PREFABRICATED SPERM-SEX SEPARATION KIT OF ANIMAL SEMEN BY USE OF NANOFIBERS FROM POLYMER-BASED MATERIAL
Field of the Invention
Biology, molecular genetics, agriculture and polymers regarding prefabricated sperm-sex separation kits of animal semen by use of nanofibers from polymer-based materials Background of the Invention
In-depth innovation and technology development are advanced continuously as a support and response to the production needs in the agricultural system of the country, leading to a great leap forward in such a system. Innovation is also applied to solving new problems, particularly in the agricultural production system, where innovation to meet agricultural needs rely on the integration of a core of knowledge and diverse skills. The core of knowledge leads to practical innovations applicable to the farm-level production, or agricultural industry. Invented innovations, therefore, have values and create changes for a better production system in terms of both production efficiency enhancement and efficient minimization of the cost of production for target groups.
As regards the cattle production system, the basic protein diet production is deemed important for populations at national and global levels. It is a production system with high-value impacts that helps drive the economy from the foundation to the industrial level of the country. The cattle production system includes beef cattle, dairy cattle, swine, and buffalo. For calf production that meets the demands of dairy and beef farmers, the dairy cattle production industry requires cows for milk production, while the beef cattle industry requires oxen for beef production and rapid growth rates. These result in a requirement that semen used in artificial insemination be capable of determining the sexes of conceived calves.
Normally, cattle semen contains gametes, which are spermatozoa (sperm) with two sexes namely 1) the Y-chromo some-bearing sperm, which makes a male calf (XY chromosome) upon fertilization with an oocyte (X-chromosome) of a breeder cow, and 2) the X-chromo some-bearing sperm), which makes a female calf (XX chromosome) upon fertilization with an ovum of breeder cow. In nature, mammal semen contains a ratio of Y-chromo some-bearing sperm to X- chromo some-bearing sperm of 50:50 percent. Calf sex determination depends on which sex chromosome is carried by the sperm fertilizing the oocyte. Therefore, an increase in the number of Y-chromo some-bearing sperm in semen increases the chance of conceiving male calves. Likewise, an increase in the number of X-chromo some-bearing sperm in semen increases the chance of conceiving female calves. The technology for sexed sperm in cattle semen was developed on the basis of weight differences, electrodes and antibodies specific to surface antigens on sperm.
The technology of sexed sperm in cattle semen widely used is cell sort flow cytometer based on staining, with Hoechst fluorescence dye, of DNA in chromosomes inside sperm. Y- chromo some-bearing sperm contain less DNA quantity than X-chromosome-bearing ones do and are less fluorescent, resulting in the cell soring flow cytometer being able to distinguish them and making it possible to select and collect each sperm type separately. This method has 85-percent accuracy for sexed sperm and can produce calves of the required sexes highly accurately, but the cell sort flow cytometer requires highly-expensive machine, produces low volume, and takes a long production time, making a very high production cost. There is no such machine in Thailand.
Therefore, sexed semen products on the market are imported merchandise only. Also, there have been reports in academic articles indicating decreased semen quality following this method, resulting in reduced quality of sperm used in artificial insemination. The fluorescent dye used in the DNA staining also results in genetic defects in calves. In addition, the sperm number contained in each breeding dose was found to be low (2-4 million sperm per dose), resulting in low reproductive performance in female breeders. There has been a recommendation that only heifer be used.
Additionally, there was a process of semen sex selection by cytotoxic reaction based on monoclonal antibodies against H-Y antigen on the Thai patent application number 0701004255 that was a process of semen sex selection using monoclonal antibodies against H-Y antigen on the surface of Y-chromo some-bearing sperm, based on an interaction between such antibodies and antigens, followed by Y-chromosome -bearing sperm digestion by the addition of complements to bind to monoclonal antibodies, causing a cytotoxic reaction. Sperm membranes began to be destroyed, and the cells were eventually lysed and ruined. X-chromosome-bearing sperm were not bound to monoclonal antibodies, thereby not digested by the cytotoxic reaction, hence their high survival. Therefore, the proportion of remaining X-chromosome-bearing sperm is higher than that of Y-chromo some-bearing ones in semen. Upon breeding with cows, female calf-yielding rates were increased to > 70%. A large amount of sex-selected semen could be produced each time. As many sperm could be contained in each dose as normal semen could (20 million sperm per dose) in a short time with low production costs and reproductive performance rates equal to those of normal semen, but a weakness of this method was the accuracy of female calf proportion yield because of the limitations of monoclonal antibodies that still had cross reactivity that could bind to X-chromosome-bearing sperm partly and had non-specific binding that interfered binding to the Fc portion of monoclonal antibodies, resulting in the female calf-yielding proportion > 70%, but < 80% because there was also a limitation in terms of monoclonal antibody specificity to each male breeder.
The Thai patent application number 1201004011 described a process for sexed sperm of cattle using monoclonal antibodies specific to cattle male-sex sperm based on an interaction of monoclonal antibodies bound to Y-chromo some-bearing sperm surface protein. Then, complements were added for a toxicity reaction and separation of female- sex sperm from dead male-sex sperm, and cattle sperm that passed through the sex separation with this process were used in in vitro fertilization (IVF). This invention can increase the chance and rates of female calf conception in the dairy cattle industry.
The Thai patent application number 1701001527 with the invention title “Sperm-sorting system using electrical field that is not uniform in the form of dielectrophoresis system for sexed sperm in microfluidic chip system” described such system in the form of dielectrophoresis system with at least one layer of the said sperm-sorting system using an electrical field that was not uniform, comprising dielectrophoresis electrode layers comprising a dielectrophoresis-electrode electric conductor made from indium-tin-oxide (ITO) in a pattern of DEP dumbbell shape electrode on a microscope slide, wherein a condition in the sorting was an alternate-current electrical field using the alternate-current voltage of 4-8 volts as sinusoidal wave pulses at the frequency of 1-20 MHz.
The Thai patent application number 2101005004 with the invention title “Tool for selecting sperm by microfluidic technology for reproduction” described such tool comprising a flow channel in the form of a long channel functioning in allowing sperm to move through. Any one side of the flow channel contained an input in the form of a spherical tube functioning as a position to introduce sperm required to be tested while the other side of the flow channel is an output in the form of a spherical tube as an exit of sperm, wherein a special feature was that the input was connected to a screening part in the form of small poles. In this part, each row was arranged kittycorner from another row and functioned in screening sediments or impurities that may have mixed in samples required to be tested. Next to the screening part was a first sorting part in the form of poles functioning as the first barrier in selecting sperm with normal appearance that moved well. Next to the first sorting part was a second sorting part in the form of poles functioning as the second barrier in selecting sperm with normal appearance that moved well.
Based on the technology of separating sperm sexes in cattle using biotechnology principles related to the immune system and using production of antibodies (Abs) specific to Y-chromo some- bearing sperm that was successful in the production of anti-Y-chromosome-bearing sperm antibodies in beef cattle and dairy cattle, namely monoclonal antibodies (MAbs) produced from hybridoma cells and recombinant single-chain fragment variable antibodies (scFv antibodies) against Y-chromosome-bearing sperm of dairy cattle as produced from recombinant E. coli, upon obtaining antibodies with high efficiency in binding to Y-chromosome-bearing sperm, a method was then developed in sperm sex separation with the application of nanotechnology in producing magnetic microbeads to single-chain fragment variable antibodies (scFv antibodies) specific to Y- chromo some-bearing sperm of cattle, leading to a new innovation in sexed sperm in cattle using magnetic-activated cell sorting combined with the produced single-chain fragment variable antibodies that could sort sexed sperm, resulting in as high as 80% X-chromo some-bearing sperm in sexed semen (Sringarm et al., 2022). This innovation is called M-Zlex, which has been used in sorting sexed sperm of beef cattle and dairy cattle more and will help in management systems in the production of both beef cattle and dairy cattle, leading to reduced production costs for agriculturists and agriculturists being able to access technology. It can be seen that the innovation of use of magnetic nanobeads capable of attaching to antibodies has very high production costs. Should there be many sperm required to be separated each time, a large amount of magnetic nanobeads will be required, also resulting in very high production costs required each time. Application to semen production size expansion has limitations as well, such as impossible simultaneous production of sexed semen with semen milked from several male breeders, short useful life of magnetic nanobeads, and low chances of application to the industry and export.
Polymer-based material films were, therefore, developed, using both synthetic polymers and naturally producible polymers capable of attaching to antibodies specific to Y-chromosome- bearing sperm, that are options that will be able to expand the production of sexed sperm in animals, reducing costs of the technique nicely and influencing industrial adoption. These polymer-based material film have a property of antibody attachment and are used for sexed sperm, namely polymers with COOH groups like ionomer and poly (lactic acid) (PLA). Polymer-based material film have the quality of having small surface areas. Upon their development for their use, there have been biological limitations in terms of quality of semen milked each time with as many as billion sperm in the mere volume of 5-10 milliliters, and sperm are short-lived in exogenous environment, leading to low efficiency of the sexed sperm procedure.
The inventor has, therefore, created and produced a polymer-based material in a suitable form used in sperm sex separation of animals together with antibodies. A suitable polymer-based material is required as regards the type, form, and size for use in the process of sperm sex separation with efficiency in terms of speed so that the semen remains in good quality and processing of sperm sex separation of animals can be expanded, leading to production of prefabricated sperm-sex separation kit of animal semen by use of nanofibers from polymer-based material that can be used in the separation of sperm in animal semen and also leads to further manufacturing production.
Summary of the Invention
In this invention, the prefabricated sperm-sex separation kit of animal semen by use of nanofibers from polymer-based material can be made using principles comprising creation and production of suitable polymer nanofibers capable of attaching to antibodies specifically bound to Y-chromosome-bearing sperm. Polymer nanofibers are small in the size of 1-1,000 nanometers and produced from an engine that creates fibers from polymer compound in a static electricity system. These polymer nanofibers have a large surface area fit and sufficient for coating with antibodies using a chemical reaction at the carboxyl group (-COOH) of the polymer in a suitable proportion for use in sperm sex separation. The invented polymer nanofibers can be used in the invention of prefabricated sperm- sex separation kit of animal semen efficiently that can separate for the purity of X-sperm enriched fraction > 80% and the purity of Y-sperm enriched fraction > 80% while sperm maintain competency and good quality in large quantity, highly efficient in the artificial insemination in both cow and heifer, eliminating limitations found with several methods.
The intent of this invention is to be the prefabricated sperm-sex separation kit of animals capable of separating sperm sexes of animals efficiently with low production costs, minimized expensive tool use, agriculturists and users able to access and operate easily and efficiently; and to be capable of separating sperm in semen for the purity > 80% while sperm maintain competency and good quality in large quantity. This process of sperm sex separation helps minimize physiological limitations of each animal type by milking the semen each time in the mere quantity of 1-10 milliliters containing as many as billion sperm. Use of this small and highly efficient sperm sex separation kit will contribute to ideal separation.
Brief Description of the Drawings
Figure 1: polymer nanofibers under an SEM with 4000x magnification.
Figure 2: preparation and mechanism of binding between polymer nanofibers and antibodies bound to Y-sperm
Figure 3: prefabricated sperm-sex separation kit of animal semen by use of nanofibers from polymer-based material
Detailed Description of the Invention
The prefabricated sperm-sex separation kit of animal semen by use of nanofibers from polymer-based material is a prefabricated sperm- sex separation kit of animals capable of separating animal sperm sexes rapidly with the process and operational procedure efficient in separating Y-sperm from X-sperm. That can provide for X-sperm more than 80% in X-sperm enriched fraction while sperm maintain competency and good quality in large quantities, highly efficient in the artificial insemination.
This invention applies principles consisting of the creation and production of suitable polymer nanofibers capable of conjugating to antibodies that specifically bind to Y-sperm. Polymer nanofibers are small in the size of 1-1,000 nanometers and produced from a machine that creates fibers from the polymer compound in a static electricity system. These polymer nanofibers have a large surface area fit and sufficient for conjugating with antibodies using a chemical reaction at the carboxyl group (-COOH) of the polymer in a suitable proportion for use in sperm sex separation. The invented polymer nanofibers can be used in the process of sperm sex separation in animal.
The process of sperm sex separation using polymer nanofibers conjugated with antibodies that bind to Y-sperm is adopted in sperm separation process of animal semen, where polymer nanofibers conjugated with antibodies function as a binder to Y-sperm, allowing unbound sperm to flow through polymer nanofibers, which are X-sperm-enriched fractions containing X-sperm more than 80%. Afterwards, Y-sperm bound to antibody on polymer nanofibers are removed by using an eluting buffer flowed down to the new tube. This fraction was called “Y-sperm enriched fraction”, which contained Y-sperm more than 80%. Sperm that have passed through the sex separation process maintain competency and good quality, fit for artificial insemination or in vitro fertilization.
This invention is made of biobased polymer-based material, with its application that is safe for users, animals - cow, heifer and conceived new born animals. The waste following use of prefabricated sperm- sex separation kit of animal semen and the biobased polymer-based material are biodegradable, leaving no waste residues in the environment.
Polymer nanofiber preparation consists of the following steps.
A. Select a suitable biobased polymer that is biodegradable, namely a biobased polymer with carboxyl group that can be selected from poly (lactic acid) (PLA) or ionomer. Prepare a pellet of biobased polymer with carboxyl group in an amount of 5-25% weight/volume. Dissolve the prepared pellet in a solvent with a ratio of dimethylformamide: acetone = 1:1. Stir well and then expose to heat at a constant temperature of 120-180 degrees Celsius until a homogeneous solution is obtained.
B. Then, form the obtained polymer into nanofibers using a machine that creates fibers from polymer compound in a system with nozzle electrospinning sized 0.61-2.27 millimeters, a roll drum collector that is 5-50 centimeters wide, clearance between the nozzle and collector in a range of 5-50 centimeters, and high voltage of 10-30 kilovolts. The nozzle and nozzle drum adjoin a solution discharge cylinder, yielding polymer fibers with a diameter size of 1-1,000 nanometers to be conjugated to antibodies.
A method of conjugating polymer nanofibers to antibodies specific to Y-sperm in various species of mammal consists of the following steps.
C. Arrange the formed biobased polymer nanofibers with carboxyl group into polymer nanofiber sheet, preferably having an area of 5-200 square centimeters.
D. Prepare antibodies specific to Y-sperm of animal for attachment to polymer nanofibers, where such antibodies can be prepared from:
- Polyclonal antibodies (PAbs) obtained from the preparation of Y-sperm of various species of animal where Y-sperm are separated from X-sperm using fluorescent dye staining by a flow cytometry sorter for the purity of Y-sperm > 85%. Then, they are injected to immunize for the antibody production from rabbits, mice, or rats aged more than 6 weeks using 50 x 106 Y-sperm per animal and 0.5 milliliter of Freund's complete adjuvant. The injection is made three times with 14 days apart. Then blood is drawn for plasma extraction and polyclonal antibody purification for use in the conjugation of polymer nanofibers.
- Monoclonal antibodies (MAbs) obtained from the preparation of Y-sperm of various species of animal where Y-sperm are sorted from X-sperm using fluorescent dye staining and then sorted with a flow cytometry sorter for purity of Y-sperm > 85%. Then, they are injected to immunize for the antibody production from mice or rats three times using 10-20 x 106 Y-sperm per animal and 0.25 milliliter of Freund's complete adjuvant. The injection is 14 days apart. Then, they produce monoclonal antibody using splenocytes fused with myeloma cells at a ratio of 2.5-5: 1, and then the hybridoma cells produce monoclonal antibodies with high efficiency capable of binding to Y-sperm. Then monoclonal antibodies are expanded and increased in quantity by culturing hybridoma cells in culture medium and producing ascitic fluid in mice or rats for antibody production and use in the conjugation of polymer nanofibers.
- Recombinant antibodies or single chain fragment antibodies (scFv antibodies) obtained from the preparation of Y-sperm of various species of animal where Y-sperm are sorted from X-sperm using fluorescent dye staining and then sorted with a flow cytometry sorter for purity of Y-sperm > 85%. Then, they are injected to immunize for the antibody production from mice or rats three times using 10-20 x 106 Y-sperm per animal and 0.25 milliliter of Freund's complete adjuvant. The injection is 14 days apart. Then, it is a production process of recombinant antibodies or single chain fragment antibodies by introducing splenocytes, bone marrow and hybridoma cells capable of producing antibodies against Y-sperm of respective mammal to RNA extraction process, and then cDNA is synthesized with first-strands. The obtained cDNA is used in constructing nucleotide sequences controlling the production of single chain fragments comprising variable heavy chain (VH) and variable light chain (VL) by having the cDNA interact with primers to construct nucleotide sequence fragments controlling the production of VH and VL using the PCR technique. The VH is produced using VH-Forward and VH-Revers primers, and VL is produced using VL-Forward and VL- Revers primers. Then a linker is used in linking both fragments together using the splicing overlap extension PCR (SOE PCR) technique together with primer set. Then the gene nucleotide sequences of single chain fragments are obtained. Single chain fragment antibodies specific to Y-sperm are selected by library screening by phage display and using the bio-panning method in selecting the phage library specific to Y-sperm, and the obtained phage library is used in classification and separation using amino acid sequences to classify VH + VL regions or CDR3 (complementary determining region) of single chain fragment antibodies specific to Y-sperm of mammals. Upon acquiring amino acid sequences of VH + VL, a suitable vector is linked, and E. coli is linked for use in the production of single chain fragment antibodies, and recombinant antibodies or single chain fragment antibodies are obtained for conjugation to polymer nanofibers.
E. Add a solution of 50-350 millimoles N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) in a coating buffer (0.05 M Carbonate-Bicarbonate, pH 9.6) containing 1% potassium chloride (KC1) to the biobased polymer nanofibers with carboxyl group, preferably with an area greater than 10 square centimeters. Then, add a solution of 50-350 millimoles N- hydroxy succinimide (NHS) in the coating buffer. Shake with a shaker at a temperature range of 4- 30 degrees Celsius for 2-12 hours. Then, add antibodies specific binding to Y-sperm at 1-100 milligrams per milliliter. Shake at a temperature range of 2-8 degrees Celsius for 6-24 hours. Then store at a temperature range of 2-8 degrees Celsius. Put into a package with aperture to allow fluid flow where each package contains a polymer nanofiber sheet of 40 square centimeters as a ready- to-use sperm-sex separation kit of semen.
Separation method of semen sperm sexes using the prefabricated sperm-sex separation kit of animal semen by use of nanofibers from polymer-based material consists of the following steps.
- Prepare semen milked from male breeder or frozen semen with a cell count of 1,000 million. Add a suitable volume of semen-diluting solution. - Prepare packages with aperture to allow fluid flow, such as syringes, with the polymer nanofiber sheet attached to antibodies specific binding to Y-sperm of 40 square centimeters. Then, add 5 milliliters of semen-diluting solution by allowing it to flow through the polymer nanofiber sheet to wash the sheet twice and disposing of the eluted fluid.
- Add the semen prepared in item 1, along with retaining the fluid exposed to the polymer nanofiber sheet, for a duration of 5-30 minutes. Then, let the fluid flow out of the polymer nanofiber sheet into a receptacle and retain the liquid, which is the portion containing X- sperm in a proportion > 80-90% that can be used in artificial insemination or introduced to a freezing process for storage.
- Sperm bound to the polymer nanofiber sheet are Y-sperm that can be removed from the sheet by adding a solution for eluting Y-sperm from polymer nanofibers (Al), and the fluid exposed to the polymer nanofiber sheet is retained for a duration of 5-30 minutes, and Y-sperm thus are eluted in the solution. Then, let the fluid flow out of the polymer nanofiber sheet into a receptacle and retain the liquid, which is the portion containing Y-sperm in a proportion > 80-90% that can be used in artificial insemination or introduced to a freezing process for storage.
The solution for eluting Y-sperm from polymer nanofibers (Al) consists of the following compositions.
Tris (hydroxymethyl) methylamine 150 - 190 millimolars
Citric acid monohydrate 55 - 70 millimolars
D-fructose 45 - 60 millimolars
Antibiotic 1000 units
Imidazole 100 - 300 millimolars
Also, adjust the pH of solution to a range of 6.8 - 7.2.
Following the use of this prefabricated sperm- sex separation kit, the polymer nanofiber sheet can be disposed of easily because the material is biodegradable without causing a problem of environmentally toxic waste. It can be manufactured and used in large quantities without impacting the environment.
Best Mode of the Invention
As described in the Detailed Description of the Invention