In ovo leptin administration accelerates post‐hatch muscle growth and changes myofibre characteristics,gene expression and enzymes activity in broiler chickens

To evaluate the effect of maternal leptin on muscle growth, we injected 0 μg (control, CON), 0.5 μg (low leptin dose, LL) or 5.0 μg (high leptin dose, HL) of recombinant murine leptin dissolved in 100 μl of PBS into the albumen of broiler eggs prior to incubation. The newly hatched chicks were all raised under the same conditions until 21 days of age (D21), when body weight was measured and samples of gastrocnemius muscle were collected and weighed. Myosin ATPase staining was applied to identify myofibre types and measure the cross‐sectional area (CSA) of myofibres. Real‐time PCR was performed to quantify leptin receptor (LEPR), insulin‐like growth factor 1 (IGF‐1), IGF‐1 receptor (IGF‐1R), growth hormone receptor (GHR) and myostatin (MSTN) mRNA expression in the gastrocnemius muscle. The activity of calpains (CAPNs) in the gastrocnemius muscle was measured using a quantitative fluorescence detection kit. Male chickens treated with both high and low doses of leptin had significantly higher (p < 0.05) body weight on D21. The high leptin significantly increased the CSA (p < 0.05) of gastrocnemius muscle in male chickens, which coincided with a 93% increase (p < 0.05) in IGF‐1 mRNA expression. Likewise, the LL dose increased the weight of gastrocnemius muscle in male chickens (p < 0.05), which was accompanied by a 41% down‐regulation (p < 0.05) of MSTN mRNA expression and a decreased activity of CAPNs. However, all these changes were not observed in female chickens. The proportion of myofibre types did not altered. No significant change was detected for LEPR and GHR mRNA expression. These results indicate that in ovo leptin treatment affects skeletal muscle growth in chickens in a dose‐dependent and sex‐specific manner. The altered expression of IGF‐1, MSTN mRNA and activity of CAPNs in skeletal muscle may be responsible for such effects.     

Effects of age and strain on the expression of leptin receptor, neuropeptide Y and pro-opiomelanocortin in the hypothalamus of young chickens

1. An experiment was conducted to study changes in the expression of the hypothalamic leptin receptor, neuropeptide Y (NPY) and proopiomelanocortin (POMC) with age during the early neonatal period in two different strains of chickens: Beijing-You (BY) and Arbor Acres (AA). 2. Compared with BY chickens, AA chickens ate more, and grew faster. Hypothalamic NPY concentrations of both strains increased with age until d 7 followed by a decline. Hypothalamic NPY of BY chickens on d 7 was lower than in AA chickens at the same age. 3. No difference with age was observed in hypothalamic α-melanocyte-stimulating hormone (α-MSH) of BY chickens, while hypothalamic α-MSH in AA chickens on d 0 was higher than on the other days. Compared with AA chickens, BY chickens showed lower hypothalamic α-MSH on d 0. 4. Similar developmental changes between two strains were observed in the expression of leptin receptor, NPY or POMC genes in the hypothalamus. Hypothalamic mRNA of leptin receptor on d 0 was higher than on d 1 and 7. Unlike NPY, hypothalamic NPY mRNA on d 0 was higher than on the other days. 5. Hypothalamic POMC mRNA decreased gradually with age until 7 d followed by a slight increase. 6. The results showed that the developmental changes of hypothalamic signal molecules varied with age and strain. NPY, α-MSH and leptin receptor might be involved in the early programming of feed intake in newly hatched chickens.     

Leptin mRNA expression and serum leptin concentrations as influenced by age, weight, and estradiol in pigs

Two experiments (EXP) were conducted to determine the roles of age, weight and estradiol (E) treatment on serum leptin concentrations and leptin gene expression. In EXP I, jugular blood samples were collected from gilts at 42 to 49 (n = 8), 105 to 112 (n = 8) and 140 to 154 (n = 8) d of age. Serum leptin concentrations increased (P < 0.05) with age and averaged 0.66, 2.7, and 3.0 ng/ml (pooled SE 0.21) for the 42- to 49-, 105- to 112-, and 140- to 154-d-old gilts, respectively. In EXP II, RNase protection assays were used to assess leptin mRNA in adipose tissue of ovariectomized gilts at 90 (n = 12), 150 (n = 11) or 210 (n = 12) d of age. Six pigs from each age group received estradiol (E) osmotic pump implants and the remaining animals received vehicle control implants (C; Day 0). On Day 7, back fat and blood samples were collected. Estradiol treatment resulted in greater (P < 0.05) serum E levels in E (9 +/- 1 pg/ml) than C (3 +/- 1 pg/ml) pigs. Serum leptin concentrations were not affected by age, nor E treatment. Leptin mRNA expression was not increased by age in C pigs nor by F in 90- and 150-d-old pigs. However, by 210 d of age, leptin mRNA expression was 2.5-fold greater (P < 0.01) in E-treated pigs compared to C animals. Serum insulin concentrations were similar between treatments for 210-d-old pigs. However, insulin concentrations were greater (P < 0.05) in E than C pigs at 90 d and greater in C than E animals at 150 d. Plasma glucose and serum insulin-like growth factor-I concentrations were not influenced by treatment. These results demonstrate that serum leptin concentrations increased with age and E-induced leptin mRNA expression is age- and weight-dependent.     

In ovo leptin administration affects hepatic lipid metabolism and microRNA expression in newly hatched broiler chickens

Background

A leptin-like immunoreactive substance has been found in chicken eggs and has been implicated in serving as a maternal signal to program offspring growth and metabolism. In the present study, we investigated the effects of in ovo leptin administration on hatch weight, serum and hepatic concentrations of metabolites and hormones, as well as on the expression of genes involved in hepatic lipid metabolism and the predicted microRNAs (miRNAs) targeting the affected genes. To this end we injected fertile eggs with either 0.5 μg of recombinant murine leptin or vehicle (PBS) before incubation.

Results

Prenatally leptin-exposed chicks showed lower hatch weight, but higher liver weight relative to the body weight, compared to the control group. In ovo leptin treatment increased the hepatic content and serum concentration of leptin in newly hatched chickens. The hepatic contents of triglycerides (TG) and total cholesterol (Tch) were decreased, whereas the serum levels of TG, Tch and apolipoprotein B (ApoB) were increased. The hepatic mRNA expression of sterol regulator element binding protein 1 (SREBP-1c), SREBP-2, hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and cholesterol 7α-hydroxylase 1 (CYP7A1) was significantly up-regulated, as was the protein content of both SREBP-1c and SREBP-2 in hepatic nuclear extracts of leptin-treated chickens. Moreover, out of 12 miRNAs targeting SREBP-1c and/or HMGCR, five were significantly up-regulated in liver of leptin-treated chicks, including gga-miR-200b and gga-miR-429, which target both SREBP-1c and HMGCR.

Conclusions

These results suggest that leptin in ovo decreases hatch weight, and modifies hepatic leptin secretion and lipid metabolism in newly hatched broiler chickens, possibly via microRNA-mediated gene regulation.     

The role of retinal and extra-retinal photostimulation in reproductive activity in broiler breeder hens

Photostimulation of retinal photoreceptors, which are sensitive to green light, appears to inhibit reproductive activity in birds, whereas photostimulation of extra-retinal photoreceptors, which are sensitive to red light, accelerates it. The objective of this study was to determine the effect of either retinal or extra-retinal photostimulation on reproductive activities of broiler breeder hens. At 23 wk of age, Cobb hens (N = 135) were divided into 9 rooms with individual cages (n = 15). At 24 wk of age, 3 rooms were photostimulated (14L:10D) with white light (Control, n = 45). Six rooms had 2 parallel lighting systems, red (660 nm) and green (560 nm), which were both on during 6 out of 14 h of the light period. Then, in 3 of these rooms, the green light was turned off and hens were exposed to a total of 14 h of red light (Red, n = 45), and in the other 3, the red light was turned off and green lighting continued for a total of 14 h (Green, n = 45). The Green group had reduced egg production; reduced plasma concentrations of ovarian steroids; reduced luteinizing hormone (LH)-β, vasoactive intestinal peptide (VIP), and prolactin mRNA expression; and greater retinal green opsin mRNA expression (P ≤ 0.05). The Red group had greater egg production; greater gonadotropin-releasing hormone-I (GnRH-I) and red opsin gene expression in the hypothalamus; and lesser green opsin gene expression in the retina (P ≤ 0.05). We suggest that selective photostimulation of extra-retinal photostimulation as opposed to retinal photostimulation is a key factor in the determination of successful reproduction of broiler breeder hens.     

Comparing mRNA levels of genes encoding leptin,leptin receptor,and lipoprotein lipase between dairy and beef cattle

Body weight and fat mass vary distinctly between German Holstein (dairy cattle) and Charolais (beef cattle). The aim of this study was to determine whether the expression of the obese (Ob) gene and lipoprotein lipase (LPL) gene in fat tissues and expression of the long isoform leptin receptor (Ob-Rb) gene in the hypothalamus were different between these two cattle breeds. Body weight and the area of longissimus muscle cross-section of German Holstein were lower (P<0.001), while body fat content, as well as the omental and perirenal fat mass were higher (P<0.001), compared to Charolais. Plasma insulin and leptin levels between two cattle breeds were determined by radioimmunoassay. Compared to Charolais, plasma insulin concentrations were significantly higher (P<0.01), and plasma leptin levels were tended to be higher (P<0.1) in German Holstein. Ob mRNA levels in subcutaneous and perirenal fat depots, but not in the omental fat depot, were significantly higher (P<0.05) in German Holstein than in Charolais. LPL mRNA expression in the perirenal fat depot of German Holstein was greater in abundance than that of Charolais. No significantly different LPL mRNA levels were found in subcutaneous and omental fat depots, and Ob-Rb mRNA levels in the hypothalamus between these two cattle breeds (P<0.05). Both Ob and LPL expression was greater in perirenal and omental fat depots than in the subcutaneous fat depot (P<0.05). Data indicated that in bovine the Ob and LPL gene expression levels in perirenal fats are an important index that is associated with body fat content, while Ob-Rb in hypothalamus is not.     

GnRH-1 mRNA, LH surges, steroid hormones, egg production, and intersequence pause days alter in birds exposed to longer wavelength of light in the later stages of production in Gallus gallus domesticus

The objective of this was to establish the effects of red spectrum of light (650 nm, treated n = 12) and normal spectrum of light (450 nm control = 12) on GnRH-I mRNA expression, amplitude and frequency of luteinizing hormone (LH), and egg production from 72-82 weeks of age in white leghorn hens. Birds exposed to red spectrum of wavelength significantly improved (P < 0.01) steroid hormone, and egg production improved over old laying 72 to 82 weeks. Weekly interval profiles followed the same pattern. At 77th weeks of age blood, samples from both the groups were collected at every 3 h for 36 h to study the pulsatile secretion of LH surges. Plasma LH concentration was higher (P < 0.01) in treated birds with more number of frequencies and amplitude LH surges in plasma of treated birds. LH frequencies were more pronounced and advanced during 36 h of sampling at 3 h interval in treated birds. Weekly interval of plasma LH, E2β, and P(4) concentrations increased (P < 0.01) in treated birds from 72 to 82 weeks of age. GnRH-I mRNA concentration was significantly (P < 0.01) higher in birds exposed to red spectrum of light compared to controls. It is hypothesized that exposure of birds to red spectrum of light-enhanced (P < 0.01) GnRH-I mRNA with more number of yellow yolky follicles was found in birds exposed to red spectrum of light during 77 days (72-82 weeks of age) of experimental period. It is concluded that higher levels of GnRH-I mRNA, LH, E2β, and P(4) concentration with lower incidence of pause days enabled the birds to lay more eggs even later in the productive period by modulating the wavelengths of light under normal husbandry conditions.     

Effects of immunisation against leptin on feed intake, weight gain, fat deposition and laying performance in chickens

1. Three experiments were conducted to study the effects of leptin on weight gain and body composition in laying hens. 2. The effects of immunisation against chicken leptin on feed intake (FI), fat deposition and laying rate were observed in laying Guangdong yellow-feathered hens. Ten hens were inoculated with leptin immunogen on d 3, 31, 63 and 84, together with 10 control hens immunised with bovine serum albumin (BSA). In the 100-d experiment, immunisation against leptin increased blood anti-leptin antibody titres, slightly reduced plasma T3 concentrations, slightly decreased FI and increased live weight; however, laying rate was significantly depressed and abdominal fat mass was increased by the end of the 100-d experiment. 3. Passive immunisation of 50-d-old pullets with yolk extract containing anti-leptin antibody IgY significantly increased FI within 6 h of treatment compared with physiological saline treated controls. 4. In growing 70-d-old pullets, inoculation with 0.5 (group 1) or 1 (group 2) ml leptin immunogen on d 1 and 28 of the experiment slightly increased FI and significantly increased daily gain compared with BSA-immunised control pullets. Abdominal fat mass on d 49 increased from 48+/-4.5 g in controls to 66+/-3.5 and 80+/-3.1 g in groups 1 and 2, respectively. 5. It was suggested that immunisation against leptin mimicked loss of leptin bioactivity and might become a novel technique to stimulate fat growth in certain types of animal production.     

Increasing daily feeding occasions in restricted feeding strategies does not improve performance or well being of fattening pigs

Background

The natural feeding behaviour of the pig is searching for feed by rooting activities throughout the day; self-feeding pigs randomly space their eating and drinking periods throughout the day consuming ten to twelve meals per day. Pigs in conventional fattening pig production are normally fed 2–3 times daily with the feed consumed within 15 minutes. The aim of this study was to determine if more frequent feedings could improve the performance of conventionally kept fattening pigs.

Methods

The experiment was carried out on 360 fattening pigs (27–112 kg live weight), weighed and assigned to pens stratified by weight and sex. Each treatment group consisted of 180 pigs, allocated to 20 pens with nine pigs in each pen. To evaluate how more feeding occasions affects performance and well-being the pigs were divided into two groups and fed three (control group) or nine (treatment group) times daily. The same total amount of liquid feed was fed to each group and the feed ration was correlated to the live weight of the pigs. All weight and slaughter recordings were made individually and recordings of feed consumption were made pen-wise. At slaughter the stomach of each pig was examined for lesions in the pars oesophagea and scored on a scale from 1–6.

Results

Frequent feeding occasions influenced both performance and status of gastric lesions of the pigs adversely. Pigs in the treatment group grew slower compared to pigs in the control group; 697 g/day (± 6.76) versus 804 g/day (± 6.78) (P < 0.001) with no difference in within-pen variation. There was also a lower prevalence of gastric lesions within pigs in the control group (2.4 (± 0.12) compared to 3.0 (± 0.12) (P < 0.01)). There was a positive correlation between gastric lesions in the treatment group and daily weight gain (r = 0.19; P < 0.01).

Conclusion

Increased daily feeding occasions among group housed pigs resulted in a poorer daily weight gain and increased mean gastric lesion score as compared with pigs fed three times daily. This may be a consequence of more frequently occurring competition for feed in the treatment group. The present study does not support increased daily feeding occasions in fattening pigs.     

Exogenous leptin advances puberty in domestic hen

The present study was undertaken to examine the effect of recombinant chicken leptin administered to fed ad libitum and feed-restricted immature chickens of a layer strain on ovarian development and the timing of sexual maturity. In the first experiment 11-week-old pullets (77 days of age) fed ad libitum were injected daily with leptin at four dose levels (4, 16, 64 and 256 microg/kg body weight) until sexual maturity (lay of the first egg). Leptin treatment at the highest dose significantly (P<0.05) advanced the onset of puberty (day 116.3+/-1.0) in comparison to controls (day 121.3+/-1.2). The rises of luteinizing hormone, estradiol and progesterone in blood plasma were also advanced by leptin treatment. In the second experiment, both full-fed and feed-restricted pullets (79 days of age) were injected daily with leptin (256 microg/kg body weight). In birds fed ad libitum, exogenous leptin again significantly (P<0.05) advanced first ovipostion (day 118.4+/-1.4 versus day 124.4+/-1.7), while abolishing the significant (P<0.05) delay caused by feed restriction (day 131.5+/-1.6) and restoring the normal onset of sexual maturity (day 125.7+/-1.6). Analysis of the ovaries in 106-day-old pullets revealed that leptin injections advanced follicular development, particularly in birds fed ad libitum, and significantly (P<0.01) reduced follicular apoptosis both in full-fed and feed-restricted birds. In conclusion, we have shown that in female chickens exogenous leptin advances the onset of puberty by attenuation of ovarian apoptosis and enhancement of folliculogenesis.     

光照周期对褪黑激素受体在母兔下丘脑-垂体-卵巢轴中分布与表达的影响

旨在观察不同光照周期对新西兰白色母兔褪黑激素受体在"下丘脑-垂体-性腺轴"中的分布和表达模式,从而进一步分析褪黑激素在不同光照周期下调控母兔发情的机制。选用5月龄未经产新西兰母兔48只,随机分成3组,每组16只,前10 d各试验组采用"12 h光照：12 h黑暗"的光照制度,后6 d分别采用长光照（16 h光照：8 h黑暗）、短光照（8 h光照：16 h黑暗）和正常光照（12 h光照：12 h黑暗,对照组）的光照制度,光照强度80 lx,试验期为16 d。试验结束后剖杀动物,取下丘脑、垂体和卵巢,用qPCR、免疫印迹、免疫组织化学方法,研究不同光照周期对母兔的下丘脑、垂体、卵巢中褪黑激素受体亚型分布与表达的影响。结果表明：在下丘脑,短光照组的MT1 mRNA表达量较长光照组高88.8%（P<0.05）,较对照组高54.9%（P<0.05）,长光照组与对照组间无显著差异;MT2 mRNA表达量在不同光周期组间差异不显著（P>0.05）。免疫组织化学染色结果显示,长光照组下丘脑室周核的MT1阳性细胞数明显较短光照组少69.4%（P<0.05）,较对照组少37.3%（P<0.05）,短光照组比对照组显著多104.8%（P<0.05）;同样,长光照组室旁核的MT1阳性细胞数明显较短光照组少52.9%（P<0.05）,对照组明显较短光照组少55.8%（P<0.05）,而长光照组与对照组间差异不显著（P>0.05）。在垂体,短光照组的MT1 mRNA表达量比长光照组显著高164%（P<0.05）,比对照组显著高49.5%（P<0.05）,而长光照组比对照组显著低43.5%（P<0.05）;但不同光周期下MT2 mRNA表达量差异不显著（P>0.05）;长光照组的卵巢MT1 mRNA表达量较对照组低33.3%（P<0.05）,较短光照组低53.6%（P<0.05）,短光照组与对照组间差异不显著（P>0.05）;卵巢中短光照组MT2的mRNA相对表达量比对照组显著高90.0%（P<0.05）,比长光照组显著高100%（P<0.05）,长光照组与对照组差异不显著（P>0.05）。短光照周期显著提高了下丘脑、垂体和卵巢中褪黑激素受体亚型MT1表达,而不是MT2受体亚型。光照周期调控母兔发情的作用机制可能是褪黑激素通过MT1受体途径实现的。     

The detection of 3 leptin receptor isoforms in crucian carp gill and the influence of fasting and hypoxia on their expression

To understand leptin signaling pathway in the crucian carp (Carassius carassius), we cloned 3 leptin receptor isoform complementary DNAs (ie, the long form [cclpr-L], the short form [cclpr-s1], and the secreted form [cclpr-s2]). Variant cclpr-L had a 3,255-bp open reading frame and a complete intracellular domain with box 1 and box 2 consensus sequences. By contrast, cclpr-s1 contained only 4 amino acids in its intracellular domain, without the “box 1” motif, which is conserved among membrane-bound leptin receptor short isoforms in mammals. Variant cclpr-s2 had no transmembrane domain, suggesting that it is a soluble form of the receptor, and alternative splicing of cclpr-s2 mRNA employs a different mechanism for the generation of soluble leptin receptor by intron retention. The fasting-treated fish showed significantly lower cclpr-L mRNA levels in gill tissue than the control group, whereas cclpr-s2 mRNA levels did not vary significantly among the groups. Treatment with hypoxia significantly increased mRNA levels of both cclpr-L and cclpr-s2 in gill tissue. To our knowledge, this is the first study of leptin receptor isoforms expression in teleosts.     

瘦素及其长型瘦素受体在雌性小鼠体内的表达

采用免疫组织化学SP染色法对长型瘦素受体在雌性小鼠生殖周期不同阶段的下丘脑、卵巢、子宫中细胞定位和分布进行了研究,并用ELISA方法对小鼠血清中的瘦素浓度进行了检测。结果显示,下丘脑神经元胞质中有棕褐色阳性颗粒,且阳性细胞数量随妊娠日龄增加逐渐增加,妊娠期与间情期阳性细胞数差异显著(P0.05);在卵巢中,卵母细胞胞质中有长型瘦素受体表达,随卵泡的发育,卵泡细胞中免疫反应阳性细胞数量逐渐增加;在胚泡附植期,瘦素受体在子宫腺和子宫内膜上皮细胞中大量表达。妊娠期血清瘦素浓度均高于间情期,且瘦素浓度从间情期到妊娠4日龄,呈上升趋势,妊娠5日龄稍下降,后随妊娠日龄逐渐增加。结果表明,瘦素及其长型受体能够促进卵泡发育,有利于小鼠胚泡的附植。     

Serum Adipokine Concentrations in Dogs with Naturally Occurring Pituitary‐Dependent Hyperadrenocorticism

Background

An excess of intra‐abdominal fat is observed frequently in dogs with hyperadrenocorticism (HAC). Adipokine dysregulation is a possible cause of complications related to visceral obesity, but little information is available on adipokine in dogs with naturally occurring HAC.

Objectives

To examine the differences in the circulating adipokines concentrations in overweight dogs with and without pituitary‐dependent HAC (PDH).

Animals

Thirty healthy dogs and 15 client‐owned dogs with PDH.

Methods

Case–controlled observational study, which enrolled 15 overweight dogs diagnosed with PDH and 30 otherwise healthy dogs of similar body condition score. Nine of 15 dogs with PDH were treated with low‐dose trilostane twice daily and reassessed after treatment.

Results

The serum leptin (P < .0001) and insulin (P < .0001) concentrations were significantly higher in the PDH group (leptin, 22.8 ± 8.8 [mean ± SD]; insulin, 9.1 ± 6.1) than the healthy group (leptin, 4.9 ± 3.7; insulin, 1.9 ± 0.9). However, there were no significant differences in the adiponectin, resistin, tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6, IL‐10, and IL‐18 levels between the 2 groups. In the PDH group, the serum cortisol concentrations had a linear association with the leptin concentrations, and there were significant decreases in the leptin (P = .0039) and insulin (P = .0039) levels after trilostane treatment. However, the leptin and insulin levels remained higher after trilostane treatment than in healthy control dogs with similar body condition score.

Conclusions and Clinical Importance

Hypercortisolemia in dogs with PDH might upregulate the circulating leptin levels. However, a large population‐based study will be necessary to determine whether the upregulation of leptin is involved directly with the complications caused by HAC.