Yingdong Ni

Layer and broiler chicks exhibit similar hypothalamic expression of orexigenic neuropeptides but distinct expression of genes related to energy homeostasis and obesity

Layer and broiler chickens demonstrate striking differences in body weight and body composition. However, the mechanism underlying such difference is elusive. Hypothalamus-pituitary-adrenal (HPA) axis regulates energy homeostasis and body size in mammals, but information in birds is scarce. Here we test the hypothesis that such breed difference is more associated with hypothalamic expression of genes related to HPA axis, rather than orexigenic neuropeptides. Broiler chicks exhibit significantly higher body weight and food intake at day (D) 7 posthatching, but the food intake relative to body weight gain was actually lower. No breed differences were observed for hypothalamic expression of neuropeptide Y (NPY), agouti-related protein (AGRP), proopiomelanocortin (POMC), orexin (ORX), leptin receptor (LEPR), acetyl-CoA carboxylase (ACC) or fatty acid synthase (FAS). However, broiler chicks expressed significantly higher glucocorticoid receptor (GR) mRNA (P<0.05) and protein (P<0.01) in hypothalamus compared to layer chicks, which is associated with lower corticotropin-releasing hormone (CRH) mRNA (P<0.05) yet higher accumulation of CRH peptide in hypothalamus, suggesting an augmented GR-mediated negative feedback regulation of CRH transcription and release in broiler chicks. Furthermore, fat mass and obesity associated (FTO) gene was also more highly expressed in hypothalamus of broiler chicks (P<0.05). These results suggest that the genes related to energy homeostasis and obesity, such as GR, CRH and FTO, rather than orexigenic neuropeptides, are impacted by the genetic selection practices and play a role in breed-specific body weight setpoint regulation in the chicken.

Embryonic exposure to corticosterone modifies aggressive behavior through alterations of the hypothalamic pituitary adrenal axis and the serotonergic system in the chicken.

Exposure to excess glucocorticoids (GCs) during embryonic development influences offspring phenotypes and behaviors and induces epigenetic modifications of the genes in the hypothalamic-pituitary-adrenal (HPA) axis and in the serotonergic system in mammals. Whether prenatal corticosterone (CORT) exposure causes similar effects in avian species is less clear. In this study, we injected low (0.2μg) and high (1μg) doses of CORT into developing embryos on day 11 of incubation (E11) and tested the changes in aggressive behavior and hypothalamic gene expression on posthatch chickens of different ages. In ovo administration of high dose CORT significantly suppressed the growth rate from 3weeks of age and increased the frequency of aggressive behaviors, and the dosage was associated with elevated plasma CORT concentrations and significantly downregulated hypothalamic expression of arginine vasotocin (AVT) and corticotropin-releasing hormone (CRH). The hypothalamic content of glucocorticoid receptor (GR) protein was significantly decreased in the high dose group (p<0.05), whereas no changes were observed for GR mRNA. High dose CORT exposure significantly increased platelet serotonin (5-HT) uptake, decreased whole blood 5-HT concentration (p<0.05), downregulated hypothalamic tryptophan hydroxylase 1 (TPH1) mRNA and upregulated 5-HT receptor 1A (5-HTR1A) and monoamine oxidase A (MAO-A) mRNA, but not monoamine oxidase B (MAO-B). High dose CORT also significantly increased DNA methylation of the hypothalamic GR and CRH gene promoters (p<0.05). Our findings suggest that embryonic exposure to CORT programs aggressive behavior in the chicken through alterations of the HPA axis and the serotonergic system, which may involve modifications in DNA methylation.

Long-term effects of subacute ruminal acidosis (SARA) on milk quality and hepatic gene expression in lactating goats fed a high-concentrate diet.

Purpose The mechanism underlying the decline in milk quality during periods of feeding high-concentrate diets to dairy ruminants is not well documented. The aim of this study was to investigate the metabolic changes in the liver that contribute to the input of substrate precursors to the mammary gland after feeding a high-concentrate diet to lactating goats for a long period. Experimental Design Eight mid-lactating goats with rumen cannulas were randomly assigned to two groups. For 9 weeks, the treatment group was fed a high-concentrate diet (60% concentrate of dry matter, HC) and the control group was fed a low-concentrate diet (40% concentrate of dry matter, LC). Ruminal fluid, plasma, and liver tissues were sampled, microarray techniques and real-time polymerase chain reaction were used to evaluate metabolic parameters and gene expression in liver. Results Feeding a 60%-concentrate diet for 9 weeks resulted in a significant decrease in rumen pH. Changes in fat and protein content also occurred, which negatively affected milk quality. Plasma levels of leptin (p = 0.058), non-esterified fatty acid (p = 0.071), and glucose (p = 0.014) increased markedly in HC group. Plasma cortisol concentration was significantly elevated in the treatment group (p<0.05). Expression of the glucocorticoid receptor protein gene was significantly down-regulated (p<0.05) in the liver. The expression of genes for interleukin 1β, serum amyloid A, C-reactive protein, and haptoglobin mRNA was significantly increased (p<0.05) in the HC group. GeneRelNet analysis showed that gene expression involved in inflammatory responses and the metabolism of lipids, protein, and carbohydrate were significantly altered by feeding a high-concentrate diet for 9 weeks. Conclusions Activation of the acute phase response and the inflammatory response may contribute to nutrient partitioning and re-distribution of energy in the liver, and ultimately lead to a decline in milk quality.

Effects of kisspeptin-10 on progesterone secretion in cultured chicken ovarian granulosa cells from preovulatory (F1–F3) follicles

The effect of kisspeptin-10 (Kp-10) on the secretion of progesterone (P(4)) was investigated in cultured granulosa cells from F(1) to F(3) follicles of hens. The results showed that granulosa cells were stained with clear signals for kisspeptin using immunocytochemistry with the specific antibody against Kp-10. Among 10, 100 and 1000 nM concentrations tested, 100 nM Kp-10 treated for 24h significantly increased P(4) secretion in granulosa cells from F(1) to F(3) follicles. After 24h and 48 h of treatment, 100 nM Kp-10 showed a significant increase in P(4) secretion, while after 72 h of treatment P(4) secretion was markedly decreased by Kp-10 compared to the control group (P<0.05). F(1) and F(2/3) cells treated with 100 nM Kp-10 for 24h showed significantly increased viability (P<0.05) and which was in parallel to a marked increase in P(4) secretion (P<0.01). Real-time PCR results showed that the gene expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450scc) and the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) in F(1) and F(2/3) granulosa cells was significantly up-regulated by 24h-100 nM Kp-10 treatment (P<0.05 versus P<0.01, respectively). However, there was no significant difference in StAR, P450scc and 3β-HSD protein content between control and the Kp-10 treated group (P>0.05). These results indicate that Kp-10 stimulates P(4) secretion in cultured chicken granulosa cells, which was associated with an up-regulation in StAR, P450scc and 3β-HSD gene transcription.

Leptin Is Involved in the Effects of Cysteamine on Egg Laying of Hens, Characteristics of Eggs, and Posthatch Growth of Broiler Offspring

Cysteamine has been reported to modulate energy homeostasis and exert significant growth-promoting effects in broiler chickens. However, little is known concerning its effects on egg production of hens and the growth rate of their offspring. In the present study, 67-wk-old broiler breeders were allotted at random to control and cysteamine-supplemented (400 mg/kg) groups for 8 wk. The hatchlings were fed under the same condition until 6 wk of age. Cysteamine significantly increased the average laying rate by 2.24% (P < 0.01), decreased dramatically the percentage of the broken eggs by 40.55% (P < 0.01), and increased that of the abnormal eggs by 20.15% (P < 0.05). Cysteamine did not alter the egg weight, egg quality, fertility, or hatch-ability but significantly increased eggshell weight (P < 0.05) and decreased albumin weight (P < 0.05). Serum concentrations of total thyroxine (P < 0.01) and leptin (P < 0.01) were significantly lower in cysteamine-treated hens, whereas total triiodothyronine (T(3)), free T(3), and glucagon were not affected. Western blot analysis with leptin-specific antibody detected a band of approximately 15 to 16 kDa in egg yolk and albumin extracts as well as in liver homogenates of hens. Cysteamine did not affect the yolk content of T(3), thyroxine, estradiol, or glucagon, but significantly increased leptin content in liver of hens (P < 0.05), as well as in yolk (P < 0.05) and albumin (P < 0.05) of eggs. These changes were accompanied by a significant downregulation of leptin receptor mRNA expression (P < 0.05) in the yolk sac of d-12 embryos. Female offspring hatched from cysteamine-treated eggs demonstrated significantly lower body weight at hatching (P < 0.01) and 42 d of age (P < 0.01). The results indicate that cysteamine improves laying performance of hens and affects the early posthatch growth of broiler offspring, in a sex-specific fashion. The modified leptin secretion and egg deposition, together with altered yolk sac leptin receptor expression, may be involved in such an effect.

Effect of early feed restriction on myofibre types and expression of growth-related genes in the gastrocnemius muscle of crossbred broiler chickens

The purpose of this study was to investigate the immediate and long-term effects of early feed restriction (ER) on morphology and gene expression of lateral gastrocnemius muscle. Newly hatched crossbred broiler chickens were allocated into control and ER groups, the latter being free-fed on alternate days from hatch to 14 days of age (14 d), followed by ad libitum feeding as the control group until 63 d. The lateral gastrocnemius muscle was taken at 14 and 63 d, respectively for myofibre typing by both myosin ATPase staining and relative quantification of myosin heavy chain (MyHC) mRNA for slow-twitch (SM), red fast-twitch (FRM) and white fast-twitch (FWM) myofibres. The body weight and lateral gastrocnemius weight were significantly lower in the ER group, accompanied by significantly reduced serum triiodothyronine. The ER group exhibited significantly higher SM and FRM MyHC expression at 14 d, but lower SM expression at 63 d. Myosin ATPase staining revealed a similar pattern. The percentage of SM was higher at 14 d while lower at 63 d in the ER group. These morphological changes were accompanied by changes of mRNA expression for growth-related genes. The ER group expressed lower insulin-like growth factor I (IGF-I) and higher IGF-I receptor (IGF-IR) at 14 d, yet significantly increased growth hormone receptor and IGF-IR mRNA at 63 d. These results indicate that ER may delay the slow to fast myofibre conversion as an immediate effect, but would result in a lower percentage of slow fibres owing to compensatory growth in the long term, which involves changes of mRNA expression for the growth-related genes in the muscle.

Effect of corticosterone on growth and welfare of broiler chickens showing long or short tonic immobility.

Tonic immobility (TI) test is commonly used to assess fear. Animals showing different TI durations demonstrate distinct behavior and biochemical responses to stress. However, less is known about how TI phenotype affects growth and welfare of domestic fowl. In this study, broiler chickens (Gallus gallus) were classified into short and long TI duration (STI and LTI) phenotypes and treated chronically with vehicle (CON) or corticosterone (CORT). STI broilers demonstrated significantly higher growth rate with higher breast muscle yield (P<0.05) and liver weight relative to BW tended to be lower (P=0.053), which was accompanied by higher serum concentration of CORT (P<0.05) and uric acid (P<0.01), but lower serum level of T4 (P=0.01). CORT severely reduced body weight, as well as the relative weight of muscle, bursa of Fabricius and spleen (P<0.001), but relative liver weight was increased (P<0.001). CORT-treated chickens had reduced serum CORT, elevated heterophile/lymphocyte ratio, and increased serum levels of total and free T3. STI broilers displayed more preening behavior (P<0.05), yet CORT elicited more walking behavior (P<0.05). No difference was observed in the welfare assessment scores between STI and LTI phenotypes under basal situation, while LTI chickens showed significantly increased incidence of pad dermatitis compared to STI under CORT exposure. The results suggest that STI broilers demonstrate better growth performance and higher adaptability to stress compared to LTI chickens.

Leptin stimulates hepatic activation of thyroid hormones and promotes early posthatch growth in the chicken.

Hepatic iodothyronine deiodinases (Ds) are involved in the conversion of thyroid hormones (THs) which interacts with growth hormone (GH) to regulate posthatch growth in the chicken. Previous studies suggest that leptin-like immunoreactive substance deposited in the egg may serve as a maternal signal to program posthatch growth. To test the hypothesis that maternal leptin may affect early posthatch growth through modifying hepatic activation of THs, we injected 5.0μg of recombinant murine leptin into the albumen of breeder eggs before incubation. Furthermore, chicken embryo hepatocytes (CEHs) were treated with leptin in vitro to reveal the direct effect of leptin on expression and activity of Ds. In ovo leptin administration markedly accelerated early posthatch growth, elevated serum levels of total and free triiodothyronine (tT3 and fT3), while that of total thyroxin (tT4) remained unchanged. Hepatic mRNA expression and activity of D1 which converts T4 to T3 or rT3 to T2, were significantly increased in leptin-treated chickens, while those of D3 which converts T3 to T2 or T4 to rT3, were significantly decreased. Moreover, hepatic expression of GHR and IGF-I mRNA was all up-regulated in leptin-treated chickens. Males demonstrated more pronounced responses. A direct effect of leptin on Ds was shown in CEHs cultured in vitro. Expression and activity of D1 were increased, whereas those of D3 were decreased, in leptin-treated cells. These data suggest that in ovo leptin administration improves early posthatch growth, in a gender-specific fashion, probably through improving hepatic activation of THs and up-regulating hepatic expression of GHR and IGF-I.

A high-concentrate diet induced colonic epithelial barrier disruption is associated with the activating of cell apoptosis in lactating goats

BackgroundIn ruminants, lower ruminal pH causes massive disruption of ruminal epithelial structure during periods of feeding high-concentrate diets. However, the influence of excessive organic fatty acids in the lumen of hindgut on the epithelial structure is unclear. In this study, twelve mid-lactating goats were randomly assigned to either a HC diet group (65% concentrate of dry matter; n = 6) or a LC diet group (35% concentrate of dry matter; n = 6) for 10 weeks. The colonic epithelial structure was detected by HE staining and transmission electron microscopy (TEM), and the apoptotic status of epithelial cells was estimated by TUNEL method and caspase activities.ResultsHC goats showed higher level of free lipopolysaccharide (LPS) in rumen fluid (p?<?0.01) but not in colonic digesta (p?>?0.05), and higher total volatile fatty acid (VFA) concentrations in rumen fluid (p?<?0.05) and in colonic digesta (p?<?0.01), and higher content of starch in colonic digesta (p?<?0.05) compared to LC goats. HC goats demonstrated profound alterations in the colonic epithelial structure and tight junctions (TJ), apparently due to damage of the epithelium with widened TJs space and nuclear breakdown and mitochondrial swelling. HC goats showed higher level of apoptosis in the colonic epithelium with higher proportion of TUNEL-positive apoptotic cells and increases of caspase-3 and ?3/7 activities, as well as the lower ratio of bcl-2/bax mRNA expression in the colonic mucosa (p?<?0.05). However, ?-defense mRNA was significantly down-regulated in the colonic mucosa of HC goats compared to LC (p?<?0.05). HC goats showed higher level of TJ proteins including claudin-1 and claudin-4 in the colonic mucosa than LC (p?<?0.05). Neither free LPS content in the colonic digesta nor NF-? B protein expression in tissues showed significant difference between HC and LC goats (p?>?0.05).ConclusionsOur results reveal that long-term feeding HC diet to lactating goats causes severe damages to the colonic mucosa barrier associated with activating cells apoptosis.

Coordinated miRNA/mRNA Expression Profiles for Understanding Breed-Specific Metabolic Characters of Liver between Erhualian and Large White Pigs

MicroRNAs (miRNAs) are involved in the regulation of various metabolic processes in the liver, yet little is known on the breed-specific expression profiles of miRNAs in coordination with those of mRNAs. Here we used two breeds of male newborn piglets with distinct metabolic characteristics, Large White (LW) and Erhualian (EHL), to delineate the hepatic expression profiles of mRNA with microarray and miRNAs with both deep sequencing and microarray, and to analyze the functional relevance of integrated miRNA and mRNA expression in relation to the physiological and biochemical parameters. EHL had significantly lower body weight and liver weight at birth, but showed elevated serum levels of total cholesterol (TCH), high-density lipoprotein cholesterol (HDLC) and low-density lipoprotein cholesterol (LDLC), as well as higher liver content of cholesterol. Higher serum cortisol and lower serum insulin and leptin were also observed in EHL piglets. Compared to LW, 30 up-regulated and 18 down-regulated miRNAs were identified in the liver of EHL, together with 298 up-regulated and 510 down-regulated mRNAs (FDR<10%). RT-PCR validation of some differentially expressed miRNAs (DEMs) further confirmed the high-throughput data analysis. Using a target prediction algorithm, we found significant correlation between the up-regulated miRNAs and down-regulated mRNAs. Moreover, differentially expressed genes (DEGs), which are involved in proteolysis, were predicted to be mediated by DEMs. These findings provide new information on the miRNA and mRNA profiles in porcine liver, which would shed light on the molecular mechanisms underlying the breed-specific traits in the pig, and may serve as a basis for further investigation into the biological functions of miRNAs in porcine liver.