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Kinetics of nuclear maturation and effect of holding ovaries at room temperature on in vitro maturation of camel (Camelus dromedarius) oocytes.

Experiments were conducted to investigate kinetics of in vitro nuclear maturation and the effect of storing ovaries at room temperature on initial chromatin configuration and in vitro maturation of dromedary camel oocytes. Cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 4-48h. At every 4h interval (starting from 0 to 48 h), groups of oocytes were fixed, stained and evaluated for the status of nuclear chromatin. Oocytes were categorized as germinal vesicle (GV), diakinesis (DK), metaphase-I (M-I), anaphase-I (A-I), metaphase-II (M-II) stage and those with degenerated, fragmented, activated or without a visible chromatin as others. At the start of culture, 74% (66/89) oocytes were at GV stage, 13% (12/89) at DK and 12% (11/89) were classified as others. Germinal vesicle breakdown started spontaneously in culture and at 20 h of culture 97% oocytes had already completed this process. After 8 and 16 h of maturation the highest proportion of oocytes (42%, 48/114 and 41%, 51/123) were at DK and M-I stage, respectively. The proportions of oocytes reaching M-II stage at 32 (42%, 50/118), 36 (45%, 47/104), 40 (49%, 57/117), 44 (52%, 103/198) and 48 h (46%, 55/120) of culture were not different from each other (P>0.05). The proportion of oocytes categorized as others, however, increased after 40 h of culture and was higher (P<0.05) at 48 h compared with other maturation periods. There was no difference (P>0.05) in the proportion of oocytes reaching M-II stage from the ovaries collected and stored in normal saline solution (NSS) at room temperature for 12h (43%, 64/148) and those collected in warm NSS (37 degrees C) and processed immediately after arrival in laboratory (49%, 57/117). However, low number of oocytes reached M-II stage from ovaries collected in warm NSS but stored at room temperature (29%, 37/128) compared with other two groups (P<0.05). It may be concluded that dromedary oocytes require 32-44h of in vitro culture to have an optimum number of oocytes in M-II stage. However, further studies are required to find out the most appropriate maturation period, which will result in the further development of these oocytes after IVF, ICSI, parthenogenetic activation or nuclear transfer. Ovaries can be collected and stored in normal saline solution at room temperature for 12h without any appreciable effect on the nuclear maturation of the oocytes.

Animals↗

Immunized camel sera and derived immunoglobulin subclasses neutralizing Androctonus australis hector scorpion toxins.

Scorpion envenoming is a real health problem. The only specific treatment is immunotherapy with antibodies from immunized horses. The severity of scorpion envenoming and the rapid diffusion of the toxins into the blood compartment require an improvement of the present antivenom therapy. In this study, we report successful immunization of dromedaries (Camelus dromedarius) against the small weakly antigenic neurotoxins of Androctonus australis hector scorpion. Camel immune sera was tested for its specific antigenic reactivity and neutralizing capacity against Aah toxic fraction and AahII toxin. We demonstrate that a substantial proportion of polyclonal heavy chain antibodies bind to Aah toxins and in particular to AahII, the most toxic one scorpion venom component. Furthermore, we show that both dromedary sera and heavy chain antibody subclasses are capable of neutralizing the toxicity of Aah toxins in mice.

Animals↗

A comparative evaluation of parasitological, serological and DNA amplification methods for diagnosis of natural Trypanosoma evansi infection in camels.

A representative number of 217 camels (Camelus dromedarius) from different areas of western Rajasthan State, India, were examined from July 2002 to May 2003 for Trypanosoma evansi infection. The tests used were parasitological (wet blood film, WBF; stained thin blood smear, TBS), immunodiagnostic (double antibody sandwich enzyme linked immunosorbent assay for antigen detection, Ag-ELISA), and DNA amplification by polymerase chain reaction (PCR). These techniques were compared and the best efficiency was found for the last named (PCR). A prevalence of T. evansi infection was detected in 17.05, 9.67, 4.60 and 4.14% by PCR, Ag-ELISA, TBS and WBF with a sensitivity of 100, 56.75, 27.02 and 24.32%, respectively. PCR revealed a specific 227bp band in positive samples. The intensity of PCR bands was variable in different test samples depending upon the level of infection in the test samples. The history of intermittent fever, emaciation, oedema, poor body condition significantly correlated with positive serological status in ELISA as well as trypanosome DNA detection by PCR.

Animals↗

Incidence, biochemical and haematological alterations induced by natural trypanosomosis in racing dromedary camels.

In this study 10.67% incidence of trypanosomosis was observed in 150 racing camels by Suratex (latex agglutination). A significant decrease (P<0.05) in RBC, Hb, PCV and lymphocyte was observed, while a significant (P<0.05) increase in WBC and neutrophils was noted in trypanosomosis positive samples. The blood chemistry parameters indicated that there was significant (P<0.05) decrease in iron and albumin, but no significant alteration was observed in alkaline phosphatase (ALP), alanine aminotransferase (ALT), asparate aminotransferase (AST), gamma glutamyltransferase (GGT), lactic dehydrogenase (LDH), urea, total protein, calcium, creatinine, phosphorous and magnesium.

Animals↗

Camel lens crystallins glycosylation and high molecular weight aggregate formation in the presence of ferrous ions and glucose.

The incubation of camel lens cortex homogenate with 100 microM ferrous ions and 5.5 mM glucose under sterile conditions caused rapid protein aggregation, but little or no reaction was seen with either 100 microM ferrous ions or 5.5 mM glucose alone. The formation of glycosylated high molecular weight (HMW) protein aggregates was confirmed by light scattering studies, a decreased level of free -SH groups, incorporation of [14C]-glucose and elution of HMW protein aggregate just after the void volume of a Sephacryl S-1000 column. The bonding involved in the formation of these aggregates was found to be mainly disulfide in nature. Isoelectric focusing (IEF) in the presence and absence of reducing conditions indicated that gamma-crystallins may be involved in the formation of HMW protein aggregates. The modifications observed were found to mimic those seen in cataractous lenses.

Animals↗

Characterization of the camel skin cell line Dubca.

A skin fibroblast cell culture was established from a 2-month-old dromedary foetus. The cells were transformed by infection with SV40 and cloned in soft agar. The established cell line is now designated Dubca cells (Dubai camel) and has been in permanent culture for 95 passages. The cell culture was examined morphologically, chromosome preparations made and DNA fingerprinting performed by hybridization with the oligonucleotide probe (GTG)5. SV40 large T antigen was detected by western blotting. The viral host range was determined by infection with viruses of different families. Camelpox virus (CaPV) bovine herpesvirus-1 (BHV-1), vesicular stomatitis virus (VSV) and border disease virus (BDV) could be propagated in these cells.

Animals↗

Clinical and subclinical colloid goitre in adult camels (Camelus dromedarius) at Kordofan region of Sudan.

Colloid goitre was diagnosed in adult camels in the Kordofan region of the Sudan. The disease is characterized by gross enlargement of the thyroid, histopathological follicular changes, reproductive disorders, low concentrations of circulating thyroid hormones (T3, T4) and normocytic normochromic anaemia. The possible cause of the condition is discussed and iodine supplementation is recommended.

Animals↗

Cervical spinal injuries caused by collision of cars with camels.

We report 16 patients who sustained spinal injuries by collision of their cars with camels. Out of the 16 patients, 10 had severe spinal injuries with neurological deficits. The pattern of injury, possible mechanism of injury and recommendations for avoiding these severe injuries are suggested.

Accidents, Traffic↗

Carnosine, anserine and taurine contents in individual fibres from the middle gluteal muscle of the camel.

High muscle carnosine und anserine contents contribute significantly to intra-cellular physico-chemical buffering. Our aim was to measure carnosine, anserine and taurine contents directly in individual type I, IIA and IIB fibres from the middle gluteus muscle of the camel. Mean carnosine contents in type I, IIA and IIB were 24.6 +/- 9.2, 39.4 +/- 11.4 and 42.8 +/- 18.8 mmol kg-1 dry weight (dw), respectively. Mean anserine contents in type I, IIA and IIB fibres were 30.0 +/- 8.4, 37.3 +/- 10.1 and 34.5 +/- 9.7 mmol kg-1 dw, respectively. Mean taurine contents in type I, IIA and IIB fibres were 42.4 +/- 15.9, 20.3 +/- 12.9 and 24.7 +/- 15.9 mmol kg-1 dw, respectively. Higher carnosine contents in type II fibres emphasise the importance of carnosine to intra-muscular acid-base regulation. A specific role for taurine in type I fibres in unclear.

Animals↗

Biochemical analysis of serum and cerebrospinal fluid in clinically normal adult camels (Camelus dromedarius).

The concentrations of total protein, glucose, cholesterol, triglyceride, urea nitrogen, creatinine, sodium, potassium, chloride, calcium, inorganic phosphorus, copper, magnesium, and iron and the activities of aspartate aminotransferase (AST), alanine aminotransferase, (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LD), creatine kinase (CK) and amylase were determined in cerebrospinal fluid (CSF) and serum from 21 clinically healthy adult camels. The concentrations of sodium, potassium and chloride in CSF were similar to those of serum; whereas the values for all other constituents were significantly higher (P<0.05) in serum than in CSF.

Analysis of Variance↗

Plasma pharmacokinetics of intravenous and intramuscular furosemide in the camel (Camelus dromedarius).

Single bolus intravenous (i.v.) and intramuscular (i.m.) doses of furosemide (1.5 mg kg(-1)) were administered in a crossover design to three female and three male adult, dromedary camels. Plasma furosemide concentrations were assayed by HPLC and sodium, potassium and chloride concentrations were measured using ion-selective electrodes. Plasma furosemide concentration-time plots indicated multi-compartment disposition, and there was considerable intersubject variability in the pharmacokinetic parameters. The mean (SD) i.v. terminal elimination half-life was 118 (67) minutes. The systemic clearance was 5.4 (1.2) ml min(-1) kg(-1). and the steady-state volume of distribution was 0.43 (0.14) litre kg(-1). The mean absorption time after i.m. dosing was 33 (62) minutes, while the absolute bioavailability was 71 (20) per cent. The glucuronide metabolite of furosemide was not detected in plasma. Clearance was lower, volume of distribution was larger, and half-life was longer compared with published data for dogs, horses, rats and humans. Plasma potassium and sodium concentrations were significantly diminished (P<0.05) by an average of 35.6 per cent (two to six hours post-dose) and 11.3 per cent (eight hours post-dose), respectively. Plasma chloride concentrations were not significantly affected by furosemide administration.

Animals↗

Retrospective study on an unusual form of ovario-bursal pathology in the camel (Camelus dromedarius).

A peculiar form of ovarian bursa pathology, called hydrobursitis, is described in the dromedary camel. This malformation is characterized by a collection of fluid within the ovarian bursa with encapsulation of the ovary. Retrospective study of all diagnosed cases in our laboratory shows that this malformation is responsible for reduced reproductive performance due to abortion, infertility and embryonic death. Hydrobursitis was diagnosed in 33 of 355 animals examined. The incidence of this lesion was higher in animals that aborted (9/24) or were barren for more than 2 years (13/37). Diagnosis by palpation per rectum and ultrasonography, is described. Gross pathological and biochemical studies on specimens collected at surgery or in the slaughterhouse showed that the fluid is hemorrhagic and that it could be a mixture of blood and follicular fluid. Bacteriological and cytological studies were inconclusive. The origin of this lesion is discussed in light of genetic, pathological and management factors. Loss of embryos in affected animals could be due to the effect of increased tension on the uterus caused by the accumulation of fluid which ranged in volume from 250 mL to 4240 mL. The condition was treated in several females by surgical ablation. Embryo transfer was used in females that were bilaterally affected and underwent a bilateral ovariectomy. Pregnancy and calving rates were 52.5% and 38.1% for unilaterally ovariectomized females bred after surgery and 33.3% and 22.2% for bilaterally ovariectomized females used as embryo recipients after surgery. These results show that involvement of the uterus in this malformation is very limited and that surgery can be considered for valuable animals if they are unilaterally affected.

Abortion, Veterinary↗

Clinical and anatomical studies of the camel (Camelus dromedarius) genitalia.

Clinical and anatomical studies of the genitalia of 294 camel (197 males and 97 females) were carried out between May 1984 and October 1985. Preslaughter (clinical) examination was followed by detailed post-mortem (anatomical) examination and dissection of genitalia at the abattoir. Measurements and weights of the various segments of genitalia were made to establish the baseline data for breeding soundness evaluation in the dromedary in Northern Nigeria.

Journal Article↗

Serum progesterone and 17 beta-estradiol concentrations during pregnancy of Bactrian camel (Camelus bactrianus).

Serum progesterone and 17 beta-estradiol during pregnancy in the Bactrian camel were measured by radioimmunoassay. Serum progesterone concentrations increased by 15 d after artificial insemination (AI) and remained elevated throughout most of gestation, the mean concentrations (3.06 +/- 0.49 to 8.51 +/- 4.80 ng/mL) were similar to those reported for many species during the same stage of pregnancy. Serum 17 beta-estradiol increased significantly from 11 m.o. of pregnancy with peak mean concentrations of 617.47 +/- 32.56 pg/mL at the 11.5 m.o.

Animals↗

Implantation and early placentation in the one-humped camel (Camelus dromedarius).

The ovaries and uteri were removed from four pregnant camels on days 14, 25, 35 and 56 after ovulation. The day 14 and 25 uteri were perfuse-fixed with 3 per cent glutaraldehyde: 3 per cent paraformaldehyde whereas the day 35 and 56 specimens were opened dorsally for photography before biopsies of allantochorion attached to endometrium were taken and fixed in either 3 per cent glutaraldehyde: 3 per cent paraformaldehyde or Bouin's fluid. Samples of each uterus were then processed and sectioned for light and transmission electron microscopy. By day 14 the majority of the trophoblast had become closely apposed to the luminal epithelium of the endometrium to form the start of an epitheliochorial placenta with microvillar interdigitation initiated in some places. By day 25 a well-developed microvillar junction had formed between the fetal and maternal tissues. The fetus was situated in the middle of the left uterine horn in the day 35 and 56 specimens and, histologically, large multinucleate giant trophoblast cells had developed at frequent but irregular intervals along the, otherwise single-cell, trophoblast. These giant cells were often situated over the mouth of an endometrial gland but their actual function in pregnancy is not yet known.

Animals↗

High-affinity binding of NADPH to camel lens zeta-crystallin.

Fluorescence spectrum of camel lens zeta-crystallin, a major protein in the lens of camelids and histicomorph rodents, showed maximum emission at 315 nm. This emission maximum is blue shifted compared to most proteins, including alpha-crystallin, and appeared to be due to tryptophan in highly hydrophobic environment. Interaction of NADPH with zeta-crystallin quenched the protein fluorescence and enhanced the fluorescence of bound NADPH. Analysis of fluorescence quenching suggested high-affinity interaction between NADPH and zeta-crystallin with an apparent Km<0.45 microM. This value is at least an order of magnitude lower than that suggested by activity measurements. Analysis of NADPH fluorescence showed a biphasic curve representing fluorescence of free- and bound-NADPH. The intersection between free- and bound-NADPH closely paralleled the enzyme concentration, suggesting one mole of NADPH was bound per subunit of the enzyme. Phenanthrenequinone (PQ), the substrate of zeta-crystallin, also was able to quench the fluorescence of zeta-crystallin, albeit weaker than NADPH. Quantitative analysis suggested that zeta-crystallin had low affinity for PQ in the absence of NADPH, and PQ binding induced significant conformational changes in zeta-crystallin.

Animals↗

Hydrophobicity of the NADPH binding domain of camel lens zeta-crystallin.

Interaction of camel lens zeta-crystallin with the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) enhanced the ANS fluorescence and quenched the protein fluorescence. Both of these events were concentration-dependent and showed typical saturation curves suggesting specific ANS-zeta-crystallin binding. Quantitative analysis indicated that 1 mole zeta-crystallin bound at most 1 mole ANS. NADPH but not 9,10-phenanthrenequinone (PQ) was able to displace zeta-crystallin-bound ANS. These results suggested the presence of a hydrophobic domain in zeta-crystallin, possibly at the NADPH binding site. alpha-Crystallin as well as NADPH protected zeta-crystallin against thermal inactivation suggesting the importance of this site for enzyme stability. The NADPH:quinone oxidoreductase activity of zeta-crystallin was inhibited by ANS with NADPH as electron donor and PQ as electron acceptor. Lineweaver-Burk plots indicated mixed-type inhibition with respect to NADPH, with a K(i) of 2.3 microM. Secondary plots of inhibition with respect to NADPH indicated a dissociation constant (K'I) of 12 microM for the zeta-crystallin-NADPH-ANS complex. The K(i) being smaller than K'I suggested that competitive inhibition at the NADPH binding site was predominant over non-competitive inhibition. Like ANS-zeta-crystallin binding, inhibition was dependent on ANS concentration but independent of incubation time.

Anilino Naphthalenesulfonates↗

Inhibition of camel lens zeta-crystallin/NADPH:quinone oxidoreductase by pyridoxal-5'-phosphate.

Camel lens zeta-crystallin was inhibited by pyridoxal-5'-phosphate (PAL-P) and o-phthalaldehyde. PAL-P inactivated zeta-crystallin in a time- and concentration-dependent manner. The initial rate of inactivation followed pseudo-first-order kinetics with the second-order rate constant of 91 M-1 s-1. The modified enzyme showed the characteristic absorption peak at 325 nm indicative of the formation of phosphopyridoxallysine. Quantitative analysis suggested the incorporation of 1 mole of PAL-P/subunit of enzyme. NADPH was able to substantially protect zeta-crystallin against PAL-P inactivation, whereas the substrate 9,10-phenanthrenequinone (PQ) did not provide any protection. Inhibition of zeta-crystallin by PAL-P was uncompetitive with NADPH (Ki=37 microM) and non-competitive with respect to the substrate (Ki=57 microM). Inhibition of zeta-crystallin by o-phthalaldehyde was used to establish the location of an essential lysine residue. Incubation of zeta-crystallin with o-phthalaldehyde resulted in the formation of an isoindole derivative that had a characteristic fluorescence spectrum. This suggested that a lysine residue is located within 3 A of a cysteine residue at the NADPH binding region. SDS-PAGE showed the o-phthalaldehyde-modified enzyme remained largely monomer (approx. 80%), although bands corresponding to dimer and tetramer forms were also present. These results suggested that an essential lysine residue is located in the vicinity of the NADPH binding site. This residue may simply ensure the proper binding of NADPH to the active site of zeta-crystallin.

Animals↗