• Asian-Australasian Journal of Animal Sciences
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• 제24권11호
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• pp.1529-1540
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• 2011

Silencing of a specific gene using RNAi (RNA interference) is a valuable tool for functional analysis of a target gene. However, information on RNAi for analysis of gene function in farm animals is relatively nil. In the present study, we have validated the interfering effects of siRNA (small interfering RNA) using both quantitative and qualitative gene silencing in buffalo granulosa cells. Qualitative gene knockdown was validated using a fluorescent vector, enhanced green fluorescence protein (EGFP) and fluorescently labeled siRNA (Cy3) duplex. While quantitatively, siRNA targeted against the luciferase and CYP19 mRNA was used to validate the technique. CYP19 gene, a candidate fertility gene, was selected as a model to demonstrate the technique optimization. However, to sustain the expression of CYP19 gene in culture conditions using serum is difficult because granulosa cells have the tendency to luteinize in presence of serum. Therefore, serum free culture conditions were optimized for transfection and were found to be more suitable for the maintenance of CYP19 gene transcripts in comparison to culture conditions with serum. Decline in fluorescence intensity of green fluorescent protein (EGFP) was observed following co-transfection with plasmid generating siRNA targeted against EGFP gene. Quantitative decrease in luminescence was seen when co-transfected with siRNA against the luciferase gene. A significant suppressive effect on the mRNA levels of CYP19 gene at 100 nM siRNA concentration was observed. Also, measurement of estradiol levels using ELISA (enzyme-linked immunosorbent assay) showed a significant decline in comparison to control. In conclusion, the present study validated gene silencing using RNAi in cultured buffalo granulosa cells which can be used as an effective tool for functional analysis of target genes.

• Asian-Australasian Journal of Animal Sciences
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• 제21권5호
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• pp.715-722
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• 2008

There are marked variations in the activity of lipoprotein lipase (LPL) among adipose depots. The aim of this study was to compare the mechanisms of 24 h of fasting on LPL regulation between epididymal (EPI) adipocytes and mesenteric (MES) adipocytes in rats. 1-Day fasting consistently decreased activities of heparin-releasable LPL, total extractable LPL and cellular LPL markedly in both EPI and MES fat pads. LPL activity in MES fat pads was relatively lower than in the EPI fat pads. Consistent with data on LPL activity, the levels of expression of LPL mRNA in both nutritional states were lower in MES than EPI adipose tissue and isolated adipocytes. The decreased LPL activity after 1 day of fasting in MES adipocytes was explained mainly by a 50% decrease in the relative abundance of LPL mRNA level and a parallel 50% decrease in relative rate of LPL synthesis. In contrast, fasting of 1 day in EPI adipocytes decreased total LPL activity by 47% but did not affect LPL mRNA level or relative rate of LPL synthesis. A decrease in overall protein synthesis contributed to the decreased LPL activity after 1 day fasting both in EPI and MES adipocytes. In MES adipocytes the decrease in LPL activity, LPL mRNA and LPL synthesis were comparable, but in EPI adipocytes the changes in LPL activity were substantially larger than the changes in LPL mRNA level and LPL synthesis. Therefore, fasting decreased fat cell size, LPL activity, LPL mRNA level and relative rate of LPL synthesis in rats, and these effects were more marked in the MES adipocytes. These results clearly demonstrate the regional variations in the metabolic response of adipose tissue and LPL functions to fasting.

• 한국발생생물학회지:발생과생식
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• 제22권2호
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• pp.175-182
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• 2018

In order to examine the effects of four different light spectra (white, red, green, and blue) on the oocyte maturation, the change of reproductive parameters, via brain-pituitary-gonad (BPG) axis in grass puffer, were investigated. After exposure four different light spectra for 7 weeks, the abundance of gonadotropin-releasing hormone (GnRH) mRNA which is a type of seabream (sbGnRH) and two different subunit of gonadotropin hormones mRNAs, follicle-stimulating hormone ($fsh{\beta}$) mRNA and luteinizing hormone ($lh{\beta}$) mRNA, were analyzed in the brain and pituitary. Histological analysis showed that the mature oocyte ratio in the green spectrum was higher than other light spectra-exposed groups. Gonadosomatic index (GSI) and oocyte developmental stage were also investigated in the gonad based on histological observations. GSI value with the presence of yolk stage oocytes was significantly increased in the green spectrum-exposed group when compared to that of the other light-exposed groups (white, red, and blue) (p<0.05). The abundances of sbGnRH mRNA and $fsh{\beta}$ mRNA in the green spectrum-exposed group were also significant higher than those of the other light spectra-exposed groups (p<0.05). These results indicate that the maturation of oocyte in grass puffer can be accelerated by exposure to the spectrum of green. To better understand the molecular mechanism for the maturation of oocyte in grass puffer, further study examining the relationship between oocyte development and its related genes is required.

• Asian-Australasian Journal of Animal Sciences
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• 제23권2호
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• pp.272-278
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• 2010

In this study, we cloned, sequenced and characterized porcine y+L Amino Acid Transporter-1 (y+LAT1). By screening a translated EST database with the protein sequence of the human $y^{+}$LAT1 and by using rapid amplification of cDNA ends (RACE), the full-length cDNA encoding porcine $y^{+}$LAT1 was isolated from porcine intestine RNA. It was 2,111 bp long, encoding a 511 amino acid trans-membrane glycoprotein composed of 12 transmembrane domains. The predicted amino acid sequence was found to be 91%, 90%, 87% and 87% identical to those of cattle, human, mouse and rat $y^{+}$LAT1 respectively. Real-time RT-PCR results indicated that the small intestine had the highest $y^{+}$LAT1 mRNA abundance and the lung had the lowest $y^{+}$LAT1 mRNA abundance. Baby hamster kidney (BHK) cells transfected with green fluorescent protein (GFP) tagged porcine $y^{+}$LAT1 cDNA indicated that the cellular localization of the gene product in BHK was on the plasma membrane.

• The Korean Journal of Physiology and Pharmacology
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• 제12권4호
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• pp.149-153
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• 2008

Endothelin-1 (ET-1) is unequivocally elevated in the kidney with ischemic acute renal failure (ARF), whereas ET receptors ($ET_AR$ and $ET_BR$) are variably expressed. Although renal functional and structural changes are similar between ischemic and nephrotoxic ARF, there are few reports on the alteration in the ET system in nephrotoxic ARF. This study was, therefore, undertaken to investigate changes in renal expression of ET-l and its receptors in nephrotoxic ARF induced by cisplatin. Mice were intraperitoneally injected with 16 mg of cisplatin/kg at a single dose, and the expression of mRNA and protein was then quantified by real-time RT-PCR and Western blot, respectively. Immunohistochemistry was conducted for localization. Three days after treatment, ET-1 transcript in cisplatin-treated mice was thirteen times higher than that in controls, whereas ET-1 peptide was increased by 1.5-fold. Cisplatin caused a 2-fold increase in the levels of ETAR mRNA and protein. Most of the increased immunoreactive ET-1 and ETAR were localized in damaged tubules. Neither the expression of ETBR mRNA nor the abundance and immunoreactive level of ETBR protein were changed. The findings suggest that the individual components of the renal ET system are differentially regulated in cisplatin-induced nephrotoxic ARF.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.99-99
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• 2003

Epidermal growth factor (EGF) induces well-documented mitegenic and differentiating effects on murine and bovine preimplantation embryos. However, the effects of EGF on apoptosis and implantation-related gene expression in bovine embryos developing in vitro have not been evaluated. The objective of this study was to determine the effects of exogenous EGF in the presence and absence of BSA on the preimplantation development of bovine embryos. In addition, we measured cell number, apoptosis, and expression of apoptosis and implantation-related genes of the blastocysts that developed in these culture conditions. In vitro produced bovine embryos were randomly cultured in the same medium containing 0 or 10 ng/$m\ell$ EGF in the presence and absence of 0.8% BSA. More 2-cell embryos developed into blastocysts at day 7 when BSA was present than when BSA was absent. The addition of 10 ng/$m\ell$ EGF into the medium did not significantly increase the developmental rate and the cell numbers per blastocyst. However, addition of EGF in the presence of 0.8% BSA significantly reduced the degree of apoptosis in the blastocysts (P< 0.01). To investigate whether EGF modulates mRNA expression of apoptosis-related genes, mRNA was prepared from single blastocysts and each preparation was subjected to RT-PCR for Bcl-2 and Bax transcripts. EGF did not alter the relative abundance of Bax gene expression in the presence of BSA, but increase Bcl-2 (P < 0.01). The relative abundance of Interferon tau expression was increased by EGF treatment in the presence of BSA. These results suggest that EGF and BSA synergistically enhance Bcl-2 and interferone tau gene expression, which may result in a net increase in viability in bovine embryos.

• The Plant Pathology Journal
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• 제38권6호
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• pp.583-592
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• 2022

Root-knot nematode disease is a widespread and catastrophic disease of tobacco. However, little is known about the relationship between rhizosphere bacterial community and root-knot nematode disease. This study used 16S rRNA gene sequencing and PICRUSt to assess bacterial community structure and function changes in rhizosphere soil from Meloidogyne incognita-infected tobacco plants. We studied the rhizosphere bacterial community structure of M. incognita-infected and uninfected tobacco plants through a paired comparison design in two regions of tobacco planting area, Yuxi and Jiuxiang of Yunnan Province, southwest China. According to the findings, M. incognita infection can alter the bacterial population in the soil. Uninfested soil has more operational taxonomic unit numbers and richness than infested soil. Principal Coordinate Analysis revealed clear separations between bacterial communities from infested and uninfested soil, indicating that different infection conditions resulted in significantly different bacterial community structures in soils. Firmicutes was prevalent in infested soil, but Chloroflexi and Acidobacteria were prevalent in uninfested soil. Sphingomonas, Streptomyces, and Bradyrhizobium were the dominant bacteria genera, and their abundance were higher in infested soil. By PICRUSt analysis, some metabolism-related functions and signal transduction functions of the rhizosphere bacterial community in the M. incognita infection-tobacco plants had a higher relative abundance than those uninfected. As a result, rhizosphere soils from tobacco plants infected with M. incognita showed considerable bacterial community structure and function alterations.

• Asian-Australasian Journal of Animal Sciences
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• 제21권8호
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• pp.1134-1142
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• 2008

Cationic amino acid transporter $b^{0,+}AT$ (HGMW-approved gene symbol SLC7A9, solute carrier family 7, member 9) plays a crucial role in amino acid nutrition. In the present study, we describe the cloning and sequencing of porcine $b^{0,+}AT$. Based on the sequence of porcine $b^{0,+}AT$ deposited in the NCBI (National Center for Biotechnological Information), we identified a putative porcine homologue. Using rapid amplification of cDNA ends (RACE), the full-length cDNA encoding porcine $b^{0,+}AT$ was isolated. The porcine $b^{0,+}AT$ cDNA was 1,680 bp long, encoding a 487 amino acid trans-membrane protein. The predicted amino acid sequence was found to have 88.9% and 87.1% identity with human and mouse $b^{0,+}AT$, respectively. Real-time RT-PCR indicated porcine $b^{0,+}AT$ transcripts expressed in heart, kidney, muscle and small intestine. The small intestine had the highest $b^{0,+}AT$ mRNA abundance while the muscle had the lowest (p<0.05). Along the longitudinal axis, the ileum had the highest $b^{0,+}AT$ mRNA abundance while the colon had the lowest (p<0.05). The $b^{0,+}AT$ mRNA level was highest on day 7 and 90 in the duodenum (p<0.05). It increased from day 1 to day 26 in the jejunum (p>0.05) and had the highest abundance on day 60 (p<0.05). There was, however, no difference between day 1, 7, 26, 30, 90 and 150 (p>0.05). The strongest $b^{0,+}AT$ expression appeared on day 7 in the ileum before weaning, and then decreased till day 30 but rose gradually again from day 60 to 150 (p<0.05).

• Asian-Australasian Journal of Animal Sciences
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• 제26권7호
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• pp.1012-1020
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• 2013

The objective of this study was to investigate the effect of dietary supplementation with thiazolidinedione (TZD) on growth performance and meat quality of finishing pigs. In Experiment 1, 80 castrated finishing pigs (Large White${\times}$Landrace, BW = 54.34 kg) were randomly assigned to 2 treatments with 5 replicates of 8 pigs each. The experimental pigs in the 2 groups were respectively fed with a diet with or without a TZD supplementation (15 mg/kg). In Experiment 2, 80 castrated finishing pigs (Large White${\times}$Landrace, BW = 71.46 kg) were divided into 2 treatments as designed in Experiment 1, moreover, carcass evaluations were performed. The results from Experiment 1 showed that TZD supplementation could significantly decreased the average daily feed intake (ADFI) (p<0.05) during 0 to 28 d, without impairing the average daily gain (ADG) (p>0.05). In Experiment 2, the ADG was significantly increased by TZD supplementation during 14 to 28 d and 0 to 28 d (p<0.05) and the feed:gain ratio (F:G) was significantly decreased by TZD supplementation during 0 to 28 d (p<0.05). Compared with the control group, TZD group had significantly higher serum triglyceride (TG) concentration at 28h and serum high-density lipoprotein (HDL) levels at 14 d (p<0.05). Moreover, there was an apparent improvement in the marbling score (p<0.10) and intramuscular fat (IMF) content (p<0.10) of the longissimus dorsi muscle in pigs treated by TZD supplementation. Real-time RT-PCR analyses demonstrated that pigs of TZD group had higher mRNA abundance of $PPAR{\gamma}$ coactivator 1 (PGC-1) (p<0.05) and fatty acid-binding protein 3 (FABP3) (p<0.05) than pigs of control group. Taken together, these results suggested that dietary TZD supplementation could improve growth performance and increase the IMF content of finishing pigs through regulating the serum parameters and genes mRNA abundance involved in fat metabolism.

• Journal of Animal Science and Technology
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• 제64권1호
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• pp.52-69
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• 2022

Ferulic acid (FA) is a phytochemical with various bioactive properties. It has recently been proposed that due to its phytogenic action it can be used as an alternative growth promoter additive to synthetic compounds. The objective of the present study was to evaluate the growth performance, carcass traits, fiber characterization and skeletal muscle gene expression on hair-lambs supplemented with two doses of FA. Thirty-two male lambs (n = 8 per treatment) were individually housed during a 32 d feeding trial to evaluate the effect of FA (300 and 600 mg d^-1) or zilpaterol hydrochloride (ZH; 6 mg d^-1) on growth performance, and then slaughtered to evaluate the effects on carcass traits, and muscle fibers morphometry from Longissimus thoracis (LT) and mRNA abundance of β₂-adrenergic receptor (β₂-AR), MHC-I, MHC-IIX and IGF-I genes. FA increased final weight and average daily gain with respect to non-supplemented animals (p < 0.05). The ZH supplementation increased LT muscle area, with respect to FA doses and control (p < 0.05). Cross-sectional area (CSA) of oxidative fibers was larger with FA doses and ZH (p < 0.05). Feeding ZH increased mRNA abundance for β₂-AR compared to FA and control (p < 0.05), and expression of MHC-I was affected by FA doses and ZH (p < 0.05). Overall, FA supplementation of male hair lambs enhanced productive variables due to skeletal muscle hypertrophy caused by MHC-I up-regulation. Results suggest that FA has the potential like a growth promoter in lambs.