Abstract: Systems and methods are provided for identification and discrimination of subpopulations within a mixture of particles. The systems and methods implement continuous calibration of the classification of particles within the mixture to provide consistency in operation and to reduce inter- and intra-batch processing variation. The systems and methods produce advantageously sorted particle products.

Abstract: Methods and systems for processing semen samples from straws for determining genetic identity, testing the purity of the sample, detecting errors or contamination, calculating an amount of contamination, and determining the identity of the contaminant. The methods herein can detect low levels of contamination, such as contamination of about 0.5%, 1%, 2%, etc.

Abstract: Disclosed is an approach to differentiating between different particle types in samples flowing through microfluidics chips. A sample may have an initial proportion of a first cell type to a second cell type. An illuminating light source may emit a coherent light at the sample, and light leaving the chip in a first direction may be detected using a first light detector, and light leaving the chip in a second direction (e.g., orthogonal to the first direction) may be detected using a second light detector. The detected light may be fluorescence. An orientational feature of a plurality of cells in the sample may be determined based on the light detected by the detectors. Based on the orientational features and the detected light, a biasing operation may be performed for each cell in the sample to obtain a modified proportion of cell types in the sample.

Abstract: A microfluidic system configured to focus particles suspended in a fluid. One general aspect includes a microfluidic system comprising one or more substrates and a focusing channel formed in the one or more substrates and spanning a length from an inlet to an outlet for receiving a flow of particles suspended in fluid, wherein the particles have a diameter (a) and the focusing channel has a hydraulic diameter (dh).

Abstract: The present invention provides rapid genomic diagnostic methods of in vitro produced embryos. Cell-free DNA can be obtained from the culture medium of blastocysts, and then added to a LAMP or PCR amplification reaction to detect the presence or absence of a certain DNA sequence. Colorimetric or fluorescent indicators such as metallochromatic dyes, intercalating dyes, or pH indicator dyes can be added to the reaction so that amplification can be tracked without additional steps. Provided are methods of sex determination and detection of a specific gene edit, as well adverse or desirable phenotypes. Methods of genotyping using cell-free DNA are also provided.

Abstract: The present disclosure relates generally to methods for improving traits or fitness in cattle, pigs, or aquatic organisms via gene editing. In particular, the methods comprise modifying genes in embryonic stem cells of livestock (e.g., cattle, pigs, or fish) for the purpose of improving a trait (e.g., promoting pathogen resistance, fertility, lactation, and native traits that support more rapid growth or feed efficiency). The methods particularly include CRISPR-CAS9 editing of porcine and bovine embryonic stem cells. Also disclosed herein are methods of producing and cultivating porcine embryonic stem cells under feeder free conditions.

Abstract: Technologies for assessing, quantifying and isolating sperm cell populations and/or subpopulations having specific genetic signatures are provided, as well as methods and systems to assess the efficacy of chromosomal differentiation processes. Compositions for identification and differentiation of X-chromosomes and Y-chromosomes in DNA are also provided.

Abstract: The present teachings provide for a method of breeding livestock in vitro. Provided are steps to create embryonic stem cells from a plurality of blastocysts, genotype the embryonic stem cells to select the best embryos for mating to create offspring, and induce the embryonic stem cells into primordial germ cell-like cells (PGCLCs). Male PGCLCs are further induced into spermatogonial stem cell-like cells, and then spermatid-like cells. Female PGCLCs are induced into oocytes, which are then matured. The resulting gametes can then be mixed with each other or with opposite sex gametes from animals with desirable genetics to create the next generation of embryos, which can then be run through the process again. This method of in vitro breeding can be used to increase the speed of genetic progress in livestock.

Abstract: Disclosed are gene edited ungulate animals and methods of editing genes that control various ungulate traits. Such methods include CRISPR, zinc fingers, or TALENS. Exemplary traits for editing include polled, sterility or fertility, milk production, growth (which increases meat production), fat production, conception rates, stillborn rates, calving ease, or content of produced milk such as the amount of protein or the amount of fat. Other traits include backfat thickness, intramuscular fat, ultrasound loin muscle area, loin muscle area and intramuscular fat content, chest circumference, withers height, body length, hip height, rump length, and heart girth. Other exemplary native traits include, but are not limited to, high altitude adaptation and response to hypoxia, cold acclimation, body size and stature, resistance to disease and bacterial infection, reproduction, milk yield and components, and feed efficiency.

Abstract: A cytometer includes an avalanche photodiode, a switching power supply, a filter, and voltage adjustment circuitry. The switching power supply includes a feedback loop. The filter is electrically connected between the switching power supply and the avalanche photodiode. The voltage adjustment circuitry adjusts a voltage on the feedback loop based at least in part on a voltage measured between the filter and the avalanche photodiode.

Abstract: A method and related apparatus for confirming whether a kill laser successfully destroys an undesired population of cells includes introducing fluorescent dye into cells, exciting the cells with a detection laser or a light emitting diode to cause the cell to fluoresce for a first time, measuring the amount of fluorescence in the cells with a detector capable of emitting a detection pulse, classifying the cells via embedded processing as undesired or desired cells based on the amount of fluorescence, firing a kill beam with a kill laser at any undesired cells, measuring the amount of fluorescence in the cells a second time to determine whether a fluorescent event was generated from the kill beam striking the cells, and providing feedback to an operator of the kill laser as to whether any fluorescent events were generated from the kill beam striking the cells.

Abstract: 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.

Abstract: A method of choosing which undesired cell to destroy in a multi-cell fluorescent event includes detecting fluorescence of cells, converting photons detected in the fluorescence into an analog voltage output signal, and identifying at least two discernable peaks associated with the cells. By looking solely at properties measured within the multi-cell fluorescent event, a decision of which cell to target for elimination can be made. Using this method with large population sizes can result in an effective sex skewed product. The sex skewed product can, for example, be formed from bull semen which is then later used to inseminate cows which results in an increased likelihood of giving birth to female cattle.

Abstract: The present teachings provide for methods of separating live and dead spermatozoa cells from non-human mammalian sex-selected semen. Sex-selected semen is first treated with proteinase K and TCEP, then applied to a density gradient media for separation. Separation is typically accomplished through centrifugation. Commercially available density gradient media include PERCOLL® and BOVIPURE®. Separation of live and dead cells in sex selected semen allows for precise quantification of sex skew using ddPCR™.

Abstract: Provided are genetically edited animals, particularly dairy animals such as cows or heifers, that lactate without having first been pregnant. Also provided are methods and reagents for creating genetically modified animals. Specifically provided are genetically edited dairy animals that have a histidine to arginine substitution in the prolactin receptor gene.

Abstract: An integrated system and method for preparing sperm cells to improve their survivability during cryopreservation are described herein. The system features a vessel, a controlled dispenser, and a dispense tube. The dispense tube has a first end fluidly connected to the dispenser and a second end disposed inside the vessel. The second end can be submerged in the sperm cell fluid. The controlled dispenser may be a syringe pump that includes a syringe for containing a cryoprotectant and a pushing mechanism for displacing the syringe. The syringe pump is configured to discharge the cryoprotectant through the dispense tube and into the vessel, thereby dispensing the cryoprotectant into the sperm cell fluid. Mixing of the fluids is achieved using a shaker table agitation.

Abstract: A cytometer includes an avalanche photodiode, a switching power supply, a filter, and voltage adjustment circuitry. The switching power supply includes a feedback loop. The filter is electrically connected between the switching power supply and the avalanche photodiode. The voltage adjustment circuitry adjusts a voltage on the feedback loop based at least in part on a voltage measured between the filter and the avalanche photodiode.

Abstract: Technologies for cryopreserving ungulate embryos for implantation into recipient females are described.