Melatonin is a key hormone that regulates circadian rhythms, metabolism, and reproduction. However, the mechanisms of melatonin synthesis and secretion have not been fully defined. The purpose of this study was to investigate the functions of the LIM homeobox transcription factor Isl1 in regulating melatonin synthesis and secretion in porcine pineal gland. We found that Isl1 is highly expressed in the melatonin-producing cells in the porcine pineal gland. Further functional studies demonstrate that Isl1 knockdown in cultured primary porcine pinealocytes results in the decline of melatonin and arylalkylamine N-acetyltransferase (AANAT) mRNA levels by 29.2% and 72.2%, respectively, whereas Isl1 overexpression raised by 1.3-fold and 2.7-fold. In addition, the enhancing effect of norepinephrine (NE) on melatonin synthesis was abolished by Isl1 knockdown. The in vivo intracerebroventricular NE injections upregulate Isl1 mRNA and protein levels by about threefold and 4.5-fold in the porcine pineal gland. We then examined the changes in Isl1 expression in the pineal gland and global melatonin levels throughout the day. The results show that Isl1 protein level at 24:00 is 2.5-fold higher than that at 12:00, which is parallel to melatonin levels. We further found that Isl1 increases the activity of AANAT promoter, and the effect of NE on Isl1 expression was blocked by an ERK inhibitor. Collectively, the results presented here demonstrate that Isl1 positively modulates melatonin synthesis by targeting AANAT, via the ERK signaling pathway of NE. These suggest that Isl1 plays important roles in maintaining the daily circadian rhythm.

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-To demonstrate the role of gonadotropin-releasing hormone (GnRH) in yaks (Bos grunniens), we characterized the expression of gonadotropin-releasing hormone receptor (GnRHR) mRNA and protein. The level of GnRHR mRNA in the hypothalamus was higher than that in the pineal gland, pituitary gland, and ovary during estrus. Immunofluorescence analysis showed that GnRHR was expressed in the pinealocyte, synaptic ribbon, and synaptic spherules of the pineal gland and that melatonin interacts with GnRHR via nerve fibers. In the hypothalamus, GnRHR was expressed in the magnocellular neurons and parvocellular neurons. In the pituitary gland, GnRHR was expressed in acidophilic cells and basophilic cells. In the ovary, GnRHR was present in the ovarian follicle and Leydig cells. Gonadotropin-releasing hormone receptor is located in the pineal gland, hypothalamus, pituitary, and gonad during estrus of yaks and is mainly expressed in the hypothalamus and ovaries during the estrus period.

-The objective of this study was to investigate mRNA by real-time PCR and protein expression by immunofluorescence of the estradiol receptors (ER) in the pineal gland, hypothalamus, pituitary gland, and gonads of yaks (Bos grunniens). The analysis showed that the level of expression of ER mRNA was greater in the pituitary gland tissue than in other glands during estrus. Immunofluorescence analyses showed that ER proteins were located in the pineal cells, synaptic ribbon, and synaptic spherules of the pineal gland. In the hypothalamus, ER proteins were located in the magnocellular and parvocellular neurons. The ER proteins were located in acidophilic cells and basophilic cells in the pituitary gland. In the ovary, ER proteins were present in the ovarian follicle, corpus luteum and Leydig cells. Estradiol exerts its main effects on the pituitary gland during estrus in yak.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant threat to the pig industry in China. However, the epidemiological characteristics of PRRSV after the outbreak of African swine fever in China were not thoroughly investigated. In the present study, the serological and epidemiological investigations of PRRSV in pigs from the Hunan and Hebei provinces of China were assessed. The results showed that 73.12% (95% CI 71.74–74.49) of pigs were positive for PRRSV-special antibody by enzyme-linked immunosorbent assay. Out of 5799 samples, 482 (8.31%, 95% CI 7.60–9.02) samples were positive for PRRSV nucleic acids. The positive rates of PRRSV in healthy pigs from farms and slaughterhouses were 2.27% (47/2072) and 7.70% (217/2818), which were lower than that in diseased pigs (23.98%, 218/909). Furthermore, the full-length OFR5 gene sequences of 43 PRRSV strains were sequenced and analysed. Phylogenetic analysis revealed that 43 isolates were classified into three lineages, namely lineage 1 (n = 24), lineage 8 (n = 15), and lineage 3 (n = 4). Lineage 1 could be further divided into sublineage 1.5 (n = 2) and sublineage 1.8 (n = 22), and lineage 8 was classified into sublineage 8.1 (n = 3) and sublineage 8.7 (n = 12). Collectively, our findings revealed the severe prevalence of PRRSV in the Hunan and Hebei provinces, where sublineage 1.8 and sublineage 8.7 predominated. The present study provides the update information of the epidemiological and genetic characteristics of PRRSV in the investigated regions, which will be beneficial for PRRS control.

The potential reproduction power of domestic animals is limited by a complicated follicular atresia process. P53, caspase-9 (Casp9), Bax, Bcl-2 and Fas play a crucial role in the ovarian mitochondrion-dependent apoptosis and death receptor pathway. In accordance with this study, the expression levels of Casp9, Bax, Bcl-2 and Fas were analysed in ovaries and oviducts of yak by immunohistochemistry (IHC). P53 and the above in ovarian granulosa cells (GCs) from atretic (3-6 mm) to healthy follicles (6-8 mm) and in oviducts were examined from the luteal phase to the follicular phase during the oestrous circle by Western blot (WB) and real-time PCR (RT-PCR). Results demonstrated that typical classic apoptotic factors Casp9, Bax, Bcl-2 and Fas were expressed in the cytoplasm and zonal pellucida of oocytes, primordial follicles, primary follicles, ovarian surface epithelium, ovarian GCs, granular lutein cells, surface epithelia in oviduct uterotubal junction and oviduct ampulla during the luteal phase. RT-PCR and WB revealed that P53 and Fas significantly increased in GCs of atretic follicles.P53 and Casp9 increased in oviduct epithelium during the luteal phase, but Fas was unchanged. A contrary tendency was noted in Bcl-2 and Bax expression. Overall, P53 and Fas play an essential role in inducing GC apoptosis, and Bax, Bcl-2, Casp9 and P53 are involved in oviduct epithelial regeneration in yak.

The aim of the present study was to establish a method of EvaGreen real time fluorescence quantitative polymerase chain reaction (qPCR) for detecting quickly bovine rotavirus (BRV) in fecal samples from calves with diarrhea. The specific primers were designed and synthesized according to BRV VP7 gene in Genbank. VP7 gene was cloned into the pMD18-T vector. The bovine viral diarrhea virus, bovine coronavirus, porcine epidemic diarrhea virus, bovine mycobacterium tuberculosis and negative control were detected with qPCR. Plasmids in five 10-fold gradients were detected using qPCR. The results show that a EvaGreen qPCR was established in the present study. Only BRV displayed amplification curve; cross-reactivity between BRV and the other viruses or bacteria was not observed. The amplification curve of pMD18-T vector plasmid (diluted in 10 -1 to 10 3 gradient) was typical S shape. The detection limit of qPCR was 8.0 copies/μL, namely 10 0 plasmid concentrations. The coefficients of variation in both intra-assay and inter-assay was less than 2%. The established EvaGreen qPCR had a high specificity, sensitivity and reproducibility. It can be applied to clinical diagnosis and epidemiological surveys of BRV.

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