Journal Abstract:

 Advances in Animal and Veterinary Sciences 2 (4): 226 – 232

 

INTRODUCTION

Background

The Food and Agricultural Organization predicts that in the next 50 years, food demand will increase by 100%, with 70% of it coming from the agricultural sector. The demand for livestock products such as meat, milk, and others continues to rise, making the sustainability of livestock farming crucial. Semen sexing or embryo technology is an effort to apply breeding technology and strategies to improve production. Determining the gender at the beginning of a breeding program reduces management costs and enhances the genetic quality of animals.

Semen sexing techniques have been modified and commercially used in several countries with success rates around 90%. Development in techniques and instruments to accurately sort spermatozoa without damaging them has accelerated the progress of this technology. This review aims to explore the advances in spermatozoa sexing technology from various perspectives.

Basic Principles of Gender Selection

Males produce two types of spermatozoa: X or Y, which determine the gender of the offspring during fertilization. The pragmatic approach to gender separation of spermatozoa involves separating X and Y chromosome-containing spermatozoa for use in artificial insemination. Some differences known between X and Y spermatozoa include X carrying more DNA on its chromosome, X being larger than Y, Y having greater motility, X having a negative charge while Y has a positive charge, and differences in antigen expression. Among these differences, DNA content difference holds the most potential as the basis for gender separation technique. The DNA content difference between X and Y in various species and breeds is presented in Tables 1 and 2.

SPERMATOZOA SEXING METHODS

Albumin Gradient or Swim Down Gradient

            This technique is based on the difference in swimming ability of X and Y spermatozoa through a gradient solution. Y spermatozoa, being smaller and more motile, have a higher likelihood of penetrating the gradient solution. Different fractions in the solution are separated to create specific X and Y spermatozoa solutions. Success rates for this method have been reported at around 75%.

Percoll Gradient

This separation method uses the difference in sedimentation characteristics between X and Y spermatozoa. X spermatozoa have a higher sedimentation density than Y spermatozoa. X settles below, while Y settles above. Success rates for this method range from 86% to 94%.

Swim Up

            Size serves as the basis for separation using the swim-up method. Y spermatozoa swim faster due to their smaller size. Success with this method is around 81%.

Free Flow Electrophoresis

This method is based on the difference in charge between X and Y spermatozoa. X spermatozoa have a negative charge, and Y spermatozoa have a positive charge. Based on this, spermatozoa are separated by applying an electric charge.

Identification of H and Y Antigens

Identifying proteins on the surface of X and Y spermatozoa through immunological methods forms the basis for separation in this method. This approach can be done on a large scale. Separating spermatozoa using H-Y antigens (expressed by Y spermatozoa) through chromatography affinity or magnetic beads yields success rates above 90%.

Sperm Selection Based on Volume Differences

This method uses interference microscopy to examine the volume size difference in spermatozoa's DNA content between X and Y spermatozoa. This technique becomes the basis for flow cytometry sexing. Success rates for this method are less than 80%.

Centrifugal Counter Current Distribution

This separation process divides spermatozoa into different phases: slow, moving, and upper phases. Centrifugation at specific speeds separates the X and Y-bearing portions.

Flow Cytometry

Flow cytometry involves separating sperm using a laser to view DNA inside cells. Specific DNA content and DNA percentages are the main basis for separation. Spermatozoa are treated with a solution that interacts with living sperm and binds to DNA. UV laser scans individual spermatozoa and observes the fluorescent light resulting from reactions with the solution. X spermatozoa exhibit more DNA staining than Y spermatozoa. Success rates for this method range between 85% and 95%.

Among the sexing methods, flow cytometry is the most popular due to its effectiveness in producing offspring and predicting their gender.

Sperm Sorting Methods and Efficiency

Sexing using flow cytometers depends on sperm heads passing through laser shots. Among various livestock, cattle are most efficiently sorted using flow cytometry due to their relatively uniform head sizes, enabling easy laser penetration. Current flow cytometry can analyze 20,000 spermatozoa per second, separating 6,000 or more spermatozoa per second with a 90% success rate. Under these conditions, 15-20 million X and Y spermatozoa can be produced in an hour.

USDA-developed sperm sorters use injection needles with sorting nozzles applied to flow cytometry. High-speed flow cytometry was also developed by Livermore National Laboratory and has been commercialized.

Impact of Sexing

Sexing has an impact on spermatozoa. Effects that may arise from the sexing process include cell death, abnormalities, and decreased motility. Influential factors include diluents, sorting speed, pressure, laser, and others.

Effect of Solutions

Solution addition can damage chromatin. Among various animals, pig chromatin is relatively more sensitive. Addition of solution can lead to heat shock.

Pressure and Speed of Sexing

High speed (55-60 mph) and high pressure (40-60 psi) weaken spermatozoa during sorting.

Laser

Laser power of 200 MW or higher negatively affects fertility rates by damaging chromatin strength.

 Efforts to Reduce Damage During Sexing

  • Lowering pressure to 50 or 40 psi
  • Using UV laser with argon
  • Adding 10% semen plasma
  • Adding diluents (tris, citric acid, fructose)
  • Further research is needed to reduce damage caused by the sorting process

 

Pregnancy Rates Using Sexed Semen

Conception Rate (CR) using sexed semen ranges from 50-60%. In other studies, CR is around 45% in heifers and 28% in lactating cows. Meanwhile, in China, the success rate of sexed semen is higher at 69.7%.

 

Factors Limiting Sexed Semen

  • Expensive sexing equipment costs
  • High maintenance costs
  • Slow process
  • Half of sperm samples are discarded
  • Efficient sexing can only be done on fresh semen

 

Conclusion

The success of semen sexing industry relies on the speed, accuracy, and fertility of sexed semen. Current technology needs improvement to support these aspects. Flow cytometry is currently the most effective method among others. It's highly likely that this sexing technique will be further developed with ongoing research. Identifying specific markers on the surface of X or Y spermatozoa could be a future development option. Adding features to eliminate unwanted sperm could be integrated into the sexing method. It's also feasible to produce males that can produce only specific X or Y sperm, but this requires in-depth research. Nevertheless, current technology demands good management and care.