Blood Plasma Of Sheep Research Articles

Short communication: Metabolic synthesis of vitamin D2 by the gut microbiome

The origin of vitamin D2 in herbivorous animals was investigated in vivo in sheep and in bovine as well as mouse gastrointestinal tracts. A high concentration of 25-hydroxyvitamin D2 in blood plasma of sheep both in summer and winter appeared to be incompatible with the undetectable level of vitamin D2 in the pasture on which the sheep were grazing. Studies with bovine rumen contents from a cow grazing the same pasture as the sheep, demonstrated an increased concentration of vitamin D2 on anaerobic incubation in a ‘Rusitec’ artificial rumen, which was further enhanced when cellulose powder was added as a fermentation substrate. The colon contents of mice that were fed from weaning on a vitamin D-free diet were found to contain vitamin D2. The results of these comparative studies in 3 animal species indicated that vitamin D2 was being generated by microbial anaerobic metabolism in the gastrointestinal tract.

Appraisal of iron accumulation in soil, forages, and blood plasma of sheep and goats: a case study in different districts of Punjab, Pakistan.

Minerals are essential for ruminants affecting significantly the production of grazing livestock. Iron level in forages, soil, and blood plasma of the small ruminants (goat and sheep) was investigated in three districts of Punjab. Atomic absorption spectrophotometer was used to determine the concentration of iron in collected samples. The results revealed that the mean Fe concentrations in soil of districts Sargodha, Mianwali, and Bhakhar were significantly varied and ranged from 21.85 to 23.78, 28.45 to 31.2, and 18.079 to 24.33 mg/kg, respectively. The Fe level in soil of Mianwali significantly varied and was higher than Sargodha and Bhakkar. The mean Fe concentration in forages which were used for feeding purpose were significantly varied and found between 10.95-14.49, 23.63-25.65, and 6.616-9.45 mg/kg for Sargodha, Mianwali, and Bhakhar, respectively. The mean Fe concentrations in blood plasma of goat which consumed the contaminated forages were 8.5026-11.763 mg/L in district Sargodha, 19.77-20.19 mg/L in Mianwali, and 5.508-5.858mg/L in Bhakkar. In blood plasma of sheep, the residual levels of Fe in districts Sargodha, Mianwali, and Bhakhar were ranged from 9.987 to 12.455, 15.8 to 19.785, and 3.425 to 6.383 mg/L, respectively. This study provides the data of metals effected by different sites and also their mobility from low to higher trophic level which enables us to study the iron toxicity in different trophic levels, and we recommend different safe limits and treatment in case of low and high metal profile.

Evaluation of Cobalt Concentration in the Forages and Small Ruminant Blood Collected from Various Agroecological Regions

Like animals plants required nutrients for their growth and existence. Cobalt is also important element for plant and animal growth, however higher levels of Co might have toxic potential. The study was aimed to investigate Co level in samples of forages, soil, and blood plasma of sheep and goat in Bhakkar, Mianwali and Sargodha district. The results showed that district Mianwali excelled in level of Co as compared to Bhakkar and Sargodha. The factors such as bio-concentration factor and pollution load index explained the movement of cobalt along the soil-plant-animal continuum.

A study on the transfer of chromium from meadows to grazing livestock: an assessment of health risk.

The present investigation was performed in different district of Punjab to determine the chromium level in soil, forages, and blood plasma of the small ruminants (goat and sheep). The results showed that the mean concentrations of chromium in the soil of Sargodha, Mianwali, and Bhakkar districts were ranged from 1.8 to 2.3, 3.01 to 4.99, and 2.04 to 2.87 mg/kg, respectively. The chromium level was higher in Mianwali compared with Sargodha and Bhakkar. The mean concentrations of chromium in forages which were used for feeding purposes were found between 0.672 and 1.631, 1.493 and 2.612, and 0.7804 and 1.047 mg/kg for Sargodha, Mianwali, and Bhakkar, respectively. The mean concentrations of chromium in blood plasma of goat which consumed the contaminated forages were between 0.666 and 1.269 mg/L in Sargodha, 0.657 and 0.752 mg/L in Mianwali, and 1.39 and 2.37 mg/L in Bhakkar. In blood plasma of sheep, the residual levels of chromium in the districts Sargodha, Mianwali, and Bhakkar were ranged from 0.673 to 1.384, 0.83 to 1.086, and 1.496 to 3.611 mg/L, respectively. In the present research, there was a higher concentration of chromium in blood plasma of sheep from Bhakkar and the lowest was in Sargodha. The chromium level in blood samples was found to be higher than the tolerable level of 1.0 mg/L in all districts. In light of these results, it was concluded that chromium levels in the soil and forages of all sites were present within the safe limit while in blood samples of sheep and goats were found to be above the acceptable limit. Sheep and goats also consume water from river, streams, and ponds and other contaminated water sources. So it might be the reason of higher level of chromium in their blood plasma. Hence, the proper monitoring of study sites will be necessary to prevent the excessive accumulation of chromium in ruminants in the near future.

Epitope mapping of an uncertain endogenous antigen implies secretogranin II peptide splicing.

Background: The search for a tissue-mass reducing reproductive hormone involved a bioassay-guided physicochemical fractionation of sheep blood plasma. This brought forth a candidate protein whose apparent mass on gels and in mass spectrometry (MS) was 7-8 kDa, implying a polypeptide of ~70 residues. Four purification runs gave Edman N-terminal sequences relating to 1MKPLTGKVKEFNNI 14. This is bioinformatically obscure and has been resistant to molecular biological investigation. The sequence was synthesized as the peptide EPL001, against which was raised a goat polyclonal antiserum, G530. Used in an antigen capture campaign, G530 pointed to the existence of a novel derivative of secretogranin II (SgII), the neuroendocrine secretory vesicle helper protein and prohormone. The proposed SgII derivative was dubbed SgII-70, yet the sequence commonality between SgII and EPL001 is essentially NNI. Methods: Immunohistochemical (IHC) labelling with G530 is reported within rat, mouse and human cerebrovasculature and in glandular elements of the mouse intestine. Epitope mapping involved IHC peptide preabsorption, allied to deductive bioinformatics and molecular modelling in silico. Results: G530 is deemed monoepitopic in regard to both its synthetic antigen (EPL001) and its putative endogenous antigen (SgII related). The epitope within EPL001 of the anti-EPL001 antibody is inferred to be the contiguous C-terminal 9KEFNNI 14. This is so because the G530 blockade data are consistent with the epitope in the mammalian endogenous antigen being part contiguous, part non-contiguous KE·F·NNI, ex hypothesi. The observed immunostaining is deduced to be due to pre-SgII-70, which has a non-C-terminal NNI, and SgII-70, which has an N-terminal MLKTGEKPV/N and a C-terminal NNI (these two motifs being in the reverse order in the SgII parent protein). Conclusion: The present data are consistent with the hypothesis that the anti-EPL001 antibody binds to an SgII-related epitope. SgII is apparently subject to peptide splicing, as has been reported for the related chromogranin A.

Epitope mapping of an uncertain endogenous antigen implies secretogranin II peptide splicing

Background: The search for a tissue-mass reducing reproductive hormone involved a bioassay-guided physicochemical fractionation of sheep blood plasma. This brought forth a candidate protein whose apparent mass on gels and in mass spectrometry (MS) was 7-8 kDa, implying a polypeptide of ~70 residues. Four purification runs gave Edman N-terminal sequences relating to 1MKPLTGKVKEFNNI 14. This is bioinformatically obscure and has been resistant to molecular biological investigation. The sequence was synthesized as the peptide EPL001, against which was raised a goat polyclonal antiserum, G530. Used in an antigen capture campaign, G530 pointed to the existence of a novel derivative of secretogranin II (SgII), the neuroendocrine secretory vesicle helper protein and prohormone. The proposed SgII derivative was dubbed SgII-70, yet the sequence commonality between SgII and EPL001 is essentially NNI. Methods: Immunohistochemical (IHC) labelling with G530 is reported within rat, mouse and human cerebrovasculature and in glandular elements of the mouse intestine. Epitope mapping involved IHC peptide preabsorption, allied to deductive bioinformatics and molecular modelling in silico. Results: G530 is deemed monoepitopic in regard to both its synthetic antigen (EPL001) and its putative endogenous antigen (SgII related). The epitope within EPL001 of the anti-EPL001 antibody is inferred to be the contiguous C-terminal 9KEFNNI 14. This is so because the G530 blockade data are consistent with the epitope in the mammalian endogenous antigen being part contiguous, part non-contiguous KE·F·NNI, ex hypothesi. The observed immunostaining is deduced to be due to pre-SgII-70, which has a non-C-terminal NNI, and SgII-70, which has an N-terminal MLKTGEKPV/N and a C-terminal NNI (these two motifs being in the reverse order in the SgII parent protein). Conclusion: The present data are consistent with the hypothesis that the anti-EPL001 antibody binds to an SgII-related epitope. SgII is apparently subject to peptide splicing, as has been reported for the related chromogranin A.

Epitope mapping of an uncertain endogenous antigen implies secretogranin II peptide splicing.

Background: The search for a tissue-mass reducing reproductive hormone involved a bioassay-guided physicochemical fractionation of sheep blood plasma. This brought forth a candidate protein whose apparent mass on gels and in mass spectrometry (MS) was 7-8 kDa, implying a polypeptide of ~70 residues. Four purification runs gave Edman N-terminal sequences relating to 1MKPLTGKVKEFNNI 14. This is bioinformatically obscure and has been resistant to molecular biological investigation. The sequence was synthesized as the peptide EPL001, against which was raised a goat polyclonal antiserum, G530. Used in an antigen capture campaign, G530 pointed to the existence of a novel derivative of secretogranin II (SgII), the neuroendocrine secretory vesicle helper protein and prohormone. The proposed SgII derivative was dubbed SgII-70, yet the sequence commonality between SgII and EPL001 is essentially NNI. Methods: Immunohistochemical (IHC) labelling with G530 is reported within rat, mouse and human cerebrovasculature and in glandular elements of the mouse intestine. Epitope mapping involved IHC peptide preabsorption, allied to deductive bioinformatics and molecular modelling in silico. Results: G530 is deemed monoepitopic in regard to both its synthetic antigen (EPL001) and its putative endogenous antigen (SgII related). The epitope within EPL001 of the anti-EPL001 antibody is inferred to be the contiguous C-terminal 9KEFNNI 14. This is so because the G530 blockade data are consistent with the epitope in the mammalian endogenous antigen being part contiguous, part non-contiguous KE·F·NNI, ex hypothesi. The observed immunostaining is deduced to be due to pre-SgII-70, which has a non-C-terminal NNI, and SgII-70, which has an N-terminal MLKTGEKPV/N and a C-terminal NNI (these two motifs being in the reverse order in the SgII parent protein). Conclusion: The present data are consistent with the hypothesis that the anti-EPL001 antibody binds to an SgII-related epitope. SgII is apparently subject to peptide splicing, as has been reported for the related chromogranin A.

Searching for an inhibitory action of blood-borne [beta]-endorphin on LH release

Concentrations of beta-endorphin were quantified in peripheral blood plasma of sheep by a radioimmunoassay that cross-reacted with beta-lipotrophin. Plasma concentrations of beta-endorphin increased abruptly after physical confinement, bacteraemia, and electroacupuncture treatment for induction of analgesia. In these experimental situations in which plasma concentrations of beta-endorphin increased, plasma concentrations of LH often decreased. To test the hypothesis that increases in blood-borne beta-endorphin actually caused the decrease in LH release, naloxone was administered to antagonize the opioid receptors at which blood-borne beta-endorphin might act. In no case did administration of naloxone disrupt the temporal correlation between experimentally induced increases in plasma beta-endorphin and decreases in plasma LH. It was concluded that the increases in blood-borne beta-endorphin did not cause the decrease in LH release. Other research investigated whether beta-endorphin might be delivered via blood from pituitary to hypothalamus in locally enriched concentrations. Even when pituitary release of beta-endorphin was acutely stimulated, it was not possible to demonstrate retrograde delivery of beta-endorphin to the hypothalamus without dilution in the systemic circulation. In conclusion, it is unlikely that blood-borne beta-endorphin inhibits the release of LH, and beta-endorphin should not be classified as a hormone until blood concentrations of the peptide can be shown to exert some effect at a location distant from its site of secretion.

Investigating hydrogen peroxide induced damage to alpha-2-macroglobulin: Biophysical and thermodynamic study

Hydrogen peroxide (H2O2) is an important signalling molecule and a non-radical reactive oxygen species (ROS) which can convert into more potent oxygen radicals leading to oxidative stress. The correlation between oxidative stress and the cellular proteinase-antiproteinase balance has been a major component in the development of several pathologies such as emphysema and cystic fibrosis. The present studies attempt to explore the functional inactivation and structural alterations induced in one of the major antiproteinase, alpha-2-macroglobulin (α2M) on interaction with H2O2. α2M, purified from sheep blood plasma is a highly abundant proteinase inhibitor which acts as a backup or reserve force of antiproteinase. Our studies provide biophysical insights into the interaction of H2O2 with sheep α2M. Result demonstrates that antiproteolytic activity of α2M was significantly lowered upon interaction with increasing concentration of H2O2. Analysis of fluorescence spectra reveals quenching of fluorescence intensity in a static manner which occurs due to ground state complex formation. Synchronous fluorescence illustrates alteration in microenvironment around tryptophan and tyrosine residues of α2M. Additionally, UV/visible spectroscopy, circular dichroism and fourier transform infrared spectroscopy results confirms the alteration in secondary structure and conformation of α2M due to H2O2 induced modification. Moreover, polyacrylamide gel electrophoresis also confirms the structural change in α2M on H2O2 interaction. Thermodynamic parameters of H2O2 binding with α2M obtained by ITC indicate the reaction to be exothermic and spontaneous. Our studies suggest that H2O2 induces functional inactivation and structural disruption of sheep α2M possibly due to perturbation of secondary structure, at concentration of oxidant achievable in patho-physiological conditions such as cancer.

Changes in novel gastrointestinal and renal injury markers in the blood plasma of sheep following increasing intravenous doses of tolfenamic acid.

The administration of high doses of non-steroidal anti-inflammatory drugs (NSAID), such as tolfenamic acid (TA), has undesirable effects on different organs. Some novel biomarkers have been reported that can determine the gastrointestinal and renal injury caused by a high dose of NSAIDs or other toxic substances. This study was aimed at determining the changes in gastrointestinal (TFF2 and HYP), renal (NGAL and KIM-1) and cardiac (cTn-I, CK-MB) injury markers after the use of increasing intravenous doses of TA in sheep. TA was administered intravenously to groups of six sheep each, at the dose levels of 0 (Group 0, i.e., G0), 2 (G2), 4 (G4), 8 (G8) and 16 (G16) mg/kg. The concentrations of the studied biomarkers were measured at 3, 9, 18 and 36 h after administration of TA. The TFF2 and NGAL concentrations in G16 were found to be significantly higher (P < 0.05) than in the other groups except for G8 at different sampling times. HYP concentration in G16 was observed to be significantly (P < 0.05) lower than that in all other groups at 36 h. KIM-1 level in G16 was significantly (P < 0.05) higher than in all other groups at different sampling times. An increase in the renal markers, KIM-1 and NGAL, in G8 was observed before any change in plasma creatinine and urea. The cardiac marker cTn-I in G16 was significantly (P < 0.05) higher than in other groups at different sampling times. The results showed that the novel biomarkers (HYP, TFF2, NGAL, and KIM-1) can be used to determine gastric and renal injury in sheep.

Comparative Study of Concentration of Some Minerals in Milk and Blood Plasma of Sheep and Goats Native and Foreign During Post-Partum Period under Climatic Conditions of Upper Egypt

Comparative Study of Concentration of Some Minerals in Milk and Blood Plasma of Sheep and Goats Native and Foreign During Post-Partum Period under Climatic Conditions of Upper Egypt

Supraspinal basis of analgesic and clinical effects of the metabotropic glutamate mGluR1 antagonist during colonic distension in sheep

We have previously demonstrated a differential role for metabotropic glutamate receptors (mGluRs) using behavioral evidence, the role of mGluRs in spinal nociception in normal sheep. It was confirmed that is particularly apparent in neuropathic, inflammatory hyperalgesia produced by unilateral intradermal injection of carrageenan into the lower forelimb and mechanical hypersensitivity following abdominal surgery in sheep intrathecal blockade of mGluRs group I antagonists inhibits this phenomenon.The present study examined contribution of group I mGluRs to development and maintenance changes in behavioural and clinical symptoms, cortisol and catecholamines blood plasma concentrations caused by visceral pain evoked by colonic distension (CD) in sheep. Intracerebroventricular (icv) administration of the group I mGluRs antagonist racemic mixture of DL-AP3 blocked development of visceral pain symptoms and neuroendocrinological changes in the blood plasma of sheep. This data clearly demonstrated that development and maintenance of visceral pain symptoms of CD is dependent on activation of group I mGluRs in supraspinal level and that these types of mGluR play a crucial role in modulating of acute colonic pain (colic). What is more, this is the first record of supraspinal analgesic effects of mGluR1 antagonist in sheep.

Bacteriolytic enzymes from the blood plasma of the sheep

In the present work the studies ofbacteriolytic factors from sheep blood plasma have been performed. Three novel enzymes have been identified and characterized. Two of them have a molecular weight 15 +/- 2 kDa and able to lyse the gram-negative Escherichia coli bacteria. The third enzyme has a molecular weight 34 +/- 4 kDa and is able to lyse both gram-negative Escherichia coli and gram-positive Micrococcus luteus bacteria. The bacteriolytic reactions have been studied for all three enzymes; particularly, pH-optima have been identified with respect to the substrate. To identify the enzymes trypsinolysis and consequent MALDI-TOF mass spectrometry studies were performed. The results were compared to data from publicly available databases, such as Swiss-Prot, NCBI, MSDB.

Influence of selenate and linseed oil on fatty-acid and amino-acid profiles in the liver, muscles, fat tissues and blood plasma of sheep

Influence of selenate and linseed oil on fatty-acid and amino-acid profiles in the liver, muscles, fat tissues and blood plasma of sheep

Multiplexed Flow Cytometric Sensing of Blood Electrolytes in Physiological Samples Using Fluorescent Bulk Optode Microspheres

Polymeric bulk optode microsphere ion sensors in combination with suspension array technologies such as analytical flow cytometry may become a power tool for measuring electrolytes in physiological samples. In this work, the methodology for the direct measurement of common blood electrolytes in physiological samples using bulk optode microsphere sensors was explored. The simultaneous determination of Na(+), K(+), and Ca(2+) in diluted sheep blood plasma was demonstrated for the first time, using a random suspension array containing three types of mixed microsphere bulk optodes of similar size, fabricated from the same chromoionophore without additional labeling. Sodium ionophore X, potassium ionophore III, and grafted AU-1 in poly(butyl acrylate) were the ionophores used in the bulk optode microsphere ion sensors for Na(+), K(+), and Ca(2+), respectively, in combination with the cation-exchanger NaTFPB (sodium tetrakis-[3,5-bis(trifluoromethyl)phenyl]borate) and the same concentration of the chromoionophore ETH 5294 (9-(di-ethylamino)-5-octadecanoylimino-5H-benzo[a]phen-oxazine) in plasticized poly(vinyl chloride). Excellent reproducibility was achieved for the sensing of potassium ions. The effect of sample pH was relatively small at near-physiological pH and followed theoretical predictions, yet the sample temperature was found to influence the sensor response to a larger extent. Multiplexed ion sensing was achieved by taking advantage of the chemical tunability of the sensor response, adjusting the sensor compositions so that the three types of ion sensors responded with distinct levels of protonation of the chromoionophore. Consequently, three well-resolved peaks were simultaneously observed in the single-channel histogram during the multiplexed calibration as well as in the subsequent measurement of the three cations in 10-fold-diluted sheep plasma. The assigned peak positions corresponded very well to the physiological range of the measured ions.

Voltammetric Detection of Heparin at Polarized Blood Plasma/1,2-Dichloroethane Interfaces

Heparin, a highly negatively charged polysaccharide, which has been used widely as an anticoagulant and antithrombotic, was detected by ion-transfer voltammetry at the interface between 1,2-dichloroethane and an aqueous buffer solution or undiluted blood plasma. Quaternary ammoniums with different numbers of methyl and long alkyl groups were examined as positively charged heparin ionophores using pipet electrodes filled with the organic electrolyte solutions of their tetrakis(4-chlorophenyl)borate salts. It was shown that octadecyltrimethylammonium most selectively facilitates interfacial heparin adsorption without interference from potential-dependent ionophore transfer into the aqueous phase. Water-filled pipet electrodes were also used to study the stoichiometry of the interfacial complex between a heparin molecule and multiple ionophore molecules, which is discussed as a counterion condensation effect. Stripping voltammetry based on facilitated heparin adsorption and desorption gives a detection limit of 0.012 unit/mL in 0.12 M NaCl buffered at pH 7.2, which is 1 order of magnitude lower than therapeutic heparin concentrations (>0.2 unit/mL) and is comparable to a detection limit of the most sensitive heparin sensor reported so far. The biomedical utility of ion-transfer voltammetry was demonstrated for the first time in an undiluted blood sample. Despite interferences by Na+, Cl-, and plasma proteins such as serum albumin, a detection limit of 0.13 unit/mL was obtained in sheep blood plasma with the stripping method.

Reversed-phase liquid chromatographic method with fluorescence detection for the simultaneous determination of albendazole sulphoxide, albendazole sulphone and albendazole 2-aminosulphone in sheep plasma

A rapid and sensitive HPLC method for the simultaneous quantification of albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO 2) and albendazole 2-aminosulphone (ABZ-SO 2NH 2) in sheep blood plasma has been developed. Plasma samples were extracted with ethyl acetate under alkaline conditions. Separation was achieved on a C 18 reversed-phase analytical column, in the presence of positively- (tetra- n-butylammonium hydrogen sulphate) and negatively-charged (octanesulphonate sodium) pairing ions, while detection was performed fluorometrically. Excitation and emission wavelengths were 290 and 320 nm, respectively. Limits of quantification were defined at 39 ng/ml for ABZ-SO, 4.95 ng/ml for ABZ-SO 2 and 4 ng/ml for ABZ-SO 2NH 2. Accuracy data, in terms of recovery efficiency showed overall values (±S.E.M.) of 85.6±1.0% for ABZ-SO, 100.0±1.0% for ABZ-SO 2 and 89.1±0.6% for ABZ-SO 2NH 2. The method was successfully applied to quantitatively determine the three albendazole metabolites in plasma samples collected from sheep that had been orally administered albendazole.

Influence of protein starvation on some lysosomal enzyme activities in blood serum of sheep

Abstract. Reactivity of none lysosomal enzymes in blood plasma of sheep has been estimated. After 48 h protein deprivation the activity of BGLU, BGAL, AAP and LAP increased significantly, NAGL and LL activity decreased, EL and KF activity remained unchanged.

Effects of lasalocid and cationomycin on the evolution of certain parameters in the blood plasma of sheep.

Six adult sheep were fed at maintenance level, successively over three experimental periods, 1100 g of a roughage-rich diet without supplement or containing 33 mg kg-1 of lasalocid or cationomycin. The feed was administered in eight equal meals daily, every three hours. Blood samples were taken in each animal from the jugular vein at 10.00 hours, 16.00 and 22.00 hours, one hour after the animals were fed. The ionophores did not affect the plasma concentrations of glucose, free fatty acids, total amino acids, insulin, acetate, Ca or Mg. They decreased beta-hydroxy butyrate content (P < 0.05) and increased that of albumin (P < 0.05). Lasalocid alone significantly decreased uremia, but the significant threshold was only reached at 16.00 hours (P < 0.01). With this exception, the two ionophores had similar effects. Samples taken in peripheral blood appear to be too far from nutrient absorption sites to give a clear indication of the effects of these molecules on the products absorbed or metabolised in the digestive tract.

Nitrate and nitrite kinetics after single intravenous dosage in sheep

Nitrite was quickly eliminated from blood plasma of sheep ( T 1 2 = 0.49 h ). Three hours after its administration, nitrite ion disappeared from blood plasma. Simultaneously the appearance of nitrate was observed. After sodium nitrite or nitrate intravenous administration, the kinetic parameters for nitrate were similar ( K el = 0.150 h −1 and 0.154 h −1, respectively). Urine excretion of nitrite amounted to only 0.29% ± 0.22 of the administered dose. Urine excretion of nitrate was 13.80% ± 5.78 of the administered dose of nitrite and was slightly lower than that observed after the administration of nitrate (16.12% ± 6.95). The quick elimination of nitrite appears to be mainly from its conversion into nitrate, because the nitrate elimination in urine, as well as the values of its kinetic parameters, were similar after the administration of nitrite or nitrate. Elimination of nitrate from blood plasma of sheep over 80% was different than its excretion in urine in the unchanged form.