US-20260125641-A1 - METHOD FOR IMPROVED SPERM CELL POPULATIONS

US20260125641A1US 20260125641 A1US20260125641 A1US 20260125641A1US-20260125641-A1

Abstract

The invention encompasses methods for reducing the proportion of sperm cells with abnormal morphology, and unviable sperm cells, in a sperm cell population using flow-cytometric processing. The methods generally entail staining the sperm cells in the population; irradiating the sperm cells in the population; detecting fluorescence emitted by the sperm cells in the population in response to the step of irradiating; differentiating the sperm cells in the population based on orientation or viability; and then collecting a subpopulation of oriented or viable sperm cells.

Inventors

Juan Moreno
Kenneth Michael Evans
Ramakrishnan Vishwanath
Clara Gonzalez-Marin

Assignees

INGURAN, LLC

Dates

Publication Date: 20260507
Application Date: 20250714

Claims (6)

1 . A method of processing sperm cells by flow cytometry comprising a) selecting a population of sperm cells, wherein 25% or more of sperm cells in the population have abnormal morphology; b) staining the sperm cells in the population; c) placing the sperm cells in the population in sheath fluid; d) subjecting the sperm cells in the population to an orienting force; e) irradiating the sperm cells in the population; f) detecting side fluorescence emitted by the sperm cells in the population in response to the step of irradiating; e) differentiating the sperm cells in the population having abnormal morphology from the sperm cells in the population having normal morphology based on their orientation; and f) collecting a subpopulation of oriented sperm cells, wherein the percentage of sperm cells in the collected subpopulation having abnormal morphology is less than the percentage of sperm cells in the population having abnormal morphology in step a).
2 . The method of claim 1 , further comprising the step of staining the population of sperm cells with a quenching dye prior to step c).
3 . The method of claim 1 , wherein in step b) the sperm cells are stained with a DNA-selective dye.
4 . The method of claim 3 , wherein the DNA-selective dye is Hoechst 33342.
5 . The method of claim 1 , wherein the step of differentiating the sperm cells in the population further comprises creating a gate based on the detected fluorescence emitted by the sperm cells in the population, wherein the gate encompasses oriented sperm cells.
6 . The method of claim 1 , further comprising the step of treating the population with magnetic particles prior to step c).

Description

REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 18/134,451 filed Apr. 13, 2023, which is a Continuation of U.S. application Ser. No. 16/823,001 filed Mar. 18, 2020, now U.S. Pat. No. 11,629,330 issued Apr. 18, 2023, which, claims the benefit of U.S. Provisional Patent Application No. 62/820,724 filed Mar. 19, 2019. The entire disclosure of which is incorporated herein by reference. BACKGROUND OF THE INVENTION Limited quantities of gametes, such as sperm and oocytes, from animals of the highest genetic merit or of elite genomic value can reduce the capacity for genetic dissemination and genetic improvement within a species. As such, there is a need within the animal breeding industry, and in particular the livestock industry, for maximizing the efficiency in the use of such gametes. SUMMARY OF THE INVENTION One embodiment of the invention comprises a method of processing sperm cells comprising: a) selecting a population of sperm cells wherein greater than 25% of sperm cells in the population have abnormal morphology; b) staining the sperm cells in the population; c) irradiating the sperm cells in the population; d) detecting fluorescence emitted by the sperm cells in the population in response to the step of irradiating; e) differentiating the sperm cells in the population based on orientation or viability; and f) collecting a subpopulation of oriented or viable sperm cells, wherein 85% or less of the sperm cells in the collected subpopulation bear an X-chromosome or 85% or less of the sperm cells in the collected subpopulation bear an Y-chromosome. In a particular embodiment, the percentage of sperm cells in the collected subpopulation having abnormal morphology is less than the percentage of sperm cells in the population having abnormal morphology in step a). In a further embodiment, in step e), the sperm cells are differentiated based on orientation and viability. In an even further embodiment, the method further comprises the step of staining the population of sperm cells with a quenching dye. In a particular embodiment, greater than 25% of sperm cells in the population have abnormal head morphology. In another embodiment, greater than 30% of sperm cells in the population have abnormal tail morphology. In yet another embodiment, greater than 35% of sperm cells in the population have abnormal morphology. In an additional embodiment, in step b) the sperm cells are stained with a DNA-selective dye. In a particular embodiment, the DNA-selective dye is Hoechst 33342. In another embodiment, the method further comprises the step of contacting the sperm cells in the population with magnetic particles. In certain embodiments, the step of differentiating the sperm cells in the population comprises creating a gated region based on the detected fluorescence emitted by the sperm cells in the population, wherein the gated region encompasses oriented or viable sperm cells. In other embodiments, the step of differentiating the sperm cells in the population comprises creating a gated region that excludes quenched, dead sperm and sperm having a higher incidence of abnormal morphology, and collecting sperm within the gated region. Another embodiment of the invention comprises a method of processing sperm cells comprising: a) selecting a population of sperm cells wherein greater than 45% of sperm cells in the population have abnormal morphology; b) staining the sperm cells in the population; c) irradiating the sperm cells in the population; d) detecting fluorescence emitted by the sperm cells in the population in response to the step of irradiating; c) differentiating the sperm cells in the population based on orientation or viability; and f) collecting a subpopulation of oriented or viable sperm cells, wherein the percentage of sperm cells in the subpopulation having abnormal morphology is at least 50% lower than the percentage of sperm cells in the population having abnormal morphology in step a). An additional embodiment of the invention comprises a method of processing sperm cells comprising: a) selecting a population of sperm cells wherein greater than 25% of sperm cells in the population have abnormal morphology; b) contacting the population of sperm cells with magnetic particles; c) staining the sperm cells in the population; d) irradiating the sperm cells in the population; c) detecting fluorescence emitted by the sperm cells in the population in response to the step of irradiating; f) differentiating the sperm cells in the population based on orientation or viability; and g) collecting a subpopulation of oriented or viable sperm cells. In a particular embodiment, the magnetic particles bind to dead or damaged sperm cells in the population through an electrical charge interaction. In a further embodiment, 90% or more of the sperm cells in the collected subpopulation bear an X-chromosome or 90% or more of the sperm cells in the collected subpopulation bear a Y-