Twin bearing ewes were randomly assigned and individually fed one of three treatments: 1) total mixed ration (TMR) (CON, n = 10), 2) 5-hydroxytryptophan (5-HTP, 2.5 mg/kg BW) and toxic tall fescue seed containing 1.77 mg ewe-1 d-1 ergovaline/ergovalinine (E+) in TMR (E+/5-HTP, n = 10), or 3) E+ tall fescue seed in TMR (E+, n = 10) from gestational day (gd) 86 to 110. At terminal necropsy (gd111), placental and fetal growth measurements were collected and analyzed as a completely randomized design. Maternal blood samples were collected over time and samples of rumen microbiome and fetal blood were collected at necropsy. Feeding E+ fescue seed reduced (P < 0.05) total fetal weight per ewe by 20% compared to CON with E+/5-HTP being intermediate. Supplementation of 5-HTP increased (P < 0.05) circulating 5-HTP and 5-HIAA but not 5-HT. Ergovaline was detected in the maternal and fetal blood samples collected at necropsy from E+ and E+/5-HTP fed ewes. Serum non-esterified fatty acid concentrations were greater (P < 0.05) in CON at the end of the study; however, serum glucose and plasma insulin concentrations did not differ. Individual caruncle and cotyledon weights differed by treatment and placentome type. Ewe rumen microbiome diversity was greater with E+/5-HTP compared to E+. Feeding E+ tall fescue seed to ewes from gd 86 to 110 reduced fetal growth; however, supplementation of 5-HTP partially mitigated the negative effects of E+. Further research is needed at higher dose levels of 5-HTP to fully mitigate the negative effects of E+ on fetal growth.
Skeletal muscle-derived signals contribute to intercellular communication with adipose tissue and may influence adipose tissue development; however, the role of exosomes in muscle–adipose crosstalk in sheep remains unclear. This study investigated whether myotube-derived exosomes regulate ovine preadipocyte proliferation and adipogenic differentiation through microRNA-mediated mechanisms. Myotube-preadipocyte interactions were examined using transwell co-culture, exosome isolation and characterization, small RNA sequencing, gain and loss function assays, dual-luciferase assays, and rescue experiments. Myotube co-culture promoted preadipocyte proliferation and suppressed adipogenic differentiation, as indicated by an increased proportion of 5-ethynyl-2′-deoxyuridine (EdU) positive cells and proliferation related markers (P < 0.05) and reduced lipid accumulation and adipogenic markers (P < 0.05). Myotube-derived exosomes, which had a mean diameter of approximately 105 nm and expressed cluster of differentiation 9 (CD9), cluster of differentiation 63 (CD63), and tumor susceptibility gene 101 (TSG101), were internalized by preadipocytes and reproduced these effects (P < 0.05). Small RNA sequencing identified microRNA-21 (miR-21) as the most abundant exosomal miRNA, accounting for approximately 20% of miRNA-mapped reads. Overexpression of miR-21 promoted preadipocyte proliferation and suppressed adipogenic differentiation, whereas miR-21 inhibition produced the opposite responses (P < 0.05). Dual-luciferase assays confirmed programmed cell death protein 4 (PDCD4) and nuclear factor I A (NFIA) as direct targets of miR-21. Rescue experiments showed that PDCD4 and NFIA overexpression attenuated the proliferative and anti-adipogenic effects of miR-21, respectively. Collectively, myotube-derived exosomal miR-21 promotes ovine preadipocyte proliferation by regulating PDCD4 and suppresses adipogenic differentiation through NFIA. These findings reveal an exosome-mediated mechanism underlying muscle-adipose crosstalk in sheep.
Feather elongation is sustained by continuous cell proliferation within the feather follicle; however, the molecular mechanisms responsible for feather growth termination remain poorly understood. In this study, we investigated molecular changes associated with feather growth termination by comparing two chicken wing feather types with contrasting growth characteristics: primary wing feathers and primary coverts. Histological and bromodeoxyuridine incorporation analyses demonstrated that primary wing feathers remained in an active growth phase, whereas primary coverts exhibited pulp regression, reduced cell proliferation, and characteristics of growth termination. Gene expression analyses showed that primary coverts exhibited reduced expression of prolactin receptor (PRLR), type 2 iodothyronine deiodinase (DIO2), and insulin-like growth factor 2 (IGF2), together with marked induction of the thyroid hormone (TH)-inactivating enzyme type 3 iodothyronine deiodinase (DIO3). In contrast, primary wing feathers exhibited relatively higher expression of PRLR, DIO2, and IGF2 than primary coverts, while maintaining consistently low DIO3 expression. Reporter assays showed increased IGF2 promoter activity following triiodothyronine (T3) treatment in all three independent experiments, although the increase did not reach statistical significance. Thus, the reporter assay provides only tentative support for T3 responsiveness of the IGF2 promoter, consistent with our previous in vivo and primary-culture findings showing T3-dependent increases in IGF2 expression. These coordinated transcriptional changes are consistent with remodeling of local TH metabolism during the transition from active feather growth to growth termination. While local TH concentrations and direct deiodinase activities remain to be quantified, and the causal relationship between PRL/PRLR signaling and DIO3 induction remains unresolved, this study provides a molecular framework for understanding how local endocrine regulation translates systemic hormonal signals into feather type-specific growth responses during avian feather development.
Plasma adrenocorticotropin (ACTH) concentration is measured to support a diagnosis of pituitary pars intermedia dysfunction (PPID). However, baseline ACTH and ACTH concentrations after IV administration of thyrotropin releasing hormone (TRH) vary with season and assay used. To compare two ACTH assays (Scantibodies immunoradiometric assay [IRMA] and Immulite 2000Xpi chemiluminescent immunometric assay [CIA]) for diagnosis of PPID, ACTH was measured before and 10 minutes after administration of TRH in three groups of horses: 1) four young horses (6 ± 3 years, no hypertrichosis and a pituitary gland [PG] histology score of 1-3/5); 2) eight aged horses (26 ± 6 years, no hypertrichosis and a PG histology score of 2-3/5); and 3) five PPID+ horses (27 ± 5 years, all with hypertrichosis and a PG histology score of 5/5). All horses underwent TRH stimulation in non-fall months with necropsy examination within 7 days. Baseline ACTH was elevated (>40 pg/mL) with IRMA in 0/4, 2/8, and 4/5 and with CIA in 0/4, 4/8, and 4/5 of young, aged, and PPID+ horses, respectively. After TRH administration, ACTH was elevated (>200 pg/mL) with IRMA in 0/4, 0/8, and 2/5 and with CIA in 1/4, 5/8, and 5/5 of young, aged, and PPID+ horses, respectively. Mean increases in ACTH after TRH administration were 13-, 18-, and 14-fold greater with CIA compared to IRMA in young, aged, and PPID+ horses, respectively. In conclusion, ACTH (or immunoreactive ACTH) concentration before and after TRH administration varies substantially with assay used and can affect PPID classification.
