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Effects of centrifugation before freezing on boar sperm cryosurvival.

Current protocols for boar sperm cryopreservation require the centrifugation of semen in order to separate sperm cells from the seminal plasma. This study evaluated the influence of different centrifugation regimes on both sperm recovery and yield (percentage of viable sperm with an intact acrosome relative to the initial sperm population) after centrifugation (experiment 1) as well as the influence of different centrifugation regimes on boar sperm cryosurvival (experiment 2). In both experiments, sperm-rich fractions from 3 boars were diluted, pooled, and cooled to 17 degrees C before centrifugation. In experiment 1, the g-forces tested were 400, 800, 1600, and 2400 x g for 3 or 5 minutes, using the standard regime (800 x g for 10 minutes) as a reference. Sperm recovery (Bürker Chamber) and yield (triple fluorescent stain of PI/R123/FITC-PNA [DNA-specific fluorochrome propidium iodide/mitochondria-specific fluorochrome rhodamine-123/acrosome-specific fluorochrome fluorescein isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin]) were calculated. The highest recovery and yield (P <.05) values were achieved using 2400 x g for 5 or 3 minutes and 1600 x g for 5 minutes, which showed no differences (P >.05) from the reference in terms of sperm yield. In experiment 2, cooled semen was centrifuged using 3 different regimes: C1 (2400 x g for 3 minutes), C2 (1600 x g for 5 minutes), and C3 (800 x g for 10 minutes). Pellets were diluted in lactose-egg yolk (LEY)-glycerol-Equex STM (1 x 10(9) cells/mL) and frozen in 0.5-mL straws. After thawing, sperm quality was assessed after 30 and 150 minutes of incubation (37 degrees C). Centrifugation regimes C1 and C2 showed significantly (P <.05) higher postthaw sperm motility (assessed with a computer-assisted semen analysis system), viability (evaluated as for experiment 1), and percentage of uncapacitated sperm (assessed with a chlortetracycline assay) than did C3. In addition, C1 had the highest (P <.05) oocyte penetrating ability (assessed with the homologous in vitro penetration test performed with immature oocytes). Malondialdehyde production, assessed with the thiobarbituric acid reactive species test, was unaffected (P >.05) by the centrifugation regime used. We conclude that high g-force (2400 x g) and short centrifugation time (3 minutes) do not affect sperm recovery and yield and that, moreover, they have a positive effect on the cryosurvival of boar sperm. Therefore, we recommend the use of short-term centrifugation with a relatively high g-force (2400 x g for 3 minutes) in boar sperm cryopreservation protocol.

Animals↗
PubMed2001

DNA-based genotyping techniques for the detection of point mutations associated with insecticide resistance in Colorado potato beetle Leptinotarsa decemlineata.

Three DNA-based genotyping techniques, bi-directional PCR amplification of specific allele (bi-PASA), single-stranded conformational polymorphism (SSCP) and minisequencing, have been developed and compared for the detection of the S291G (insensitive acetylcholinesterase) and L1014F (insensitive sodium channel) mutations associated with azinphos-methyl and permethrin resistance, respectively, in the Colorado potato beetle (Leptinotarsa decemlineata). Extraction of genomic DNA from individual neonates that were hatched from previously collected egg masses is the most efficient and reliable means to obtain suitable templates in terms of convenience, economy, speed and DNA quality. Bi-PASA, employing two allele-specific primers, appears to be the most efficient and rapid genotyping method for the simultaneous detection of both resistant/susceptible homozygous (SS, RR) and heterozygous (SR) alleles. Its resolution, however, is strongly dependent on the quality of template genomic DNA. SSCP also allows unambiguous genotyping, including the detection of heterozygous alleles, and is less dependent on template DNA quality, but requires a longer processing time. Minisequencing is amenable to a 96-well microtiter plate format for the processing of a large number of samples and allows direct detection of resistant/susceptible homozygous alleles but is not as efficient as the PASA and SSCP in detecting heterozygous alleles. In considering the advantages and disadvantages of each technique, DNA-based genotyping is best employed in combinations, with the bi-PASA as the primary method and the SSCP and minisequencing as the secondary validating methods. These methods are rugged, rapid, cost-effective and capable of resolving SS, RR and SR individuals. The availability of such DNA-based genotyping techniques, using neonate genomic DNA as templates, will enable the precise monitoring of the resistant and susceptible allele frequencies, including those of heterozygote individuals, in field populations of L. decemlineata.

Acetylcholinesterase↗
PubMed1980

A new and simple technique for chromosomal preparations from peripheral blood lymphocytes, amniotic cell cultures, skin fibroblasts, bone marrow and single cell clones when the yields from harvests are low.

A technique for chromosomal preparations from low yields harvest is presented using Poly-L-Lysine (300,000-400,000 MW). The procedure can be successfully used for peripheral blood lymphocytes, amniotic cell cultures, skin fibroblasts, direct preparations from bone marrow, cloned clusters and colonies from bone marrow grown on agar or methyl cellulose and eggs and blastocysts, with applications to other tissues. The technique is simple with minimal cell loss, and chromosomal preparations of good quality can be obtained which are amenable to all banding techniques.

Amniotic Fluid↗
PubMed2006

Seminal plasma improves cryopreservation of Iberian red deer epididymal sperm.

We tested the protective action of seminal plasma on epididymal spermatozoa from Iberian red deer, especially considering cryopreservation, as a means for germplasm banking improvement. We obtained seminal plasma by centrifuging electroejaculated semen, and part of it was thermically inactivated (denatured plasma; 55 degrees C 30 min). Epididymal samples (always at 5 degrees C) were obtained from genitalia harvested after regulated hunting, and pooled for each assay (five in total). We tested three seminal plasma treatments (mixing seminal plasma with samples 2:1): no plasma, untreated plasma and denatured plasma; and four incubation treatments: 32 degrees C 15 min, 5 degrees C 15 min, 5 degrees C 2h and 5 degrees C 6h. After each incubation, samples were diluted 1:1 with extender: Tes-Tris-Fructose, 10% egg yolk, 4% glycerol; equilibrated for 2h at 5 degrees C, extended down to 10(8) spz./mL and frozen. Sperm quality was evaluated before 1:1 dilution, before freezing and after thawing the samples, assessing motility (CASA) and viability (percentage of viable and acrosome-intact spermatozoa; PI/PNA-FITC and fluorescent microscopy). Plasma treatment, both untreated and denatured, rendered higher viability before freezing and higher results for most parameters after thawing. The improvement was irrespective of incubation treatment, except for viability, which rendered slightly different results for untreated and denatured plasma. This may be due to the presence of thermolabile components. We still have to determine the underlying mechanisms involved in this protection. These results might help to improve the design of cryopreservation extenders for red deer epididymal sperm.

Acrosome Reaction↗
PubMed2004

Tropical winter habitat limits reproductive success on the temperate breeding grounds in a migratory bird.

Identifying the factors that control population dynamics in migratory animals has been constrained by our inability to track individuals throughout the annual cycle. Using stable carbon isotopes, we show that the reproductive success of a long-distance migratory bird is influenced by the quality of habitat located thousands of kilometres away on tropical wintering grounds. For male American redstarts (Setophaga ruticilla), winter habitat quality influenced arrival date on the breeding grounds, which in turn affected key variables associated with reproduction, including the number of young fledged. Based on a winter-habitat model, females occupying high-quality winter habitat were predicted to produce more than two additional young and to fledge offspring up to a month earlier compared with females wintering in poor-quality habitat. Differences of this magnitude are highly important considering redstarts are single brooded, lay clutches of only three to five eggs and spend only two-and-a-half months on the breeding grounds. Results from this study indicate the importance of understanding how periods of the annual cycle interact for migratory animals. Continued loss of tropical wintering habitat could have negative effects on migratory populations in the following breeding season, minimizing density-dependent effects on the breeding grounds and leading to further population declines. If conservation efforts are to be successful, strategies must incorporate measures to protect all the habitats used during the entire annual cycle of migratory animals.

Animal Migration↗
PubMed2005

Anaerobic reactor/high rate pond combined technology for sewage treatment in the Mediterranean area.

Two high-rate, anaerobic/aerobic units were used to treat the sewage of the Institut Agronomique st Vétérinaire Hassan II (Morocco) campus in a 1,100 m2-plant designed for 1,500 e.p. and receiving 63 m3 per day. The anaerobic pre-treatment consisted of a two-step up-flow anaerobic reactor (TSUAR) comprising two reactors and one external settler all in series. The aerobic line, or post-treatment, consisted of a high-rate algal pond (HRAP) and one maturation pond in series. The system totalized a hydraulic retention time (HRT) of 9 days. A gravel filter (GF) was constructed behind the TSUAR to trap low-density particles. The TSUAR removed 80% of COD and 90% of SS within 48 h. Solids retention time in the reactors averaged 32 d with a specific sludge production of 0.28 g SS g(-1) COD removed. Almost 93% of the sludge evacuated from the settler was stabilized. Specific biogas production from both reactors was 0.25m3 kg(-1) COD removed. Used in this configuration, the HRAP lost its BOD removal activity and increased its nutrients and pathogens removal capabilities (tertiary treatment). Results showed that 85% of total nitrogen and 48% of total phosphorus were removed by the HRAP. Land area requirement of this combination was less than 1 m2 per capita and filtered final effluent was of excellent quality (COD, 82 mg/l; TKN, 8.3 mg/l; total P, 2.7 mg/l, faecal coliforms, 2.4 10(3)/100 ml and zero helminths eggs).

Ammonia↗
PubMed1995

[Structural changes in the poultry production industry and in the tasks of veterinarians].

The rapid expansion and improvement in poultry production began during the mid-1930s. These enhanced by innovation in technology, science and management on different levels such as on artificial incubation, genetics, breeding, rearing, nutrition, disease control and processing technology. During the past 30 years the total egg production as well as poultry meat production has increased tremendously. The modern poultry industry oriented for high production and better quality at low cost, together with increasing claim of consumers for poultry meat and products, demand continuous efficient and goal oriented veterinary care. For this purpose, in many countries poultry health services supervised by veterinarians in co-operation with a central veterinary diagnostic laboratory, are established. Today the main duties of the poultry veterinarians have changed from therapeutic to prevention objectives. The tasks of the modern veterinary poultry specialists are described.

Animal Husbandry↗
PubMed1992

Egg and milk allergy in adults: comparison between fresh foods and commercial allergen extracts in skin prick test and histamine release from basophils.

The ability of skin prick test (SPT) and histamine release from basophils (HR) to diagnose clinical type I allergy to egg and milk was investigated as compared with double-blind, placebo-controlled food challenge (DBPCFC) in 17 adults suspected of type I egg and/or milk allergy. In both SPT and HR, commercial allergen extracts commonly used for SPT were compared with fresh, standardized foods. With commercial extracts the overall sensitivities of SPT and HR were 0.75 and 0.56 respectively, and none of the tests showed concordance with DBPCFC. With fresh, standardized foods the overall sensitivities of SPT and HR were 1.00 and 0.89 respectively, and both tests now showed a significant concordance with DBPCFC (P < 0.05). Specificity was only slightly improved in SPT, and unchanged in HR. Thus, the use of fresh, standardized foods significantly improved the outcome of both tests, as regards to sensitivity and concordance with DBPCFC. The diagnostic ability of SPT and HR appear to be strongly influenced by the allergen quality.

Adult↗
PubMed2004

Unpredictable food supply modifies costs of reproduction and hampers individual optimization.

Investment into the current reproductive attempt is thought to be at the expense of survival and/or future reproduction. Individuals are therefore expected to adjust their decisions to their physiological state and predictable aspects of environmental quality. The main predictions of the individual optimization hypothesis for bird clutch sizes are: (1) an increase in the number of recruits with an increasing number of eggs in natural broods, with no corresponding impairment of parental survival or future reproduction, and (2) a decrease in the fitness of parents in response to both negative and positive brood size manipulation, as a result of a low number of recruits, poor future reproduction of parents, or both. We analysed environmental influences on costs and optimization of reproduction on 6 years of natural and experimentally manipulated broods in a Central European population of the collared flycatcher. Based on dramatic differences in caterpillar availability, we classified breeding seasons as average and rich food years. The categorization was substantiated by the majority of present and future fitness components of adults and offspring. Neither observational nor experimental data supported the individual optimization hypothesis, in contrast to a Scandinavian population of the species. The quality of fledglings deteriorated, and the number of recruits did not increase with natural clutch size. Manipulation revealed significant costs of reproduction to female parents in terms of future reproductive potential. However, the influence of manipulation on recruitment was linear, with no significant polynomial effect. The number of recruits increased with manipulation in rich food years and tended to decrease in average years, so control broods did not recruit more young than manipulated broods in any of the year types. This indicates that females did not optimize their clutch size, and that they generally laid fewer eggs than optimal in rich food years. Mean yearly clutch size did not follow food availability, which suggests that females cannot predict food supply of the brood-rearing period at the beginning of the season. This lack of information on future food conditions seems to prevent them from accurately estimating their optimal clutch size for each season. Our results suggest that individual optimization may not be a general pattern even within a species, and alternative mechanisms are needed to explain clutch size variation.

Adaptation, Physiological↗
PubMed2001

The effect of liming on the microbiological quality of urban sludge.

Treatments applied to sludge in order to stabilise and dehydrate them may give notable inactivation of microorganisms. This is observed when sludge is exposed either to high temperature or drastic pH when residual sludge is limed. The control of virological, parasitological and bacteriological sludge quality by detecting pathogenic microorganisms is slow and too expensive to be commonly practised. Thus, it is possible to replace pathogenic microorganisms detection by that of contamination indicators. The aim of this study was to determine the influence of liming on the behaviour of pathogenic microorganisms detected in urban sludge. The detection of Salmonella and helminth eggs was carried out in liquid sludge (2-3% dryness) and solid sludge (23% dryness) with added lime (0-45% weight/dry weight) and stored for 24 h-46 weeks. The results showed that liming modified some characteristics such as temperature, dryness and pH of the sludge. It appeared that, whatever the percentage of added lime, the temperature of liquid sludge did not change while it increased by about 9 degrees C when 30-45% lime was added to solid sludge. In the same way, the dryness of liquid sludge did not change during the liming, whereas the dryness of 45% limed solid sludge increased from 23% to 31%. Finally, 15%, 30% and 45% liming gave a pH of at least 10, 11.5 and 12, respectively, although the pH increase depended on the sludge type. The efficiency of liming was considered to be related to the pH and not to the percentage of added lime. Three factors determined the efficiency of pathogen elimination: (a) the pH reached by the sludge, (b) the period of liming activity and (c) the dryness of the sludge. Salmonella were eliminated from liquid sludge in 24 h at pH 10.7 and from solid sludge in 24 h at pH 10.0. Viable helminth egg concentration decreased to 3 eggs/10 g DM in liquid sludge in 14 d at pH 11.9 and 60 d at pH 11.6. In solid sludge, egg reduction was achieved in 24 h at pH 12.5, 7 d at pH 12.0 and 14 d at pH 11.5. From this study, it appeared that liming resulted in a much better microbiological quality of liquid sludge if its pH was maintained at 11.6 over 60 d or at pH 11.9 for 14 d. Solid sludge needed to be maintained at pH 11.5 for 14 d, pH 12.0 for 7 d or pH 12.5 for 24 h to achieve similar results.

Animals↗
PubMed1982

An analysis of sperm function in cases of unexplained infertility: conventional criteria, movement characteristics, and fertilizing capacity.

A detailed analysis of semen quality was carried out in 85 couples with unexplained infertility by the use of conventional criteria of semen analysis, time-exposure photomicrography, and the zona-free hamster egg penetration test. According to the latter, 34.1% of the male partners exhibited evidence of defective sperm function, although only 4% of these patients were devoid of any demonstrable fertilizing capacity. Of the conventional parameters of semen analysis examined, the most revealing was the morphologic character of the sperm, which was significantly poorer (P less than 0.001) in the group with unexplained infertility than in the normal fertile control group and also showed a significant relationship (P less than 0.001) with the presence of subnormal fertilizing capacity in the hamster egg assay. A majority of movement characteristics measured by time-exposure photomicrography were significantly depressed in the group with unexplained infertility, compared with the normal fertile control group. In addition, certain of the movement characteristics investigated were significantly related to the fertilizing capacity of the spermatozoa; and, in this respect, a progressive swimming speed (greater than 25 micrometers/sec), a straight swimming mode of progression, and a small amplitude of lateral head displacement (Ah) all appeared to be important qualities.

Animals↗
PubMed1984

Thawing optimums for bovine spermatozoa processed by three methods and packaged in Continental and French straws.

A modified two-dimensional central composite rotatable design was used to study the effect of thaw bath temperature and time on acrosomal integrity in Continental (.3 ml) and French (.5 ml) straws. Ejaculates from 18 bulls were packaged in both containers. Semen from 6 of the 18 bulls was processed by each of three methods: 6 bulls for egg yolk-Tris-glycerol, 6 bulls for egg yolk-citrate-glycerol, and 6 bulls for skim milk-glycerol. Thaw bath temperatures ranged from 18 to 92 degrees C, and thaw times ranged from 4.5 to 50 s. Interaction of thaw bath temperature with thaw time was detected within each processing procedure and package. Maximum cell quality, measured by intact acrosomes after incubation of semen 3 h at 37 degrees C for .3-ml and .5-ml straws, was predicted to be achieved by thaw bath temperatures and times (degrees C/s) as follow: egg yolk-Tris-glycerol 40/32, 49/16; egg yolk-citrate-glycerol 43/30, 37/19; skim milk-glycerol 50/24, 36/24. Thawing procedures to maintain best acrosomal retention, regardless of processing method, were 44 degrees C for 29 s for .3-ml straws and 40 degrees C for 20 s for .5-ml straws.

Acrosome↗
PubMed2001

[A new concept for integration of image databanks into a comprehensive patient documentation].

Image processing and archiving are of increasing importance in the practice of modern medicine. Particularly due to the introduction of computer-based investigation methods, physicians are dealing with a wide variety of analogue and digital picture archives. On the other hand, clinical information is stored in various text-based information systems without integration of image components. The link between such traditional medical databases and picture archives is a prerequisite for efficient data management as well as for continuous quality control and medical education. At the Department of Orthopedic Surgery, University of Berne, a software program was developed to create a complete multimedia electronic patient record. The client-server system contains all patients' data, questionnaire-based quality control, and a digital picture archive. Different interfaces guarantee the integration into the hospital's data network. This article describes our experiences in the development and introduction of a comprehensive image archiving system at a large orthopedic center.

Computer Systems↗
PubMed2006

Sperm competition in a fish with external fertilization: the contribution of sperm number, speed and length.

The role of sperm number and quality in male competitiveness was investigated using in vitro fertilization experiments with bluegill (Lepomis macrochirus). Bluegill males use one of three mating tactics: 'sneakers', which streak spawn; 'satellites', which mimic females; and 'parentals', which are territorial. The in vitro experiments mimicked natural spawning by incorporating these males' mean proximity to eggs and timing of sperm release. Using a maximum-likelihood algorithm, raffle equations were fit to paternity data, which revealed a strong effect of sperm number on male competitiveness. There was no difference in sperm flagellum length, curvilinear swim speed or path linearity among the three male mating types, and these traits did not explain any additional variation in male competitiveness. It was estimated that, given closer proximity to eggs, satellites need release only 0.34 times as many sperm as parentals to obtain equal paternity. Despite being farther from the eggs and releasing sperm about half a second after parentals, sneakers need only release 0.58 times as many sperm as parentals to obtain equal paternity. Thus, the increased competitiveness of sneakers' sperm must come from a component of sperm quality other than speed or length.

Animals↗
PubMed1994

Cadmium toxicity to rainbow trout Oncorhynchus mykiss Walbaum and brown trout Salmo trutta L. over extended exposure periods.

The toxicity of cadmium in water is well known, but there have been few reports on the long-term effects of this metal on fish at concentrations near the European inland water standard of 5 micrograms Cd liter-1. This paper describes experiments in which adult rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta were exposed to cadmium concentrations near water quality standard levels for periods of up to 90 weeks. The survival and growth of these fish were assessed, and sperm and eggs were stripped from them to conduct early-life-stage tests. Continuous exposure of rainbow trout adults to cadmium concentrations of up to 5.5 micrograms Cd liter-1 did not affect their survival or growth. However, eggs obtained from rainbow trout exposed to 1.8 and 3.4 micrograms Cd liter-1 failed to develop to the fry stage. Oogenesis appeared to be delayed in brown trout exposed to 9.3 and 29.1 micrograms Cd liter-1, but the eggs and fry that were produced developed normally after fertilization. Adult brown trout suffered considerable mortality in the 29.1 micrograms Cd liter-1 treatment, with a median period of survival of 54 weeks.

Animals↗
PubMed1993

The use of MDCK, MEK and LLC-MK2 cell lines with enzyme immunoassay for the isolation of influenza and parainfluenza viruses from clinical specimens.

Primary Monkey Kidney (PMK) epithelial cells or egg inoculation have been traditionally used for the culture of influenza and parainfluenza viruses. The high cost and variability of obtaining high quality PMK cells prompted us to investigate the use of other cell strains for the growth of these viruses. For this study we investigated three cell lines viz. MDCK, MEK and LLC-MK2 for the culture of influenza A and B and parainfluenza 1, 2 and 3 viruses. Clinical specimens were spun onto cell monolayers in microtitre wells. The growth of these viruses was then identified by specific antibodies in an enzyme immunoassay (EIA). The LLC-MK2 and MDCK cell lines were found to provide optimal growth of parainfluenza and influenza viruses respectively. During the period from November, 1990 to July, 1992, 6501 respiratory specimens were tested. There were 100 influenza A, 36 influenza B and 261 parainfluenza virus isolates. The influenza isolates were further subtyped by the WHO Influenza Reference Centre. The use of these cell lines and the EIA provided an effective method for the routine culture of these viruses.

Animals↗
PubMed2001

Evaluation of validity of items for a food behavior checklist.

OBJECTIVES: To evaluate the validity of food behavior items, using a biological measure (serum carotenoids) as the criterion for validity of fruit and vegetable intake, and the results from multiple 24-hour recalls to test convergent validity with nutrient intake. DESIGN: Participants responded to 39 food-behavior questions and later completed three 1-day dietary recalls. Serum carotenoid levels were determined for a 59% randomly selected subsample. SUBJECTS/SETTING: A convenience sample of 100 English-speaking, low-income women participating in a Food Stamp Nutrition Education Program in 8 California counties. Statistical analyses Spearman correlation coefficients were calculated between responses to the food behavior items and (a) serum carotenoid levels and (b) mean nutrient intakes from the 24-hour recalls. Cronbach's coefficient alpha was determined for items within broad food behavior topics. RESULTS: Responses to 10 food behavior items were significantly correlated with serum carotenoid levels (correlations greater than 0.45 were found for choosing low-fat foods and a self-evaluation of overall dietary quality). An additional 12 items showed hypothesized associations with the 24-hour recall data (with a maximum correlation 0.50 for number of eggs per week and dietary cholesterol). Cronbach's coefficient alpha ranged from 0.28 (for 5 fat and cholesterol items) to 0.79 (for 9 fruit and vegetable items). APPLICATIONS/CONCLUSIONS: Nutrition professionals can use these methods to validate items for food behavior checklists for specific populations. The items described here may be useful when designing instruments to administer to low-income women in a community setting.

Adult↗
PubMed1982

The correlates of fertilizing capacity in normal fertile men.

An investigation has been carried out in normal fertile men on the correlates of fertilizing capacity as defined by the zona-free hamster egg penetration test. The results demonstrate that the apparent fertility of these men is compatible with a wide range of intrinsic sperm quality as reflected by penetration rates ranging from 14% to 90% and differences in the minimum concentration of motile spermatozoa required to initiate penetration. These differences in sperm function could not be correlated with any of the conventional parameters of semen analysis or any of a large number of sperm movement characteristics analyzed by time-exposure photomicrography, with two exceptions: (1) the percentage of progressively (greater than 25 micron/sec) motile spermatozoa exhibiting an amplitude of lateral head displacement (Ah) of less than 10 micron, which was positively correlated with penetrating capacity (P less than 0.01), and (2) the percentage of progressively (less than 25 micron/sec) motile spermatozoa exhibiting an Ah of greater than 10 micron, which was negatively correlated with penetration rate (P less than 0.05). The information obtained in this study should provide a useful basis against which to compare the properties of spermatozoa in cases of suspected infertility.

Adult↗