Expression and localization of angiopoietin family in corpus luteum during different stages of oestrous cycle and modulatory role of angiopoietins on steroidogenesis,angiogenesis and survivability of cultured buffalo luteal cells

The objective of this study was to document the expression and localization of angiopoietin (ANGPT) family members comprising of angiopoietin (ANGPT1 and ANGPT2), and their receptors (Tie1 and Tie2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle, and the modulatory role of ANGPT1 and ANGPT2 alone or in combinations on progesterone (P₄) secretion and mRNA expression of phosphotidylinositide‐3kinase‐protein kinase B (PI3K‐AKT), phosphoinositide‐dependent kinase (PDK), protein kinase B (AKT), Bcl2 associated death promoter (BAD), caspase 3 and von willebrand factor (vWF) in luteal cells obtained from midluteal phase (MLP) of oestrous cycle in buffalo. Real‐time RT‐PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors whereas, the P₄ secretion was assessed by RIA. The mRNA and protein expression of ANGPT1 and Tie2 was maximum (p < .05) in mid luteal phase (MLP) of oestrous cycle. The ANGPT2 mRNA and protein expression was maximum (p < .05) in early luteal phase, decreased in MLP and again increased in late luteal phase of oestrous cycle. ANGPT family members were localized in luteal cells and endothelial cells with a stage specific immunoreactivity. P₄ secretion was highest (p < .05) with 100 ng/ml at 72 hr when luteal cells were treated with either protein alone. The mRNA expression of PDK, AKT and vWF was highest (p < .05) and BAD along with caspase 3 were lowest (p < .05) at 100 ng/ml at 72 hr of incubation period, when cultured luteal cells were treated with either protein alone or in combination. To conclude, our study explores the steroidogenic potential of angiopoietins to promote P₄ secretion, luteal cell survival and angiogenesis through an autocrine and paracrine actions in buffalo CL.     

Presence of melatonin‐catabolizing non‐specific enzymes myeloperoxidase and indoleamine 2,3‐dioxygenase in the ram reproductive tract

The melatonin catabolism is very complex and not completely understood. Melatonin can be metabolized by free radical interaction, but also pseudo‐enzymatically or by enzymatic pathways. We have previously detected the existence of melatonin‐synthesizing enzymes and melatonin receptors MT1 and MT2 in the ram reproductive tract; thus, in order to start to elucidate melatonin catabolism in these organs, we have investigated the presence of the melatonin‐catabolizing enzymes indoleamine 2,3‐dioxygenase (IDO, both IDO1 and IDO2 isoforms) and myeloperoxidase (MPO) in testis, epididymis and accessory glands. Gene expression analyses by real‐time PCR showed the presence of MPO, IDO1 and IDO2 in all the organs of the ram reproductive tract and revealed that MPO is the main melatonin‐catabolizing enzyme, which is mainly expressed in the testis and the bulbourethral glands (p < .05). These results were further corroborated by immunohistochemical staining, and by Western blot. Likewise, MPO was also evidenced in epididymal and ejaculated spermatozoa by indirect immunofluorescence and Western blot. In conclusion, melatonin‐catabolizing enzymes MPO, IDO1 and IDO2 are expressed in the ram reproductive tract, and MPO is the most expressed one, mainly in the testis and the bulbourethral glands. The presented results warrant further studies on the function of these enzymes and their melatonin‐metabolizing activity.     

Hepatic steroid inactivating enzymes,hepatic portal blood flow and corpus luteum blood perfusion in cattle

Production from the corpus luteum (CL) and/or hepatic steroid inactivation impacts peripheral concentrations of P4, which can alter reproductive performance. Our primary objective was to examine hepatic steroid inactivating enzymes, portal blood flow, and luteal blood perfusion at 10 days post‐insemination in pregnant versus non‐pregnant beef and dairy cows. Twenty early lactation Holstein cows and 20 lactating commercial beef cows were utilized for this study. At day 10 post‐insemination, hepatic portal blood flow and CL blood perfusion were measured via Doppler ultrasonography. Liver biopsies were collected and frozen for later determination of cytochrome P450 1A (CYP1A), 2C (CYP2C), 3A (CYP3A), uridine diphosphate‐glucuronosyltransferase (UGT) and aldo‐keto reductase 1C (AKR1C) activities. Pregnancy was determined at day 30 post‐insemination and treatment groups were retrospectively assigned as pregnant or non‐pregnant. Data were analyzed using the mixed procedure of SAS. Steroid metabolizing enzyme activity was not different (p > .10) between pregnant versus non‐pregnant beef or dairy cows. Hepatic portal blood flow tended (p < .10) to be increased in pregnant versus non‐pregnant dairy cows. Luteal blood perfusion was increased (p < .05) in pregnant versus non‐pregnant dairy cows. Pregnant dairy cows appear to have an increased rate of hepatic clearance of P4 in combination with increased synthesis from the CL. This could account for the lack of difference in peripheral P4 concentrations between pregnant and non‐pregnant dairy cows. This study highlights the relevance of further investigation into steroid secretion and inactivation and their impact on the maintenance of pregnancy in cattle.     

Follicular dynamics and colour Doppler vascularity evaluations of follicles and corpus luteum in relation to plasma progesterone during the oestrous cycle of Surti buffaloes

With an objective to evaluate the follicular dynamics and vascularity changes in follicles and corpus luteum, the ovaries of cyclic Surti buffaloes (n = 9) were examined daily sequentially by transrectal B‐mode and colour flow mode (CFM) ultrasonography starting from the day of oestrus till the onset of next oestrus. Higher proportion of buffaloes evidenced one‐wave cycle (66.66%) compared to two‐wave cycle (33.34%) with none showing a three‐wave cycle. The dominant follicle of the first follicular wave was the ovulatory follicle and persisted for 19.70 ± 0.50 days compared to its persistence for 16.5 ± 1.45 days in a two‐wave cycle. The maximum diameter of the ovulatory follicle in a one‐wave and two‐wave cycle did not differ yet their linear growth rates were significantly lower (p < 0.01) in a one‐wave cycle. Colour flow mode examination of follicles revealed that the percentage of follicles with detectable blood flow in the subsequently determined largest follicle (dominant follicle) was not different from that in the second largest follicle before follicle deviation. The blood flow in the dominant follicle increased significantly on the day of oestrus. The mean diameter and blood flow to the corpus luteum (CL) increased linearly and significantly from Day 5 of oestrus till Day 13 after which both parameters started declining. At or around Day 16, there was precipitous fall in the blood supply to the CL and CL diameter that continued declining thereafter to reach the lowest around Day 20 of the oestrous cycle. Rise in plasma progesterone concentrations was synchronous to CL diameter and vascularity and showed significant and positive correlations. It was concluded that Surti buffaloes evidence a preponderance of one‐wave follicular growth pattern with a significant increase in the vascularity of ovulatory follicle on the day of oestrus and corpus luteum on Day 13 of the oestrous cycle.     

Age‐related changes in the bovine corpus luteum function and progesterone secretion

Decreased fertility associated with maternal ageing is a well‐known critical problem, and progesterone (P4) concentration decreases during the menopause transition in women. The corpus luteum (CL) secretes P4, thereby supporting the implantation and maintenance of pregnancy. It is proposed that a bovine model is suitable for studying age‐associated decline of fertility in women because the physiology of cows is similar to that of women and cows have a greater longevity compared with other animal models. Thus, we investigated the age‐dependent qualitative changes and inflammatory responses in the bovine CL. In vivo experiment: Cows were divided into three groups, namely, young (mean age: 34.8 months), middle (80.1 months) and aged (188.9 months). Blood samples were collected on days 7 and 12 during the estrous cycle. In vitro experiments: Cows were divided into young (mean age: 27.6 months) and aged (183.1 months). The CL tissues of these groups were collected from a local slaughterhouse and used for tissue culture experiments. An in vivo experiment, plasma P4 concentration in aged cows was significantly lower than that in young cows, whereas no difference was found regarding the area of CL. An in vitro examination in the bovine CL tissues showed that the luteal P4 concentration, P4 secretion, and mRNA expression of StAR and 3β‐HSD were lower in aged cows compared with young cows, especially in the early luteal phase. However, no differences were detected in the mRNA expression of inflammation‐ and senescence‐related factors and inflammatory responses to lipopolysaccharides between the CL tissues from young and aged cows, indicating that an age‐dependent increase in inflammation is not involved in the luteal function. P4 production and secretion from the bovine CL diminish in old cows, especially during the early luteal phase, suggesting that senescence may affect the luteal function in cows.     

Polymorphisms of the melatonin receptor 1A gene that affects the reproductive seasonality and litter size in Small Tail Han sheep

Previous researches have shown that MTNR1A plays an essential role in sheep reproduction. However, most researches focused more on the reproductive seasonality of sheep, and few scientists had studied the association of polymorphisms of the MTNR1A gene with ovine litter size and reproductive seasonality. Therefore, we chose MTNR1A gene to detect its novel sequence polymorphisms and population genetics and analyse their association with seasonal reproduction and litter size in ewes. The mRNA expression level in hypothalamus, pituitary and ovary was also detected. In this study, five polymorphisms (g.15118664G > T, g.15118683C > T, g.15118756C > T, g.15118774C > T and g.15118951G > A) were identified in exon 2. Most importantly, the g.15118683C > T and g.15118951G > A were significant difference between year‐round oestrous sheep and seasonal oestrous sheep (p < .01), and g.15118756C > T had a great effect on litter size of Small Tail Han sheep (p < .05). In addition, the mRNA expression level of MTNR1A in the hypothalamus of polytocous Small Tail Han sheep was significantly higher than that in monotocous Small Tail Han sheep (p < .01) and the expression of MTNR1A in the hypothalamus of year‐round oestrous sheep was significantly higher than that in seasonal oestrous sheep (p < .01). Polymorphisms in exon 2 may regulate the reproductive seasonality and litter size of ewes by influencing gene expression to regulate the reproductive seasonality and litter size of ewes. Our studies provided useful guidance in marker‐assisted selection of the litter size in Small Tail Han sheep.     

Supplementation of Slow‐Release Melatonin Improves Recovery of Ovarian Cyclicity and Conception in Summer Anoestrous Buffaloes (Bubalus bubalis)

The role of melatonin as a protective neurohormone against restoring cyclicity in summer anoestrous animals in photoperiod species has gained wider acceptance. This study was designed to uncover the evidence the slow‐release melatonin (MLT) has on initiation of ovarian cyclicity and conception rate (CR) in summer anoestrous buffaloes. Thus, buffaloes diagnosed as summer anoestrous (absence of overt signs of oestrus, concurrent rectal examination and radioimmunoassay for serum progesterone at 10 days interval) were grouped as untreated (Group I, sterilized corn oil, n = 8) and treated (Group II, single subcutaneous injection of MLT @18 mg/50 kg bwt in sterilized corn oil, n = 20). Animals treated and detected in oestrus were artificially inseminated (AI) followed by division into Group III (second dose of MLT on 5th day post‐AI, n = 8) and Group IV (no melatonin administration, n = 10). Blood samples were collected at 4 days interval for estimation of serum MLT, progesterone and oestrogen using radioimmunoassay kit. Mean oestrous induction rate (OIR), oestrous induction interval (OII), interoestrous interval (IOI) and CR were estimated. Compared to control, concentration of melatonin was significantly (p < 0.05) higher in treated group ranging from 14.34 ± 1.72 to 412.31 ± 14.47 pg/ml whereas other two hormones did not show any concentration difference. Melatonin‐administered buffaloes showed significantly (p < 0.05) higher (90%) OIR with OII of 18.06 ± 1.57 days. Results showed improvement in conception rate in buffaloes administered with post‐insemination melatonin. It can be concluded from the study that slow‐release melatonin supplementation restored cyclicity in summer anoestrous animals resulting in improvement in conception rate in buffaloes.     

Effect of melatonin on regulation of apoptosis and steroidogenesis in cultured buffalo granulosa cells

This study was aimed to address melatonin receptor expression, mRNA level of hypothalamus and hypophysis hormone receptors (GnRHR, FSHR, and LHR), steroidogenesis, cell cycle, apoptosis, and their regulatory factors after addition of melatonin for 24 hr in cultured buffalo granulosa cells (GCs). The results revealed that direct addition of different concentrations of melatonin (100 pM, 1 nM, and 100 nM) resulted in significant upregulation (p < 0.05) of mRNA level of melatonin receptor 1a (MT1) without affecting melatonin receptor 1b (MT2). Melatonin treatment significantly downregulated (p < 0.05) mRNA level of FSH and GnRH receptors, whereas 100 nM dose of melatonin significantly increased mRNA level of LH receptor. Treatment with 100 nM of melatonin significantly decreased the basal progesterone production with significant decrease (p < 0.05) in mRNA levels of StAR and p450ssc, and lower mRNA level of genes (Insig1, Lipe, and Scrab1) that affect cholesterol availability. Melatonin supplementation suppressed apoptosis (100 nM, p < 0.05) and enhanced G2/M phase (1 nM, 100 nM, p < 0.05) of cell cycle progression which was further corroborated by decrease in protein expression of caspase‐3, p21, and p27 and increase in bcl2. Our results demonstrate that melatonin regulates gonadotrophin receptors and ovarian steroidogenesis through MT1. Furthermore, the notion of its incorporation in apoptosis and proliferation of buffalo GCs extends its role in buffalo ovaries.     

Immunolocalization of Melatonin and Follicle‐Stimulating Hormone Receptors in Caprine Ovaries and their Effects During in vitro Development of Isolated Pre‐Antral Follicles

The expression of melatonin type 1 (MT1) and FSH (FSHR) receptors in caprine ovaries and the effects of these hormones on the in vitro development of isolated pre‐antral follicles were evaluated. Follicles (≤200 μm) were cultured for 12 days in α‐MEM (control) or melatonin (100 or 1000 pg/ml) or sequential melatonin medium (100 pg/ml: from day 0 to day 6; 1000 pg/ml: from day 6 to day 12; experiment 1) and in control or sequential FSH (100 ng/ml from day 0 to day 6; 500 ng/ml from day 6 to day 12) or sequential melatonin or this latter plus sequential FSH (experiment 2). MT1 and FSHR expressions were observed in granulosa cells from secondary and antral follicles. The oocytes from primordial and primary follicles also express FSHR. Sequential melatonin increased the percentage of normal follicles and oocyte recovery compared with the control or melatonin (1000 pg/ml) at day 12. In experiment 2, all the treatments increased the normal follicles and growth compared with the control. In conclusion, this study demonstrated the presence of MT1 and FSHR in caprine ovaries. The addition of increased concentrations of melatonin (sequential medium) or FSH can be used to promote the in vitro development of caprine pre‐antral follicles.     

The influence of the ovulation rate on ultrasonically determined ovine corpus luteum morphometry and progesterone concentrations in cyclic and early pregnant sheep

The objective of the present study was to describe morphology and function of the Corpora lutea (CL) during the oestrous cycle and early pregnancy in sheep with different ovulation rates. In total 40 Booroola. Mutton Merino crosses [heterozygous carriers (FecBFec+) and non-carriers (Fec+Fec+) of the Booroola-fecundity gene (FecB)] with ovulation rates 1 to 4 were examined. During the oestrous cycle (n = 20) and the first month of pregnancy (n = 20) blood samples were taken daily (radioimmunoassay of progesterone) and an ultrasonic ovary diagnosis was conducted. The ewes were scanned transrectally with a 7.5 MHz linear probe lying in a dorsal position. During every examination the CL could be detected. The number and the diameter of the CL were documented and the total volume of luteal tissue per ewe was calculated. The effect of the ovulation rate on CL-morphology (diameter and total volume of luteal tissue per ewe) and peripheral progesterone concentrations were assessed by one-way ANOVA. On day 6 and 7 post ovulationem in cyclic and early pregnant sheep 42% of the diagnosed CL had a cavity. On day 11 (cyclic sheep) and day 10 post ovulationem (early pregnant sheep) this number decreased to 22% (p < 0.05). Both conditions of the CL (compact or with a central cavity) are similar in function and should be regarded as appearances of the same basic process. From the third day onwards the ovulation rate influenced significantly (p < 0.05) the development of the outside diameters of the CL. However, the ovulation rate had no effect on the total volume of the luteal tissue per sheep and on the progesterone concentrations. Yet, in sheep with the ovulation rate 1 significantly lower progesterone concentrations were determined than in sheep with the ovulation rates 2 to 4. In sheep with the ovulation rates 2 to 4 the peripheral progesterone concentrations did not differ significantly. In cyclic and pregnant sheep there is a positive correlation (r = 0.75, p < 0.05) between the progesterone concentration and the total volume of luteal tissue. Considering the smaller diameters of the preovulatory follicles it seems that the development of the CL continues until a threshold-value of progesterone and/or of the total luteal tissue is exceeded. Ewes with low ovulation rates reach this threshold-value with only a few but large CL. With increasing ovulation rate the CL tend to have smaller diameters.     

Melatonin and CIDR improved the follicular and luteal haemodynamics,uterine and ovarian arteries vascular perfusion,ovarian hormones and nitric oxide in cyclic cows

This study hypothesizes that melatonin with exogenous progesterone (CIDR) can improve follicular, luteal, ovarian and uterine haemodynamic of heat-stressed cows. Holstein cows (N = 12) studied for two spontaneous oestrous cycles during winter then divided equally during summer into the CIDR group received CIDR for 7 days and the melatonin group (Mel) received three injections of melatonin (75 mg/head) at the CIDR insertion, removal and ovulation days. Blood samples were collected to assay oestradiol (E2), progesterone (P4) and nitric oxide (NO). On day 0 (Ovulation), Mel had more small follicles (p < .05), higher ipsilateral and contralateral ovarian arteries (Ov.A.) peak systolic velocity (PSV), higher ipsilateral uterine artery (Ut.A.) PSV (p = .031) and blood flow volume (BFV), also Mel elevated contralateral Ut.A. PSV and BFV (p < .0001) but lowered contra Ut.A. pulsatility index (PI, p < .0001), E2 (p < .01) and NO (p < .0001). Mel increased the corpus luteum diameter (CL, p < .001), coloured area (p < .007) and P4 (p < .0001) on day 5 and reduced them (p < .05; p < .01) on Day 14. On day 10, Mel obtained CL diameter (p < .03) and coloured area (p < .002) of spontaneous that was higher than CIDR and decreased P4 (p < .003). Mel increased CL diameter, area and coloured area and decreased them thereafter. Mel increased the ipsilateral ovarian and uterine arteries PSV and BFV before ovulation and until day 8. Mel increased P4 and decreased NO until days 6 and 14. In conclusion, the improvement in follicular, luteal, ovarian and uterine haemodynamic and the decrease of NO production proved our hypothesis Melatonin doses higher than 75 mg/head is recommended to improve the heat-stressed cow's fertility.     

Status of autophagy,lysosome activity and apoptosis during corpus luteum regression in cattle

Corpus luteum (CL) regression is required during the estrous cycle. During CL regression, luteal cells stop producing progesterone and are degraded by apoptosis. However, the detailed mechanism of CL regression in cattle has not been fully elucidated. The aim of this study was to evaluate autophagy, lysosome activity, and apoptosis during CL regression in cattle. The expression of autophagy-related genes (LC3α, LC3β, Atg3, and Atg7) and the protein LC3-II was significantly higher in the late CL than in the mid CL. In addition, autophagy activity was significantly increased in the late CL. Moreover, gene expression of the autophagy inhibitor mammalian target of rapamycin (mTOR) was significantly lower in the late CL than in the mid CL. Lysosome activation and expression of cathepsin-related genes (CTSB, CTSD, and CTSZ) showed significant increases in the late CL and were associated with an increase in cathepsin B protein. In addition, mRNA expression and activity of caspase 3 (CASP3), an apoptotic enzyme, were significantly higher in the late CL than in the mid CL. These results suggest simultaneous upregulation of autophagy-related factors, lysosomal enzymes and apoptotic mediators, which are involved in regression of the bovine CL.     

Use of oxytocin to attain cervical dilation for transcervical embryo transfer in sheep

The aim of this work was to determine whether a cervical dilation protocol (CDP) composed of only oxytocin can be used to perform transcervical (non-surgical) embryo transfer in sheep (NSET) without affecting the viability of the corpus luteum (CL). Likewise, we evaluated whether a cervical transposing test with a Hegar dilator (CT Hegar test), performed at oestrous time, could be used to screen ewes for NSET (greater or lower chances to transpose the cervix). For that, oestrous and ovulation synchronization was performed in 25 Santa Inês ewes to induce the dioestrous condition. Animals went through the following CDP in a crossover design: E + OX, oestradiol benzoate (100 µg intravenously [IV]) and oxytocin (100 IU IV); OX, oxytocin (100 IU IV); and SAL, saline solution (IV). Using a Hegar dilator, cervical transposing attempts were performed at oestrous (D0) and dioestrous time (D8). The viability of the CL (morphology, luteal blood flow and progesterone values) was evaluated by ultrasonography (colour Doppler and B-mode) and by serum progesterone measurement from D7 to D13. The cervical transposing rate was lower for the SAL group (64%; 16/25; p < .05) and did not differ between the E + OX (88%; 22/25, p > .05) and OX (84%; 21/25, p > .05) groups. No treatment affected the CL viability. The CT Hegar test showed a high sensitivity (85.7%–93.3%), satisfactory accuracy (72%–84%), low false-negative rate (6.7%–14.6%), but high false-positive rate (46%–66.7%). In conclusion, a CDP protocol composed exclusively of oxytocin can lead to good cervical transposing rates and does not affect the viability of the CL. In addition, a screening test (CT Hegar) performed at oestrus can identify ewes for which cervical transposing will likely not occur at NSET.     

Melatonin improves cryopreservation of ram sperm by inhibiting mitochondrial permeability transition pore opening

Cryopreservation damages permeability of sperm mitochondrial membranes, with formation of a mitochondrial permeability transition pore (mPTP). Mitochondria are both a primary synthesis site and principle target for melatonin, which can directly inhibit mPTP formation. The objective was to determine effects of melatonin on mPTP opening of frozen-thawed ram sperm and elucidate underlying pathways by antagonist and agonists of melatonin receptors (MTs), and antagonists of PI3K and GSK 3β treatments; furthermore, plasma membrane integrity, mitochondrial membrane potential (ΔΨm), mitochondrial cytochrome c (Cyt c) release and fertilization were analysed to assess the effect of mPTP status mediated by melatonin on quality of frozen-thawed sperm. Fresh ram semen was diluted in glucose-egg yolk buffer with 0 or 10^–7 M melatonin (frozen and frozen + melatonin groups, respectively) and slow-frozen. In frozen-thawed sperm, melatonin added at initiation of 4°C equilibration was most effective for inhibiting mPTP opening, decreasing peptidyl-prolyl-cis/trans isomerase activity of cyclophilin D and increasing plasma membrane integrity, ΔΨm, mitochondrial Cyt c concentration and fertilizing ability (p < .05). In a mechanistic study, the melatonin receptor (MT)1 antagonist eliminated inhibition of melatonin on mPTP opening, whereas MT1 agonist had opposite effects (p < .05). Neither MT2 antagonist nor agonist had significant effect, but PI3K and/or GSK 3β antagonist decreased inhibition of MT1 agonist on mPTP opening (p < .05). In conclusion, melatonin improved sperm cryopreservation, perhaps by acting on MT1 via the PI3K-Akt-GSK 3β pathway to inhibit mPTP opening.     

Effect of melatonin on visceral fat deposition,lipid metabolism and hepatic lipo-metabolic gene expression in male rats

Melatonin (MT) influences lipid metabolism in animals; however, the mechanistic effect of melatonin on liver fat and abdominal adipose deposition requires further clarity. In order to study the effects of melatonin on lipid metabolism, and hepatic fat and abdominal adipose deposition in animals, twenty Sprague–Dawley (SD) rats of 6 weeks of age with similar bodyweight were randomly divided into two groups: control (CTL) and MT-treated (10 mg/kg/day). During a 60-day experiment, food intake and bodyweight were measured daily and weekly respectively. At the end of treatment, blood samples were collected to collect plasma to quantify hormones and metabolic indicators of lipid metabolism. In addition, organ and abdominal adipose depots including liver, and omental, perirenal, and epididymal fat were weighed. Liver tissue was sampled for sectioning, long-chain fatty acid (LCFA) quantification, and gene chip and Real-time quantitative PCR (qPCR) analyses. The results showed that liver weight and index (ratio of liver weight to body weight) in MT group reduced by 20.69% and 9.63% respectively; omentum weight and index reduced by 59.88% and 54.93% respectively, and epididymal fat weight reduced by 45.34% (p = 0.049), relative to CTL. Plasma lipid indices, triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and total cholesterol (TC) with MT treatment decreased significantly compared with the control. Fat and 8 LCFA content in liver in MT group also decreased. Gene chip and qPCR demonstrated that there were 289 genes up-regulated and 293 genes down-regulated by MT. Further analysis found that the mRNA expression of lipolysis-related genes increased, while the mRNA expression of lipogenesis-related enzymes decreased (p < 0.05) with MT. This study concluded that melatonin greatly affected fat deposition, and hepatic LCFA supply and the expression of genes associated with lipogenesis and lipolysis.     

Extended day length in late winter/early spring,with a return to natural day length of shorter duration,increased plasma testosterone and sexual performance in rams with or without melatonin implants

Sixteen rams were used to quantify the effects of long days, imposed during late winter/early spring, with or without exogenous melatonin, on plasma testosterone concentrations and ram serving capacity. Rams were assigned to two groups: photoperiod‐treated rams (Artificial Photoperiod, AP; n = 8), exposed to 2 months of long days (16 hr of light/day) between 22 December and 22 February, and control rams (Natural Photoperiod, NP; n = 8). At the end of the long‐day period, AP rams were returned to the natural photoperiod, and each ram in the two groups either did (+M) or did not (‐M) receive three subcutaneous melatonin implants. Four groups were created as follows: AP+M (n = 4), AP‐M (n = 4), NP+M (n = 4) and NP‐M (n = 4). Thirty days after of the onset of photoperiodic treatment, AP rams (13.5 ± 2.8 ng/ml) had significantly (p < .05) lower testosterone levels than NP rams (36.7 ± 1.0), and similar differences were not apparent at the end of the photoperiod treatment. A month later, AP rams (24.3 ± 7.9) had higher (p < .10) testosterone levels than NP rams (13.1 ± 5.0), with no effect of melatonin treatment. Fifty days after melatonin implantations, rams were exposed for 20 min to three oestrous ewes. AP rams (2.50 ± 0.42) exhibited significantly (p < .05) more serves than did NP rams (1.11 ± 0.39), and melatonin treatment had no significant effect; however, the interaction between treatments was significant. Time to first serve was significantly (p < .05) shorter in AP (2.30 ± 1.20 min) than it was in NP rams (5.58 ± 0.68 min). In conclusion, exposure to 2 months of long days in late winter/early spring, with a return to natural day length of shorter duration, increased plasma testosterone concentrations and sexual performance in rams with or without exogenous melatonin. This particular management is an option if a non‐hormonal reproductive strategy is scheduled; yet, if the use of exogenous hormones is feasible, melatonin implants increase the mating efficiency of rams.