Sexed Semen Technology
The goal of sexed semen technology is to generate a calf of a specific sex. Because of this, sex preselection has a significant impact on the profitability of livestock-based industry.
Females are required for milk production and the production of calves, while male calves are usually chosen for meat production because of their better feed conversion efficiency and lean-to-fat ratio. Furthermore, males of superior genetic quality are utilized as sires in artificial insemination programs.The principle behind the sexed semen technology is the inherent differences between X and Y chromosome-bearing spermatozoa in mass and motility, surface charges, swimming patterns, volumetric differences, centrifugal countercurrent distribution, and immunological properties (Table 24.1).
24.3.1 Procedure
The flow cytometry method of sperm sorting is the only approach that has shown to be economically feasible and
Table 24.1 Differences between X- and Y-bearing spermatozoa
| Properties | X-bearing sperm | Y-bearing sperm | Methods of separation |
| Size | Larger | Smaller | Percoll gradient |
| Motility | Slower | Faster | Swim-up technique |
| Surface charge | Faster migration to cathode | Slower migration to cathode | Free flow electrophoresis |
| Sperm surface antigen | Absence of HY surface antigen | Presence of HY surface antigen | Immunological sexing |
| DNA content | More DNA | Less DNA | Flow cytometry |
Fig. 24.1 DNA content differences of X and Y chromosome-bearing spermatozoa in different domestic animal species. (Graphical representation drawn from Garner and Siedel 2003)
DNA content difference (%)
yielded encouraging results thus far.
The difference in DNA content between X and Y chromosome-bearing spermatozoa is utilized for separating two types of spermatozoa. Figure 24.1 represents differences in DNA content in domestic animal species.Semen sample to be sorted is first diluted with bis-benzimide (Hoechst 33342) dye in standard dilution. The dye Hoechst 33342 diffuses across an intact sperm membrane and binds to the A/T base pairs in the minor groove of DNA. Hoechst 33342 has a 350/460 nm absorption and fluorescence emission spectra, which makes it a highly useful marker for determining the precise quantity of DNA in sperm cells. The flow cytometer measures the DNA content difference between two types of sperm using two fluorescence detectors that measure the strength of the signal from the Hoechst 33342 attached to the sperm DNA when excited by a laser. The diluted DNA is allowed to pass through the flow cytometer channel and a vibrating crystal breaks the stream into droplets. An argon laser beam of 351 and 364 nm wavelength illuminates the stained sperm. X chromosome-bearing sperm with more DNA content glows brighter than the Y chromosome-bearing sperm. The two separated populations are deflected into opposite streams for collection due to the presence of charged plates at the discharge point. On the flow cytometer, fluorescence histograms differentiate the sorted populations, and the software also allows for the gating out of dead and moribund sperm. An enriched population of flow-sorted sperm is collected by relative gating of the individual population (Fig. 24.2).
Due to multiple processes involved with the conventional method of sperm sorting there occurs irreversible alteration in sperm which ultimately leads to the compromised fertility potential of sex-sorted semen. Insemination with sex-sorted semen shows 10% less fertility compared to unsorted semen and increasing the number of sex-sorted sperm per inseminate cannot bridge this gap. The fertility of sex-sorted semen is compromised by irrevocable biochemical changes occurring due to additional subprocesses during sorting procedure including a prolonged holding time prior to staining, exposure to a laser beam, separation into X- and Y-sperm and finally exposure to an electrical field for enrichment of sorted population. Drastic changes in the sperm environment in every subprocess are caused due to mechanical, physical, and biochemical stresses to the sperm cells.
24.3.2 Next Generation Sperm Sorting Technologies
Advanced methods of sperm sorting technologies involve several modifications of different steps in the original technique. Pretreatment of sperm, optimization of the medium in which sperms are maintained, sperm staining medium, sheath fluid, and freezing medium are such modifications that have been integrated to maintain the physiological pH of the sperm
Fig. 24.2 Flow cytometric separation of X- and Y-bearing spermatozoa. Plot 1 identifies live/ dead sperm populations and gates only the cells within the oriented region to plot 2 and plot 3. All the dead and moribund sperm are removed from the sorting process. Plot 2 allows for the gating of the desired sex (X or Y or both) and Plot 3 evaluates resolution by measuring peak to valley ratio (PVR). (Source: Vishwanath and Moreno 2018)
throughout the procedure and ensure low-dose freezing after sorting. Furthermore, the introduction of microfluidics- and nanotechnology-based tools has aided in the rapid progress of sperm sorting technologies recently.
24.3.2.1 Microfluidics Dielectrophoretic Chip-Based Sperm Sorting
Microfluidics is a technique for controlling tiny volumes of fluid on the micro- and nanoscale through channels that are less than a thousand micrometers in diameter. It may be used to separate a variety of cells, including sperm. Dielectrophoresis (DEP) is a noninvasive cell separation technology that uses nonuniform electric fields in a suitable solution to regulate cell mobility. Cells with different electrical surface properties are separated by being attracted toward (positive DEP; pDEP) or repelled from (negative DEP; nDEP) the location of the greatest electric field. DEP is frequently amalgamated with the microfluidic chip method to achieve high performance for sorting applications. The flagellum pattern and velocity of human X and Y sperm differ, and this difference is depending on the dielectrophoretic field and medium employed.
Based on this difference the combined technology, the microfluidic dielectrophoretic system (MF-DEP) has been preliminarily applied for the enrichment of X chromosome-bearing sperms in bull. The technique is safe for sperm and the efficiency of sorting depends upon several factors like electrical voltage applied, sorting cycle, sorting buffer, flow rate, and frequency. Further efforts are much required for large-scale application of this technology in livestock sector.24.3.2.2 Magnetic Nanoparticle (MNP)-Based Sperm Sorting
MNP-based sperm sorting relies on the difference in zeta potential between X and Y chromosome-bearing sperm. Zeta potential is a negative electro-kinetic potential of about -16—20 mV acquired by the sperm membrane during spermatogenesis and epididymal maturation as a result of sperm surface coating with sialic acids. Semen sample is mixed with negatively charged nanosize magnetic microbeads (about 50 nm in diameter), incubated for 10 min, and then exposed to a magnetic field for around 20 min to isolate X- or Y-bearing sperm. The Zeta potential of Y-bearing spermatozoa is -16 mV, whereas the Zeta potential of X-bearing spermatozoa is -20 mV; hence, the Y-bearing sperm population will form complexes more easily by binding to the MNP. The complexes will stick to the inner wall of the test tube if a magnetic force is applied to it, while the X-bearing population will remain suspended in the medium and may be recovered by gentle aspiration. Preliminary studies in equines have revealed that MNP-based sperm sorting does not alter the sperm qualitative attributes like viability, motility, and chromatin integrity, and, thus, maybe a good alternative to traditional flow cytometrybased sorting.
24.4