DESC:
[0001] This application claims priority from the provisional application numbered 202311082391 filed with the Indian Patent Office, Delhi on 4th December 2023 post-dated to 4th February 2024 entitled “A process for ligand-based semen sorting for mammalian sperms”, the entirety of which is expressly incorporated herein by reference.
Preamble to the description
[0002] The following specification specifically describes the invention and the manner in which it is to be performed:
Description of the invention
Technical field of the invention
[0003] The present invention relates to a ligand-based semen sorting process for mammalian sperms into X and Y chromosome bearing sperms. More particularly, the invention relates to a process, which is capable of separating large number of fresh ejaculated spermatozoa simultaneously compared to the available convention methods.
Background of the invention
[0001] Compared to female calves, the male calves are not much desired and wanted in the industry today. The female calves are valued primarily because they have a vital role to play in the dairy industry. Unfortunately, male calves are either sold for beef or they are sent to veal facilities or shot soon after they're born. In most of the countries in the world, the male calves don’t live longer due to lack of care, ill treatment and slaughter for meat. Management of male calves is an emerging welfare issue in the dairy industry today.
[0002] To produce the female calves, semen sexing is one of the popular techniques which is used, wherein X and Y chromosome bearing sperms are segregated and female egg is fertilized only by X chromosome bearing sperm (s). Sexed semen inserted into a female calf by artificial insemination is helpful to produce female calves with high success rate. Generally, semen sexing is a very challenging process due to high cost of the technology, low efficiency of machines, lack of skilled professionals to operate the sexing machines, wastage of sperms during the process of sexing and reduced conception rate compared to conventional semen.
[0003] Various methods have been developed for sperm sexing, including albumin gradient, Percoll gradient, gradient swim-down, free-flow electrophoresis, H-Y antigen identification, centrifugal counter-current distribution, and genetic approaches. Among these, flow cytometry remains the most widely employed technique. However, it is characterized by several significant limitations, including slow processing speed, high operational costs, and an inability to produce sufficient quantities of sexed semen required for practical applications such as artificial insemination. These challenges underscore the need for more efficient, cost-effective, and scalable solutions for sperm sexing.
[0001] Flow cytometry-based methods for sperm sexing require substantial processing time, resulting in low throughput. Attempts to improve throughput using high-speed flow cytometry have proven unsuccessful, as the high pressure exerted during the process significantly compromises sperm viability and functional potency. Moreover, the requirement for fluorescent staining in these methods further reduces sperm motility and viability, adversely impacting the success rates of artificial insemination.
[0002] The Patent Application No. US20140182005A1 entitled “Sperm cell separation methods and compositions containing sperm cell targeting ligands for use therein” discloses sperm cell targeting ligands, including DNA-binding proteins, that bind target molecules on the surface of, accessible from the surface of, or inside mammalian sperm cells and methods for producing the sperm cell targeting ligands. The sperm cell targeting ligands are used to separate sperm cells based upon sperm cell qualities, such as whether the cells contain X chromosomes or Y chromosomes. The invention also provides methods of sperm cell purification using targeted radiofrequency absorption enhancers and transgenic animals with sex-skewed ejaculate.
[0003] The Patent Application No. US20060147894A1 entitled “Jacketed vessel for holding semen for sex biasing mammals through artificial insemination and systems and methods for enhancing the probability of sex biasing using the same” discloses a jacketed container for holding a semen sample from a mammalian donor at a predetermined temperature for a predetermined period of time prior to incubation to enhance the probability of obtaining offspring of a selected sex through artificial insemination is disclosed. In one embodiment, the semen sample is collected and insulated by a high-heat capacity material that has been preconditioned to a temperature between about 30° C. to about 40° C. before the semen is cooled to a temperature of about 12° C. In a second embodiment, the semen sample is collected and insulated by a high-heat capacity material that has been preconditioned to a temperature between about 4° C. to about 20° C. before the semen is cooled to a temperature of about 12° C.
[0004] The Patent Application No. US20220163438A1 entitled “Modular flow cytometry systems and methods of processing samples” disclosed Modular flow cytometry systems and methods for processing samples are described herein. The systems include automated or semi-automated modules that are replaceable and removable. A sample pathway module may be removed and placed in a microfluidic device cleaning module for cleaning and then reinstalled or stored for later use. The systems further include optical modules, electronics modules, and mixing and collection modules. The optical module includes a photo-damaging assembly and detection laser assembly that may be on the same side relative to a plane or surface of a flow cytometry device and opposite of a detection assembly. The laser beam may have a beam waist that is wider in a direction perpendicular to a flow direction than in the flow direction. The mixing and collection module can automatically mix a sample being collected in a sample tube and switch to another sample tube when the other tube is full.
[0005] Despite the development of various methods for sperm sexing for applications in artificial insemination and in vitro fertilization (IVF), most have proven to be ineffective, labor-intensive, or impractical, leading to their discontinuation. A critical limitation of the currently available techniques is their suitability primarily for frozen sperm, rendering them ineffective for sorting freshly ejaculated spermatozoa. This poses a significant challenge in achieving efficient, scalable, and viable sex-sorting solutions for fresh sperm, which is essential for improving fertility outcomes.
[0006] Hence, there is a need for effective and efficient method for semen sexing for dairy and beef farms to ensure the birth of animals of the desired sex, leading to reduced costs and improved environmental sustainability.
Summary of the Invention
[0007] The process disclosed in the present invention overcomes the drawbacks of reduced viability and motility and ensures that the sperm retains biological properties throughout the sorting process. The process disclosed in the present invention results in simultaneous separation of a large number of mammalian spermatozoa into X and Y chromosome-bearing populations without compromising the viability, motility, or functional integrity.
[0008] The present invention discloses a process for ligand-based semen sorting for mammalian sperms. The ligands used in the process are TLR 7 and TLR 8 (Toll-like receptor) ligands. The process comprises steps of diluting the fresh semen sample in the modified media, evaluating the motility of sperms in semen, dispensing of sorting media supplemented with TLR7/8 ligand, adding the sperm cells in respective tubes and subjecting to incubation, aspirating the precipitate from lower layer of suspension and adding on media supplemented with TLR 7/8, aspirating the top layer of 1 to 1.5 ml in transferring to 2 ml media in 15 ml falcon tube and incubating.
[0009] Further, transferring the cloudy bottom layer containing the desired X- sperm population to 1.5 ml microcentrifuge tubes, centrifuging the tubes to remove the supernatant, adding TLR ligand free media with additives to pellet obtained, centrifuging the tubes and resuspending the pellets in media for artificial insemination, analyzing the pre-freeze motility of the sperm cells, freezing the X sorted sperm cells, thawing the straws and removing the non-motile cells from the sample and isolating DNA of sorted sperms and analyzing the purity of X-sperms in sorted samples by quantification of copy numbers of X and Y sperms.
[0010] The process disclosed in the present invention is efficient, scalable, and preserves the viability and motility of sperm. The process is utilized in laboratory settings and reproductive applications, including artificial insemination and in vitro fertilization (IVF). The process exhibits significant advantages over conventional techniques, such as high throughput, reduced stress on sperm, and ease of adaptation to standard laboratory practices, thereby providing a cost-effective and biologically safe approach for targeted sperm selection.
Brief Description of the Drawings
[0011] FIG 1 illustrates a process for sorting the sperms into X- and Y-chromosome-bearing sperm.
[0012] FIG 2 illustrates the kinematic parameters of sex sorted semen and unsorted semen.
[0013] Detailed Description of the Invention
[0014] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.
[0015] The term “Toll-like receptors” refers to cell surface and intracellular molecules that detect and respond to microbial antigens.
[0016] The term “Toll-like receptor (TLR) ligand” refers to substances that bind to TLRs.
[0017] The term “Artificial insemination” refers to a reproductive technology that involves manually depositing sperm from a male animal into the reproductive tract of a female animal.
[0018] The present invention discloses a process for ligand-based semen sorting for mammalian sperms. The process of is capable of separating a large number of sperms simultaneously without compromising on the viability of the sperms. The present invention relates to a process for separating mammalian spermatozoa into X chromosome-bearing and Y chromosome-bearing populations using ligands targeting Toll-like receptor 7 (TLR7) and/or TLR8.
[0019] FIG 1 illustrates a flowchart for the process for sorting the sperms into X- and Y-chromosome-bearing sperm. The process (100) of separation of the semen according to an embodiment of the present invention starts with step (101) by diluting the fresh mammalian semen sample in the production media at a ratio of 1:1. The diluted semen is maintained at a temperature of 370C to prevent loss of motility and functionality of the sperm.
[0020] The quality and viability of spermatozoa used for sexing are significantly influenced by whether the sperm is fresh or frozen, as fresh sperm exhibits distinct physiological and behavioral characteristics compared to preserved sperm. The present invention addresses this challenge by optimizing a growth medium and associated parameters specifically for fresh spermatozoa. The medium incorporates TLR7 ligand to facilitate the targeted separation of X and Y chromosome-bearing sperm. The growth medium aids in maintaining the viability, motility, and functional integrity of fresh sperm, thereby ensuring enhanced efficiency and reliability in sex-sorting processes.
[0021] At step (102), the motility of the sperms in semen is evaluated to assess the progressive motility by examining sperm trajectories. The evaluation is achieved through computer–assisted sperm analysis (CASA) and microscopy. At step (103), 3ml of sorting media supplemented with different concentrations of TLR7/8 ligand with or without creatine is dispensed in fresh 15 ml conical bottom centrifuge tubes and 2 ml in 96 well deep well plates. Creatine is added at a concentration range of 200µM to 500µM to enhance the motility of the sperms. The addition of creatine significantly increases the swim-up sperm percentages by enhancing glycolytic ATP production and promoting a zigzag motility pattern. Additionally, creatine enhances the rate of fertilization and the formation of XY embryos.
[0022] TLR ligands are innate immune sensors capable of detecting single-stranded ribonucleic acid (ssRNA). The treatment of semen with TLR7/8 ligands with synthetic compounds such as imidazoquinolines or RNA analogs facilitates the selective precipitation of X spermatozoa, enabling their separation from Y spermatozoa.
[0023] The sorting media comprises resiquimod (R848) at a concentration range of 0.01µM to 3µM to enhance cell functionality during the sorting process, sodium chloride (NaCl) at a concentration range of 90mM to 150mM, potassium chloride (KCl) is added at a concentration range of 2 mM to5 mM to support ionic balance and cellular membrane potential, disodium hydrogen phosphate (Na2HPO4) is added at a concentration range of 0.2 mM to 1mM as a buffering agent to stabilize pH, magnesium chloride hexahydrate (MgCl2·6H2O) is added at a concentration range of 0.05mM to 2mM for enzymatic activity and structural stabilization of macromolecules, calcium chloride dihydrate (CaCl2·2H2O) is added at a concentration range of 0.2mM to 1.5mM for cell signaling and structural integrity, fructose at 30 to 100 mM as an energy source, HEPES buffer is added at a concentration range of 10mM to 50mM to maintain a stable pH in non-CO2 incubators, sodium bicarbonate (NaHCO3), is added at a concentration range of 5mM to 30mM, lactic acid (88%) is added at a percentage range of 0.02% to 0.1% to support metabolic pathways and mimic physiological conditions.
[0024] Bovine Serum Albumin (BSA) is added to the enhance cell stability and reduce oxidative stress at a percentage range of 0.2% to 1.0%, L-glutamine is added at a concentration range of 2mM to 10mM to support cellular metabolism and protein synthesis, glycerol (100%) is added at a percentage range of 5% to 10%, serves as a cryoprotectant, and egg yolk (100%) is added at a percentage range 10% to 30%, provides lipoproteins and phospholipids critical for membrane integrity during handling and storage.
[0025] At step (104), cells are added in respective microcentrifuge tubes and subjecting to incubation for a duration of 15 minutes to 60 minutes at a temperature of 37°C in 5% carbon dioxide in humidified conditions. In the incubator, the tubes are maintained at an angle of 45-degree slanting position to enhance motility. At step (105), the precipitate from the lower layer of the suspension is aspirated and added on the top of 2 ml sorting media with TLR ligand in another 15 ml tube. The upper layer comprising motile Y-sperms are transferred to new tube, followed by centrifugation at 400g at a temperature of 37°C for a duration of 5 minutes. The supernatant is removed without disturbing the pellet and resuspend in the appropriate media for further applications.
[0026] At step (106), 1ml to 1.5ml of top layer of suspension is aspirated and added on the top of 2 ml media with TLR ligand in a 15 ml falcon tube. The falcon tube is incubated for a duration of 5 minutes to 30 minutes. At step (107), the cloudy bottom layer containing the desired X- sperm population is transferred to 1.5 mL microcentrifuge tubes. At step (108), the tubes are subjected to centrifugation at a rotation speed of 400g, at a temperature of 37 °C for a duration of 5 minutes to 30 minutes. The supernatant is removed completely without disturbing the pellet.
[0027] At step (109), 1ml to 5ml of TLR ligand free media is added to the pellet obtained after centrifugation to recover the motility of the sperms. At step (110), the tubes are subjected to centrifugation at a rotation speed of 400g, at a temperature of 37°C for a duration of 5 minutes. The pellet is resuspended in the media suitable for artificial insemination. At step (111), the pre-freeze motility of the sperm is analyzed using CASA and microscopy techniques.
[0028] At step (112), the sorted X-enriched spermatozoa is subjected to freezing in freezing media and incubating at a temperature of 4oC. The freezing media comprises NaCl at a concentration range of 90mM to 150 mM, KCl at a concentration range of 2mM to5mM, Na2HPO4 at a concentration range of 0.2mM to 1mM, MgCl2·6H2O and CaCl2·2H2O at a concentration range of 0.05mM to 2mM and 0.2mM to 1.5 mM respectively, fructose at a concentration range of 30Mm to 100mM. HEPES at a concentration range of 10mM to 50 mM, NaHCO3 at a concentration range of 5mM to 30 mM, lactic acid (88%) at a percentage range of 0.02% to 0.1% to support cellular metabolism under hypothermic conditions, cryoprotective agents such as glycerol (100%) at a percentage range of 5% to 10%, which penetrates cells to reduce ice crystal formation, and egg yolk (100%), at a percentge range of 10% to 30%, providing lipoproteins and phospholipids for membrane protection, BSA at a percentage range of 0.2% to 1.0%, L-glutamine, at a concentration range of 2mM to 10 mM to support cellular energy metabolism and protein synthesis during the freezing process.
[0029] The freezing media comprises additives such as sodium pyruvate at a concentration of 1mM and Sodium D Lactate at a concentration of 0.018% enabling enhanced sperm survival during cryopreservation.
[0030] At step (113), the straws are thawed, and the non-motile sperm cells are removed and isolating DNA of sorted sperms. Finally, at step (114), the purity of X-sperms in sorted samples is analyzed by quantification of copy numbers of X and Y sperms.
[0031] The present invention discloses a process for ligand-based semen sorting for mammalian sperms. The treatment of mammalian semen with TLR7/8 ligands with synthetic compounds facilitates the selective precipitation of X spermatozoa, enabling their separation from Y spermatozoa. The process aids in maintaining the viability, motility, and functional integrity of fresh sperm, thereby ensuring enhanced efficiency and reliability in sex-sorting processes. With enhanced viability and motility of the sorted sperms, the rate of artificial insemination for reproductive applications is further improved. The process is time efficient and cost-effective, enabling ease of accessibility.
[0032] Having generally described this invention, a further understanding can be 10 obtained by reference to certain specific examples, which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

Example 1: Analysis of semen kinematics characteristics of sex-sorted semen vs. unsorted semen
[0033] Sex-sorted semen exhibits distinct kinematic characteristics compared to unsorted semen, primarily due to the mechanical and chemical stresses encountered during the sorting process. FIG 2 illustrates the kinematic parameters of sex sorted semen and unsorted semen.
[0034] Total and progressive motility are reduced in sex-sorted semen as a result of reduction of glucose utilization due to increase in expression of TLR7/8. The spermatozoa are further revived after centrifugation to regain full functionality of spermatozoa. Velocity parameters, including curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP), are consistently lower in sex-sorted semen, reflecting compromised movement and reduced energy efficiency. In contrast, unsorted semen demonstrates higher motility and velocity due to the preservation of structural and functional integrity. Kinetic ratios, such as linearity (LIN), straightness (STR), and wobble (WOB), tend to be slightly affected in sex-sorted semen, while unsorted semen exhibits superior values indicative of coherent and directional motility. Functional competence, such as fertilization potential and capacitation, is retained in sex-sorted semen, albeit at slightly lower levels than in unsorted semen. Sex-sorted semen is advantageous for selective breeding and offspring sex control.

[0035] The present invention discloses a process for ligand-based semen sorting for mammalian sperms. The present invention addresses the limitations of existing methods, offering a scalable, efficient, and viable solution for both artificial insemination and external fertilization, ultimately improving reproductive outcomes across various mammalian species. The separation process enhances the precision and efficacy of sperm sexing and is suitable for a wide range of reproductive applications. The segregated X and Y chromosome-bearing spermatozoa is directly utilized for artificial insemination, thereby enabling targeted production of offspring with the desired sex. Furthermore, the process is compatible with external fertilization techniques, such as in vitro fertilization (IVF), where the separated sperm populations is selectively used to fertilize female eggs under controlled conditions The process exhibits high throughput, reduced stress on sperm, and ease of adaptation to standard laboratory practices, thereby providing a cost-effective and biologically safe approach for targeted sperm selection.
,CLAIMS:We Claim:
1. A process for ligand-based semen sorting for mammalian sperms, the process (100) comprising steps of:
a) diluting a fresh mammalian semen sample in a production media at a temperature of 370C to maintain the sperm functionality and motility (101);
b) evaluating the motility of sperms in the semen to assess the progressive motility by examining sperm trajectories to enable effective sorting of sperms (102);
c) dispensing 3 ml of sorting media supplemented with Toll-like receptors (TLR7/8) ligand with or without creatine in a fresh 15 ml conical bottom falcon tube and 2 ml in 96 well deep well plate for the selective precipitation of X sperm cells (103);
d) adding the precipitated X sperm cells into the microcentrifuge tubes and subjecting the said tubes to incubation for a duration of 15 minutes to 60 minutes at a temperature of 37 °Cto maintain motility of the sperms (104);
e) aspirating the precipitate comprising X sperm cells from the lower layer of suspension and adding on the top of 2 ml sorting media comprising TLR 7/8 ligand in a 15 ml falcon tube (105);
f) aspirating the top layer comprising Y sperm cells of 1 ml to 1.5 ml in transferring to 2 ml media in a 15 ml falcon tube and incubating for a duration of 5 minutes to 30 minutes (106);
g) transferring the cloudy bottom layer containing the desired X- sperm cells to 1.5 ml microcentrifuge tube (107);
h) subjecting the microcentrifuge tubes to centrifugation at 400g at a temperature of 37°C for a duration of 5 minutes and removing the supernatant without disturbing the pellet (108);
i) adding 1 ml to 5 ml of TLR ligand free media with the additives to a pellet obtained after centrifugation to recover motility of the sperms (109);
j) subjecting the microcentrifuge tubes to centrifugation at 400g at a temperature of 37°C for a duration of 5 minutes and resuspending the pellet in the media for artificial insemination (110);
k) analysing the pre-freeze motility of the sperm using Computer Assisted Sperm Analysis (CASA) and microscopy (111);
l) freezing the sorted X-enriched sperm cells in freezing media and subjecting the frozen sorted X-enriched sperm cells to incubation process at a temperature of 4oC (112);
m) thawing the straws and removing the non-motile sperm cells and isolating deoxy ribose nucleic acid (DNA) of the sorted sperms (113); and
n) analysing the purity of X-sperms in sorted sperms by quantification of copy numbers of X and Y sperms (114).
wherein the process facilitates effective separation of X sperms and Y sperms using TLR 7/8 ligand and maintains viability and motility of the sperms.

2. The process as claimed in claim 1, wherein the fresh semen sample is diluted at a ratio of 1:1 with production media

3. The process as claimed in claim 1, wherein the sperm cells in the microcentrifuge tubes are maintained at an angle of 45-degree slanting position during incubation to enhance motility of the sperms.

4. The process as claimed in claim 1, wherein creatine added is at a concentration range of 200µM to 500µM to enhance the motility of the sperms.

5. The process as claimed in claim 1, wherein the sorting media comprises resiquimod (R848), sodium chloride, potassium chloride, disodium hydrogen phosphate, magnesium chloride hexahydrate, calcium chloride dihydrate, fructose, HEPES buffer, sodium bicarbonate, lactic acid, Bovine Serum Albumin (BSA), L-glutamine, glycerol, and egg yolk aiding effective sorting of X and Y sperm cells.

6. The process as claimed in claim 1, wherein the sorting media comprising resiquimod at a concentration of 0.01µM to 3µM.

7. The process as claimed in claim 1, wherein the freezing media comprises sodium chloride, potassium chloride, disodium hydrogen phosphate, magnesium chloride hexahydrate, calcium chloride dihydrate, fructose, HEPES buffer, glycerol, egg yolk sodium bicarbonate, lactic acid, Bovine Serum Albumin (BSA) and L-glutamine.

8. The process as claimed in claim 1, wherein the freezing media comprises one or more additives such as sodium pyruvate, Sodium D Lactate.