Abstract In many seasonally breeding species， photoperiod is an important factor involved in reproduction. Capra hircus breeds are normally considered shortday breeders. An experiment was carried out to evaluate whether or not photoperiodic regimens influence circulating hormone levels （prolactin， melatonin， thyrotropin stimulating hormine， luteinizing hormone，follicle stimulating hormone） of Henan huai local goat exposing to different photoperiodic regimens. The results showed that plasma levels of prolactin， melatonin， thyrotropin stimulating hormine， luteinizing hormone and follicle stimulating hormone were higher in the 8 L∶16 D photoperiodic regimen than that in the 16 L∶8 D photoperiodic regimen. Levels of those hormones decreased on day 7 and day 28 when the 16 L∶8 D photoperiodic regimen was used continuously. It was concluded that serum concentrations of the hormones increased in summer when short day （8 L∶16 D） was used and decreased when the goats were transferred from short day for 42 d to long day exposure for 28 d. We suggest that in summer， levels of hormones changing may be due to short day animals. Changing of related hormones is still determined by the mechanism of internal regulation of short day animals exposure to light changes. Shortening light may also help increase the reproductive activity of goat and thus increase productivity.
　　Key words Photoperiod； PRL； MLT； TSH； FSH； LH； Goat Introduction
　　As we know that changes in daylength induce strong physiological changes in seasonal mammals. The pars tuberalis （PT） of the pituitary has been known as the central tissue conveying the external photoperiodic signal into an internal signalling pathway which drives seasonal physiology. A very well described seasonal effect is the rise of blood prolactin concentrations which occurs on increasing spring photoperiods， and in turn induces a cascade of physiological events including a seasonal moult[1]. Seasonally breeding mammals such as goat and sheep named shortday animals use photoperiod， encoded by the nocturnal secretion of the pineal hormone melatonin， as a critical cue to drive hormone rhythms and synchronize reproduction to the most optimal time of year[2-3]. Melatonin acts directly on the pars tuberalis of the pituitary， regulating expression of thyrotropin， which then relays messages back to the hypothalamus tocontrol reproductive circuits [4-5].
　　Prolactin （PRL）， was discovered in nonhuman animals around 1930 by Oscar Riddle[6]and confirmed in humans in 1970 by Henry Friesen[6]. Prolactin is a peptide hormone， encoded by the PRL gene[7]. It is known as luteotropic hormone or luteotropin， which is a protein that in humans is best known for its role in enabling mammals， usually females， to produce milk. It is influential in over 300 separate processes in various vertebrates[8]. Prolactin is secreted from the pituitary gland in response to eating， mating， estrogen treatment， ovulation and nursing. Prolactin is secreted in pulses in between these events. Prolactin plays an essential role in metabolism， regulation of the immune system and pancreatic development. 　　In animals， melatonin is involved in the entrainment （synchronization） of the circadian rhythms including sleepwake timing， blood pressure regulation， seasonal reproduction， and many others[9]. Many of its biological effects in animals are produced through activation of melatonin receptors[10]， while others are due to its role as an antioxidant[11]， with a particular role in the protection of nuclear and mitochondrial DNA[12]. Thyroidstimulating hormone （also known as thyrotropin， thyrotropic hormone， TSH， or hTSH for human TSH） is a pituitary hormone that stimulates the thyroid gland to produce thyroxine （T4）， and then triiodothyronine （T3） which stimulates the metabolism of almost every tissue in the body[13]. Folliclestimulating hormone （FSH） is a gonadotropin， a glycoprotein polypeptide hormone. FSH is synthesized and secreted by the gonadotropic cells of the anterior pituitary gland， and regulates the development， growth， pubertal maturation， and reproductive processes of the body. FSH and luteinizing hormone （LH） work together in the reproductive system[14]. FSH is a 35.5 kDa glycoprotein heterodimer， consisting of two polypeptide units， alpha and beta. Its structure is similar to those of luteinizing hormone （LH）， thyroidstimulating hormone （TSH）， and human chorionic gonadotropin （hCG）. The alpha subunits of the glycoproteins LH， FSH， TSH， and hCG are identical and consist of about 96 amino acids， while the beta subunits vary[15].
　　Therefore， the relationship among PRL， MLT， TSH， LH and FSH is close， especially in aspect of regulation animals reproduction. Some of the internal regulation mechanisms are not very clear. Most studies are focused on human medicine， especially in the field of human reproduction[16]and sheep and goat reproduction[17-19]， but there are not many mechanisms to be understood. In our study， we aimed at detecting serum levels of PRL， MLT， TSH， LH and FSH in goat exposing in different duration of light and at investigating the relationship between this rhythm of hormones and the animal reproduction.
　　Materials and Methods
　　Eeighteen goats were maintained in single pens in a temperaturecontrolled photoperiod room under continuous artificial short photoperiod （SP： 6 h light∶18 h dark） for a total of 42 d. After short photoperiod， three goats were killed either 4 h after light onset or 4 h after dark onset. The rest of the cohort （n = 12） was transferred to LP （long photoperiod： 16 h light∶8 h dark）， twelve animals were killed either 7 or 28 d after the transfer （again， 4 h after light onset and 4 h after dark onset）. All goats were supplied 800 g of a pelleted diet comprising lucerne hay： oats （60∶40 w/w） daily and water ad libitum. 　　Approximately 5 ml of blood was collected every two hours before killing for twelve times via venipuncture in metalfree tubes and allowed to clot for 30 minutes at room temperature， and then centrifuged at 3 000 rpm for 30 min. Serum was checked for hemolysis by visual inspection and separated into five aliquots for PRL， MLT， TSH， LH and FSH assessment. It was stored frozen until analysis.
　　Hormones determination
　　The PRL quantitative test was based on a solidphase enzymelinked immunosorbent assay. The assay system utilized one mouse monoclonal antiPRL antibody for solidphase （micro titer wells） immobilization and another in the antibodyenzyme （horseradish peroxidase） conjugate solution. The test sample was allowed to react simultaneously with the antibodies， resulting in PRL molecules being sandwiched between the solid phase and enzymelinked antibodies. After 45 min of incubation at room temperature， the wells were washed with water to remove unbound， labeled antibodies. A solution of tetramethylbenzidine （TMB） reagent was added and the mixture was incubated at room temperature for 20 min， resulting in development of a color. The color development was stopped with the addition of stop solution， which changed the color to yellow， and the developed color was measured spectrophotometrically at 450 nm. The concentration of PRL was directly proportional to the color intensity of the test sample [20]， and other hormones were tested with the methods provided by Elisa kit form Shanghai Enzymelinked Biotechnology Co.， Ltd.
　　Statistical analysis
　　The collected data were tabulated and analyzed using SPSS version 16 software （SPSS Inc， Chicago， IL， USA）. Categorical data were presented as number and percentages， while quantitative data were expressed as median， interquartile range， and range. Spearmans correlation coefficient （ρ）， and Studentstest， and MannWhitney Utest were used as tests of significance. The accepted level of significance in this work was stated at 0.05 （P<0.05 was considered significant）[21].
　　Results
　　In this study， it was found that the secretion of PRL hormone gradually decreased with the prolonged light time. The PRL hormone increased slightly on the 28th day of long light， higher than the 7th day of long light， but the difference was not significant （P>0.05）. The PRL hormone was significantly higher in short day （P<0.05）， and the difference was not significant in different periods of long light （P>0.05）， as shown in Fig. 1. 　　Agricultural Biotechnology2018
　　Discussion
　　More and more researches have shown that photoperiod and some hormones play an important role in effecting livestock reproduction. In nature， many animals are reproductive seasonally. Animals such as equine， donkey and other animals are estrous and mated when the gradual extension of daylight in the spring， which are named longday animals. Sheep and goats are estrous and mated when the shortening of daylight in autumn， which are named shortday animals[22-24].
　　PRL has a unique seasonal signature in all vertebrate species in that it is consistently elevated by LP stimulation[25]. To illustrate this phenomenon， we first look at the main mechanism of lights effects on animal reproduction. In general， prolonged light is beneficial to the reproduction of longday light animals. Horse， for example， has the highest semen quality and the most obvious sexual response during the long sunshine season in spring and summer[26-27]. In contrast to longday animals， prolonged light inhibits shortday animals reproductive activities. A large number of studies have shown that shortening of light can increase the fecundity of short day animals. Shortening the light of sheep from 13 to 8 h/d can increase sperm activity and normal acrosome by 16.6% and 27%. Semen from ram treated with short day can increase the pregnancy rate of ewes and the yield of lamb by 35% and 150% respectively compared with natural light group. At the beginning of summer， reducing the length of light to 8 h/d can bring forward the breeding season of Ewe 27-45 d， extend the length of light to 16 h/d in autumn and winter， and end the estrous activity of ewe appear 15 d earlier than usual. Based on this theorists， it is eluted that when goats are exposed to long day in summer and then to artificial short day， the level of hormones increasing is reasonable in this study[27-28].
　　It is worth noting that， although shortening light time can induce estrus in animals such as sheep， goats and deer， it does not mean that long light can adversely affect the development of their reproductive organs. It was found that the longlight treatment of the shortsunshine animals was more obvious and the pregnancy rate was higher. For example， Bon Durant（1981） found that 19 dairy goats were treated with 70 d long light （19 h/d）， at the end of the light 62 d， 16 ewes were estrus， 15 of them were ovulating， 11 of them were pregnant and none of the natural light group was estrus. For example， Steuflug et al.（1982） found that ewe was administered 20 h of long daily light for 70 d before three months prior to the mating season， and the results showed that pregnancy rate was 55.3 %， and was 1.21 times higher than that of the control group[29]. 　　It is interesting that this study showed the levels of hormones such as PTL， MLT， TSH， LH and FSH decreased when the goats were exposed to long photoperiod （L16∶D8）， and they continued to decrease with the day prolonged. In the study， Trends in hormones in serum were similar. We need to interpret them from the interaction of hormones and the functional characteristics of each hormone. There are many classic studies on relationship among photoperiod and hormones secretion. The changes of female sheep reproductive activity were observed by damaging the anterior pituitary area. The results show that the anterior pituitary is an important nerve center involved in regulating seasonal reproduction[30]. Lincoln[31]found that treatment with melatonin embedded in pituitary nodules affected the FSH and PRL secretion induced by photoperiod. It is speculated that melatonin can at least regulate the induction of photoperiod to seasonal reproduction by PT. Lincoln and Clarke[32]studied HPD （hypothalamopituitary disconnection） ram， and suggest that melatonin acts in part on the pituitary gland， independent of the hypothalamus， to regulate PRL secretion.
　　At the beginning of the long day and the short night （such as spring）， the teeth had active reproductive function， and the concentration of MLT decreased. At the beginning of the short day and night （autumn）， cloven hooves increased their reproductive activity and MLT concentration[33]. In this experiment， the short day mechanism in autumn was simulated in summer， hormone levels increased， and the condition of long day was restored， hormone levels decreased. And the results are credible. The role of MLT from pineal in regulating the reproductive hormones such as prolactin， luteinizing hormone and folliclestimulating hormone in the anterior pituitary is important. Hormone levels in shortlight， dark， and blinding mice showed that the pituitarysecreted PRL， LH and FSH decreased， it was especially obvious in PRL. The high level of MLT directly affects the pituitary gland， and regulates the secretion of gonadotropin， thus regulating the secretion of lutein. It also directly affects the release of GnRH regulated by the nucleus of optic chiasmi and hypothalamus[34].
　　The study showed that it is necessary of long day before summer solstice for the normal starting of seasonal estrus. Malpaus reported that in condition of regime of the short sunshine system was maintained from the beginning of the winter solstice until the end of the summer solstice[35]， and the beginning time of estrus in autumn of Suffolk was significantly delayed， which shows that it is necessary of long day periods between winter solstice and summer solstice for ewe to begin to estrous in autumn. If the start time of the long day light advancing， the time that the ewe starts rutting in autumn also advancing which shows that the beginning time of long day is very necessary for the ewe to be estrus on time in autumn and is an important regulatory signal. The light system after summer solstice is not very important for the ewe to be normal estrus in autumn. 　　Another reported that raising sheep in constant short day system for several months or longer， sheep are not kept in estrus and will eventually enter the state of anestrous. On the other hand， if the ewe is raised for a long day system， the ewe will eventually enter the estrus period. This is said a "tolerance of photoperiod". Jane et al. divided adult Suffolk sheep from winter solstice into two groups， one fed under natural light and the other under a constant regime of short day （10 L∶14 D）. The results showed that the two pairs of ewes almost stopped estrus at the same time， and the same blood was collected twice a week， and the level of LH in the blood began to drop at the same time. This indicated that the ewes were anestrous because of "tolerance of short day" and not because of long day gradually prolonged[36]. To determine whether sheeps tolerance to induced short sunlight was due to changes in MLT secretion patterns， the researchers transferred Safuke ewes from long day to short day for 150 d， making all ewes "tolerant" to induced short sunlight from estrus to anestrous stage. During this period， MLT levels were measured on a continuous blood collection every 2 weeks， and it was found that the circadian rhythm of MLT did not change and was always consistent with the circadian changes in the light cycle， suggesting that sheeps tolerance to induced short sunlight was not due to changes in MLT secretion patterns[37].
　　Therefore， the long day has a posterior effect on the promotion of short day animal reproduction. The reason may be that long light treatment stimulates large amounts of prolactin （PRL） from the pituitary gland， which inhibits the production of reproductive hormones in the hypothalamus and pituitary gland， resulting in decreased levels of luteinizing hormone （LH）， folliclestimulating hormone （FSH） and estrogen in the blood of animals. The inhibition of hypothalamus and pituitary was decreased rapidly， and the low level of LH， FSH and estrogen feedback enhanced the secretion of gonadotropin， LH， FSH and estrogen to induce animals estrus and ovulation[38]. It can also be explained that the serum hormone level of goats decreased after artificial short light and then long light， perhaps the next step would be to observe the goats in detail in terms of estrus， ovulation and mating， and the results would be more convincing.
　　It is worth noting that the effects of light on the reproduction of shortday animals should not contradict the fact that shortening light induces estrus. Although it can be estrous in short light， it is not as good as that in short light after long light treatment. Some people observe the reproduction performance of sheep after natural light， constant light and long and short light （undulatory light）. It is found that undulatory light is better than constant light. This means that the treatment of short day animals before short day treatment can enhance the effect of short day treatment， promote the development of ovarian follicles， so that shortlight animals show more obvious estrus symptoms. This conclusion can explain the research results of this experiment. 　　Many animals use the variation in duration of melatonin production each day as a seasonal clock. In animals including humans[39]， the profile of melatonin synthesis and secretion is affected by the variable duration of night in summer as compared to winter. The change in duration of secretion thus serves as a biological signal for the organization of day lengthdependent （photoperiodic） seasonal functions such as reproduction， behavior， coat growth， and camouflage coloring in seasonal animals[38]. In seasonal breeders that do not have long gestation periods and that mates during longer daylight hours， the melatonin signal controls the seasonal variation in their sexual physiology， and similar physiological effects can be induced by exogenous melatonin in animals including mynah birds[40]and hamsters[41]. Melatonin can suppress libido by inhibiting secretion of luteinizing hormone and follicle stimulating hormone from the anterior pituitary gland， especially in mammals that have a breeding season when daylight hours are long. The reproduction of longday breeders is repressed by melatonin and the reproduction of shortday breeders is stimulated by melatonin. During the night， melatonin regulates leptin， lowering its levels. Exogenous melatonin has acute sleepiness inducing and temperaturelowering effects during biological daytime， and when suitably timed （it is most effective around dusk and dawn）， it will shift the phase of the human circadian clock （sleep， endogenous melatonin， core body temperature， cortisol） to earlier （advance phase shift） or later （delay phase shift） times.
　　In this study， after only 42 d artificial short day in summer， and after 7 and 28 d long day， the serum hormone content of goats was determined. The results showed that in summer， the light level could be increased by shortening the light level. So shortening light in summer and increasing reproductive hormone levels in short day animals may enhance their reproductive function， and we can increase the productivity of ewes by shortening light time if needed. Finally， compared with previous studies and the results of this experiment， we found that the deficiency of this study was that there were not enough kinds of hormones， and the analysis of diurnal light conversion was not very detailed. What would be the result of artificial long day test under short sunshine？ These are worth studying in the future， we will analyze the data from the researches on changing of hormones related with the changing of Circadian rhythm. 　　Conclusions
　　In summary， when the goat is in artificial short light condition， the prolactin level is higher， while when the artificial short light was changed to long light， the hormone level decreases， and the hormone level continued to decrease with the prolonged light time. Visible， for the goat of the short sunshine animal， in the long light period， artificial short light， can improve the hormone level and promote the animal reproductive activities， and after short light and long light treatment， goat hormone levels were not elevated， but decreased， indicating that reproductive activities are not active. The next step of this study is to explore the changes of goat hormone levels in light and dark light to further explore the regulation of light and reproductive hormone in goat reproduction.
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