Use of a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly in a staining or sorting process

A composition and cell technology, applied in the field of sorting and staining sperm cells, can solve the problems of reduced forward motility and the like

Active Publication Date: 2012-07-04
INGURAN LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Every step of the staining and sorting process is stressful for sperm cells, resulting in reduced sperm cell viability or motility, especially forward motility

Method used

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  • Use of a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly in a staining or sorting process
  • Use of a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly in a staining or sorting process
  • Use of a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly in a staining or sorting process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Bull semen was collected from sexually mature bulls using an artificial vagina, and the samples were diluted with 2 parts of carbonate buffer and transferred to the staining facility in a temperature-controlled container at 25°C. Once received, the semen was analyzed for concentration, motility, and motility using the Hamilton-Thorn Motility Analyzer (IVOS) according to standard and well-known methods (Farrell et al., Theriogenology, 49(4):871-9 (March 1998)). sex and forward motility. Depending on the semen concentration, pipette a small aliquot of the carbonated sperm suspension, centrifuge it at 500Xg for 5 minutes, remove the supernatant and resuspend the pellet in TCA buffer, pH 7.3 at 41°C, to prepare 1 mL of 150×10 6 Sperm / ml of suspension. An additional 1 mL of 150 × 10 6 Sperm / ml suspension. Aliquots of 10 mM Hoechst in water were added to the sperm suspension to give a dye concentration of 400 μM Hoechst. The sperm suspension was left in a 41°C water bath ...

Embodiment 2

[0093] Sperm samples were obtained and prepared in the same manner as in Example 1 except for the following differences: the buffers used to suspend sperm for staining and IVOS analysis were TCA and TCA containing 10 μM vitamin K. The results of the IVOS analysis are summarized in figure 2 .

Embodiment 3

[0095] Sperm samples were obtained and prepared in the same manner as in Example 1 except for the following differences: the buffers used to suspend sperm for staining and IVOS analysis were TCA and TCA containing 100 μM vitamin K. The results of the IVOS analysis are summarized in image 3 .

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Abstract

The present invention provides staining mixtures comprising viable spermatozoa, a composition which regulates oxidation / reduction reactions intracellularly or extracellularly, and a DNA selective dye are disclosed. The cells contained in such suspensions tend to have a greater capacity for enduring the various process steps typically associated with the sorting of sperm cells into gender enrichedpopulations, thereby resulting in post-sort compositions with an increased number of viable or motile sperm. Processes for staining sperm cells comprising the formation of a staining mixture are alsodisclosed.

Description

field of invention [0001] The present invention generally relates to methods of sorting stained spermatids. More specifically, the present invention relates to a method of sorting sperm cells, wherein a sperm cell suspension is formed containing a composition that modulates intracellular and / or extracellular oxidation / reduction reactions. Herein, sperm refers to non-human sperm. Background of the invention [0002] Fertilization of animals by artificial insemination (AI) and embryo transfer after in vitro fertilization is a well-established technology. In the livestock industry, there are clear advantages in being able to influence reproductive outcome so that offspring possess one or more desired characteristics. For example, in the dairy industry, if the offspring can be pre-selected in favor of females and the production of cows can be guaranteed, it will bring economic benefits. Separating sperm into enriched cell populations with X and Y chromosomes, the so-called se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/50C12N5/06C12N5/00C12N5/071G01N1/30G01N33/48
CPCC12N5/0612G01N1/30
Inventor C·L·路德维格J·A·格雷厄姆K·S·克劳利M·安扎尔
Owner INGURAN LLC
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