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Endodermal epithelial cells (EECs) within the yolk sac membrane (YSM) of avian embryos are responsible for the absorption and utilization of lipids. The lipids in the yolk are mostly composed of very low density lipoprotein (VLDL), uptake mainly depends on clathrin-mediated endocytosis (CME). The CME relies on vesicle formation through the regulation of dynamin (DNM). However, it is still unclear whether DNMs participate in avian embryonic development. We examined mRNA expression levels of several genes involved in lipid transportation and utilization in YSM during Japanese quail embryonic development using qPCR. The mRNA levels of DNM1 and DNM3 were elevated at incubation d 8 and 10 before the increase of SOAT1, CIDEA, CIDEC, and APOB mRNA's. The elevated gene expression suggested the increased demand for DNM activity might be prior to cholesteryl ester production, lipid storage, and VLDL transport. Hinted by the result, we further investigated the role of DNMs in the embryonic development of Japanese quail. A DNM inhibitor, dynasore, was injected into fertilized eggs at incubation d 3. At incubation d 10, the dynasore-injected embryo showed increased embryonic lethality compared to control groups. Thus, the activity of DNMs was essential for the embryonic development of Japanese quail. The activities of DNMs were also verified by the absorptions of fluorescent VLDL (DiI-yVLDL) in EECs. Fluorescent signals in EECs were decreased significantly after treatment with dynasore. Finally, EECs were pretreated with S-Nitroso-L-glutathione (GSNO), a DNM activator, for 30 min; this increased the uptake of DiI-yVLDL. In conclusion, DNMs serve a critical role in mediating lipid absorption in YSM. The activity of DNMs was an integral part of development in Japanese quail. Our results suggest enhancing lipid transportation through an increase of DNM activity may improve avian embryonic development., (Copyright © 2021. Published by Elsevier Inc.)

Fatty liver diseases, common metabolic diseases in chickens, can lead to a decrease in egg production and sudden death of chickens. To solve problems caused by the diseases, reliable chicken models of fatty liver disease are required. To generate chicken models of fatty liver, 7-week-old ISA female chickens were fed with a control diet (17% protein, 5.3% fat, and 1,300 mg/kg choline), a low protein and high fat diet (LPHF, 13% protein, 9.1% fat, and 1,300 mg/kg choline), a high cholesterol with low choline diet (CLC, 17% protein, 7.6% fat with additional 2% cholesterol, and 800 mg/kg choline), a low protein, high fat, high cholesterol, and low choline diet (LPHFCLC, 13% protein, 12.6% fat with additional 2% cholesterol, and 800 mg/kg choline) for 4 wk. Our data showed that the CLC and LPHFCLC diets induced hyperlipidemia. Histological examination and the content of hepatic lipids indicated that the CLC and LPHFCLC diets induced hepatic steatosis. Plasma dipeptidyl peptidase 4, a biomarker of fatty liver diseases in laying hens, increased in chickens fed with the CLC or LPHFCLC diets. Hepatic ballooning and immune infiltration were observed in these livers accompanied by elevated interleukin 1 beta and lipopolysaccharide induced tumor necrosis factor mRNAs suggesting that the CLC and LPHFCLC diets also caused steatohepatitis in these livers. These diets also induced hepatic steatosis in Plymouth Rock chickens. Thus, the CLC and LPHFCLC diets can be used to generate models for fatty liver diseases in different strains of chickens. In ISA chickens fed with the CLC diet, peroxisome proliferator-activated receptor γ, sterol regulatory element binding transcription factor 1, and fatty acid synthase mRNAs increased in the livers, suggesting that lipogenesis was enhanced by the CLC treatment. Our data show that treatment with CLC or LPHFCLC for 4 wk induces fatty liver disease in chickens. These diets can be utilized to rapidly generate chicken models for fatty liver research., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Geese have a short egg-laying period and a low egg production rate. To induce and maintain egg laying, genes related to generating hepatic lipid for yolk deposition should be adequately expressed. Liver mRNA from 6 laying geese was extracted and used for construction of a full-length enriched cDNA library. About 2,400 clones containing gene sequences were determined and National Center for Biotechnology Information Gallus gallus Gene Index databases were used to compare and analyze these sequences. Ten highly expressed genes were selected to determine the differential expression between laying and prelay goose liver. Tissue distribution data showed that very low density apolipoprotein II, liver type fatty acid binding protein, vitellogenin I, and vitellogenin II transcripts were specifically expressed in the liver of laying geese. Ovoinhibitor, preproalbumin, alpha-2-hs-glycoprotein, and vitamin D binding protein mRNA were highly expressed in the liver and to a lesser extent in other tissues. Ovotransferrin mRNA was expressed in liver, ovary, oviduct, shell gland, brain, and adipose tissues. The concentration of transthyretin mRNA was high in the liver and brain. The mRNA concentrations of liver type fatty acid binding protein, alpha-2-hs-glycoprotein, and transthyretin in the livers of laying and prelay geese were not different. The concentrations of hepatic ovotransferrin, ovoinhibitor, preproalbumin, very low density apolipoprotein II, vitellogenin I, vitellogenin II, and vitamin D binding protein mRNA were higher in the liver of laying geese than in prelay geese, suggesting that these genes may be involved in laying function or lipid metabolism related to egg formation.

Suppression subtractive hybridization was used to detect differential expression of genes in the livers of laying and prelaying geese. Liver tissues from prelaying and laying geese were dissected for mRNA extraction. The cDNA, reverse transcribed from liver mRNA of prelaying geese, was subtracted from the cDNA generated from the laying geese (forward subtraction). Five hundred seventy-six clones with possible differentially expressed gene fragments were observed by forward subtraction hybridization. After differential screening using the reverse and forward subtraction cDNA, 164 clones were subjected to gene sequence determination and further analysis. Using Northern analysis, 5 known and 8 unknown genes were shown to be highly expressed in the livers of laying geese compared with prelaying geese. Vitellogenin I, apoVLDL-II, ethanolamine kinase, G-protein gamma-5 subunit, and leucyl-tRNA synthase were highly expressed in the livers of laying geese compared with that from the prelaying geese (P<0.05). The expression of these known genes suggests that their function in the liver of laying geese is primarily involved in lipid and lipoprotein metabolism. Several of these differentially expressed genes were found to be responsive to estrogen stimulation, confirming the involvement of these genes in the egg-laying function of the goose.

The purpose of this study was to detect differential expression of genes in the pituitary gland in laying geese by suppression subtractive hybridization (SSH). Pituitary glands from prelaying and laying geese were dissected for mRNA extraction. The cDNA from pituitary glands of prelaying geese was subtracted from the cDNA from the pituitary glands of laying geese (forward subtraction); the reverse subtraction was also performed. We screened 384 clones with possible differentially expressed gene fragments by differential screening. Sixty-five clones from the differential screening results were subjected to gene sequencing and further analysis. We found that at least 19 genes were highly expressed in the pituitary glands of laying geese compared with prelaying geese. Among these, 6 genes (including 4 novel genes) were confirmed by virtual Northern analysis. We found that prolactin and visinin-like protein were highly expressed in the pituitary glands of laying geese compared with prelaying geese (P < 0.05). Further investigation is needed to demonstrate specific functions of the novel genes discovered in the current study.

Sterol regulatory element binding protein 1 (SREBP1) drives the expression of several lipogenic genes, whereas SREBP2 dictates the expression of every gene involved in cholesterolgenesis in mammals. In the current study, we cloned the cDNA fragments for SREBP1, SREBP2, fatty acid synthase (FAS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), and very low density apolipoprotein-II (apoVLDL-II), the genes associated with lipid metabolism. Fifteen ducks immediately before the first egg was laid (18 wk old) and 15 ducks from the same population at an egg production rate of 80% were killed. Total RNA was extracted from liver and used to amplify the targeted genes by reverse transcription-PCR and screening of a cDNA library. The sequence data showed that Tsaiya duck SREBP1, SREBP2, FAS, and HMG-CoA reductase were highly homologous to that of chicken. Tsaiya duck SREBP1 mRNA was expressed in adipose tissue, cardiac muscle, skeletal muscle, liver, and ovary. The SREBP2 mRNA concentration was highest in liver and ovary. Concentrations of FAS and HMG-CoA reductase mRNA were high in liver and lower in other tissues. The apoVLDL-II mRNA was specifically expressed in the liver. The differences between mRNA concentrations of SREBP1, SREBP2, and FAS in the livers of laying and prelay ducks were not significant. However, the concentrations of hepatic HMG-CoA reductase and apoVLDL-II mRNA were higher in the laying ducks than in prelay ducks. Therefore, laying may affect particular aspects of lipid metabolism, especially biochemical pathways that involved apoVLDL-II and HMG-CoA reductase.

Liver basic fatty acid (FA)-binding protein (Lb-FABP) cDNA was cloned from the livers of laying Tsaiya ducks and used to generate probes for quantification of the Lb-FABP mRNA in Tsaiya ducks. The full-length Lb-FABP cDNA of the Tsaiya duck was highly homologous with that of the mallard (99%), chicken (88%), and iguana (73%). The amino acid sequence was also highly homologous to Lb-FABP found in birds and reptiles, indicating a similar function of the Tsaiya duck Lb-FABP to those species. The calculated molecular weight for the cloned duck Lb-FABP was 14,043g/mol. The Lb-FABP was highly expressed in the liver of laying Tsaiya ducks and not detectable in heart, ovary, intestine, or adipose tissues. The expression of Tsaiya duck Lb-FABP in the skeletal muscle was also detected, and the sequence was confirmed. The greater expression of the hepatic Lb-FABP in the egg-laying Tsaiya ducks than the prelaying ducks paralleled the higher FA use by the laying ducks. These results suggest that hepatic Lb-FABP may be needed for egg production when FA metabolism is high for the ducks. Feeding laying Tsaiya ducks with diets enriched with 2% docosahexaenoic acid (DHA) oil for 2 wk significantly increased hepatic DHA content compared with in ducks fed a 2% butter basal diet. There was no effect of dietary DHA enrichment on the expression of Lb-FABP in the liver of Tsaiya ducks. The results suggest that even though the Lb-FABP may be involved in hepatic FA metabolism, the effect of individual FA on liver Lb-FABP in laying Tsaiya ducks needs to be further studied.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the formation of cholesterol esters (CE) from free cholesterol and fatty acyl-coenzyme A. This experiment was conducted to study the ontogeny of ACAT activity in the yolk sac membrane, liver, and intestine during embryonic development and early posthatch growth of turkeys. The ACAT activity was measured on tissue samples collected at 3-d intervals from embryonic Day (ED 13) 13 through 6 d posthatch (PD 6). The ACAT activity (pmol/mg microsomal protein per min) in the yolk sac membrane increased form 840 pmol at ED 13 to 2,497 pmol at ED 22, and subsequently declined to a very low level by PD 3. The high level of enzyme activity at ED 22 is concomitant with the large quantity of CE formed within the yolk sac membrane at this developmental age. Liver ACAT activity increased from 60 pmol at ED 13 to 242 to 243 pmol at ED 25 and PD 3, followed by a decline to 130 pmol by PD 6, mirroring the peak in hepatic CE concentration. This suggests that even during incubation, the liver plays a significant role in lipid metabolism. Intestinal ACAT specific activity increased from 14 pmol (ED 16) to 44 pmol (ED 25), and then declined to 23 pmol by hatch (ED 28), with no further decline through PD 6. Total intestinal ACAT activity (pmol per intestine/min) increased, however, from ED 16 through PD 6. This increase in activity suggests that the total capacity for cholesterol esterification increases during the course of incubation and shortly after hatching.

In two studies, changes in lipids and fatty acids from the yolk sac and liver of turkeys were determined during incubation and early postnatal growth. In Experiment 1, embryos were collected from Day 13 through 6 d posthatch. Embryos weighed 4.25 g at Day 13, 52.8 g at hatch (Day 28), and 104 g at 6 d. Total yolk lipid (grams per yolk sac) decreased from 9.48 g at Day 13 to 2.52 g at hatch (Day 28). Almost half (47%) of the yolk lipid decreased during the last week of incubation. In Experiment 2, embryos were collected daily from Day 22 through 2 d posthatch. Embryonic weight increased from 32.5 g at Day 22 to 55.8 g at hatch (Day 28), 3 g more than in Experiment 1. On Days 22 and 25, total yolk lipid (grams per yolk sac) was slightly less in Experiment 2 than in Experiment 1, but at hatch (Day 28) there were considerable differences (Experiment 1, 2.52 g; Experiment 2, 0.63 g), concomitant with increased hatch weight in Experiment 2. Liver DM percentage and ether extractable lipid increased as incubation proceeded. Oleic acid comprised the largest proportion of total yolk fatty acids and of liver fatty acids. There was a small but significant increase in yolk sac oleic acid (percentage of total fatty acids) between Day 22 (40%) and hatch (Day 28; 45.4%) and a much larger increase in liver oleic acid (46.6% to 56.5%). The absolute amount of all yolk sac fatty acids declined greatly during the second half of incubation through 6 d posthatch.

Lipid changes in the yolk sac of the embryo were studied in a randombred population of turkeys (RBC2) and a subline of the RBC2 selected for increased BW at 16 wk (F line). Comparisons of yolk sac and embryonic development were made between 22 d of incubation and hatch (28 d). Poults from the F line had heavier yolk sacs from 24 to 28 d and yolk free body weight was also heavier at hatch (61.1 vs 52.8 g). Yolk sac lipid (percentage of DM) declined faster in F line embryos (69 to 39%) compared with the RBC2 line (62 to 48%). In both lines, embryonic liver dry matter and lipids (percentage of DM) were similar. Yolk sac neutral lipids increased from 22 to 28 d (70 to 80% total lipid) in both lines and there was a concomitant decline in phospholipids (30 to 20%). The direction of the changes was similar for embryonic liver lipid. At 26 and 28 d, there were significantly increased neutral lipids (94 vs 88%) and decreased phospholipids (6 vs 12%) in RBC2 compared with F line embryonic livers. The concentration of cholesterol esters (percentage of total lipid) increased in the yolk sac and embryonic liver during the course of incubation. At 26 and 28 d, livers from RBC2 embryos had increased cholesterol ester concentration compared with livers from the F line.(ABSTRACT TRUNCATED AT 250 WORDS)