Search results for: porcine follicular fluid

Authors: Panomporn Wisuthseriwong, Hatairuk Tungkasen, Siyaporn Namsongsan, Chanikarn Srinark, Mayuva Youngsabanant-Areekijseree

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The objective of the present study was to evaluate the morphology of porcine cumulus-oocyte complexes (pCOCs) and follicular fluid during follicular development. The samples were obtained from local slaughterhouses in Nakorn Pathom Province, Thailand. Pigs were classified as either in the follicular phase or luteal phase. Porcine follicles (n = 3,510) were categorized as small (1-3 mm in diameters; n=2,910), medium (4-6 mm in diameters; n=530) and large (7-8 mm in diameters; n=70). Then pCOCs and follicular fluid were collected. Finally, we found that the oocytes can be categorized into intact cumulus cells layer oocyte, multi-cumulus cells layer oocyte, partial cumulus cells layer oocyte, completely denuded oocyte and degenerated oocyte. They showed high percentage of intact and multi-cumulus cells layer oocytes from small follicles (54.68%) medium follicles (69.06%) and large follicles (68.57%), which have high potential to develop into matured oocytes in vitro. Protein composition of the follicular fluid was separated by SDS-PAGE technique. The result shows that the protein molecular weight in the small and medium follicles are 23, 50, 66, 75, 92, 100, 132, 163, 225 and >225 kDa. Meanwhile, protein molecular weight in large follicles are 12, 16, 23, 50, 66, 75, 92, 100, 132, 163, 225 and >225 kDa. All proteins play an important role in promotion and regulation on development, maturation of oocytes and regulation of ovulation. We conclude that the results of discovery can be used porcine secretion proteins for supplement in IVM/IVF technology. Acknowledgements: The project was funded by a grant from Silpakorn University Research and Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: porcine follicles, porcine oocyte, follicular fluid, SDS-PAGE

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Authors: Hatairuk Tungkasen, Somrudee Phetchrid, Suwapat Jaidee, Supinya Yoomak, Chantana Kankamol, Mayuree Pumipaiboon, Mayuva Areekijseree

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Ovaries of reproductive female pigs were obtained from local slaughterhouses in Nakorn Pathom Province, Thailand. Follicular fluid of medium follicle (5-6 diameters) and large follicles (7-8 mm and 10 mm in diameter) were aspirated and collected by sterile technique and analyzed protein pattern. The follicular fluid protein bands were found by SDS-PAGE which has no protein band in difference compared to standard protein band. So we chose protein band molecular weight 50, 62-65, 75-80, 90, 120-160, and >220 kDa were analyzed by LC/MS/MS. The result was found immunoglobulin gamma chain, keratin, transferrin, heat shock protein, and plasminogen precursor, ceruloplasmin, and hemopexin, and protease, respectively. All proteins play important roles in promotion and regulation on growth and development of reproductive cells. The result of this study found many proteins which were useful and important for in vitro oocyte maturation and embryonic development of cell technology in animals. The further study will be use porcine follicular fluid protein of medium and large follicles as feeder cells in in vitro condition to promote oocyte and embryo maturation.

Keywords: follicular fluid protein, LC/MS/MS, porcine oocyte, SDS-PAGE

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Authors: Mayuva Youngsabanant-Areekijseree, Chanikarn Srinark, S. Sengsai, Mayuree Pumipaiboon

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Our project was aimed at morphology of oocytes, follicle cells and follicular fluid proteins study of Large White pig (at local slaughter house in Nakhon Pathom Province). The porcine oocytes and follicular fluid of healthy small follicles (1-2 mm), medium follicles (3-6 mm in diameters) and large follicles (7-8 mm and 10 mm in diameter) were aspirated and collected from the ovary by sterile technique. Then, the oocytes and the follicle cells were separated from the fluid. The oocytes were round shape and surrounded by zona pellucida with numerous layers of cumulus cells. Based on the number of cumulus cell layers surrounding oocytes, the oocytes were classified into 5 types, which were intact-, multi-, partial-cumulus layer oocyte, completely denuded oocyte and degenerative oocyte. The collected oocytes showed high percentages of intact- and multi- cumulus cell layers in the small follicles (53.48%) medium follicles (56.94%) and large follicles (56.52%) which have high potential to develop into mature oocytes in vitro. Proteins from follicular fluid of 3 size follicles were separated by SDS-PAGE and LC/MS/MS. The molecular weight of follicular fluid proteins from the small follicles were 24, 60-65, 79, 110, 140, 160, and > 220 kDa. Meanwhile, the follicular fluid protein from medium and large follicle contained 52, 65, 79, 90, 110, 120, 160, 190 and > 220 kDa. Almost all proteins played important roles in promoting and regulating growth and development of oocytes and ovulation. This finding was an initial tool for in vitro testing and applied biotechnology research. Acknowledgements: The project was funded by a grant from Silpakorn University Research & Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: follicular fluid protein, LC/MS/MS, porcine oocyte, SDS-PAGE, reproductive biology

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Authors: Heba F. Hozyen, Hodallah H. Ahmed, S. I. A. Shalaby, G. E. S. Essawy

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Biochemical and hormonal changes that occur in both follicular fluid and blood are involved in the control of ovarian physiology. The present study was conducted on follicular fluid and serum samples obtained from 708 buffaloes. Samples were examined for estradiol, progesterone, and cholesterol concentrations in relation to seasonal changes, ovarian follicular size, and stage of estrous cycle. The obtained results revealed that follicular fluid and serum levels of estradiol, progesterone, and cholesterol were significantly lower during summer and autumn when compared to winter and spring seasons. With the increase in follicular size, the follicular fluid levels of progesterone and cholesterol were significantly decreased, while estradiol levels were significantly increased. Estradiol and progesterone levels were significantly higher in follicular fluid than blood, while cholesterol was significantly lower in follicular fluid than serum. In conclusion, the current study threw a light on the hormonal changes in the follicular fluid and blood under the effect of heat stress which could be related to the low fertility of buffalo in the summer.

Keywords: buffalo, follicular fluid, folliculogenesis, seasonal changes, steroids

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Authors: Mayuva Youngsabanant, Chanikarn Srinark, Supanyika Sengsai, Soratorn Kerdkriangkrai, Nongnuch Gumlungpat, Mayuree Pumipaiboon

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The follicular fluid proteins of healthy medium size follicles (4-6 mm in diameters) and large size follicles (7-8 mm in diameter) of large white pig ovaries were collected by using sterile technique. They were used for testing the effect on primary in vitro cell culture growth of porcine oviductal epithelial cells (pOEC). Porcine oviductal epithelial cells of luteal phase was culture in M199 and added with 10% fetal calf serum 2.2 mg/mL, NaHCO₃, 0.25 mM pyruvate, 15 µg/mL and 50 µg/mL, gentamycin sulfate at high humidified atmosphere with 5% CO₂ in 95% air atmosphere at 37°C for 96 h before testing. The optimized concentration of pFF of two follicle sizes (at concentration of 2, 4, 20, 40, 200, 400, 500, and 600 µg proteins) in culture medium was observed for 24 h using MTT assay. Results were analyzed with a one-way ANOVA in SPSS statistic. Moreover, pOEC was also studied in morphological characteristic on long-term culture. The results of long-term study revealed that pOEC showed 70-80 percentage of healthy morphology on epithelial-like character and contained 30 percentage of an elongated shape (fibroblast-like morphology) at 4 weeks of culture time. MTT assay reviewed an increase in the percentage of viability of pOEC in 2 treated of follicular fluid groups. Two treatment concentration groups were higher than control group (p < 0.05) but not in positive control group. Interestingly, at 200 µg protein of 2 treated follicular fluid groups were reached the highest cell viability which is higher than a positive control and it is significantly different form control group (P < 0.05). These cells are developed and had fibroblast elongate shape which is longer than the cells in control group and positive control group. This report implies that pFF of medium follicle size at 200 µg proteins and large follicle size at 200 and 500 µg proteins could be optimized concentration for using as a supplement in culture medium to promote cell growth and development instead of growth hormone from fetal calf serum. It could be applied in cell biotechnology researches. Acknowledgements: The project was funded by a grant from Silpakorn University Research and Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: in vitro, porcine follicular fluid protein (pFF), porcine oviductal epithelial cells (pOEC), MTT

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Authors: Mayuva Youngsabanant, Suphaphorn Rabiab, Hatairuk Tungkasen, Nongnuch Gumlungpat, Mayuree Pumipaiboon

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The porcine follicular fluid proteins (pFF) of healthy small size ovarian follicles (1-3 mm in diameters) of Large White pig ovaries were collected by sterile technique. They were used for testing the effect on cell viability and characterization of Vero cell lines using MTT assay. Two hundred microliter of round shape Vero cell lines were culture in 96 well plates with DMEM for 24 h. After that, they were attachment to substrate and some changed into fibroblast shape and spread over the surface after culture for 48 h. Then, Vero cell lines were treated with pFF at concentration of 2, 4, 20, 40, 200, 400, 500, and 600 µg proteins/mL for 24 h. Yields of the best results were analyzed by using one-way ANOVA. MTT assay reviewed an increasing in percentage of viability of Vero cell lines indicated that at concentration of 400-600 µg proteins/mL showed higher percentage of viability (115.64 ± 6.95, 106.91 ± 5.27 and 116.73 ± 20.15) than control group. They were significantly different from the control group (p < 0.05) but lower than the positive control group (DMEM with 10% heat treated fetal bovine serum). Cell lines showed normal character in fibroblast elongate shape after treated with pFF except in high concentration of pFF. This result implies that pFF of small size ovarian follicle at concentration of 400-600 µg proteins/mL could be optimized concentration for using as a supplement in Vero cell line culture medium to promote cell viability instead of growth hormone from fetal bovine serum. This merit could be applied in other cell biotechnology researches. Acknowledgements: This work was funded by a grant from Silpakorn University and Faculty of Science, Silpakorn University, Thailand.

Keywords: cell viability, porcine follicular fluid, MTT assay, Vero cell line

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Authors: H. F. Hozyen, A. M. Abo-El Maaty

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Heat stress is a recognized problem causing huge economic losses to the buffalo breeders as well as dairy industry. The aim of the present work was to study the pattern of vitamin C and nitric oxide in follicular fluid of buffalo during different seasons of the year considering phase of estrous cycle. This study was conducted on 208 cyclic buffaloes slaughtered at Al-Qaliobia governorate, Egypt, over one year. The obtained results revealed that vitamin C in follicular fluid was significantly lower in summer than winter and spring. On the other hand, nitric oxide (NO) was significantly higher in summer and autumn than winter and spring. Both vitamin C and NO did not differ significantly between follicular and luteal phases. In conclusion, the present study revealed that alterations in concentrations of follicular fluid vitamin C and NO that occur in summer could be related to low summer fertility in buffalo.

Keywords: Buffalo, follicular fluid, vitamin C, nitric oxide, heat stress

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Authors: Tery Nehemia Nugraha Joseph, J. D. P. Wisnubroto

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Thyroid cancer is a healthcare problem with high morbidity and mortality. Follicular adenoma and follicular carcinoma are thyroid tumors from the thyroid follicular cells differentiation with a microfollicular pattern that consists of follicular cuboidal cells. vascular endothelial growth factor (VEGF) is a potent and powerful mitogen for endothelial cells and increases vascular permeability. Therefore, due to an increase in thyroid-stimulating hormone (TSH), VEGF production is activated in the thyroid that leads to the end of mitogenic TSH stimulation and initiation of angiogenesis. The differences in VEGF levels in the follicular carcinoma of thyroid tissue with follicular adenoma thyroid can be used as a basis in differentiating the two types of neoplasms. This study aims to analyze VEGF in the serum so that it can be used to differentiate the types of thyroid carcinoma before surgery. This study uses a cross-sectional research design. Samples were carried out by taking serum samples, and the VEGF levels were calculated. Data were analyzed using the Mann-Whitney test. The results found a significant difference between VEGF levels in the follicular carcinoma thyroid group and VEGF levels in the follicular adenoma thyroid group with a value of p = 0.007 (p < 0.05). The results obtained are 560,427 ± 160,506 ng/mL in the type of follicular carcinoma thyroid and 320.943 ± 134.573 ng/mL in the type of follicular adenoma thyroid. VEGF levels between follicular adenoma and follicular carcinoma are different. VEGF levels are higher in follicular carcinoma thyroid than follicular adenoma thyroid.

Keywords: follicular adenoma thyroid, follicular carcinoma thyroid, thyroid, VEGF

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Authors: Al-Mutary M., Alhimaidi A., Al-Ghadi M. Iwamoto D., Javed Ahmad. Abdulaziz A. Al-Khedhairy

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The aim of the present study was to evaluate the effect of ovine follicular fluid supplementation during IVM of sheep oocytes on the resumption of meiosis, glutathione (GSH) content and expression of Bax, Bcl-2, and HSPB1 genes. Sheep ovaries were collected from Riyadh slaughterhouse, KSA. Oocytes were aspirated from 3-6 mm follicles. Ovine oocytes were cultured in maturation medium with 0% (control), 10%, 20%, 40% of ovine follicular fluid for 24 h. Results indicated that the rate of oocyte maturation was significantly (P≤0.05) decreased in 40% OFF (36.87%) versus the control (61.3%), 10% OFF (63.95%) and 20% OFF (64.08%). Supplementation of 10% OFF to IVM medium induced an intra-oocyte GSH concentration significantly higher than that found in ovine oocytes cultured with 20% OFF and 40% OFF and similar to the GSH content in oocytes cultured without FF. Real time polymerase chain reaction analysis for gene expression revealed no differences in Bax, Bcl-2, HSPB1 genes between control and 10% OFF group, whereas they were strongly expressed in 20% OFF and 40% OFF (P < 0.05) when compared to the control and 10% OFF. In conclusion the addition of 10% OFF to the IVM culture of sheep oocytes is recommended to support cytoplasmic maturation and increase oocytes competence.

Keywords: IVM, oocyte maturation, gene expression, follicular fluid

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Authors: Hamidreza Khodaei, Behnaz Mahdavi, Leila Karshenas

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Nitric oxide (NO) is a small fast acting neurotransmitter, which is synthesized From L-arginine by nitric oxide synthase. Studies show that NO affects a wide range of reproductive functions. Steroidal hormones synthesis, LH surge during ovulation, follicular growth and ovulation are all affected by NO. Therefore, the objective of this study was to evaluate the relationship between NO and estradiol (E2) production in ovarian follicles and cysts in bovines. Two experiment groups were formed and serum and follicular fluid levels Of NO and estradiol (E2) was measured. In the first group, follicular fluids were obtained from 30 slaughtered cows. Follicles were divided into three groups according to follicular diameter: Small follicles, <5 mm, medium-sized follicles, 5 to 10 mm, and large follicles, >10 mm. 30 follicles were randomly selected within each group. Blood samples were obtained via jugular vein. NO concentrations in blood and ovarian follicular fluids were measured by Griess reaction method and radio-immunoassay respectively. In the second group: 12 cows in follicular phase and with cystic follicles were selected and a cystic follicle was obtained from each. NO and E2 levels were measured as done for the first experiment group. The data were analyzed by SAS software using ANOVA and Duncan’s test. NO concentrations of follicular fluids from large follicles were significantly higher than those of the medium and small-sized ones. There were significant differences in the concentrations of nitrite and nitrate (Stable metabolites of NO) between large and cystic follicles, with extremely low NO and high E2 levels in cystic follicles (p<0.01).The results suggest that paracrine effects of NO may play an important role in the control of ovarian follicle growth and development of cystic follicles in bovines. It seems that NO dictates its effects through inhibition of ovarian steroidal synthesis.

Keywords: nitric oxide, estradiol, cystic follicle, cow, oogenesis, oocyte maturation, follicular fluid

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Authors: Mohd Sukri Hassan, Ahlam Inayatullah Badrul Munir, M. Husaini A. Rahman

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Halal authentication and verification in supplement capsules are highly required as the gelatine available in the market can be from halal or non-halal sources. It is an obligation for Muslim to consume and use the halal consumer goods. At present, real-time polymerase chain reaction (RT-PCR) is the most common technique being used for the detection of porcine and bovine DNA in gelatine due to high sensitivity of the technique and higher stability of DNA compared to protein. In this study, twenty samples of supplements capsules from different products with different Halal logos were analyzed for porcine and bovine DNA using RT-PCR. Standard bovine and porcine gelatine from eurofins at a range of concentration from 10^-1 to 10^-5 ng/µl were used to determine the linearity range, limit of detection and specificity on RT-PCR (SYBR Green method). RT-PCR detected porcine (two samples), bovine (four samples) and mixture of porcine and bovine (six samples). The samples were also tested using FT-IR technique where normalized peak of IR spectra were pre-processed using Savitsky Golay method before Principal Components Analysis (PCA) was performed on the database. Scores plot of PCA shows three clusters of samples; bovine, porcine and mixture (bovine and porcine). The RT-PCR and FT-IR with chemometrics technique were found to give same results for porcine gelatine samples which can be used for Halal authentication.

Keywords: halal, real-time PCR, gelatine, chemometrics

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Authors: M. Areekijseree, W. Pongsawat, M. Pumipaiboon, C. Thepsithar, S. Sengsai, T. Chuen-Im

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Morphological interaction of porcine cumulus-oocyte complexes (pCOCs) was investigated on in vitro condition using electron microscope (SEM and TEM). The totals of 1,923 oocytes were round in shape, surrounded by zona pellucida with layer of cumulus cells ranging between 59.29-202.14 µm in size. They were classified into intact-, multi-, partial cumulus cell layer oocyte, and completely denuded oocyte, at the percentage composition of 22.80% 32.70%, 18.60%, and 25.90 % respectively. The pCOCs classified as intact- and multi cumulus cell layer oocytes were further culturing at 37°C with 5% CO2, 95% air atmosphere and high humidity for 44 h in M199 with Earle’s salts supplemented with 10% HTFCS, 2.2 mg/mL NaHCO3, 1 M Hepes, 0.25 mM pyruvate, 15 µg/mL porcine follicle-stimulating hormone, 1 µg/mL LH, 1µg/mL estradiol with ethanol, and 50 µg/mL gentamycin sulfate. On electron microscope study, cumulus cells were found to stick their processes to secrete substance from the sac-shape end into zona pellucida of the oocyte and also communicated with the neighboring cells through their microvilli on the beginning of incubation period. It is believed that the cumulus cells communicate with the oocyte by inserting the microvilli through this gap and embedded in the oocyte cytoplasm before secreting substance, through the sac-shape end of the microvilli, to inhibit primary oocyte development at the prophase I. Morphological changes of the complexes were observed after culturing for 24-44 h. One hundred percentages of the cumulus layers were expanded and cumulus cells were peeling off from the oocyte surface. In addition, the round-shape cumulus cells transformed themselves into either an elongate shape or a columnar shape, and no communication between cumulus neighboring cells. After 44 h of incubation time, diameter of oocytes surrounded by cumulus cells was larger than 0 h incubation. The effect of hormones in culture medium is exerted by their receptors present in porcine oocyte. It is likely that all morphological changes of the complexes after hormone treatment were to allow maturation of the oocyte. This study demonstrated that the association of hormones in M199 could promote porcine follicle activation in 44 h in vitro condition. This culture system should be useful for studying the regulation of early follicular growth and development, especially because these follicles represent a large source of oocytes that could be used in vitro for cell technology.

Keywords: cumulus cells, electron microscopy, in vitro, porcine oocyte

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Authors: Ahlam Inayatullah Badrul Munir, M. Husaini A. Rahman, Mohd Sukri Hassan

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Real-time PCR is a molecular biology technique that is currently being widely used for halal services to differentiating between porcine and bovine DNA. The useful of technique become very important for student or workers (who works in the laboratory) to learn how the technique could be run smoothly without fail. Same concept with conventional PCR, real-time PCR also needed DNA template, primer, enzyme polymerase, dNTP, and buffer. The difference is in real-time PCR, have additional component namely fluorescent dye. The most common use of fluorescent dye in real-time PCR is SYBR green. The purpose of this study was to find out how sensitive, specific and efficient real-time PCR technique was combined with SYBR green method and specific primers of CYT b. The results showed that real-time PCR technique using SYBR Green, capable of detecting porcine and bovine DNA concentrations up to 0.0001 µl/ng. The level of efficiency for both types of DNA was 91% (90-110). Not only that in specific primer CYT b bovine primer could detect only bovine DNA, and porcine primer could detect only porcine primer. So, from the study could be concluded that real-time PCR technique that was combined with specific primer CYT b and SYBR green method, was sensitive, specific and efficient to detect porcine and bovine DNA.

Keywords: sensitivity, specificity, efficiency, real-time PCR, SYBR green, Cytochrome b, porcine DNA, bovine DNA

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Authors: Duaa Mughal, Syeda Areeba Nadeem, Shakil Ahmed, Ishtiaq Ahmed Khan

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The identification of porcine DNA in Halal food items is critical to ensuring compliance with dietary restrictions and religious beliefs. In Islam, Porcine is prohibited as clearly mentioned in Quran (Surah Al-Baqrah, Ayat 173). The purpose of this study was to compare two DNA extraction procedures for detecting 0.001% of porcine DNA in processed Halal food sample mixtures containing chicken, camel, veal, turkey and goat meat using the TaqMan Real-Time PCR technology. In this research, two different commercial kit protocols were compared. The processed sample mixtures were prepared by spiking known concentration of porcine DNA to non-porcine food matrices. Afterwards, TaqMan Real-Time PCR technique was used to target a particular porcine gene from the extracted DNA samples, which was quantified after extraction. The results of the amplification were evaluated for sensitivity, specificity, and reproducibility. The results of the study demonstrated that two DNA extraction techniques can detect 0.01% of porcine DNA in mixture of Halal food samples. However, as compared to the alternative approach, Eurofins| GeneScan GeneSpin DNA Isolation kit showed more effective sensitivity and specificity. Furthermore, the commercial kit-based approach showed great repeatability with minimal variance across repeats. Quantification of DNA was done by using fluorometric assay. In conclusion, the comparison of DNA extraction methods for detecting porcine DNA in Halal food sample mixes using the TaqMan Real-Time PCR technology reveals that the commercial kit-based approach outperforms the other methods in terms of sensitivity, specificity, and repeatability. This research helps to promote the development of reliable and standardized techniques for detecting porcine DNA in Halal food items, religious conformity and assuring nutritional.

Keywords: real time PCR (qPCR), DNA extraction, porcine DNA, halal food authentication, religious conformity

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Authors: Marcela Capcarova, Adriana Kolesarova, Marina Medvedova, Peter Petruska, Alexander V. Sirotkin

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The aim of this study was to determine the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), total antioxidant status (TAS) and accumulation of Hsp70 in porcine ovarian granulosa cells after deoxynivalenol (DON) and zearalenone (ZEA) exposure in vitro. Porcine ovarian granulosa cells were incubated with DON/ZEA administrations as follows: group A (10/10 ng/mL), group B (100/100 ng/mL), group C (1000/1000 ng/mL), and the control group without any additions for 24h. In this study mycotoxins developed stress reaction of porcine ovarian granulosa cells and increased accumulation of Hsp70 what resulted in increasing activities of SOD and GPx in groups with lower doses of mycotoxins. High dose of DON and ZEA had opposite effect on GPx activity than the lower doses. Slight increase in TAS of porcine granulosa cells was observed after mycotoxins exposure. These results contribute towards the understanding of cellular stress and its response.

Keywords: deoxynivalenol, zearalenone, antioxidants, Hsp70, granulosa cells

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Authors: M. Youngsabanant-Areekijseree, C. Thepsithar, K. Sribuddhachart, J. Tananantayot

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Porcine oviductal fluid (pOF) from oviduct, an unused organ from the slaughterhouse, was effectively used for biotechnology studies. The fluid components consisted of micro- and macro-nutrients, amino acids, carbon source and proteins that played important roles in animal cell and embryo development. This was our knowledge on investigating pOF as growth promoting substance in culture medium of an orchid, Dendrobium mirbelianum. Two-leaf shoots were cultured in liquid Vacin and Went (VW) medium as a standard medium supplemented with 2 g/L peptone (Pe) or 100 g/ L boiled-potato water (Po) alone or in combinations, and added with 0, 1, 3 or 5 ml/L pOF. All explants were cultured in a stationary condition for 8 weeks. It was found that medium added with 100 g/L Po and 1 ml/L pOF provided the best results (1.02 g fresh weight, 4.2 shoots, 0.53 cm shoot height, 4.4 protocorms, 11.0 leaves and 5.7 roots with 100% survival) when compared to other medium, but not statistically significant difference from medium added with 100 g/L Po (0.86 g fresh weight, 4.3 shoots, 0.51 cm shoot height, 4.6 protocorms, 12.4 leaves and 6.6 roots with 100% survival). However, VW medium supplemented with 1 or 3 ml/L pOF alone showed the higher percentage of survival (100%) than VW medium (86.67%). It was shown the potential role of pOF as an organic supplement for promoting growth of plants. Acknowledgements—The project was funded by a grant from Silpakorn University Research & Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: Dendrobium mirbelianum, pig, oviductal fluid, in vitro growth

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Authors: Li Yuchen, Wu Qingxin, Jin Yuxing, Yang Qian

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Interleukin-11 (IL-11), a well-known anti-inflammatory factor, helps to protect against intestinal epithelium damage caused by physical or chemical factors. However, little is known about the role of IL-11 during viral infection. Herein, high mRNA and protein levels of IL-11 were found in epithelial cells and jejunum of piglets during porcine epidemic diarrhea virus (PEDV) infection, and IL-11 expression was positively correlated with the level of viral infection. Pretreatment with recombinant porcine IL-11 (pIL-11) suppressed PEDV replication in Vero E6 cells, while IL-11 knockdown promoted viral infection. Furthermore, pIL-11 inhibited viral infection by preventing PEDV-mediated apoptosis of cells through activating the IL-11/STAT3 signal pathway. Conversely, application of a STAT3 phosphorylation inhibitor significantly antagonized the anti-apoptosis function of pIL-11 and counteracted its inhibition of PEDV. Our data suggested that that IL-11 is a novel PEDV-inducible cytokine, and its production enhances the anti-apoptosis ability of epithelial cells against PEDV infection. The potential uses of IL-11 as a novel therapeutic against devastating viral diarrhea in piglets deserves more attention and study.

Keywords: Interleukin-11, Porcine epidemic diarrhea virus, STAT3, anti-apoptosis

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Authors: Orose Rugchati, Sarawut Wattanawongpitak

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This research studied the analysis of the thermal conductivity and heat transfer in porcine meat due to the electric current flowing between the electrode plates in parallel. Hot-boned pork sample was prepared in 2*1*1 cubic centimeter. The finite element method with ANSYS workbench program was applied to simulate this heat transfer problem. In the thermal simulation, the input thermoelectric energy was calculated from measured current that flowing through the pork and the input voltage from the dc voltage source. The comparison of heat transfer in pork according to two voltage sources: DC voltage 30 volts and dc pulsed voltage 60 volts (pulse width 50 milliseconds and 50 % duty cycle) were demonstrated. From the result, it shown that the thermal conductivity trends to be steady at temperature 40C and 60C around 1.39 W/mC and 2.65 W/mC for dc voltage source 30 volts and dc pulsed voltage 60 volts, respectively. For temperature increased to 50C at 5 minutes, the appearance color of porcine meat at the exposer point has become to fade. This technique could be used for predicting of thermal conductivity caused by some meat’s characteristics.

Keywords: thermal conductivity, porcine meat, electricity, finite element method

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Authors: Siti Aishah Hasbullah

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Studies on identification of pork content in food have grown rapidly to meet the Halal food standard in Malaysia. The used mitochondria DNA (mtDNA) approaches for the identification of pig species is thought to be the most precise marker due to the mtDNA genes are present in thousands of copies per cell, the large variability of mtDNA. The standard method commonly used for DNA detection is based on polymerase chain reaction (PCR) method combined with gel electrophoresis but has major drawback. Its major drawbacks are laborious, need longer time and toxic to handle. Therefore, the need for simplicity and fast assay of DNA is vital and has triggered us to develop DNA biosensors for porcine DNA detection. Therefore, the aim of this project is to develop electrochemical DNA biosensor based on ruthenium (II) complex, [Ru(bpy)2(p-PIP)]2+ as DNA hybridization label. The interaction of DNA and [Ru(bpy)2(p-HPIP)]2+ will be studied by electrochemical transduction using Gold Screen-Printed Electrode (GSPE) modified with gold nanoparticles (AuNPs) and succinimide acrylic microspheres. The electrochemical detection by redox active ruthenium (II) complex was measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The results indicate that the interaction of [Ru(bpy)2(PIP)]2+ with hybridization complementary DNA has higher response compared to single-stranded and mismatch complementary DNA. Under optimized condition, this porcine DNA biosensor incorporated modified GSPE shows good linear range towards porcine DNA.

Keywords: gold, screen printed electrode, ruthenium, porcine DNA

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Authors: T. A. Rosy, M. R. I. Talukdar, N. S. Juyena, F. Y. Bari, M. N. Islam

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The ovarian follicular dynamics, vaginal electrical resistance (VER) and progesterone (P4) and estrogen (E2) profiles were investigated during estrus cycle in four indigenous ewes. Daily VER values were recorded with heat detector. The follicles were observed and measured by trans-rectal ultrasonography. Blood was collected daily for hormonal profiles. Results showed a significant variation in VER values (P<0.05) at estrus in regards to ewes and cycles. The day difference between two successive lower values in VER waves ranged from 13-17 days which might indicate the estrus cycle in indigenous ewes. Trans-rectal ultrasonography of ovaries revealed the presence of two to four waves of follicular growth during the study period. Results also showed that follicular diameter was negatively correlated with VER values. Study of hormonal profiles by ELISA revealed a positive correlation between E2 concentration and development of follicle and negative correlation between P4 concentration and development of follicle. The concentrations of estradiol increased at the time of estrus and then fall down in a basal level. Development of follicular size was accompanied by an increase in the concentration of serum estradiol. Inversely, when follicles heed to ovulation concentration of progesterone starts to fall down and after ovulation it turns its way to the zenith and remains at this state until next ovulatory follicle comes to its maximum diameter. This study could help scientists to set up a manipulative reproductive technique for improving genetic values of sheep in Bangladesh.

Keywords: ovarian follicle, hormonal profile, sheep, ultrasonography, vaginal electrical resistance

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Authors: Antonio Munar-Frau, Sascha Klismoch, Manfred Schmolz, Federico Hernandez-Alfaro, Jordi Caballe-Serrano

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Introduction: Biocompatibility of biomaterials has been proposed as one of the main criteria for treatment success. For guided bone regeneration (GBR), barrier membranes present a conflict given the number of origins and modifications of these materials. The biologic response to biomaterials is orchestrated by a series of events leading to the integration or rejection of the biomaterial, posing questions such as if a longer occlusive property may trigger an inflammatory reaction. Whole blood cultures are a solution to study the immune response to drugs or biomaterials during the first 24-48 hours. The aim of this study is to determine the early immune response of different origins and chemical modifications of barrier membranes. Materials & Methods: 5 different widely used barrier membranes were included in this study: Acellular dermal matrix (AlloDerm, LifeCell®), Porcine Peritoneum (BioGide, Geistlich Pharma®), Porcine Pericardium (Jason, Botiss Biomaterials GmbH®), Porcine Cross-linked collagen (Ossix Plus, Datum Dental®) and d-PTFE (Cytoplast TXT, Osteogenics Biomedical®). Blood samples were extracted from 3 different healthy donors and incubated with the different samples of barrier membranes for 24 hours. After the incubation time, serum samples were obtained and analyzed by means of biocompatibility assays taking into account 42 markers. Results: In an early stage of the inflammatory response, the Acellular dermal matrix, porcine peritoneum and porcine cross-linked collagen expressed similar patterns of cytokine expression with a great manifestation of ENA 78. Porcine pericardium and d-PTFE presented similar cytokine activation, especially for MMP-3 and MMP-9, although other cytokines were highlighted with lower expression. For the later immune response, Porcine peritoneum and acellular dermal matrix MCP-1 and IL-15 were evident. Porcine pericardium, porcine cross-linked collagen and d-PTFE presented a high expression of IL-16 and lower manifestation of other cytokines. Different behaviors depending on an earlier or later stage of the inflammation process were observed. Barrier membrane inflammatory expression does not only differ depending on the origin, variables such as treatment of the collagen and polymers may also have a great impact on the cytokine expression of the studied barrier membranes during inflammation. Conclusions: Surface treatment and modifications might affect the biocompatibility of the membranes, as different cytokine expressions were evidently depending on the origin of the biomaterial. This study is only a brushstroke regarding the biocompatibility of materials, as it is one of the pioneer studies for ex vivo barrier membranes assays. Studies regarding surface modification are needed in order to clarify mystifications of barrier membrane science.

Keywords: biomaterials, bone regeneration, biocompatibility, inflammation

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Authors: Norhidayu Muhamad Zain

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Gelatin, a purified protein derived mostly from porcine and bovine sources, is used widely in food manufacturing, pharmaceutical, and cosmetic industries. However, the presence of any porcine-related products are strictly forbidden for Muslim and Jewish consumption. Therefore, analytical methods offering reliable results to differentiate the sources of gelatin are needed. The aim of this study was to differentiate the sources of gelatin (porcine and tilapia fish) using Fourier transform infrared- attenuated total reflection (FTIR-ATR) combined with two dimensional infrared (2DIR) correlation analysis. Porcine gelatin (PG) and tilapia fish gelatin (FG) samples were diluted in distilled water at concentrations ranged from 4-20% (w/v). The samples were then analysed using FTIR-ATR and 2DIR correlation software. The results showed a significant difference in the pattern map of synchronous spectra at the region of 1000 cm⁻¹ to 1100 cm⁻¹ between PG and FG samples. The auto peak at 1080 cm⁻¹ that attributed to C-O functional group was observed at high intensity in PG samples compared to FG samples. Meanwhile, two auto peaks (1080 cm⁻¹ and 1030 cm⁻¹) at lower intensity were identified in FG samples. In addition, using 2D correlation analysis, the original broad water OH bands in 1D IR spectra can be effectively differentiated into six auto peaks located at 3630, 3340, 3230, 3065, 2950 and 2885 cm⁻¹ for PG samples and five auto peaks at 3630, 3330, 3230, 3060 and 2940 cm⁻¹ for FG samples. Based on the rule proposed by Noda, the sequence of the spectral changes in PG samples is as following: NH₃⁺ amino acid > CH₂ and CH₃ aliphatic > OH stretch > carboxylic acid OH stretch > NH in secondary amide > NH in primary amide. In contrast, the sequence was totally in the opposite direction for FG samples and thus both samples provide different 2D correlation spectra ranged from 2800 cm-1 to 3700 cm⁻¹. This method may provide a rapid determination of gelatin source for application in food, pharmaceutical, and cosmetic products.

Keywords: 2 dimensional infrared (2DIR) correlation analysis, Fourier transform infrared- attenuated total reflection (FTIR-ATR), porcine gelatin, tilapia fish gelatin

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Authors: Kayode O. Afolabi, Benson C. Iweriebor, Anthony I. Okoh, Larry C. Obi

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Porcine circovirus type 2 (PCV2) is the major etiological viral agent of porcine multisystemic wasting syndrome (PWMS) and other porcine circovirus-associated diseases (PCVAD) of great economic importance in pig industry globally. In an effort to determine the status of swine herds in the Province as regarding the ‘small but powerful’ viral pathogen; a total of 375 blood, faecal and nasal swab samples were obtained from seven pig farms (commercial and communal) in Amathole, O.R. Tambo and Chris-Hani District Municipalities of Eastern Cape Province between the year 2015 and 2016. Three hundred and thirty nine (339) samples out of the total sample were subjected to molecular screening using PCV2 specific primers by conventional polymerase chain reaction (PCR). Selected sequences were further analyzed and confirmed through genome sequencing and phylogenetic analyses. The data obtained revealed that 15.93% of the screened samples (54/339) from the swine herds of the studied areas were positive for PCV2; while the severity of occurrence of the viral pathogen as observed at farm level ranges from approximately 5.6% to 60% in the studied farms. The Majority, precisely 15 out of 17 (88%) analyzed sequences were found clustering with other PCV2b reference strains in the phylogenetic analysis. More interestingly, two other sequences obtained were also found clustering within PCV2d genogroup, which is presently another fast-spreading genotype with observable higher virulence in global swine herds. This finding confirmed the presence of this all-important viral pathogen in pigs of the region; which could result in a serious outbreak of PCVAD and huge economic loss at the instances of triggering factors if no appropriate measures are taken to curb its spread effectively.

Keywords: pigs, polymerase chain reaction, porcine circovirus type 2, South Africa

Procedia PDF Downloads 176

Authors: Ferreira RC, Simons HZ, Thompson WS, Cutler AJ, Dopico XC, Smyth DJ, Mashar M, Schuilenburg H, Walker NM, Dunger DB, Wallace C, Todd JA, Wicker LS, Pekalski ML

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Type 1 diabetes is caused by autoimmune destruction of insulin-secreting beta cells in the pancreas. T cells are known to play an important role in this immune-mediated destruction; however, there is no general consensus regarding alterations in cytokine production or T cell subsets in peripheral blood of patients with type 1 diabetes. Using polychromatic flow cytometry of peripheral blood mononuclear cells (PBMCs), we assessed production of the proinflammatory cytokines IL-21, IFN-γ and IL-17 by memory CD4 T effector (Teff) cells in 69 patients with type 1 diabetes and 61 healthy donors. We found a 21.9% (95% CI 5.8, 40.2; p = 3.9 × 10(-3)) higher frequency of IL-21(+) CD45RA(-) memory CD4(+) Teffs in patients with type 1 diabetes (geometric mean 5.92% [95% CI 5.44, 6.44]) compared with healthy donors (geometric mean 4.88% [95% CI 4.33, 5.50]). In a separate cohort of 30 patients with type 1 diabetes and 32 healthy donors, we assessed the frequency of circulating T follicular helper (Tfh) cells in whole blood. Consistent with the increased production of IL-21, we also found a 14.9% increase in circulating Tfh cells in the patients with type 1 diabetes (95% CI 2.9, 26.9; p = 0.016). Analysis of IL-21 production by PBMCs from a subset of 46 of the 62 donors immunophenotyped for Tfh showed that frequency of Tfh cells was associated with the frequency of IL-21+ cells (r2 = 0.174, p = 0.004). These results indicate that increased IL-21 production is likely to be an aetiological factor in the pathogenesis of type 1 diabetes that could be considered as a potential therapeutic target.

Keywords: T follicular helper cell, IL-21, IL-17, type 1 diabetes

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Authors: Ibrahim Abdulla Labib, Medhat Mohamed Morsy, Gamal Hosny, Hanan Dawood Yassa, Gaber Hassan

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Amiodarone is a very effective drug, widely used for arrhythmia. Unfortunately it has many side effects involving many organs especially thyroid gland. The current work was conducted to elucidate the effect of amiodarone on the thyroid gland and the possible protective role of vitamin E. Fifty adult male albino rats weighed 200 – 250 grams were divided into five groups; ten rats each. Group I (Control): Five rats were sacrificed after three weeks and five rats were sacrificed after six weeks. Group II (Sham control): Each rat received sunflower oil orally; the solvent of vitamin E for three weeks. Group III (Amiodarone-treated): each rat received an oral dose of amiodarone; 150 mg/kg/day for three weeks. Group IV (Recovery): Each rat received amiodarone as group III then the drug was stopped for three weeks to evaluate recovery. Group V (Amiodarone + Vitamin E-treated): Each rat received amiodarone as group III followed by 100 mg/kg/day vitamin E orally for three weeks. Thyroid gland of the sacrificed animals were dissected out and prepared for light and electron microscopic studies. Amiodarone administration resulted in loss of normal follicular architecture as many follicles appeared either shrunken, empty or contained scanty pale colloid. Some follicles appeared lined by more than one layer of cells while others showed interruption of their membrane. Masson's Trichrome stained sections showed increased collagen fibers in between the thyroid follicles. Ultrastructurally, the apical border of the follicular cells showed few irregular detached microvilli. The nuclei of the follicular cells were almost irregular with chromatin condensation. The cytoplasm of most follicular cells revealed numerous dilated rough endoplasmic reticulum with numerous lysosomes. After three weeks of stopping amiodarone, the follicles were nearly regular in outline. Some follicles were filled with homogenous eosinophilic colloid and others had shrunken pale colloid or were empty. Some few follicles showed exfoliated cells in their lumina and others were still lined by more than one layer of follicular cells. Moderate amounts of collagen fibers were observed in-between thyroid follicles. Ultrastructurally, many follicular cells had rounded euchromatic nucleui, moderate number of lysosomes and moderately dilated rough endoplasmic reticulum. However, few follicular cells still showing irregular nucleui, dilated rough endoplasmic reticulum and many cytoplasmic vacuoles. Administration of vitamin E with amiodarone for three weeks resulted in obvious structural improvement. Most of the follicles were lined by a single layer of cuboidal cells and the lumina were filled with homogenous eosinophilic colloid with very few vacuolations. The majority of follicular cells had rounded nuclei with occasional detection of ballooned cells and dark nuclei. Scanty collagen fibers were detected among thyroid follicles. Ultrastructurally, most follicular cells exhibited rounded euchromatic nuclei with few short microvilli were projecting into the colloid. Few lysosomes were also noticed. It was concluded that amiodarone administration leads to many adverse histological changes in the thyroid gland. Some of these changes are reversible during the recovery period however concomitant vitamin E administration with amiodarone has a major protective role in preventing many of these changes.

Keywords: amiodarone, recovery, ultrastructure, vitamin E.

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Authors: Caighley Logan

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The outcome of fire related fatalities, along with other research, has found fires can have a detrimental effect to the mineral and crystalline structures within bone. This study focused on the mineral and crystalline structures within porcine bone samples to analyse the changes caused, with the intent of effectively ‘reverse engineering’ the data collected from burned bone samples to discover what may have happened. Using Fourier Transform Infrared (FT-IR), and X-Ray Fluorescence (XRF), the data collected from a controlled source of intense heat (muffle furnace) and an open fire, based in a living room setting in a standard size shipping container (8.5ft x 8ft) of a similar temperature with a known ignition source, a gasoline lighter. This approach is to analyse the changes to the samples and how the changes differ depending on the heat source. Results have found significant differences in the levels of remaining minerals for each type of heat/burning (p=<0.001), particularly Phosphorus and Calcium, this also includes notable additions of absorbed elements and minerals from the surrounding materials, i.e., Cerium (Ce), Bromine (Br) and Neodymium (Ne). The analysis techniques included provide validated results in conjunction with previous studies.

Keywords: forensic anthropology, thermal alterations, porcine bone, FTIR, XRF

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Authors: M. Yadegari

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The thyroid gland is the largest classic endocrine organ that effects many organs of the body and plays a significant role in the process of Metabolism in animals. The aim of this study was to investigate the prevalence of thyroid disorders diagnosed by ultrasound and microscopic Lesions of the thyroid during the slaughter of apparently healthy One Humped Camels (Camelus dromedarius) in Iran. Randomly, 520 male camels (With an age range of 4 to 8 years), were studied in 2012 to 2013. The Camels’ thyroid glands were evaluated by sonographic examination. In both longitudinal and transverse view and then tissue sections were provide and stained with H & E and finally examined by light microscopy. The results obtained indicated the following: hyperplastic goiter (21%), degenerative changes (12%), follicular cysts (8%), follicular atrophy (4%), nodular hyperplasia (3%), adenoma (1%), carcinoma (1%) and simple goiter colloid (1%). Ultrasound evaluation of thyroid gland in adenoma and carcinoma showed enlargement and irregular of the gland, decreased echogenicity, and the heterogeneous thyroid parenchyma. Also, in follicular cysts were observed in the enlarged gland with no echo structures of different sizes and decreased echogenicity as a local or general. In nodular hyperplasia, increase echogenicity and heterogeneous parenchymal were seen. These findings suggest the use of Ultrasound as a screening test in the diagnosis of complications of thyroid disorders. Pathology also to be used for the diagnosis of thyroid problems and other side effects.

Keywords: thyroid gland, one humped camel, sonography, pathology

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Authors: W. G. Kim, J. M. Chang, W. S. Kim

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Immunologically non-treated and acellularized porcine xenografts were implanted as an arterial graft in goats and comparatively analyzed for the explanted grafts with gross observation, as well as light microscopy and immunohistochemistry, following the predetermined periods. For immunologically non-treated xenografts, bilateral porcine carotid arteries were harvested, and after short-term freezing at -70°C, were implanted into goats. The preparation of acellularized xenograft vessels has been performed with Nacl-SDS solution and stored at the freezer until use. The goats were randomly assigned for three periods of observation (3, 6, and 12 months after implantation), four animals were observed at each of these times. Periodic ultrasonographic examinations were performed during observation period. Following the predetermined periods, the explanted grafts were analyzed. Among 12 animals, one goat died prematurely, and a total of 22 grafts were evaluated. Gross observations revealed non-thrombotic patent smooth lumens. Microscopic examinations of the explanted grafts showed satisfactory cellular reconstruction up to the 12-month observation period. The proportions of CD3 positive T lymphocytes among inflammatory cells infiltrations were very low. In conclusion, these findings, as a whole, suggest that porcine vessel xenografts can be clinically acceptably implanted in the goats as a form of small-diameter vascular graft, regardless of the acellularized xenograft or immunologically non-treated xenograft.

Keywords: xenograft, arterial graft, long-term survival animals, immunology

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Authors: Samira Laidaoui, Mohammed Djermane, Nazihe Terfaya

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The fluid-structure coupling is a natural phenomenon which reflects the effects of two continuums: fluid and structure of different types in the reciprocal action on each other, involving knowledge of elasticity and fluid mechanics. The solution for such problems is based on the relations of continuum mechanics and is mostly solved with numerical methods. It is a computational challenge to solve such problems because of the complex geometries, intricate physics of fluids, and complicated fluid-structure interactions. The way in which the interaction between fluid and solid is described gives the largest opportunity for reducing the computational effort. In this paper, a problem of fluid structure interaction is investigated with two-way coupling method. The formulation Arbitrary Lagrangian-Eulerian (ALE) was used, by considering a dynamic grid, where the solid is described by a Lagrangian formulation and the fluid by a Eulerian formulation. The simulation was made on the ANSYS software.

Keywords: ALE, coupling, FEM, fluid-structure, interaction, one-way method, two-way method

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Authors: Namfon Samsalee, Rungsinee Sothornvit

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Turmeric is a natural plant herb and generally extracted as essential oil and widely used in food, cosmetic, pharmaceutical products including insect repellent. However, turmeric oil is a volatile essential oil which is easy to be lost during storage or exposure to light. Therefore, biopolymers such as protein and polysaccharide can be used as wall materials to encapsulate the essential oil which will solve this drawback. Approximately 60% plasma from porcine blood contains 6-7% of protein content mainly albumin and globulin which can be a good source of animal protein at the low-cost biopolymer from by-product. Microencapsulation is a useful technique to entrap volatile compounds in the biopolymer matrix and protect them to degrade. The objective of this research was to investigate the different ratios of two biopolymers (PPP and maltodextrin; MD) as wall materials at 100:0, 75:25, 50:50, 25:75 and 0:100 at a fixed ratio of wall material: core material (turmeric oil) at 3:1 (oil in water) on the qualities of microencapsulated powder using freeze drying. It was found that the combination of PPP and MD showed higher solubility of microencapsules compared to the use of PPP alone (P < 0.05). Moreover, the different ratios of wall materials also affected on color (L*, a* and b*) of microencapsulated powder. Morphology of microencapsulated powder using a scanning electron microscope showed holes on the surface reflecting on free oil content and encapsulation efficiency of microencapsules. At least 50% of MD was needed to increase encapsulation efficiency of microencapsulates rather than using only PPP as the wall material (P < 0.05). Microencapsulated turmeric oil powder can be useful as food additives to improve food texture, as a biopolymer material for edible film and coating to maintain quality of food products.

Keywords: microencapsulation, turmeric oil, porcine plasma protein, maltodextrin

Procedia PDF Downloads 141