Flow microfluorometric analysis of living spermatozoa stained with Hoechst 33342

Keeler, KD; Mackenzie, N.M.; Dresser, D. W.

doi:10.1530/jrf.0.0680205

Cited by 15 publications

(13 citation statements)

References 12 publications

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“…This two-dimensional analysis technique was first implemented on Ortho Cytofluorograph machines and was used by Johnson et al (1989) for the analysis and sorting of mammalian spermatozoa. Our implementation of the technology has enabled us to make detailed analyses of spermatozoa and confirm some of our earlier conclusions (Keeler et al, 1983;Morrell et al, 1988). It is clear that there is a considerable and largely uncontrollable variation in the results of analyses of bull spermatozoa which stem from factors both within and between individuals of the same breed.…”

Section: Discussionsupporting

confidence: 60%

“…For a limited number of experiments, described in the results section, a similar modifi¬ cation was made to the Coulter Epics V by grinding one of the standard sample injection needles to a 20°(from the long axis of the needle) chisel shape. The effectiveness of this modification was verified by a standard forward angle scatter analysis of glutaraldehyde fixed turkey erythrocytes as described by Keeler et al (1983). The injection needle could be rotated so that the ribbon was at any desired position between 0°and 90°with respect to the laser beam.…”

Section: Flow Cytometrymentioning

confidence: 99%

“…Subsequently, less accurate but biologically more relevant measurements were made of viable motile spermatozoa stained with bis-benzimidazole dyes such as Hoechst 33258 and 33342 (H.33258; H.33342) (Keeler, et al, 1983). Live spermatozoa stained with H.33342 were found to have a major low fluorescent peak with a narrowly separated bimodal distribution: the peak with the greater fluorescence was assumed to represent X-bearing spermatozoa, the lower peak Y-bearing spermatozoa.…”

Section: Introductionmentioning

confidence: 99%

“…Introduction Accurate estimates of the DNA content of fixed spermatozoa stained with fluorochromes such as ethidium bromide, mithramycin or acridine orange, using flow cytometric techniques, were first made over a decade ago using fixed spermatozoa (Van Dilla et al, 1977;Gledhill et al, 1979). Subsequently, less accurate but biologically more relevant measurements were made of viable motile spermatozoa stained with bis-benzimidazole dyes such as Hoechst 33258 and 33342 (H.33258; H.33342) (Keeler, et al, 1983). Live spermatozoa stained with H.33342 were found to have a major low fluorescent peak with a narrowly separated bimodal distribution: the peak with the greater fluorescence was assumed to represent X-bearing spermatozoa, the lower peak Y-bearing spermatozoa.…”

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confidence: 99%

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Analyses of DNA content of living spermatozoa using flow cytometry techniques

Dresser¹,

Atkins²,

Pinder³

et al. 1993

Reproduction

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A two-dimensional fluorescence analysis of spermatozoa stained with Hoechst 33342 was carried out using an Epics V flow cytometer. This analysis involved the measurement of fluorescence both in the conventional manner at 90 degrees and at a narrow forward angle (nominally 0 degree) to the direction of the interrogating laser beam. The arrangement provided an optical means of distinguishing the orientation of flattened particles passing through the interrogation system and enabled the verification and reinterpretation of one-dimensional (90 degree) fluorescence histogram data obtained previously. We now consider that the distinction between live and dead spermatozoa was over simplified and that the observed differences are due partly to an artefact of orientation. It has been confirmed that our earlier use of the low fluorescence peak, consisting of (bull, rabbit, sheep and pig) spermatozoa passing through the interrogation point obliquely to both detectors, as criterion for sorting X- from Y-bearing spermatozoa, is more practical than the use of a very much smaller subpopulation, based on limits imposed by selecting only spermatozoa accurately orientated with their narrow edges to one detector, while measurement of total fluorescence (DNA content) was made simultaneously from a broad face by the other detector. We report marked differences in the rate of staining and in attainment of a staining equilibrium between spermatozoa from different animals or from different ejaculates collected on the same or different days from the same animal. This variation introduces an element of subjectivity into the use of flow cytometry for sorting X- and Y-spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionsupporting

confidence: 60%

Section: Flow Cytometrymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Analyses of DNA content of living spermatozoa using flow cytometry techniques

Dresser¹,

Atkins²,

Pinder³

et al. 1993

Reproduction

Self Cite

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“…Although sperm sexing would not allow gender selection of poultry (where the female is the heterogametic sex), it would be effective for mammals and has therefore received a lot of attention in the last few decades. The only method of sexing spermatozoa, which has been shown to work reliably so far, is the flow sorting of spermatozoa [21,22] stained with the vital dye, H33342 [23]. However, the process of sorting sufficient sperm numbers for an insemination dose takes a long time, since the stained spermatozoa must pass individually through a laser beam for detection of their DNA content.…”

Section: Sperm Sexingmentioning

confidence: 99%

Animal Reproduction Technologies—Future Perspectives

Morrell¹,

Humblot²

2016

JAST-A

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Abstract:Reproductive biotechnologies, such as artificial insemination, sperm selection and embryo technologies, offer possibilities for animal producers to increase reproductive efficiency. There have been many significant developments in reproductive biotechnologies over the last few decades, e.g., in sperm handling and preservation, in vitro embryo production and preservation, sexing and cloning. This review discusses some of the key changes that have occurred and explores their potential for increasing the reproductive efficiency of domestic animals in the future. As a consequence, they also offer opportunities to facilitate or accelerate genetic selection. If properly used, they may contribute to increase the sustainability of animal production. The role of epigenetics in influencing phenotype is also considered.

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Sexing sperm of domestic animals

Espinosa-Cervantes

Córdova-Izquierdo

2012

Trop Anim Health Prod

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The ability to preselect or predetermine the sex of offspring prior to conception is a highly desired technological tool for assisted female breeding programs specifically for milk production, and in males, for meat production and increasing livestock numbers. The current technology is based on the well-known differences in X- and Y-sperm in the amount of DNA. The technology uses modified flow cytometric instrumentation for sorting X- and Y-bearing sperm. The method can be validated on the basis of live births, laboratory reanalysis of sorted sperm for DNA content, and embryo biopsy for sex determination. Currently, the sex of animals has been predetermined with 90 % accuracy by sexing spermatozoa. In the bovine breeding industry, flow cytometric sperm sexing has not fulfilled its original promise. Sexed sperm doses are too expensive for widespread application while the fertility of sexed sperm doses is lower than unsexed ones. Essentially all bovine sexed semen is frozen and then applied through artificial insemination (AI) or in vitro fertilization. There is still a need in the animal breeding industry to develop a technique for sperm sexing that provides sufficient spermatozoa for AI doses, does not compromise sperm fertility, and is widely applicable to a range of species. In this review, we will summarize the current state-of-the-art in sex preselection in domestic animals and some wildlife species using flow cytometric sperm-sorting of X from Y sperm based on DNA differences.

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Flow microfluorometric analysis of living spermatozoa stained with Hoechst 33342

Cited by 15 publications

References 12 publications

Analyses of DNA content of living spermatozoa using flow cytometry techniques

Analyses of DNA content of living spermatozoa using flow cytometry techniques

Animal Reproduction Technologies—Future Perspectives

Sexing sperm of domestic animals

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