427 Late-Breaking: Milk extracellular vesicles endogenously labeled with ZsGreen1 are bioavailable to the testis in vivo.

Abstract Boar fertility is critical for swine reproductive efficiency. Recent data demonstrates that neonatal colostrum intake is associated with enhanced sperm counts in adult boars. These data support the “Lactocrine Hypothesis,” which states that bioactive factors from maternal milk directly promote neonatal tissue development, including reproductive organs. Notably, maternal milk consumption increased the number of Sertoli cells within the testes of nursed versus formula-fed boars. However, the biological mechanisms mediating this effect are unknown. We hypothesized that milk extracellular vesicles (EVs) may contribute because EVs are abundant in milk, carry bioactive cargo (e.g., miRNAs), and protect these cargo from digestion before uptake by the intestinal mucosa and entry into the neonatal circulatory system. Our group previously demonstrated that maternal milk EVs bioaccumulate in distant neonatal tissues (e.g., brain), but testes were not evaluated. Recently, we reported that acute consumption of milk EVs alters the testicular proteome in neonatal boars, including key Sertoli cell markers (e.g., AMH, GATA-4, SOX-9). Together, these data suggest that milk EVs directly alter testis biology. The objective of this study was to determine the bioavailability of milk EVs to the neonatal testis using a transgenic swine model. Hemizygous transgenic sows ubiquitously expressing the fluorescent protein, ZsGreen1, and their male offspring, were utilized for this study. Milk was collected and pooled from 3 lactating (20–22 days post-partum) transgenic sows. Milk EVs were isolated via ultracentrifugation and filtration (220 nm). Milk EVs were validated via nano-flow cytometry (nFCM; size and concentration) and transmission electron microscopy (TEM; size and structure). The presence of ZsGreen1 (488 nm) in milk EVs was evaluated via nFCM and confocal microscopy. At necropsy, blood and testes were collected from wild type (WT) boars nursed (8–14 days) by transgenic (TSG) sows (n=4), WT boars nursed by WT sows (negative control; n=2) and a TSG boar nursed by a TSG sow (positive control; n=1). Serum was isolated for subsequent EV collection, validation, and fluorescent evaluation as described above for milk EVs. Testis samples were snap frozen and protein was extracted for HPLC/MS-MS. Results indicate that transgenic sows produce milk EVs endogenously labeled with ZsGreen1 compared with WT sow milk EVs. In addition, WT boars nursed by transgenic sows exhibited ZsGreen1-labeled milk EVs in their serum. Finally, ZsGreen1 was detectable in testes of WT boars nursed by TSG sows. The present study is the first to evaluate if maternal milk EVs are bioavailable to the neonatal testis. Using a novel swine model, these data suggest the transfer of maternal milk EVs to offspring blood and testes after a natural ingestion route (ad libitum suckling). Hence, milk EVs are bioavailable to the testis and may help mediate lactocrine programming.