Melatonin Regulates The Rhythmic Laying Behavior By Circadian Clock Genes In Geese

The goose has low egg yield with 30-50 egg in every year,which not only hinders the development of goose industry,but also leads to the high cost of goslings and low raising benefits.Therefore,it has become one of the most difficult problems in goose production.The period of ovulation in geese is long with significant time preference.Melatonin is an indole hormone secreted by the pineal gland.It acts as an important regulator of endogenous biological clock,and involves in the regulation of various physiological processes of rhythmic behavior.However,whether melatonin mediate clock genes and affect goose egg production behavior or not is unclear.In our study,yangzhou goose was selected,egg production rhythm and melatonin secretion regularity were investigated,and the clock genes expressed in the endocrine tissue such as pineal gland and ovine were detected.Our research was aimed to reveal the preliminarily melatonin regulating goose egg nest granulosa cell proliferation and circadian rhythms mechanism.Finally,we can find a way to shorten the goose egg production cycle,improve the performance of egg production,and provide a theoretical method for breeding high egg yield geese.1 Egg laying rhythm and circadian secretion of melatonin in Yangzhou geeseIn order to reveal the rhythm of laying and the circadian secretion of melatonin in Yangzhou geese,the laying behavior of Yangzhou geese in peak period was observed continuously for 24 hours(once per hour).The results showed that the Yangzhou geese has time preference,mainly from 4:00 am to 7:00 am,lasts 46 to 48 hours.At the same time,the secretion of melatonin in blood was detected,and the results were consistent with expectations.Melatonin secretion was low in the day and high in the night.The mRNA and protein MEL1A of melatoninreceptor in pineal gland,hypothalamus,pituitary gland and ovary were measured by real-time fluorescence quantitative PCR and immunohistochemistry,respectively.The results showed that the expression trend of melatonin was same with the melatonin.2 Expression of clock genes in pineal gland and endocrine tissueThe pineal gland and the hypothalamus-pituitary gonadal axis(HPG)both exist Clock genes and the pineal gland is the master Clock oscillator.In order to determine the position of biological Clock that exist in the clockwork mechanism in the pineal gland and endocrine,real-time fluorescent quantitative PCR technique was used to detect the Clock genes(Clock,Bmal1 and Bmal2)and negative Clock genes(Cry1,Cry2,Per1 and these forms)in yangzhou goose in the pineal gland,hypothalamus,pituitary and ovarian within 48 h.Subsequently,cosine theorem was adopted to determine whether the expression appears significant rhythm.The results showed that clock genes showed up during the day,fell during the night,and the negative control clock genes showed the opposite trend.The goose laying behavior concentrated at 4 in the morning-7:00 in the morning.Its expression oscillated in the peak phase.Cosine characteristics test showed that genes in the pineal gland showed significantrhythmicity expect for Bmal1(P<0.05),genes in the ovarian show significant rhythmicity except for Cry1,Per2(P<0.05).However,while in the hypothalamus and pituitary,there existed multiple genes without rhythmic.We concluded so there are obvious biological clocks in the pineal gland and ovary molecular oscillation mechanism,while in the hypothalamus and pituitary,the biological molecular oscillation mechanism is relatively weak.3 The mechanism of melatonin regulating granulosa cell proliferation and circadian rhythm in gooseTo explore the roles of melatonin in goose granulosa cell proliferation and circadian rhythms,we cultivated ovarian granular cell in vitro with 20 μmol/L melatonin treatment 24 h.The result showed that the melatonin can improve the Clock,Bmal1,Bmal2,Per2,and Cry1 Clock gene expression and rhythmic amplitude,promoted the proliferation gene C-myc rhythmic.The transcriptome was adopted to analyze the mechanisms of melatonin regulating granulosa cell proliferation in goose.The genes were significantly enriched in the second messenger cAMP and cGMP-PKG signaling pathways.Also,the differential expressed gene CNGB1 was detected to be involved in rhythmic regulation.Taken together,melatonin inhibits the expression of CNGB1,promotes the expression of clock genes Per2 and Per3,and regulates granulosa cell proliferation.In conclusion,Yangzhou geese had a preference of laying period with 4:00 am--7:00 am and the secretion character of melatonin with low in the day and high in the night.It was confirmed that there existed significant circadian clock molecular oscillation in the pineal gland and ovary,and the expression of clock genes in geese oscillated at the peak phase during the peak period of laying.At the same time,it was preliminarily clarified that melatonin may mediate the expression of CNGB1 and promote the expression of rhythm genes,further regulate the laying behavior of geese.