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Tutorials : Apr 1, 2011 ( )
Real-Time Cytometric Assay for Gender Bias
New Methodology Makes It Faster and Easier to Selectively Bias Births of Female Dairy Cows
The 2011 Final Report of the Foresight Global Food and Farming Futures Project predicted that global population size will increase from nearly seven billion today to eight billion by 2030, and probably to over nine billion by 2050. With these increases in population, global food production will need to increase without the use of substantially more land and with diminishing impact on the environment.
Technologies that support increased, sustainable productivity and which are practical for the poorest farms to adopt can increase the supply of diverse foods at affordable prices. Dairy products are one of the most efficiently produced and safely transported protein foods. However, to meet the demands of population growth, the current 277 million worldwide dairy cow population needs to expand. This is not an easy feat: dairy farmers are already in constant need of more female calves to grow their herds and replace their aging cows.
Artificial insemination (AI) is widely used for breeding dairy cattle and has made bulls of high genetic merit available to all. A normal ejaculate from a dairy bull will contain 5 to 10 billion sperm, which can be diluted and used to inseminate 300 to 1,000 cows. The natural bias leans slightly toward bull calves as with most mammal species. While the basic principles controlling the sex of mammalian offspring are well known, only recently has technology allowed the advent of commercialization of sexed semen.
Female sperm have 3–4% more DNA than male sperm. Traditional sexed semen techniques rely on this difference in DNA content to separate female, male, and undefined sperm on a fluorescence-activated cell sorter. The process sorts sperm with high (approximately 90%) purity but is inefficient and requires extensive manipulation of sperm prior to cryopreservation.
In addition cell sorters are expensive, both in terms of instrument acquisition and day-to-day operation. Extensive training and a dedicated operator are typically required. Recovery of female sperm after sorting is only about 15%. This, combined with slow sorting speeds, has limited the application of sex-sorted semen to low-dose inseminations of about 2 million sperm, instead of the 10–20 million sperm used in conventional doses. Female birth rates do increase (up to 80%) but with drawbacks—lower conception rates, process variability, a steep learning curve, and high instrumentation costs. Easy-to-perform, cost-effective complimentary alternatives are needed to make widespread commercialization of sexed-sperm techniques viable.
Natural (Sex) Selection
The EnGender™ biomarker assay from Arex Life Sciences is used in real time to guide the processing of semen from elite dairy bull sires into doses of sperm for the artificial insemination of cows and heifers. The EnGender assay uses the Cohen biomarker to monitor cell surface changes that have different temporal occurrences post collection on female and male sperm.
X-bearing sperm (female sperm) gain expression of the biomarker and ability to fertilize slightly later than male sperm (Figure 1). Expression of the biomarker is measured in small samples of the ejaculate in real time to adjust the processing time to bias fertilization by female sperm.
Results from three trials, containing over 957 calf births and 300 fetal ultrasounds, on working dairy farms, demonstrated an 8–20% increase in female calvings to both heifers and cows. This supports Cohen’s hypothesis that temporal, rather than physical, separation can be used to obtain female-biased doses for AI, which eliminates the concerns of sperm loss and damage.
The EnGender biomarker assay does not rely on complex, expensive cell sorting for gender bias; rather biomarker expression is monitored by fluorescence measurement. Early work during assay development used fluorescence microscopy to score the biomarker fluorescence. However, this was so labor intensive in a real-time environment that it caused process deviations; it was too difficult to collect enough assay points and as a result, quality was compromised.
Analysis of Samples
Flow cytometry allows analysis of thousands of sperm per sample, achieving a much higher precision than is possible with microscopic assessment, and removes the subjectivity and labor intensity affiliated with microscopic scoring. However, flow cytometers are not common commodities at stud farms, and the high cost, complexity, large footprint, and special installation requirements of most cytometers preclude them from becoming standard equipment at small farms.
In order to measure enough cells in real time, Arex is using the Accuri® C6 Flow Cytometer® System. The small footprint and computer workstation make the C6 (Figure 2) highly portable. Portability combined with ease-of-use and low acquisition and operating costs make the C6 a good choice for quick, real-time use in a crowded bull hall.
Samples for the biomarker assay are prepared by transferring a few microliters of semen from the ejaculate to a tube containing EnGender biomarker assay reagents. The sample is incubated for 20 minutes and then analyzed on the C6 to measure the percentage of sperm that are positive for the Cohen biomarker.
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.
Erin Gatza, Ph.D., is applications scientist and MaryAnn Labant (MLabant@accuricytometers.com) is marketing communications manager at Accuri Cytometers. Ron Parkinson is CEO and Barb A. Cohen, Ph.D., is founder and CSO at Arex Life Sciences.
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