Elimination of apoptotic boar spermatozoa using magnetic activated cell sorting

Abstract

One of the features of apoptosis is the externalization of phosphatidylserine which could be used to remove apoptotic cells from semen preparations. Magnetic-activated cell sorting using annexin V-conjugated microbeads which bind to phosphatidylserine could be used to enhance semen quality. Twelve boar semen samples after 3 days of liquid storage at 16 -17 °C were subjected to magnetic-activated cell sorting. Bound and unbound fractions and control samples were subjected to flow cytometry following the staining of spermatozoa with Annexin V conjugated with Alexa Fluor 488 and propidium iodide. Four subpopulations were obtained: live, early apoptotic live, late apoptotic, early necrotic dead and late necrotic dead. The frequency of early apoptotic and late necrotic spermatozoa was significantly higher (P < 0.05) in bound (14.1 ± 10.6% and 24.1 ± 10.2%, respectively) than in unbound fractions (3.4 ± 2.1% and 12.7 ± 3.1%) and control (3.5 ± 1.6% and 12.0 ± 5.0%). The lowest concentration of live spermatozoa was found in the bound fraction (10.6 ± 8.0 %), which differed significantly (P < 0.05) from the control. In unbound fractions there was a significantly higher concentration (P < 0.05) of morphologically normal spermatozoa (31.8 ± 12.6%) compared to bound ones (5.9 ± 7.3%). A significantly (P < 0.05) lower proportion of morphologically normal spermatozoa was observed in both fractions compared to control (67.2 ± 17.0%). Boar spermatozoa were separated by the above method for the first time, however, the results showed this method to be inappropriate for boar semen separation under the tested conditions. Semen, short-term storage, MACS, apoptosis

Magnetic-activated cell sorting of non-apoptotic spermatozoa improves the quality of embryos according to female age: a prospective sibling oocyte study

“…2 The outcome of the ICSI cycles in terms of oocytes and embryo development according to the sperm selection method among women aged 31 years or more. The only observed statistically significant difference (marked with asterisk) was a higher proportion of good quality day 5/6 blastocysts following MACS-ICSI treatment (33.3 and 75.0% for the ICSI and MACS-ICSI procedures, respectively; P = 0.028) [34]. This study also showed that MACS selected nonapoptotic fraction contains significantly lower proportions of normal spermatozoa as a control [34].…”

“…The only observed statistically significant difference (marked with asterisk) was a higher proportion of good quality day 5/6 blastocysts following MACS-ICSI treatment (33.3 and 75.0% for the ICSI and MACS-ICSI procedures, respectively; P = 0.028) [34]. This study also showed that MACS selected nonapoptotic fraction contains significantly lower proportions of normal spermatozoa as a control [34]. The authors of this study suggest that boar spermatozoa are more sensitive than humans, or maybe the magnetic field could have some negative influence.…”

Optimization of Facs Sorting for the Improvement of Livestock Semen Quality

“…However, at the present sorting of apoptotic cells using YO-PRO-1 staining is possible only via FACS as there is no available similar commercial kit conjugated with nanoparticles as for example the Annexin V MicroBead kit (Miltenyi Biotec, Germany) for the MACS removal of apoptotic cells. Although, the latter mentioned kit is successfully used in human assisted reproduction (Said et al, 2006); the largescale application of this method for the improvement of livestock semen quality has been proven as unsatisfying for rabbit (Vasicek et al, 2014) or boar (Mrkun et al, 2014). On the other hand, the MACS technique offers a much bigger yield of sorted cells if automatic instrument is used for a comparable time as FACS that usually resulted with a lower concentrated cell fraction.…”

“…In recent years, the Magnetic-activated cell sorting (MACS) has been used to improve the quality of human or livestock semen based on the elimination of apoptotic spermatozoa via binding of Annexin V-conjugated nanoparticles to externalized phosphatidylserine (Grunewald et al, 2001). This method can enhance the reproductive outcomes in human clinical practice (Said et al, 2006;Oseguera-López et al, 2019), but not in livestock production as reported in rabbits (Vasicek et al, 2014) or boar (Mrkun et al, 2014). On the other hand, it was observed that bull sperm cells with damaged membrane can be effectively removed using propidium iodide-conjugated magnetic nanoparticles (Fox et al, 2012).…”