited with AAALAC International. Adult male nine-week-old sexually mature Sprague–Dawley rats were exposed to THC via oral gavage at a dose of 2 mg/kg/d (n = 8) versus vehicle (ethanol) control (n = 7) for 12 days (Fig. 6A). These rats were mated with adult female THC-naïve rats, and their offspring (n = 51) were raised for 24–30 days, sacrificed, and tissues harvested (Fig. 6B). For the original THC-exposed males, epididymal harvest was performed, and the sperm were collected via the swim-out method in PBS to enrich for mature motile sperm. The sperm were stored at −80 °C until used for nucleic acid extraction.

Experimental design. (A) Exposure of sperm via gavage: adult male rats were exposed to either vehicle or THC (2 mg/kg/d) via oral gavage. Exposed sperm were isolated, DNA extracted, and reduced representation bisulfite sequencing (RRBS) performed for candidate gene identification. Bisulfite pyrosequencing of the sperm was then carried out to confirm RRBS findings. (B) Heritability: Oral gavage-exposed males were mated with exposure-naive females, and pyrosequencing was then performed on the cerebral cortex of the F1 generation. (C) Exposure via injection: adult male rats were exposed to either vehicle, THC (2 mg/kg/d), or THC (4 mg/kg/d) via subcutaneous injection. Sperm were then isolated from these exposed males, and bisulfite pyrosequencing was carried out.