Influence of EDTA and magnesium on DNA extraction from blood samples and specificity of polymerase chain reaction

This study consisting of two trails conducted to examine the impact of initial EDTA level added to blood samples on quantity and quality of genomic DNA isolated from avian fresh blood and the influence ofinitial EDTA level with various levels of MgCl2 added to polymerase chain reaction (PCR) final volume on amplification pattern. EDTA level added to collected blood samples had no significant impact onquantity as well as quality of extracted genomic DNA. However, higher levels of EDTA increased the OD260 and enhanced the OD260/OD280 ratio beyond 1.8-1.9 which is broadly accepted as an indicator of high quality DNA. To avoid such an error, EDTA level in initial blood sample must not exceed 9 ìg/ìl blood. The initial amount of EDTA has no influence on PCR process if a valid DNA isolation protocol is used. Addition of MgCl2 from 1.0 to 2.4 ìl in a final volume of 25 ìl could support the amplification properly. Low levels of MgCl2 results in incomplete amplification but levels higher than 2.4 ìl impedes the amplification by negative interference to the whole reactions

Optimizing factors influencing DNA extraction from fresh whole avian blood

A study was conducted to optimize the efficient combination of lysis buffer, proteinase K, incubation time, phenol-chloroform-isoamyl alcohol (PCI) volume, spinning rate (rpm), and precipitation agent onquantity and quality of DNA extracted from various volumes of avian blood. Blood samples were collected in EDTA and swiftly transferred to a laboratory for DNA extraction. The lysis buffer used hadcomposition 5 M NaCl, 1 M Tris (pH=8.0), 0.5 M EDTA and20% SDS. The effect of various levels for each factor concerned was examined using General Linear Models or t-test procedures of SAS® software. The volume removed from the top aqueous part following the first and the second PCI washings was included in the models as a continuous variable; the variables of interest were OD280, OD260, OD260/OD280 (as quality criterion), total extracted DNA, extraction efficiency (ìg DNA/ìl blood), assay scores for easiness of removing the top aqueous phase after the first (assay 1) and the second (assay 2) spinning. The optimum level of factors significant for DNA extraction from fresh avian blood was found to be lysis buffer : blood sample ratio of 31:36 (ìl : ìl), incubation time of 60-70 min at 58oC, two washings with PCI at 1.2:1.3 PCI : top aqueous phase (ìl : ìl) for the first and 1.4 for the second washing, centrifuge of homogenised sample at 2000 - 2500 rpm for 20 min, precipitation of DNA with 1.5 – 2.0 volume ofabsolute ethanol

Hypolipidemic effects of carvacrol in relation with sex hormones in broiler chicken

Two experiments were conducted to evaluate the effects of high and low doses of carvacrol on blood lipid constituents and sex hormones in broiler chicks. Inclusion of carvacrol into the drinking water at 0.5 and 0.3 g/ L in Experiments 1 and 2, respectively, modulated serum cholesterol and high density lipoproteins (HDL) levels, albeit the differences were not significant when compared to the corresponding control groups (P> 0.05). Carvacrol at 0.5 g/ L significantly decrease (16%) abdominal fat percentage of the birds at 28 d (Trial 1; P 0.05). Serum testosterone, however, increased in the birds received carvacrol at doses greater than 0.2 g/ L in comparison with the control birds (P<0.05). The results propose the possibility of testosterone-coupled hypolipidemic properties for carvacrol in broiler chicken

Physiological Adaptive Indicators in Fasted Neonate Broiler Chicks in Response to Calcium Gluconate Injection

Four hundred and eighty mixed-sex broiler chicks aged 3 hrs after hatching were allotted according to a completely random design in a 6 × 2 × 2 factorial schedule into 2 groups of 12 replications of 20 chicks each. The main experimental factors were fasting for 0, 6, 12, 24, 36, and 48 hrs after chick placement, calcium gluconate (Ca-glu) injection (0 and 0.6 mL) and sex (male and female). Independent of sex, live body weight (BW) of chicks decreased linearly (Y=43.36-0.109BW0h, r2=0.876) as neonatal fasting extended. Injection of 0.6 mL Ca-glu at 3 hrs post hatching did not affect weight loss of chicks. Yolk residuals (YR) utilized linearly (Y=5.75-0.062YR, r2=0.956) by 0.062 g/hr in neonate fasted chicks showing no effect for Ca-glu injection. Neonatal fasting periods longer than 12 hrs increased liver weight (P). The mean absolute and proportional (% of BW0h) breast and leg weight were reduced linearly as neonatal fasting extended (P). Serum glucose concentration in both sexes increased up to 6 hrs fasting, then reduced linearly to 150 mg/dL after 48 hrs feed withdrawal. The Ca-glu treatment influenced serum glucose level for a short period up to 6 hrs of fasting. Serum Ca concentration sharply increased up to three-fold in the birds received Ca-glu injection resulting in acute hypercalcemia, then decreased to the initial level after 24 hrs feed withdrawal. The mean serum level for creatinine, uric acid, cholesterol, HDL, albumins and total proteins significantly increased during the fasting periods of 6 to 48 hrs and significantly elevated in the birds received 0.6 mL Ca-glu injection compared with the non treated chicks. It was concluded that subcutaneous administration of 0.6 mL Ca-glu in the chick's neck did not suitably support the increased metabolic demands for glucose and calcium in feed deprived neonate chicks

Sexual Dimorphism in Lori Sheep Vomeronasal Organ dimensions and their relationships with external body measurements

This experiment was carried out to study the effect of gender on anatomy of vomeronasal organ (VNO) and their correlations with some external body measurements in Lori sheep. Six external body characteristics were measured on 21 Lori sheep (10 ewes and 11 rams). Heads of the animals were collected and several transverse sections were made through the rostral region of incisive tubercle and repeated at every 1.5 cm up to the point that macroscopic signs of the VNO disappeared. The mean length of VNO was not differ between males and females (p&lt;0.05). The average width of VNO in the middle and rostral third parts, the mean height of the organ in the middle third part and the mean distance between left and right VNO cartilaginous capsule in the middle third part were significantly greater in males compared with females. The width of the tubercle on incisive was significantly greater in males than females (9.99 vs. 8.42 mm). Average width of VNO in rostral third was significantly correlated with trunk length (p&gt;0.05). The mean height of VNO lumen in middle and rostral third was negatively correlated with chest height (r = -0.476) and testis circumference (r = -0.731), respectively (p&gt;0.05). The average width of the distance between left and right VNO lumen in middle third segment was significantly correlated with external nose circumference (p&gt;0.05). Width and length of the tubercle on incisive were both significantly related with trunk length, and the former showed also a significant positive association with heart girdle (p&gt;0.05). In conclusion, minor macroscopically differences exist especially in the middle third segment of VNO in Lori ram and ewe. The VNO anatomical parameters show significant correlations with common external body measures which their physiological interpretation needs further  investigation.Keywords: Vomeronasal organ, Hard palate, External body measures, Lori shee

Influence of EDTA and magnesium on DNA extraction from blood samples and specificity of polymerase chain reaction

This study consisting of two trails conducted to examine the impact of initial EDTA level added to blood samples on quantity and quality of genomic DNA isolated from avian fresh blood and the influence of initial EDTA level with various levels of $MgCl_2$ added to polymerase chain reaction (PCR) final volume on amplification pattern. EDTA level added to collected blood samples had no significant impact on quantity as well as quality of extracted genomic DNA. However, higher levels of EDTA increased the $OD_{260}$ and enhanced the $OD_{260}/OD_{280}$ ratio beyond 1.8-1.9 which is broadly accepted as an indicator of high quality DNA. To avoid such an error, EDTA level in initial blood sample must not exceed $9 \mu g/ \mu l$ blood. The initial amount of EDTA has no influence on PCR process if a valid DNA isolation protocol is used. Addition of $MgCl_2$ from 1.0 to $2.4 \mu l$ in a final volume of $25 \mu l$ could support the amplification properly. Low levels of $MgCl_2$ results in incomplete amplification but levels higher than $2.4 \mu l$ impedes the amplification by negative interference to the whole reactions

Acidic stress caused by dietary administration of citric acid in broiler chickens

Citric acid (CA) is commonly used in poultry diets to promote growth by acidifying the gastrointestinal contents, improving nutrient digestibility, and reducing pathogen loads; therefore, this study was conducted to demonstrate the effects of 0, 30 and 60 g of CA per kilogramme of diet on productive performance, selected blood metabolites, immune response and certain gut-related variables in broiler chickens using 150, 7-day-old Ross 308 male broiler chicks in five replicates of 10 birds each per diet. Growth performance, daily feed intake and tibia phosphorous (P) retention were significantly improved by the diets containing 30 g kg⁻¹ of CA (<i>P</i> < 0.01) but were suppressed as CA increased to 60 g kg⁻¹. Dietary CA increased proventriculus, gizzard and ileum percentage and villus length, crypt depth and goblet cell number in duodenum, jejunum and ileum as well as ileal digestibility of crude protein, apparent metabolisable energy and total phosphorus while it decreased the pH of contents in the gut segments concerned (<i>P</i> < 0.01). Diets containing 60 g kg⁻¹ of CA significantly reduced plasma P and Fe levels as well as cholesterol level and Alkaline phosphatase activity (<i>P</i> < 0.05) while increasing the aspartate aminotransferase and lactate dehydrogenase activities (<i>P</i> < 0.01) in the blood serum of the birds at day 42 of age. The percentage of bursa and thymus was greater in the birds fed on diets containing 60 and 30 g kg⁻¹ of CA, respectively (<i>P</i> < 0.01). It was concluded that inclusion of 60 g kg⁻¹ of CA in the diet resulted in a severe reduction in performance, nutrient digestion and absorption and liver dysfunctions in broiler chickens, a phenomenon we call as acidic stress