Ceramide accumulation and up-regulation of proinflammatory interleukin-1β exemplify lipotoxicity to mediate declines of reproductive efficacy of broiler hens

Feed Intake Alters Immune Cell Functions and Ovarian Infiltration in Broiler Hens: Implications for Reproductive Performance1

“…We were the first to recognize that feed-satiated broiler hens also developed obesityassociated dyslipidemia similar to humans with metabolic syndrome, and exhibited some ovarian manifestations that resembled those of women with polycystic ovary syndrome (PCOS); namely enlarged cystic ovarian follicles in association with inflammation and follicle atresia [8][9][10]. We next showed that feed-satiated broiler hens experienced in situ lipotoxic and proinflammatory changes including ceramide accumulation, increased IL-1β production, immune cell infiltration in conjunction with reduced Akt activity in the ovarian hierarchical follicles [11,12]. Based on these observations we hypothesized that immune cell functions in broiler hens could also be influenced by feed intake and may contribute to ovarian dysfunction observed in feed-satiated broiler hens.…”

“…All bird husbandry and tissue collections were conducted in accordance with an approved animal care protocol in the National Chung University, Taiwan. Details of the management of the flock were described previously [11]. Starting at age 26 wk, 30 birds were continued with breeder recommended restricted feeding protocol [13] and were designated as Restricted; R, while the remaining 60 birds were provided with sufficient feed for ad libitum consumption and were designated as Ad libitum; AL.…”

“…Cellular and plasma ceramide content were determined spectrofluorometrically following alkaline hydrolysis of sample ceramide to free sphingoid bases at 100° C for 150 min and derivatization with naphthalene-2, 3-dicarboxyaldehyde [11,21,22]. The derivatized compounds were separated using an HPLC system comprised a pump (LC-10AD, Shimadzu), reverse phase column (LiChroCART100RP, Merck, Germany), fluorescence detector (F-4500, Hitachi, Japan), and data analysis system (FL solution, Hitachi, Japan).…”

“…Total ceramide content was calculated by subtracting the sphingoid base content of unhydrolzyed samples from the amounts found in hydrolyzed samples. A TLC-GC (thin layer chromatographygas chromatography) method was used for cellular and plasma sphingomyelin separation and quantification exactly as described in our previous report [11] Respiratory Burst and Phagocytosis Analysis Generation of oxidative radicals was measured using a luminol-dependent chemiluminescence method [23]. Briefly, isolated heterophils or monocytes (2 × 10 6 cells) were incubated with 1 mL of freshly made luminol (4 × 10 -4 M, in HBSS containing 0.05 M Na 2 CO 3 , 0.3 M NaHCO 3 , 5 mM NH 4 CO 3 , and 1.5 mM CuSO 4 ) in the absence or presence of phorbol 12-myristate 13-acetate (PMA, final concentration 5 ng/mL) (Sigma, St. Louis, MO).…”

“…The concentrated medium or pieces of collected follicle walls devoid of the granulosa layer were used for total protein extraction using RIPA buffer (0.15 M NaCl, 0.1% SDS,0.5% sodium deoxycholate, 1% NP-40, 50 mM Tris-HCl; pH 7.4, protease inhibitor cocktail comprising 2 mM PMSF, 50 mM NaF, 10 mM Na 4 P 2 O 7 ,and 2 mM Na 3 VO 4 ). Western blot analysis was conducted as described previously [11,12]. A rabbit anti-chicken IL-1β primary antibody was purchased from Abcam (Cambridge, UK).…”