Sex-sorted sperm exhibiting a dose response and methods of producing sex-sorted sperm exhibiting a dose response

A dose-response, sperm technology, used in sexual diseases, germ cells, animal cells, etc., can solve the problem that sperm cannot fully compensate for low energy

Active Publication Date: 2020-04-24
INGURAN LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Further, the increased number of sex-sorted sperm used in the AI ​​cannot fully compensate for the low insemination effort

Method used

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  • Sex-sorted sperm exhibiting a dose response and methods of producing sex-sorted sperm exhibiting a dose response
  • Sex-sorted sperm exhibiting a dose response and methods of producing sex-sorted sperm exhibiting a dose response
  • Sex-sorted sperm exhibiting a dose response and methods of producing sex-sorted sperm exhibiting a dose response

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] collect - Sperm were collected from five different bulls using artificial vaginas according to a routine collection protocol. Each bull was collected two or three times a day. Of the five bulls, two are Jersey bulls and three are Holstein bulls. All ejaculates contained greater than 60% progressive motility, with sperm concentrations ranging from 875 million sperm / ml to 2,480 million sperm / ml. Ejaculates collected from the same bull were pooled and then divided into nine sperm samples for collection and staining.

[0073] Sperm Processing and Staining - Sections of each bull ejaculate were treated and dyed in nine different ways, each of which is described below.

[0074] (A) Control (no normalization, two-step staining) - A control was established that did not include the step of normalizing the collected ejaculate and in which spermatozoa were stained in two steps. Prior to staining, the sperm samples were concentrated to between 1.700 million sperm / ml and 1.80...

Embodiment 2

[0092] collect - Sperm were collected from six different Jersey bulls using artificial vaginas according to a routine collection protocol. All ejaculates contained greater than 65% forward motility, with sperm concentrations ranging from 765 to 1,710 million sperm / ml. Each sperm sample was divided into two portions in 15 mL tubes for two collections and staining processes. pH measurements were taken at collection and at each subsequent processing step.

[0093] Sperm Processing and Staining - For comparison, portions of each bull ejaculate were treated and dyed by two methods.

[0094] Control (no normalization, two-step staining) - A control was created that did not include the step of normalizing the collected ejaculate and in which sperm were stained in two steps. Prior to staining, the sperm samples were concentrated to between 1.700 million sperm / ml and 1.800 million sperm / ml by centrifugation or by addition of tris-egg yolk dilution at pH 6.8, depending on the samp...

Embodiment 3

[0105] collect - Sperm were collected from three different Jersey bulls and three different Holstein bulls according to a routine collection protocol for a total of 17 collections. Each ejecta was split for the two treatments.

[0106] Sperm Processing and Staining - For comparison, portions of each bull ejaculate were treated and dyed by two methods.

[0107] Control (no normalization, two-step staining) - A control was created that did not include the step of normalizing the collected ejaculate and in which sperm were stained in two steps. Sperm in the control group were diluted to 160 × 10 in the modified TALP buffer as described in Table 1 at pH 7.4. 6 sperm / ml. Each sperm sample in the control group was then incubated with 16-17 μL Hoechst 33342 / ml (64-68 μM) of sample for 45 minutes at 34°C. After incubation, an equal volume of the second modified TALP was added, reducing the concentration to 80×10 6 sperm / ml. A second modified TALP included the ingredients desc...

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Abstract

Embodiments of the claimed invention relate to sex sorted sperm having an improved dose response which may be produced by the steps of extending a sperm sample with a buffering holding media and adjusting the concentration of the extended sperm sample to a target concentration range. The extended sperm may then be stained with a DNA selective dye and sex sorted with a flow cytometer into a catch fluid. During the extending and sorting of the sperm sample, the pH may be maintained at a target pH. Additionally, at least one of the buffering holding media, the DNA selective dye and the catch fluid may be supplemented with at least one antioxidant.

Description

technical field [0001] In general, the present technology relates to staining and sorting methods, such as those employed for sorting sperm, and more particularly to sperm sorting methods and flow cytometry methods, which improve the efficiency and Recovery, and improved dose-response in sex-sorted spermatozoa. Background technique [0002] The most widely used sperm sorting methods rely on detecting quantifiable differences in the DNA content of X-chromosome bearing and Y-chromosome bearing sperm. Various modifications of flow cytometers for this purpose are described in US Pat. In many species, differences in DNA content may be small. For example, among cattle, Holstein bulls have about 3.8% difference in DNA content, while Jersey bulls have about 4.1% difference. The imprecise nature of stoichiometric DNA staining makes determination of such small differences difficult and requires robust staining protocols. [0003] Although the fluorescent dye Hoechst 33342 is suita...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/076
CPCA61P15/08C12N5/0612
Inventor R·维什瓦纳特K·M·埃文斯T·B·吉利根J·莫雷诺R·楞次C·冈萨雷斯-马琳
Owner INGURAN LLC
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