Light-scatter signals from the sperm are initially plotted on a 2-D dot plot of forward scatter versus side scatter, and a gate (Figure 3A, gate R1) is drawn to exclude debris. Biomarker expression by sperm within the R1 gate is determined using a ligand conjugated to AlexaFluor® 488, detected in the FL1 channel of the C6, and represented in histogram plots (Figure 3B and C).
Early studies indicated that assessing biomarker expression at four or more time points is important for defining the peak of biomarker expression for a particular ejaculate. While the timing from ejaculate collection to peak-biomarker expression can vary between ejaculates, early studies indicated that the time to progress from peak biomarker expression to the point of obtaining female bias is relatively constant. Therefore, to allow straightforward sample preparation and analysis in real time, biomarker expression is currently the only parameter monitored.
The samples run during the course of incubation detect the biomarker change that dictates the appropriate time for further processing of the ejaculate into frozen doses for shipment to dairy farmers. Incorporating the EnGender biomarker assay utilizing the C6 into semen processing allows production of 500–1,000 standard doses (10–20 million sperm per dose) of semen from a single ejaculate, just as with unsexed semen—with the added benefit of gender bias.
To meet the increased food demands caused by population growth, the worldwide dairy cow population will need to expand dramatically over the next two decades. Technologies that selectively bias conception of female dairy cows may enable efficient expansion of milk production while allowing smaller herds.
The EnGender bioassay, along with the C6, can be used to increase births of female calves while maintaining conception rates typically observed following AI with unsexed sperm. Maintaining high conception rates after AI is important; only cows that have been pregnant produce milk.