Lauren Chen

Turkey breeder hens are artificially inseminated on a weekly basis throughout their production lifespan. After insemination, the sperm that is deposited in the vagina travels to the uterovaginal junction (UVJ) of the reproductive tract where it is stored in the sperm storage tubule (SST). Eventually, it travels through the oviduct to the infundibulum where fertilization of the oocyte occurs. Despite the repeated weekly inseminations, turkey hen fertility starts to decline around 8 to 10 weeks of production. The current study investigates the transcriptomic response of the UVJ to artificial insemination across the production lifespan of turkey breeder hens. Turkey hens were divided into 2 groups, sham and semen insemination, and were inseminated weekly for 27 weeks. UVJ tissues were collected from both groups (n=4/group) at the start of lay, peak lay (8 week of lay, WOL), and end of lay (27 WOL). Total RNA was extracted and sent for sequencing. Differential gene expression was performed through pairwise comparisons (Bonferroni and False Discovery Rate corrected) between sham and semen inseminated groups and among laying periods. For all comparisons, q-values <0.05 and log2 fold change >2 were considered significant. A significant increase of avian beta-defensin (AvBD2, AvBD4, and AvBD11) and cathelicidin (CATH2 and CATH3), together with a decrease in the interferon regulatory factor (IRF7) were observed in semen-inseminated group at peak lay compared to semen-inseminated groups at the end of lay. Interferon-inducible proteins (IFIT5 and IFI27L2B) were increased in semen-inseminated group at the end of lay compared to semen-inseminated group at peak lay. Defensins and cathelicidins are antimicrobial peptides. Despite their importance in innate immunity, defensins have been reported to play a role in sperm function and fertility. There is evidence showing that interferons impair sperm viability and induces sperm apoptosis. Further, immunohistochemical staining of CD3 antibody was carried out to determine CD3+ T cells count in the SST of those samples (n=5/group). Using ImageJ software, the number of T cells were counted and calculated per area of mucosal epithelium and submucosa and per number of SSTs. Data were analyzed using a two-way ANOVA with lay and insemination status as treatment levels. Results show that the number of T cells in the mucosal epithelium and submucosa tended to be lower in peak of lay compared to end of lay (289 ± 29 to 844 ± 250, p=0.1) and the number of T cells in SSTs significantly lowed in peak lay compared to end of lay (10.5 ± 8.4 to 29.2 ± 7.6 cells/50 SST) of semen-inseminated groups (p=0.01) while there are no significant changes observed in sham-inseminated groups across laying periods. Taken together, the increase in avian defensins, the decrease in interferon-related factors, and the low number of T cells in the SSTs during the peak lay may be a key response of the UVJ assisting sperm function and storage. Whereas their increase at peak lay suggests that local immune cells in the UVJ may mount a T-cell mediated attack on sperm in SSTs.