Effects Of Photoperiod Change On Reproductive Hormone Secretion,Hair Follicle Development,Immune And Antioxidant Functions Of Cashmere Goats

The physiological state of cashmere goats is regulated by light.In this experiment,photoperiod regulation is carried out in non-reproductive and no-growing cashmere seasons to explore the effects of photoperiod change on melatonin(MLT)secretion,reproduction-related hormone secretion,hair follicle development-related index and immune and antioxidant states of cashmere goats.The potential mechanism of photoperiod change affecting immune and antioxidant functions is explored by combining in vivo and in vitro methods.The main research contents and results are as follows.Experiment 1:Effects of Photoperiod Change on Melatonin Secretion in Cashmere GoatsIn the experiment,a single-factor experimental design was adopted.Eighteen multiparity female goats and 18 6-month-old female goats were selected and divided into,respectively,three groups according to their age,weight and parity(multiparity goats),namely,multiparity goat control group(MCG),multiparity goats short-day photoperiod group(MSDPP)and multiparity goats shortening-day photoperiod group(MSIPP),and 6-month-old goat control group(YCG),6-month-old goat short-day photoperiod group(YSDPP)and 6-month-old goat shortening-day photoperiod group(YSIPP).Each group has 6 goats.There was no significant difference in body weight between groups(P>0.05).Goats in MCG group and YCG group received natural light cycle treatment.Goats in MSDPP group and YSDPP group received 8 light:16 dark(8L:16D)constant short-day photoperiod treatment from 10:00 am to 6:00 pm every day.The MSIPP group and YSIPP group received shortening-day photoperiod treatment:the illumination in the house started from 16L:8D per day,and the illumination time was shortened by 1 h per week until 8L:16D per day at the end of the experiment,and the daylight was supplemented by fluorescent lamps after the natural light disappeared in the early stage of the experiment.The experiment was divided into early stage and late stage,each for 30 days.Blood samples were collected every 2 hours from 6:00 pm on the first day of the early stage of the experiment until 6:00 pm on the second day to explore the daily variation of MLT and determine the best time for blood collection.After determining the best time for blood collection,blood should be collected once a week in the early stage of experiment,and in the late stage of the experiment,blood was collected every 2 days.The results indicated that MLT of cashmere goats showed wave-like secretion in the natural light cycle,increased at 16:00 every day,reached the secretion peak at 00:00 in the middle of the night,maintained until 2:00,and then began to decline,with the lowest concentration at 12:00.Short-day photoperiod and shortening-day photoperiod significantly increased the MLT concentration in serum of cashmere goats compared with the control group.Short-day photoperiod increased the MLT concentration in serum of multiparity goats at d 26 and 6-month-old goats at d 32,respectively(P<0.05).The effects of shortening-day photoperiod treatment was relatively late,and the serum MLT concentrations of multiparity goats and 6-month-old goats were increased on d 40 and 42 respectively(P<0.05).Short-day photoperiod treatment and shortening-day photoperiod treatment could up-regulate the gene expression of MLT synthetase arylalkylamine N-acetyltransferase(AANAT)in leukocytes(P<0.05),but have no significant effect on hydroxyindole-oxygen-methyltransferase(HIOMT).Among them,short-day photoperiod treatment increased the gene expression of AANATE in multiparity goats and 6-month-old goats(P<0.05)in the early stage of the experiment,while shortening-day photoperiod treatment group increased the gene expression only in the late stage of the experiment(P<0.05).Experiment 2:Effects of Photoperiod Changes on Reproductive Hormone Secretion of Cashmere GoatsThe design and sample collection were the same as experiment 1.The results showed that there was no difference in the concentrations of gonadotropin-releasing hormone(GnRH),luteinizing hormone(LH),follicle stimulating hormone(FSH),estrogen(E2)and Kisspeptin between different light treatments in the early stage(P>0.05).In the later stage of the experiment,compared with the control group,the short-day photoperiod treatment increased the serum GnRH concentration of the multiparity goats and the 6-month-old goats on d 40 and 44 respectively(P<0.05),while the shortening-day photoperiod treatment increased the serum GnRH concentration of the multiparity goats and the 6-month-old goats on d 48 and 50 of the experiment(P<0.05),and maintained until the end of the experiment.Compared with the control group,the serum LH concentration in MSDPP group increased at d 46(P<0.05)and that in MSIPP group increased at d 52(P<0.05).However,photoperiod change did not affect serum LH concentration of 6-month-old goats group(P>0.05).Short-day photoperiod treatment and shortening-day photoperiod treatment increased the serum FSH concentration of multiparity goats at d 50 and 54 respectively(P<0.05).The photoperiod change had little effect on the 6-month-old goats,only on d 58 and 60,the shortening-day photoperiod treatment increased the serum FSH concentration of the goats(P<0.05),while the short-day photoperiod treatment had no effect on FSH secretion of the 6-month-old goats(P>0.05).Short-day photoperiod treatment and shortening-day photoperiod treatment increased the serum E2 concentration of the multiparity goats on d 46 and 54 respectively(P<0.05).For the 6-month-old goats group,short-day photoperiod treatment and shortening-day photoperiod treatment both increased E2 concentration at d 54(P<0.05).Kisspeptin concentration was significantly affected by photoperiod change at the late stage of the experiment,in which short-day photoperiod treatment and shortening-day photoperiod treatment increased the Kisspeptin concentration in the serum of multiparity goats(P<0.05)on the d 40 and 48 of the experiment respectively.Short-day photoperiod treatment and shortening-day photoperiod treatment increased Kisspeptin concentration of goats at d 46 and 52 respectively(P<0.05).The above results suggest that short light treatment can affect the secretion of reproductive hormones of cashmere goats.On the one hand,it can affect the release of GnRH into blood by regulating the secretion level of MLT,and finally affect the secretion status of FSH,LH and E2.On the other hand,short light treatment improved the secretion level of Kisspeptin in cashmere goats,and high concentration of Kisspeptin can directly stimulate the secretion of GnRH and eventually affect the secretion of reproductive hormones.For 6-month-old goats,short illumination can also affect the concentrations of serum GnRH and Kisspeptin by changing the secretion of MLT,but the secretion of FSH,LH and E2 does not form a regular pattern.Experiment 3:Effects of Photoperiod Changes on Hair Follicle Development,Hormone Secretion and Gene Expression in Cashmere Goats.The design of the experiment was the same as experiment 1.On the basis of the experiment 1,skin samples were collected on d 30 and 60 of the experiment.The results showed that,short-day photoperiod treatment and shortening-day photoperiod treatment deepened the depth of primary hair follicle and secondary hair follicle(P<0.05),widened the width of secondary hair bulb(P<0.05),in multiparity goats,but had no effect on the width of primary hair bulb.For 6-month-old goats,short-day photoperiod treatment and shortening-day photoperiod treatment deepened the depth of secondary hair follicle(P<0.05),widened the width of primary hair bulb and secondary hair bulb(P<0.05),but had no effect on the depth of primary hair follicle.From the results of hormone secretion,compared with the control group,the short-day photoperiod treatment and the shortening-day photoperiod treatment reduced the prolactin(PRL)concentration in serum of the multiparity goats at d 46 and 56 respectively(P<0.05).For the 6-month-old goats,the PRL concentration in serum was decreased at d 44 and 56 by short-day photoperiod treatment and shortening-day photoperiod treatment respectively(P<0.05).Short-day photoperiod treatment increased insulin-like growth factor 1(IGF-1)concentration in the serum of the multiparity goats at d 44(P<0.05),but the secretion fluctuated afterwards,which was higher than that of the control group at d 54(P<0.05).The concentration of IGF-1 in the serum of the multiparity goats was increased at d 56 by shortening-day photoperiod(P<0.05).Photoperiod change had no effect on IGF-1 concentration in 6-month-old goats(P>0.05).The multiparity goats concentration of epidermal growth factor(EGF)in serum was increased at d 42 and 50 by short-day photoperiod and shortening-day photoperiod respectively(P<0.05).For 6-month-old goats,short-day photoperiod treatment and shortening-day photoperiod treatment increased EGF concentration in serum at d 48 and 54 respectively(P<0.05).The photoperiod change had no effect on T4(P>0.05),but it changed T3 secretion:short-day photoperiod treatment and shortening-day photoperiod treatment increased T3 concentration of multiparity goats(P<0.05)at d 48 and 52 respectively;both short-day photoperiod treatment and shortening-day photoperiod treatment increased T3 concentration of 6-month-old goats on d 56(P<0.05).According to the results of gene expression related to hair follicle development,on d 30 of the experiment,photoperiod change had no effect on gene expression.On d 60,compared with the control group,the short-day photoperiod treatment increased the gene expression of β-catenin and platelet-derived growth factor A(PDGFA)in the skin of the multiparity goats and the gene expression of β-catenin in the skin tissue of the 6-month-old goats(P<0.05).Shortening-day photoperiod treatment increased the gene expression of β-catenin,bone morphogenetic protein(BMP2)and PDGFA in the skin tissue of the multiparity goats(P<0.05)and increased the gene expression of β-catenin and PDGFA in the skin tissue of 6-month-old goats(P<0.05).Expriment 4:Effects of Photoperiod Changes on Immune and Antioxidant Functions of Cashmere GoatsTest design and sample collection were the same as experiment 1 and 3.The results showed that on d 30 of the experiment,short-day photoperiod increased the concentrations of immunoglobulin G(IgG),interleukin-1β(IL-1β)and interleukin-2(IL-2)in the serum of the goats.On d 60 of the experiment,short-day photoperiod and shortening-day photoperiod both increased the concentration of serum IgG,IL-1β and IL-2 in multiparity goats(P<0.05),and increased the concentration of serum IgG and IL-1β in 6-month-old goats(P<0.05),short-day photoperiod also increased the concentration of IL-2 and tumor necrosis factor-α(TNF-α)(P<0.05).In addition,on d 30 of the experiment,compared with the control group,the short-day photoperiod treatment increased the activity of total superoxide dismutase(T-SOD),catalase(CAT)and glutathione peroxidase(GPx)in the serum of the goats(P<0.05),and decreased the content of malondialdehyde(MDA)(P<0.05).The above indexes in the shortening-day photoperiod treatment groups had no significant change.On d 60 of the experiment,short-day photoperiod and shortening-day photoperiod increased the activity of T-SOD,CAT and GPx in the serum of the goats(P<0.05),and decreased the content of MDA(P<0.05).In terms of gene expression,on d 30 of the experiment,short-day photoperiod increased the gene expression of SOD1,CAT,GPx4,Nrf2,IL-1β,IL-2 and TNFa in multiparity goats(P<0.05),and increased the gene expression of SOD1,GPx1,GPx4,CAT,Nrf2,IL-1β and IL-2 in 6-month-old goats(P<0.05),while shortening-day photoperiod had no effect on gene expression.On d 60 of the experiment,short-day photoperiod increased the gene expressions of CAT,GPx4,IL-1β and IL-2 in multiparity goats(P<0.05),and increased the gene expressions of SOD1,CAT,GPx4,IL-1β and IL-2 in 6-month-old goats(P<0.05).Decreasing illumination increased the gene expression of SOD1,GPx4,CAT,Nrf2,IL-1β,IL-2 and TNFa in multiparity goats(P<0.05),and increased the gene expression of SOD1,GPx1,CAT,IL-1β and IL-2 in 6-month-old goats(P<0.05).According to the results of skin antioxidant indexes,on d 30 of the expeiment,short-day photoperiod increased the activity of T-SOD and GPx in the skin tissue of multiparity goats(P<0.05),and increased the activity of T-SOD in the skin tissue of 6-month-old goats(P<0.05),while shortening-day photoperiod had no effect on the skin antioxidant indexes of goats.On d 60 of the experiment,short-day photoperiod and shortening-day photoperiod both increased the activity of T-SOD,CAT,total antioxidant capacity(T-AOC)and GPx in the skin tissue of multiparity goats(P<0.05),and decreased the content of MDA(P<0.05).In the 6-month-old goat groups,short-day photoperiod and shortening-day photoperiod increased the activity of CAT and GPx in skin tissue and reduced the content of MDA(P<0.05).From the results of skin antioxidant enzyme gene expression,on d 30 of the experiment,photoperiod change did not affect the expression of related genes.On d 60 of the experiment,short-day photoperiod and shortening-day photoperiod both increased the gene expression of SOD 1,GPx4 and CAT in skin tissue(P<0.05).Experiment 5:Effects of Melatonin on Immune and Antioxidant Functions of Peripheral Blood Lymphocytes in Cashmere GoatsIn this experiment,the lymphocyte culture method in vitro was used,and a single factor experiment design was adopted.Six MLT concentration treatments(0,10,20,40,80,160 pg/mL)were set,with 6 replicates for each treatment.The results showed that the proliferation rate of lymphocytes increased with the increase of MLT concentration(P<0.05).For immune indexes,the content of IgM was significantly increased when the MLT dosage was 40,80 and 160 pg/mL(P<0.05),the content of IgA was significantly increased when the MLT dosage was 10,20,40,80 and 160 pg/mL,and the content of IgG,IL-2 and TNFa was significantly increased when the MLT dosage was 20,40,80 and 160 pg/mL(P<0.05).The content of IL-1β was significantly increased when the dosage was 10 and 160 pg/mL(P<0.05),and the content of IL-1βwas significantly increased when the dosage was 10 and 160 pg/mL(P<0.05).Addition of MLT to cell culture fluid significantly improved the antioxidant and immune levels,among which,when the addition amounts of MLT were 20,40 and 80pg/mL,the T-SOD activity of lymphocyte culture fluid was significantly improved(P<0.05).The activity of CAT and T-AOC was significantly increased when the dosage was 20,40,80 and 160 pg/mL(P<0.05).When the dosage was 40,80 and 160 pg/mL,the activity of GPx was significantly increased(P<0.05).When the dosage was 40,80 and 160 pg/mL,MDA content was significantly reduced(P<0.05).Experiment 6:Study on the Mechanism of Melatonin Regulating Immune and Antioxidant Functions of Peripheral Blood Lymphocytes of Cashmere Goats via Nrf2 PathwayIn this experiment,a two-factor experimental design was adopted by using in vitro lymphocyte culture method.The main effects included two MLT addition levels(with or without 80 pg/mL MLT)and two Nrf2 inhibitor addition levels(with or without 5μmol/L ML385 inhibitor).The experiment consisted of four treatments with 6 replicates per treatment.The results showed that ML385 reduced the lymphocyte proliferation rate of cashmere goats(P<0.05),and the addition of MLT could alleviate this trend(P<0.05).For immune indexes,MLT increased the concentration of IgM,IL-1β and IL-2 in lymphocytes(P<0.05),and up-regulated the gene expression of IL-2 and down-regulated the gene expression of nuclear-factor kappa B(NF-κB)in non-inhibitor addition group.In inhibitor addition group,MLT had no effect on immune indexes and related gene expression(P>0.05).For antioxidant indexes,MLT improved antioxidant enzyme activity of lymphocytes,reduced MDA concentration,and increased gene expression of SOD1,GPx1,GPx4,CAT and Nrf2 in non-inhibitor addition group(P<0.05).In the inhibitor addition group,MLT had no effecct on the antioxidant level of lymphocytes(P>0.05).The above results suggest that MLT may be used as proteasome inhibitor to reduce the degradation of Nrf2 and ikbkinase and accumulate them,thus increasing the concentration of Nrf2 and IkBa in nucleus,then:increasing the expression of Nrf2 and inhibiting the gene expression of NF-κB,and affecting the downstream gene expression.