Extending the interval between second vaccination and slaughter: I. Effects on growth, scrotal size and stress responses of immunocastrated ram lambs.

Immunocastration provides a less invasive means of castrating lambs. Considering increasing consumer awareness, the efficacy of this technique on commercial slaughter lambs needs to be further investigated and its effects on growth and stress responses need to be established. This study compared the growth rate, testes size and stress responses of immunocastrated lambs with that of lambs physically castrated with a Burdizzo clamp, as well as intact rams. A total of 40 Dohne Merino ram lambs (average live weight = 45.4±3.68 kg) were randomly allocated to the following four treatment groups: control (intact; R), Burdizzo-castrated (on day 2; B), immunocastrated with a 4-week (ICS4), or a 6-week (ICS6) interval between the second immunocastration vaccination and slaughter. Within the immunocastration treatments, the reaction to vaccination was assessed through injection site scoring, recording the local injection site surface temperature and assigning a walking score. The response to Burdizzo castration was assessed by scoring the reaction during the procedure, testes palpation reaction, walking gait and measuring testis temperature. Additional parameters recorded included BW, serum cortisol concentration, scrotal circumference and rectal temperature. Pain behaviours were described for the short-, medium- and long-term effects after the two methods of castration. Predominantly, tissue-hardening and bruising occurred at the injection sites of immunocastrates, but little effect was observed on walking comfort and no effect on injection site temperature or rectal temperatures. After Burdizzo castration, lambs spent more time in abnormal postures, and from day 3 (D3) to D8 of the trial, discomfort was observed during testes palpation and walking in B lambs. Serum cortisol concentrations were elevated in B lambs on D3 and D15, indicating physiological stress. Thus, immunocastration improved the welfare of castrated lambs as assessed by cortisol secretion, scrotal swelling and pain behaviours, without influencing growth rate.

Extending the interval between second vaccination and slaughter: II. Changes in the reproductive capacity of immunocastrated ram lambs.

Immunocastration improves the welfare of castrated commercial slaughter lambs; however, the time-point at which this technique influences semen quality and sperm production has not yet been established for various vaccination schedules. Furthermore, the effect of extended intervals between second vaccination and slaughter needs to be investigated regarding continued testosterone suppression in immunocastrated lambs. The effect of extending the interval between second immunocastration vaccination and slaughter from four to six weeks on the reproductive capacity of Dohne Merino lambs was examined. A total of 40 Dohne Merino lambs were stratified according to initial weight (45.4±3.68 kg) and randomly assigned to four treatments that included intact control rams (R), Burdizzo-castrated lambs (B) and lambs immunocastrated with either four (ICS4) or six (ICS6) weeks between second vaccination and slaughter. Blood and semen samples were collected throughout the study period to determine serum testosterone concentrations, evaluate semen quality and assess sperm viability. Semen samples from R showed improvement over the trial. Throughout the collection period, B lambs had low serum testosterone concentrations, poor sperm motility and sperm viability, as expected. However, a slight increase in the percentage of live sperm in semen samples from B lambs towards the end of the collection period indicated poor success rates of the technique in some lambs. Burdizzo-castration also caused testes tissue necrosis and abscessing, indicating physiological stress. Semen appearance scores varied for both immunocastrated treatments, but the mass motility scores decreased over time. The ICS6 lambs showed a consistent and continuous decline in serum testosterone concentrations and sperm viability, with an increased percentage of dead abnormal sperm in the semen samples at the end of the study. The ICS4 treatment was successful in interrupting serum testosterone production and reducing semen quality; however, not as consistently as the ICS6 treatment. Primary immunocastration vaccination influenced serum testosterone concentrations but consistently low levels were only realised for both treatments after secondary vaccination. Although all castration treatments influenced testes size and colour, the six-week vaccination-to-slaughter interval caused a greater decrease in testes cut surface L* (lightness) colour values and in seminiferous tubule circumference. Extending the interval between second immunocastration vaccination and slaughter resulted in a more consistent and reliable influence on reproductive capacity of immunocastrated lambs. Thus, immunocastration is a suitable alternative to Burdizzo-castration regarding the interruption of testosterone production and testis functioning.

The role of apoptotic cell death in the radiosensitising effect of gemcitabine.

BACKGROUND: The aim of this study was to evaluate the radiosensitising effect of gemcitabine, in terms of cell-cycle progression, induction of apoptosis, and to investigate the molecular events regulating apoptosis. METHODS: Tumour cells were treated with gemcitabine, radiation, or the combination. 0-72 h after treatment, cells were collected for cell-cycle analysis and apoptosis determination. Caspase 8 and 9, Bid and tBid expression were determined by western blot. The mitochondrial membrane potential was determined using flow cytometry. An RT(2) Profiler PCR Array for human apoptotic genes was performed after the combination or TRAIL treatment. RESULTS: Gemcitabine and radiation resulted in an early S-phase block immediately after treatment, after which the cells moved synchronously through the cell cycle. When cell-cycle distribution returned to pre-treatment levels, an increased induction of apoptosis was observed with activation of caspase 8 and 9 and a reduction of the mitochondrial membrane potential. Gene expression after treatment with radiosensitising conditions was comparable with expression after the TRAIL treatment. CONCLUSION: A role for the cell-cycle perturbations and the induction of apoptosis could be attributed to the radiosensitising effect of gemcitabine. Apoptosis induction was comparable with the apoptotic pathway observed after the TRAIL treatment, that is the involvement of the extrinsic apoptosis pathway.

Feasibility of collecting self-sampled vaginal swabs by mail: quantity and quality of genomic DNA.

Vaginal self-sampling may be valuable as an alternative method of cervical cancer screening in areas of poor resources, to enroll women who, otherwise, would not participate in population-based cervical cancer screening and in epidemiological follow-up studies. We assessed the reliability of mailed vaginal samples by evaluating the quantity and quality of genomic DNA in the samples. Mailed swabs (n = 201) were compared with freshly collected samples (n = 200) for DNA concentration (45.1 versus 50.9 ng/microl, respectively) and purity (mean optical density [OD] 260/280 ratio 1.88 versus 1.78, respectively). A small, non-significant, decrease in DNA yield with longer transport time was noted. The DNA yield of mailed samples was significantly lower compared to fresh samples (P < 0.002), but this lower yield had little effect on polymerase chain reaction (PCR) amplification. In conclusion, the large majority of mailed self-sampled vaginal swabs resulted in DNA of adequate purity and concentration for further research.

Variance components for live weight, body measurements and reproductive traits of pair-mated ostrich females.

Estimates of genetic parameters for reproductive traits, live weight and body measurements were obtained using data from a pair-mated ostrich flock at Oudtshoorn in South Africa. Reproductive traits included total egg and chick production, along with hatchability percentage. Live weight, chest circumference and tail circumferences were recorded at the commencement and cessation of breeding. Heritability estimates (h(2)) were 0.23 for egg production, 0.20 for chick production, 0.10 for hatchability, 0.20 to 0.34 for live weight, 0.12 for chest circumference and 0.30 to 0.38 for tail circumference. Female permanent environmental effects (c(2)) amounted to 0.18 for egg production, 0.18 for chick production, 0.21 for hatchability, 0.32 to 0.36 for live weight and 0.23 to 0.32 for chest circumference. Service sire exerted significant effects only on hatchability (0.22) and subsequently chick production (0.09). Genetic correlations of reproductive traits with live weight were low to moderate, variable in sign, and did not differ significantly from zero. Correlations between live weight recorded at the beginning and end of the breeding season were unity for additive genetic and permanent environmental effects. Egg and chick production were highly correlated genetically and phenotypically, with the genetic correlation exceeding the theoretical limit. In unconstrained analyses, hatchability was positively related to chick production, including at the service sire level. Selection gains in the current flock and future generations are likely. No significant adverse relationships were found between live weight, body measurements and reproductive traits.

In vitro interaction between ecteinascidin 743 (ET-743) and radiation, in relation to its cell cycle effects.

Ecteinascidin 743 (ET-743) is a new marine-derived agent with promising activity against a number of solid tumours. In four human tumour cell lines, the interaction between ET-743 and radiation was investigated in relation to the effects of ET-743 on the cell cycle, in vitro. Cell survival was measured based on quantitative staining of cellular protein by sulforhodamine B. A 24 h treatment with ET-743 before radiation resulted in a moderate increase in radiosensitivity in three out of four cell lines. Dose enhancement factors > or =1.8 were observed for concentrations resulting in 52, 46 and 30% cell kill in ECV304, H292 and CAL-27, respectively, whereas in A549 no radiosensitisation was observed (no significant increase in radiosensitivity). According to the combination index analysis, synergism was observed only in ECV304 and CAL-27 cells. A 24 h incubation with ET-743 resulted in a concentration-dependent G2/M block, which might explain the moderate radiosensitising effects in ECV304 and H292. The lack of radiosensitisation in A549 might be due to the S phase delay preceding the G2/M block at the moment of radiation, which only occurred in this cell line. In conclusion, ET-743 has moderate cell line-dependent radiosensitising properties; however, only when cytotoxic concentrations of ET-743 are used. In one of the four cell lines tested, no radiosensitisation was observed.

The radiosensitising effect of gemcitabine and the influence of the rescue agent amifostine in vitro.

In this study, the radiosensitising effect of different concentrations of gemcitabine and the combination of gemcitabine/radiotherapy with the rescue agent amifostine was investigated in different human tumour cell lines. The cells were treated with gemcitabine (0-8 nM) for 24 h prior to radiation (0-8 Gy). Amifostine (ami) and alkaline phosphatase (AP) were added 30 min before radiation. Cell survival was determined 7 or 8 days after radiation treatment by the sulforhodamine B (SRB) test. For ECV304 cells, the dose enhancement factor (DEF) varied from 1.39 to 2.98 after treatment with 1-6 nM gemcitabine. FaDu, H292, A549 and CAL-27 seemed to be less sensitive, with DEFs ranging from 1.02 to 2.67. These cells were also less sensitive to the cytotoxic effects of single-agent gemcitabine. Amifostine with AP clearly showed a protective effect in combination with gemcitabine/radiotherapy. In H292 cells, the protection factor (PF) of amifostine after treatment with gemcitabine and radiotherapy varied from 1.64 to 1.86. In ECV304 cells, the PF varied from 2.20 to 2.29. In conclusion, a clear concentration- and cell line-dependent radiosensitising effect of gemcitabine was observed in all cell lines. Amifostine with AP showed protection against the radiosensitising effect of gemcitabine. If the protection in vivo indeed occurs selectively in normal tissues, then amifostine could prevent or strongly minimise the increased toxicity resulting from the radiosensitising effect of the combination of gemcitabine and radiotherapy, without influencing the antitumour effect.

Preliminary results on the use of diagnostic ultrasonography as a management tool to quantify egg production potential in breeding ostrich (Struthio camelus australis) females.

An ostrich breeding flock, joined as individual breeding pairs (n = 136 pairs), was used to investigate the possibility of diagnostic ultrasonography as a method to predict the reproductive performance of ostrich females during a breeding season. Follicular activity was easily detected and quantified by using diagnostic ultrasonography. One to 8 follicles were recorded in 25% of females scanned at the beginning of the 9-month breeding season. At the end of the breeding season, 1-3 follicles were observed in 28.7% females. Females in which follicular activity was observed came into production earlier than those in which no follicles were observed, with the mean (+/- SE) number of days to the production of the 1st egg being 22.3 +/- 12.5 and 87.4 +/- 7.2 days, respectively. Females in which follicular activity was observed at the beginning of the breeding season, produced on average 181% more eggs during the 1st month of the breeding season (P < 0.01) than females in which no follicular activity was observed (6.67 +/- 0.70 vs 2.37 +/- 0.41 eggs). Egg production over the first 2 months of breeding and over the entire breeding season were similarly affected (P < 0.01), with the mean number of eggs produced over the first 2 months of the breeding season being 14.7 +/- 1.5 for females with observed follicular activity and 7.4 +/- 0.9 eggs for females with no observed follicular activity. Females in which follicular activity was observed at the end of the breeding season produced on average 108% more eggs (P < 0.01) during the last month of the breeding season than females in which no follicular activity was observed (2.77 +/- 0.43 vs. 1.33 +/- 0.27 eggs). There was a tendency (P = 0.06) for egg production over the last 2 months to be similarly affected (6.10 +/- 0.85 vs 4.19 +/- 0.54 eggs). No relationship with egg production over the entire breeding season was found for the end-of-the-breeding-season observations. Diagnostic ultrasonography can thus be used as a management tool to identify reproductively healthy ostrich females and also females with a higher egg production potential over a period of 2 months after or prior to assessment. Future studies should focus on the development of the technique to predict reproductive performance over entire breeding seasons for selection purposes.

Prevalence of human papillomavirus in elderly women with cervical cancer.

To investigate the relation between the prevalence of human papillomavirus (HPV) and age in cervical cancer patients, material from 93 patients with Ia-IIb cervical carcinoma was analyzed for the presence of HPV by both type-specific and general primer polymerase chain reaction. Patients were divided into 2 groups: 64 years or younger, and 65 years and older. There was no statistically significant difference in either the prevalence of HPV DNA or distribution of genotypes amongst the 2 groups. Therefore, HPV detection can be equally well used in the management and follow-up of elderly cervical cancer patients.

Factors related to high levels of ostrich chick mortality from hatching to 90 days of age in an intensive rearing system.

Ostrich chick mortality was studied in 2522 chicks that were hatched artificially during the 1999/2000 breeding season. High levels of mortality were observed, with 1978 (78.4 %) of these chicks dying before 90 days after hatching. A total of 46.7 % (1,177) of these chicks died before 28 days of age, and a further 30.7% (801) died between 28 and 90 days post-hatching. Chick mortality to 28 days of age could not be conclusively related to sex, day of external pipping or breeder diet. Mortality rates were higher (P< 0.05) at the beginning and end of the breeding season than in the middle months. Differences in mortality levels of chicks incubated in different incubators could be related to the time of the breeding season during which the incubator was mostly used. The regression of chick mortality to 28 days of age on day-old chick mass followed a 2nd-degree polynomial. Chicks with day-old masses below 762.5 g were particularly at risk of dying before 28 days after hatching. Chicks hatching from eggs where excessive water loss to 35 days of incubation (>18%) was recorded were also at risk of succumbing before 28 days of age. Chick mortality percentages for the period from 28 to 90 days of age exceeded 80 % in chicks weighing an average of 1.050 g at 28 days. Mortality percentages declined sharply at higher live masses, to between 20 and 30 % in chicks weighing > or = 1,950 g. This 'core' level of mortality remained throughout, even in the heaviest chicks. It was concluded that the high levels of chick mortality could be related to stress in chicks, resulting from an inability to adapt to the rearing environment. The high subsequent mortality percentages of low live mass chicks that survived to 28 days after hatching could probably be attributed to residual setbacks suffered earlier. Abetter understanding of the underlying principles involved in ostrich chick mortality in intensive rearing environments is required for progress in this field, resulting in more predictable survival rates under these conditions.

Sperm viability and morphology of two genetically diverse Merino lines.

Microscopically evaluated sperm parameters, as well as computer-aided sperm motility analysis (CASMA), were used to assess sperm quality and the effect of cryopreservation on ram semen obtained from two genetically diverse Merino lines. These lines were divergently selected on maternal ranking values for multiple rearing ability from the same base population since 1986. Replacements in the high (+) line were preferentially the progeny of ewes rearing >1 lamb per joining. Progeny of ewes rearing <1 lamb per joining was preferred as replacements in the low (-) line. Sperm quality, as assessed by percentages of live, abnormal and acrosome-intact spermatozoa as well as by motility, was independent (P < or = 0.20) of line, time of sampling and their interaction in ejaculated samples obtained from the eight rams used as sires in 1995. Sperm quality of frozen-thawed samples was adversely affected (P < or = 0.01) by cryopreservation and thawing at 35 degrees C for 30 s relative to fresh ejaculated samples. No consistent differences between lines were found in epididymal sperm samples obtained from 12 slaughtered rams (6 from each line). The adverse effect (P < or = 0.05) of cryopreservation and thawing at 35 degrees C for 30 s on sperm viability and motility was also demonstrated for these samples.

The effect of cryopreservation on the survivability, viability and motility of epididymal African buffalo (Syncerus caffer) spermatozoa.

The effect of cryopreservation on the viability and motility of epididymal African buffalo spermatozoa was studied in samples obtained from 17 and 13 animals in 1995 and 1996, respectively. Cryopreservation significantly reduced the viability and motility of the epididymal spermatozoa. The average percentage of live (+/- SE) spermatozoa declined significantly from 90.4 +/- 2.0% (1995) and 84.4 +/- 1.1% (1996) in fresh epididymal samples, to 57.0 +/- 2.0% and 56.3 +/- 1.1%, respectively, in frozen-thawed samples. The acrosomal integrity (+/- SE) of spermatozoa declined from 89.3 +/- 2.3% (1995) and 93.3 +/- 2.2% (1996) to 50.2 +/- 2.3% and 37.5 +/- 2.2%, respectively. In 1995, this effect was largely associated with the thermal equilibration prior to cryopreservation.

Elevated reversible and irreversible lipid peroxidation in human oesophageal cancer.

BACKGROUND: Cancer of the oesophagus is the most common gastrointestinal malignancy in South African blacks. The aim of this study was to determine the reversible and irreversible lipid peroxidation in cancer of the oesophagus; a feature that has not been assessed before in this or other cancer tissue. METHODS: Biopsies and plasma from 39 patients with cancer of the oesophagus and 22 biopsies and plasma from non-cancer patients were analysed for the irreversible lipid peroxide product malondialdehyde (MDA) and other reversible lipid peroxide products (LPO) by the thiobarbituric acid-reactive substances (TBARS) method. RESULTS: The mean (+/- SEM) for MDA in plasma from normal patients was 1.697 (0.149), and for cancer patients 4.23 (0.417) nmol MDA/ml. The tissue MDA for normal patients was 0.807 (0.154), and for cancer patients 2.530 (0.379) nmol MDA/mg protein. The mean (+/- SEM) for LPO in plasma from normal patients was 1.929 (0.281), and for cancer patients 12.607 (1.451) nmol MDA/ml. The tissue LPO for normal patients was 2.957 (0.306), and for cancer patients 16.320 (1.868) nmol MDA/mg protein. All the MDA and LPO values for cancer patients were significantly elevated (p < 10(-4)). In oesophageal cancer 85% of the lipid which was peroxidized, was of the reversible type. CONCLUSIONS: This elevated reversible LPO levels in plasma and oesophageal tissue of cancer patients support the notion that the oxy-radicals in cancer are not under proper metabolic control. Therefore, human oesophageal cancer does not progress to self regression or destruction but rather to more mutagenic changes probably due to high reversible lipid peroxidations.

The effect of pH on chemiluminescence of different probes exposed to superoxide and singlet oxygen generators.

The compromised optima for high intensity chemiluminescence (CL), using superoxide generators, were all above pH 9.0 for the CL probes luminol and lucigenin. With luminol the optima were at pH 9.0 and 9.4 for the generators KO2 and hypoxanthine/xanthine oxidase (HX/XO), respectively. Lucigenin, with the same generators, produced optima at pH 9.5 and 10.0, respectively. The probe methyl-Cypridina-luciferin analogue (MCLA) produced optima closer to neutral pH, which is preferred for physiological assessments. MCLA had optima at pH 6.0, 8.7 and 9.5 with KO2 and with HX/XO optima at pH 4.8, 6.0, 7.0 and 8.7. When CL was assessed at physiological pH, MCLA observed superoxide radicals with a sensitivity of 100- and 330-fold more than luminol or luicigenin respectively. For singlet oxygen, the sensitivity of MCLA at this pH was 45- and 5465-fold more than for the said probes respectively. H2O2 did not elicit CL between pH 4 and 9.5 with any of the probes and did not influence the production of superoxide or singlet oxygen when co-assessed. Therefore CL could only be obtained when enzymes were used as converters. The optima for the enzyme-conversion system horseradish peroxidase (HRP)/H2O2, and luminol, were at pH 8.0 and 9.2. Lucigenin and HRP/H2O2 also had a biphasic CL profile with optima at pH 7.4 and 9.6. MCLA and HRP/H2O2 had five optima, with the major ones at pH 6.1 and beyond 10. The optima for the myeloperoxidase/H2O system were at 8.6 and beyond 10.0 when luminol and 0.15 mol/L NaBr were used.

Carbohydrate Status of Tulip Bulbs during Cold-Induced Flower Stalk Elongation and Flowering.

The effect of a cold treatment on the carbohydrate status of the scales and flower stalk of Tulipa gesneriana L. cv Apeldoorn bulbs during growth after planting was studied and compared with bulbs not given cold treatment. Bulbs were stored dry for 12 weeks at 5[deg]C (precooled) or 17[deg]C (noncooled). Only the 5[deg]C treatment led to rapid flower stalk elongation and flowering following planting at higher temperatures. Precooling enhanced mobilization of starch, fructans, and sucrose in the scales. The cold-stimulated starch breakdown was initially accompanied by increased [alpha]-amylase activity per scale. In noncooled bulbs, [alpha]-amylase activity slightly decreased or remained more or less constant. Cold-induced flower stalk elongation was partially accompanied by a decrease in the sucrose content and an increase in the glucose content and invertase activity per g dry weight. The starch content in internodes initially decreased and subsequently increased; [alpha]-amylase activity per g dry weight of the lowermost internode showed a peak pattern during starch breakdown and increased thereafter. The internodes of noncooled bulbs, on the contrary, accumulated sucrose. Their glucose content and invertase activity per g dry weight remained low. Starch breakdown was not found and [alpha]-amylase activity per g dry weight of the lowermost internode remained at a low level. Precooling of tulip bulbs thus favors reserve mobilization in the scales and flower stalk and glucose accumulation in the elongating internodes.

Portable medical records on microchip cards: the Tournai experiment.

A test of the use of microchip cards as portable medical records has been organized and evaluated in a small Belgian town. The portable record was accepted by patients and could be a useful innovation for emergency situations. A comparative evaluation of other portable media is needed.

Properties of the isoenzyme forms A-1, A-2 and B of N-acetyl-beta-D-glucosaminidase purified from baboon kidneys.

Three forms of N-acetyl-beta-D-glucosaminidase (NAG: A, B and I) were separated from baboon kidney using Con A-Sepharose and DEAE-Trisacryl chromatography. 2. The A form was further purified into two forms A-1 and A-2 using hydroxylapatite chromatography and anodic PAGE. Both were homogeneous on SDS-PAGE and anodic PAGE but microheterogeneous on PAG-IEF, which could be eliminated by prior treatment with endoglycosidase H or glycopeptidase F. 3. The carbohydrate content accounted for some of this microheterogeneity since it varied from 31 for A-1 to 17% for A-2 and the sialic acid was 6 and 1%. Deamidation may also contribute since the acidic amino acids (29 mol%) and ammonia were high following acid hydrolysis. 4. The mol. wt for A-1, determined by SDS-PAGE, was 52.1 K. 5. The pH optimum was 4.55 and the pI4.97. 6. The optimum temperature for NAG A and B was 50 degrees and 42 degrees C, but B retained more activity above 55 degrees C. 7. The Km for N-acetyl-beta-D-glucosamine and -galactosamine for both isoforms was 0.497 and 0.627 mM respectively. 8. Several ions were found to be uncompetitive inhibitors. Ag+ and Pb2+ were the most potent having Ki values of 3.6 and 8.5 mM respectively. Acetate acted as a competitive inhibitor.