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Biomedical subjects

R N Clarke

Publications and source records attributed to R N Clarke.

At least 37 records · Page 2Linked to original sources
PubMed1989

Development of a noninvasive ultramicrofluorometric method for measuring net uptake of glutamine by single preimplantation mouse embryos.

A noninvasive ultramicrofluorometric method for measuring net uptake of glutamine by single preimplantation mouse embryos is described. A linear relationship was found between fluorescence intensity of NADH produced and glutamine concentration (R2 = 0.985). Single embryos were placed in 20 nl drops of medium containing 0.5 mM glutamine, and the medium was sampled after 2 hr incubation at 37 degrees C. Changes in net glutamine uptake were determined in one-cell, two-cell, and eight-cell embryos and blastocysts incubated in medium containing no energy substrates (glucose, pyruvate, and lactate). The median glutamine uptake increased significantly from 0.480 and 0.270 pmoles/embryo/2 hr at the one-cell and two-cell stages, respectively, to 1.610 pmoles/embryo/2 hr at the blastocyst stage. Mean glutamine uptake was compared in the presence or absence of energy substrates at several developmental stages. A highly significant reduction of glutamine uptake in the presence of substrates was observed at the one-cell and two-cell stages of development. At the eight-cell stage, glutamine uptake was only marginally reduced in the presence of substrates, and no effect was found at the blastocyst stage. These data may partially explain the beneficial effect of glutamine on the culture of early mouse embryos through the two-cell block of development.

Animals↗
PubMed1989

Use of ultramicrofluorometric methods for the study of single preimplantation embryos.

Ultramicrofluorescence and video fluorescence microscopy are highly sensitive techniques for studying embryo metabolism and developmental potential. These techniques are attractive in that they allow one to study single embryos and thus to detect more subtle changes in embryo metabolism or intracellular pH which, using less sensitive techniques or batches of embryos, would likely go undetected. This is especially important in those species where few embryos can be recovered from a single mother. An additional advantage of ultramicrofluorescence techniques lies in their lack of invasiveness to the embryo under study. When measuring nutrient uptake, several measurements can be made on a single embryo that can then be placed back into culture for evaluation of its developmental potential. Alternatively, test embryos can be transferred back into a recipient for determination of embryo viability. This flexibility further illustrates the beneficial aspects of using ultramicrofluorescence techniques for studying embryo metabolism and development.

Animals↗
PubMed1988

Flow sorting of X and Y chromosome-bearing mammalian sperm: activation and pronuclear development of sorted bull, boar, and ram sperm microinjected into hamster oocytes.

Flow cytometric techniques were used to measure relative DNA content of X and Y chromosome-bearing bull, boar, and ram sperm populations and to separate the two sex-determining populations. Neat semen was prepared for flow cytometric analysis by washing, light sonication, and staining with 9 microM Hoechst 33342. Computer analysis of the bimodal histograms showed mean X-Y DNA differences of 3.9, 3.7, and 4.2% for bull, boar, and ram, respectively. Flow cytometric reanalysis of sorted bull, boar, and ram sperm showed purities greater than 90%. Bull, boar, and ram sperm nuclei were microinjected into hamster oocytes. Microinjected sperm were either unsorted, sorted, unsorted plus dithiothreitol (DTT) exposure, or sorted plus DTT exposure. Following microinjection, eggs were incubated 3 hr, fixed, and stained. A total of 579 eggs was observed for sperm activation (decondensation or formation of a male pronucleus). A lower percentage of sorted than unsorted (3 vs. 23%) boar sperm was activated (P less than .05). However, sorted and unsorted DTT-exposed boar sperm or sorted and unsorted bull or ram sperm, regardless of DTT treatment, did not differ significantly. Sorted sperm nuclei of both rams and bulls exhibited higher activation rates than sorted boar sperm (P less than .05). Treatment of sperm with DTT increased the activation rate (P less than .05) for sorted boar sperm but not for bull or ram sperm.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Factors related to successful sperm microinjection of hamster eggs: The effect of sperm species, technical experience, needle dimensions, and incubation medium on egg viability and sperm decondensation following microinjection.

A series of experiments was designed to identify factors in a sperm microinjection system that could influence egg viability and decondensation of sperm nuclei after microinjection. Egg viability and sperm decondensation rates were not different among eggs microinjected with rodent sperm. The microinjection of ram sperm required a larger diameter needle for injection, which resulted in low egg viability and sperm decondensation in the first 3 mo of the study but improved greatly after 9 mo of technical experience. The degree of technical experience (3 vs 9 mo) also improved (P<0.05) egg viability after microinjection with rodent sperm; however, the rate of sperm decondensation remained unaffected. Altering the dimensions of the injection needle from a tapered needle barrel to a more uniform needle barrel increased egg viability from 61 to 96% and sperm decondensation from 3 to 27%. The use of medium 199 for incubating microinjected eggs further increased (P<0.05) the percentage of eggs containing decondensed sperm nuclei (52%) compared to eggs incubated in Holmes defined medium (28%). By altering the dimensions of the injection needle, by selecting an appropriate incubation medium, and by gaining technical experience in microinjection, the efficiency of a sperm microinjection system was improved for both rodents and domestic animals.

Journal Article↗
PubMed1987

Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro.

The effect of liquid storage and cryopreservation of boar spermatozoa on sperm motility, acrosomal integrity, and the penetration of zona-free hamster (ZFH) ova was examined. The sperm penetration assay (SPA) provides valuable information on specific events of fertilization and is a potentially useful indicator of sperm fertility. Ejaculated semen from 4 boars was subjected to 3 treatments: fresh (FRE, no storage), liquid-stored (LIS, stored at 18 degrees C for 3 days), and frozen (FRO, frozen by pellet method and stored at -196 degrees C for 3 days). A highly motile sperm population was isolated by the swim-up procedure (1 hr). FRE and LIS were incubated an additional 3 hr at 39 degrees C in a Tris-buffered medium to elicit capacitation and the acrosome reaction. Sperm motility and acrosomal integrity were assessed before and after incubation. For the SPA, sperm and eggs were incubated at 39 degrees C for 3 hr in Hams F-10 medium. Each egg was assessed for sperm penetration, sperm binding, and stage of development. Percentages of sperm motility and sperm with a normal apical ridge (NAR) prior to incubation were 78 and 78 (FRE), 75 and 69 (LIS), and 28 and 50 (FRO). After incubation, percentages of motility, NAR, and acrosome-reacted sperm were 34, 10, and 73 (FRE); 43, 24, and 51 (LIS); and 18, 13, and 59 (FRO). A somewhat higher (P less than .05) percentage of ZFH ova was penetrated by FRE (45.8) than by LIS (42.0). Penetration of ZFH ova by FRO was markedly (P less than .05) reduced (30.2). Sperm penetration was not significantly correlated with motility or acrosomal integrity before or after incubation, regardless of treatment. These data suggest that the SPA can be used in conjunction with conventional measures of semen analysis in assessing the potential fertilizing capacity of boar sperm and that liquid storage is superior to frozen storage with respect to preserving sperm fertility.

Acrosome↗
PubMed1987

Age-related changes in testicular development and reproductive endocrinology associated with aflatoxicosis in the male chicken.

A study was conducted to investigate endocrine and testicular changes in the male chicken associated with the ingestion of 10 or 20 ppm aflatoxin at 3 different stages of development. Weekly body weight gain, absolute and relative combined testes weights, and plasma testosterone concentrations were reduced in aflatoxin-fed males as compared to controls, with the greatest differences seen at 12 wk of age. The effect of dietary aflatoxin on levels of plasma luteinizing hormone (LH) was dependent on age at exposure. Concentrations of plasma LH in 6-wk-old control males were significantly higher than in aflatoxin-fed birds, whereas no treatment differences were observed in older males. Additionally, few changes were observed in static levels of monoamines in any of the brain regions assayed, regardless of age at exposure. The delay in peak levels of LH, as well as the suppression of plasma testosterone and testicular weight, indicate a possible delay in the onset of sexual maturation associated with aflatoxicosis in this species.

Aflatoxins↗
PubMed1987

The response of the anterior pituitary and testes to synthetic luteinizing hormone-releasing hormone (LHRH) and the effect of castration on pituitary responsiveness in the maturing chicken fed aflatoxin.

The responsiveness of the anterior pituitary to exogenous luteinizing hormone-releasing hormone (LHRH; 20 micrograms/kg body weight) and the subsequent stimulation of testosterone secretion by the testes was studied after administration of dietary aflatoxin (10 ppm) to 9-wk-old male chickens. In both control and aflatoxin-treated males, there were significant (p less than 0.05) increases in plasma luteinizing hormone (LH) concentrations following LHRH administration, which peaked at 5 min post injection and declined thereafter. Plasma testosterone levels increased soon after the LHRH injection in control males, secondary to elevated LH levels in the peripheral circulation, and continued to increase throughout the experimental period. In contrast, this LH-induced elevation in plasma testosterone was delayed in aflatoxin-treated males, with no substantial increase until 20 min post-LHRH injection. In a subsequent experiment, castration of aflatoxin-fed males resulted in an altered response to exogenous LHRH, as compared to their intact counterparts. Based on these data, it appeared that while the LH-secretory capacity of the anterior pituitary was not diminished in birds receiving aflatoxin, the testicular response to exogenous LHRH was altered during aflatoxicosis. Additionally, the effect of castration on plasma LH profiles after LHRH administration provides preliminary evidence for extra-testicular effects of dietary aflatoxin on reproduction in the avian male.

Aflatoxins↗
PubMed1987

Health care marketing: lots of talk, any action?

Although health care managers pay a great deal of attention to marketing, there appears to be limited substantive action. As more organizations successfully implement the marketing concept, other organizations will follow suit and marketing will become more prevalent throughout the health care industry.

Administrative Personnel↗
PubMed1986

Depression of plasma luteinizing hormone concentration in quail by the anticholinesterase insecticide parathion.

To examine the effects of parathion on basal plasma luteinizing hormone (LH) concentration, male Japanese quail (Coturnix japonica) were orally intubated with 0, 5 or 10 mg/kg parathion and sacrificed after 4, 8 and 24 hr. At the 5 mg/kg dose, plasma LH levels were reduced at 4 and 8 hr, but returned to control values by 24 hr. Brain acetylcholinesterase activity was substantially reduced by 10 mg/kg parathion (52, 75 and 37% inhibition at 4, 8 and 24 hr, respectively) and plasma LH concentration remained depressed through the 24-hr period. These findings suggest that the organophosphorus insecticide parathion may alter plasma LH concentration in a manner which might impair reproductive activity, and provide indirect evidence for a cholinergic component in the regulation of LH secretion in quail.

Acetylcholinesterase↗
PubMed1986

Comparison of monoamine concentrations in the brains of adult male and female Japanese quail.

A fluorometric assay measuring brain tissue concentrations of norepinephrine, dopamine, and serotonin has been validated for Japanese quail. Accuracy, precision, specificity, and parallelism were determined. The sensitivity of the assays was 6 ng/tube, which allowed individual assay of 1 to 2 mg hypothalamic tissue. In Experiment 1, relatively large areas of brain from adult, reproductively active males and females were found to differ significantly in norepinephrine content in optic lobes and for dopamine in right telencephalon. A microdissection technique was used in Experiment 2 to sample small portions of hypothalamic tissue. Sex differences were observed for norepinephrine in the sections containing the lobus paraolfactorius and the preoptic, anterior, and medial hypothalamus. Differences in monoamine content were most apparent when smaller areas dissected by microdissection were analyzed. These results give evidence for sex differences in the monoamine content in specific areas of the brain.

Animals↗
PubMed1986

Relative importance of dietary aflatoxin and feed restriction on reproductive changes associated with aflatoxicosis in the maturing White Leghorn male.

A study was conducted to investigate the relative contribution of dietary aflatoxin and feed restriction on reproductive and endocrine changes associated with aflatoxicosis. Pair-feeding was used to factor out nutritional and toxicological effects of aflatoxin on reproduction in the maturing White Leghorn male. Birds were randomly assigned to treatment groups at 9 weeks and fed experimental diets for 3 weeks. Experiment 1 involved the partitioning of toxicological and nutritional effects of aflatoxin on both physiological and reproductive parameters. Aflatoxin-fed males had significantly greater relative liver weights and liver lipid content than control or pair-fed males; no differences in testicular weights were found among treatments. Plasma testosterone levels in controls were significantly higher than in pair-fed birds; aflatoxin-treated males exhibited the lowest concentrations. Pituitary and testicular responsiveness to synthetic luteinizing hormone-releasing hormone (LHRH) were monitored in Experiment 2. Plasma luteinizing hormone (LH) levels in aflatoxin-fed males were significantly higher than in control or pair-fed males prior to LHRH administration. Additionally, the LH response of pair-fed birds to exogenous LHRH was qualitatively different from that observed in the other two treatments; this indicates that hormonal changes observed during aflatoxicosis and feed restriction are a result of different physiological mechanisms. Plasma testosterone also increased after LHRH injection. This response was significantly greater in controls than in pair-fed males, and nonexistent in aflatoxin-treated birds.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Morphological changes in chicken and turkey spermatozoa incubated under various conditions.

The viability and morphology of undiluted and diluted chicken and turkey spermatozoa were compared when incubated at either 41, 25, 15, or 5 C for 0 (control), 3, or 6 hr. Increasing the incubation time to 6 hr, raising the incubation temperature to 41 C, or both resulted in higher numbers of dead spermatozoa in semen from both species. Conversely, dilution (1:5 with Beltsville Poultry Semen Extender) of chicken or turkey semen resulted in a significant (P less than .05) decrease in the number of dead spermatozoa as compared to their respective undiluted counterparts, especially in samples incubated at 41 C. Turkey semen exhibited a somewhat lower percentage of dead spermatozoa, bent spermatozoa, and sperm tail coiling than did chicken semen incubated under similar conditions. No significant (P greater than .05) differences were observed in chicken and turkey semen samples when measuring other sperm cell abnormalities regardless of incubation conditions. These results indicate selective species differences in sperm morphological changes in response to incubation temperature, incubation time, and semen dilution.

Animals↗
PubMed1983

Strategies for a crowded marketplace.

Today's crowded marketplace demands that health care organizations use sophisticated marketing techniques. One technique is for the organization to differentiate itself in order to attract clients away from competing organizations. Another is for the organization to focus its resources on a limited number of market segments with the understanding that it will not devote resources to segments that have not been specifically designated as target markets.

Consumer Behavior↗
PubMed1982

Effects of holding temperature and storage time on respiratory rate, motility, and fertility of chicken and turkey semen.

A series of experiments was conducted to measure the respiratory activity, motility, and fertility of chicken and turkey sperm in undiluted and diluted (1 part semen to 5 parts Beltsville Poultry Semen Extender) semen held at either 41, 25, 15, or 5 C for 3 or 6 hr. Sperm respiration was temperature dependent, increasing with increasing temperature. When incubated under identical conditions, the respiratory rate of spermatozoa in diluted semen of both species was significantly (P less than .05) higher than in undiluted semen. In undiluted and diluted chicken and turkey semen, sperm mortality was lowest at 41 C. No differences in the motility of spermatozoa from undiluted and diluted semen of either species were observed in the unstored controls. Sperm motility in samples held at 15 or 5 C was similar to that of the unstored controls. The fertility of diluted chicken semen was highest (75%) after 6 hr of storage at 5 C. Fertility levels (71 to 83%) of chicken semen was highest (75%) after 6 hr of storage at 5 C. Fertility levels (71 to 83%) of chicken semen held for 3 hr at 25, 15, or 5 C did not differ from the unstored control. In contrast, the fertilizing capacity of diluted turkey semen (0 to 45%) was significantly lower than the unstored controls (68%), regardless of holding times and temperatures. The fertility of undiluted turkey semen was significantly (P less than .05) lower than the unstored control in all cases, with the exception of semen held at 25 C for 3 hr.

Animals↗