Author : Rini Widyastuti

Summary : Sperm separation is the process of altering the proportion of sperm obtained with X or Y chromosomes using a specific method to determine the sex outcome of the offspring. There have been several sperm separation methods developed. It includes albumin columns, density gradient centrifugation, electrophoresis, H-Y antigen, filtration with a Sephadex column, and flow cytometry. Flow cytometry is the only method with an accuracy 85%-95%. This flow cytometry technique is reasonably expensive, takes a long time, and damages the separated sperm, which causes a decrease in sperm fertility and abnormalities in the fertilized embryo. Therefore, there is still a need to develop recognized techniques for a successful natural strategy for sorting X- and Y-chromosome-bearing sperm.

Scientists believed X and Y sperm could be selected based on different migration rates in acidic and basic media. The vagina has a moderately acidic pH that ranges from 3.8 to 4, which may play a role in the selection of X- or Y-bearing sperm and thus affect the sex of the offspring. Furthermore, the previous research reported that Y sperm could not survive long in acidic media, high temperatures, or environments with a high reactive oxygen species (ROS) content. Our study employed to perform sperm sexing based on the hypothetical existence of physiological differences between X- and Y-bearing sperm in extreme pH media, as well as the idea that these differences are significant enough to allow sperm separation. This research aims to provide comprehensive information on cellular and molecular changes affecting sperm separation by incubating extreme pH media and the fertilizing of the selected sperm.

The study was divided into three steps: (1) Sperm characteristics in different pH media, (2) Sperm cellular and molecular investigations in incubation using extremely acidic and alkaline medium, and (3) Sperm separation and sperm fertility and embryo development potential. For the first step, we determine the range of pH values tolerable for sperm and the effect on sperm quality. The 250 x 10 to the 6th power sperm/ml frozen-thawed Pasundan Cattle sperm was divided into ten equal aliquots, and each was diluted in the medium within a particular pH value. HCL or NaOH was added to buffer media to create ten different solutions with varying pH values of 3, 4, 5, 6 as acidic, 7.2-7.4 as a control, and 8, 9, 10, 11, 12 as alkali. Furthermore, the samples were incubated for 5 minutes at room temperature within a particular pH medium before being immediately supplemented with a buffered medium to achieve a pH of 7.2-7.4. After 10 minutes of incubation at room temperature, all of the parameters were assessed. For the second step, we do the same procedure for thawing and preparing the incubation media as in the first research step. The sperm were exposed to acid (pH: 4), alkali (pH: 11), and control medium (pH: 7.2 -7.4) for 5 minutes, then returned to physiological media and incubated for 10 minutes at room temperature. Later, sperm motility, sperm viability, membrane integrity, acrosome intact, morphology, and DNA integrity were observed at 0, 15, and 30 minutes after incubation at room temperature. The level of H2O2, mitochondrial activity, and intracellular Ca2+ was analyzed by using CLSM. The fertility of sperm was evaluated by the ICSI method using a selected sperm exposed to pH 4 and pH 11 compared to control. In addition, the qPCR was used to analyze the mRNA abundance of PLP, ZFY, BAX, and PLC for sperm and PLP, ZFY, and GLUT3 for embryos resulting from ICSI for sperm fertility.

The results showed that sperm is still tolerable in pH 4-pH 11, but the sperm quality degrades as the acidity or alkaline level increases. The sperm separation by incubated bovine sperm in pH 4 and pH 11 media decreases sperm motility, viability, membrane integrity, acrosome intactness, normal morphology, induced apoptosis, and DNA damage. Furthermore, it also impaired the mitochondrial activity and intracellular Ca2+, which reduced sperm fertility leading to low cleavage rate and zygote arrest. The incubation of sperm in pH 4 seems effective to separate X-chromosome-bearing sperm, while pH 11 media seems effective in separating Y-chromosome-bearing sperm. According to the abundance level of the GLUT3 gene, the fertilized oocytes using selected sperm incubated in both pH 4 and pH 11 media have the same capacity to develop into blastocyst as the control group.

H202, one ROS that slows motility and interferes with ATP synthesis, is generated when pH changes arise during incubation. The excessive rise in ROS induces oxidative stress that damages the sperm membrane, disturbance of motility, and alters sperm morphology. Furthermore, apoptosis is carried on by the activation of the BAX gene, which damages sperm DNA, and the stimulation of mitochondria to release pro-apoptotic substances that enhance mitochondrial activity and raise intracellular Ca2+ levels via the Catsperm channel. Sperm treated in pH 4 or pH 11 environments reveal hyperactivation, capacitation, and spontaneous acrosomal responses due to alterations in sperm motility patterns due to elevated Ca2+ and mitochondrial activity. The lower cleavage rate in embryos fertilized with separated sperm may be due to the fragmentation of sperm DNA. The embryo, however, can develop into a blastocyst because it has passed the cell block stage, which takes place during the 8-16 cell stage. Due to the low percentage of sperm motility obtained, sperm separation by incubation in pH 4 and pH 11 medium can be used in assisted reproductive technology (ART) through the ICSI procedure.