Effect of Feed Restriction and Photoperiod on Reproduction and LEPR, MELR mRNA Expression of Layers

Photoperiod and nutrition are major factors that affect the reproductive efficiency particularly in female animals. In this study we examined the interaction of photoperiod and food restriction on growth, sexual maturation and receptor mRNA expressions of leptin, melatonin, and estrogen in abdominal fat and the ovary of pullets. There were no interaction effects between photoperiod and feeding level on body weight, abdominal fat weight, ovary weight at both 14 wk and 18 wk. Abdominal fat weight of feed restriction group was significantly lower compared with the control group at the age of 14 wk, 18 wk, and age of the first egg (AFE) (p<0.05). Ovary LEPR (Leptin receptor) gene expression showed an interaction effect of the first egg. Restricted feeding significantly inhibited ovary ER (Estrogen receptor), LEPR and MELR1B (Melatonin 1B receptor) gene expression at 14 wk, 18 wk and the first egg. At 14-week-old, abdominal fat LEPR gene expression was significantly lower in long photoperiod group compared with the short photoperiod group. At the first egg, short photoperiod and feed restriction group reduced abdominal fat LEPR gene expression. The results indicated that the reproductive activity of pullets is sensitive to feed intake and photoperiod. Feed restriction down regulated the ER, LEPR, MELR1A (Melatonin 1A receptor) and MELR1B mRNA expression of the ovary at 14 wk, 18wk and AFE. Long photoperiod enhanced the LEPR, MELR1A and MELR1B mRNA expression of abdominal fat at AFE.


INTRODUCTION
Laying hens, like many other birds, rely heavily on vision, and light is an important factor within their natural environment (Huber-Eicher et al., 2013). Lighting is the factor that most affects the performance of the production and reproduction of birds, sexual maturation, feeding behavior and productivity of eggs and egg weight (Lewis et al., 2010;Lewis Gous, 2006;Schweanlardner et al., 2012). Nutrition is another major factor that affects the reproductive efficiency particularly in female animals (Brecchia et al., 2006;Walzem Chen, 2014). Various nutritional methods have been employed in breeder pullets for attempting to reduce body weight at the onset of egg laying in order to improve performance during the laying period (Bozkurt et al., 2014;De Coon, 2007;Proudfoot, 1979). Feed restriction has been justified as a means of controlling body weight, improving subsequent reproduction to achieve greater production efficiency without inflicting severe adverse effects on the birds' nutritional requirements (Crouch et al., 2002;De Coon, 2007;Hocking, 2004). Photoperiod cues plays important roles in the regulation of seasonal variations in body mass (BM) and energy balance for many small mammals (Zhao & Wang, 2006).
Leptin, a 146-amino acid protein, is mainly secreted by adipocytes (Paczoska-Eliasiewicz et al., 2006;Sirotkin & Grossmann, 2015) and is eRBCA-2019eRBCA- -1042 implicated in the regulation of metabolic status, feed intake, reproduction, immune function and body condition in rodent and primates (Bouloumie et al., 1998;Fantuzzi & Faggioni, 2000;Zieba et al., 2005). Gallus Gallus leptin cDNA was first cloned by Taouis , which led to a controversy whether a leptin gene exists in the chicken genome (Pitel et al., 2010). Although the sequence of the chicken leptin gene is controversial, cloning of the chicken leptin receptor gene provides evidence of the existence of the leptin homologue in birds (Horev et al., 2000;Liu et al., 2007). Melatonin (N-acetyl-5-methoxytryptamine), an indole hormone, regulates circadian rhythm, hibernation, feeding pattern, thermoregulation, and neuroendocrine function in birds through three different receptor subtypes (MELR1A, MELR1b, and MELR1c) (Adachi et al., 2002;Sinkalu et al., 2015). In mammals, melatonin also influences the reproductive function via activation of receptor sites within the hypothalamic-pituitary-gonadal axis (Malpaux et al., 2001). In birds, melatonin binding sites have been identified in the ovaries, suggesting a possible role of melatonin regulating ovarian functions (Sundaresan et al., 2009).

Effect of Feed Restriction and Photoperiod on Reproduction and LEPR, MELR mRNA Expression of Layers
Following the attainment of minimum age and body weight thresholds, the present study was undertaken to investigate the relationship between photoperiod and feed restriction, and the possible mechanism about how the photoperiod, nutrition or both impact on the adipose store and the sexual maturity in pullets.

Experimental Design, Birds, and Management
Female Gray Hy-line chicks were purchased from Hebei Huayu Poultry Breeding Company. Chicks were raised according to the management protocols established by Hy-line International. At 10 wk of age, 480 healthy pullets were selected and allotted randomly to one of the 6 treatments, i.e., a 3 (photoperiod: 8L:16D, 12L:12D, or 16L:8D) × 2 (ad libitum or feed restriction) factorial design. The feed restriction was 80% of the ad libitum. The diet contained 11.72 MJ/kg energy, 16.3% crude protein, 0.33% methionine and 0.74% lysine. The specific feeding and photoperiod schedule for the birds were given in Table 1. Each treatment had four replicates comprising 20 pullets each, 4 pullets per cage. Water was provided ad libitum throughout the study. Illumination was provided by 2 15-W compact fluorescent lamps producing a mean illuminance of 15 ± 2.4 lx. Pullets' beaks were trimmed at 7 d of age, and all pullets were wing-banded at 6 wk. The present study was performed in accordance with Hebei Agricultural University Institutional Animal Care and Use Commit tee Policies for Animal Use under an approved animal.

Sample Collection
Samples (n = 8 per feeding × photoperiod combination) were collected at the age of 14 wk, 18 wk, and at first egg (AFE), respectively. Body weight was recorded, then pullets were killed by cervical dislocation. Weight of the abdominal fat (including the fat surrounding the gizzard) and the ovaries were measured, and then abdominal fat and ovaries were snap-frozen in liquid nitrogen, and stored at -80º until assayed. Also at the age of 18 wk, 2 pullets from each replicate were selected, weighed, and randomly placed in individual, illuminated, standard laying cages. The age and egg weight at first egg were recorded.

Isolation of Total mRNA and qPCR
Total RNA was isolated from the ovarian cortex and abdominal fat using the RNAeasy mini kit (Omega Bio-Tek, Inc.). Equal amounts of total RNA (1µg) were reverse transcribed into cDNA using the Reverse Transcription kit (TransGen Biotech, Inc). Amplification of specific transcripts was conducted using gene specific primers (Table 2). For each primer pair, only a single product of the predicted size was identified. All amplification products were sequenced to confirm specificity of the reaction. Abundance of specific mRNAs was analyzed by real -time PCR using the 2-ΔΔCt method (Livak Schmittgen, 2001). Values shown for transcript abundance are the Mean ± SEM.

Effect of Feed Restriction and Photoperiod on Reproduction and LEPR, MELR mRNA Expression of Layers
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Statistical Analysis
The data were analyzed by two factors analyses of variance using the General Linear Models procedures of SPSS. When significant differences were determined for the main effects, comparison among means were made using the Duncan procedure. Unless otherwise stated, all statements of significance were assessed using p<0.05.

Body Weight, Ovary Weight and Abdominal Fat Weight
There were no interaction effects between photoperiod and feeding level on body weight, abdominal fat weight, and ovary weight at 14 wk, 18 wk, and at first egg (Table 3). However, treatment effects were detected. Abdominal fat weight of pullets from the feed restricted group was significantly lower compared with pullets from the ad libitum group at the age of 14wk, and AFE (p<0.05). Pullets' ovary weight in the feed restricted group was lower compared to the ones in ad libitum group at the age of 14 wk (p<0.05). Lighting program effects were found on ovary weight at AFE only, it was lower in the 16L:8D group compared with the 12L:12D group but not the group of 8L:16D (p<0.01).

Age of First Egg and First Egg Weight
There were no interaction effects between photoperiod and feed restriction on the age of the first egg and first egg weight (Table 4). However, long photoperiod significantly reduced the age of the first egg compared with the short photoperiod (p<0.05). The average age at the first egg was 146 d for the In the same row, values with no letter or the same letter superscripts mean no significant difference (p>0.05), while with different small letter superscripts mean significant difference (p<0.05), and with different capital letter superscripts mean significant difference (p<0.01). The same as below.

LEPR, MELR1A, MELR1B Gene Expression
There were no photoperiod and feeding level interaction effects on abdominal fat LEPR, MELR1A, and MELR1B gene expression at 14 wk and 18 wk except LEPR at AFE (Table 5). At 14-week-old, abdominal fat LEPR and MELRIA gene expressions were significantly lower in the 16L:8D photoperiod group compared with the 8L:16D photoperiod group (p<0.05); feed restriction increased the abdominal fat LEPR expression compared with the ad libitum group at the age of 14 wk (p<0.05). However, at first egg, both LEPR and MELRIA gene expressions were higher in the 16L:8D long photoperiod group than in the 8L:16D short photoperiod (Table 5). There were also no photoperiod and feeding level interaction effects on the ovary ER, LEPR, MELR1A and MELR1B gene expression at 14 wk, 18 wk and first egg (Table 6). Feed Restriction significantly inhibited ovary ER, LEPR, MEIRIB, and MELR1B gene expression at 14 wk, 18 wk, and first egg, except MEIRIB at 18 wk . Photoperiod did not show significant differences on all measured genes at all the examined time periods (Table 6).

DISCUSSION
It has been suggested that there is a BW or body composition threshold for the onset of sexual maturation (Brody et al., 1980;Brody et al., 1984). Chen (2007) reported that all lighting programs were effectively able to stimulate the sexual maturation process, however, photoperiod had no effect on BW or absolute abdominal fat weight at first egg. Results from the current study showed that photoperiod had no effect on BW and absolute abdominal fat, but the 16L:8D photoperiod group reduced ovary weight in chickens at first egg compared with the 12L:12D group. Feed restriction early in life has been proposed as a strategy for improving feed efficiency and reducing body fat in broilers (Akande Atteh, 2016;Xu et al., 2017). Previous studies have shown that feed restriction (75% of control ad libitum) delayed the broilers age of sexual maturity and significantly reduced ovary weight, number of yellow follicles, number of atretic yellow follicle, incidence of double hierarchy, internal ovulation as compared to control from 19 to 25 wk of age (Madnurkar et al., 2014). During egg production, feed restriction resulted in significantly lower body and abdominal fat pad weights compared with unrestricted feeding (Richards et al., 2003). The data of the present study showed that feed restriction could reduce abdominal fat weight at the age of 14 wk and first egg, and there was an interaction effect between photoperiod and feed restriction on ovary weight at first egg. The age at first egg (AFE) was affected in a curviform by the lighting intensity and length of the photoperiod (Lewis et al., 1997). Exposure to photoperiods of 17L:7D, 15L:9D, 13L:11D or 11L:13D significantly affected the age at first egg (Chen et al., 2007). The average age at first egg was 144.8 d for the 17L:7D group and 150.5 d for the 11L:13D group. In the present study, the age of the first egg in the 16L:8D photoperiod group was 10.45 d earlier than in the 8L:16D photoperiod group.

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There was no interaction effect between photoperiod and food restriction on the age of the first egg and first egg weight. The current data further evidences that the photoperiod remains the primary mediator of regulating AFE in birds.
A study of Japanese quail in which leptin was injected in ovo enhanced the growth rate during embryonic and postembryonic development and led to earlier hatching and puberty (Lamosova et al., 2003). Furthermore, whereas a single injection of leptin in chickens resulted in attenuation of feed intake (Cassy et al., 2004;Denbow et al., 2000;Lohmus et al., 2003;Raver et al., 1998;Taouis et al., 2001), but not chronic leptin injections lasting several weeks. It is now clear that reproductive maturation will not take place in the complete absence of leptin signaling (i.e. in mammals lacking either functional leptin or its receptor) but leptin is not necessarily the rate-limiting determinant for puberty onset, it acts rather as a permissive factor or 'metabolic gate' (Foster Nagatani, 1999). For example, leptin will not advance the timing of normal puberty in ad libitum fed rats, but in moderately feed restricted prepubertal rats, puberty is delayed. This delay can be prevented by simultaneous treatment with leptin, leading to the result that puberty occurs at a similar time to ad-libitum fed rats (Cheung et al., 1997). This study suggested that feed restriction significantly inhibited ovary LEPR gene expression at 14 wk, 18 wk and at first egg, but did not significantly affect abdominal fat LEPR gene expression at 18 wk and at first egg. The current data evidences that photoperiod mainly mediates the abdominal fat LEPR gene expression, while feed restriction mostly mediated the ovary LEPR gene expression.
In birds, melatonin binding sites have been identified in the ovaries, suggesting a possible role of melatonin regulating ovarian functions (Sundaresan et al., 2009). The present findings are in line with the hypothesis that melatonin directly acts on the gonads (Ayre Pang, 1994). In the current study, we observed two main subtypes of melatonin receptors expression in the ovary. The differential distribution of MELR1A and MELR1B in ovarian tissues suggests that these receptors mediate distinct downstream cellular functions of melatonin in these tissues. There was a trend towards feed restriction reducing ovary expression of LEPR and MELR1B mRNA in treated chicken.
The role of estrogens in hen reproduction has been well established (Hrabia et al., 2008). Therefore, the mRNA expression of estrogen receptors under different photoperiod and feed restriction was examined within  the ovaries of pullets. The current study showed that there was a trend towards feed restriction reducing ovary expression of ER mRNA in treated chickens, but photoperiod did not affect ER mRNA expression.

CONCLUSION
Taken together, the results of this study suggest that feed restriction down regulated the ER, LEPR, MELR1A and MELR1B mRNA expression of the ovary at 14 wk, 18 wk, and AFE. Long photoperiod enhanced the LEPR, MELR1A and MELR1B mRNA expression of abdominal fat at AFE. Moreover, a better understanding of the mechanisms governing the partitioning of leptin and melatonin between adipose and ovarian tissue were reached, thereby enabling strategies to effectively control the threshold of sexual maturation in chickens.