Journal of Animal Reproduction and Biotechnology (한국동물생명공학회지)

The Korean Society of Animal Reproduction and Biotechnology (한국동물생명공학회)
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Magnetic-activated cell sorting improves high-quality spermatozoa in bovine semen

  • Received : 2021.03.22
  • Accepted : 2021.06.06
  • Published : 2021.06.30
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Abstract

The objective of this study was to establish a selection process for high quality sperm in bovine semen using sperm separation by magnetic activation (MACS). For this, semen from 21 Nellore bulls was collected using an artificial vagina. To guarantee the presence of pathologies in the ejaculate, animals previously declassified in four consecutive spermiogram were used. Semen was analyzed in five statuses: (1) fresh semen (fresh); (2) density gradient centrifugation (DGC), percoll column; (3) non-apoptotic fraction after separation by MACS (MAC); (4) apoptotic fraction from the separation (MACPOOR); and (5) MAC followed by DGC (MACDGC). Using a computerized analysis system (CASA), motility was measured. The sperm morphology was evaluated by phase contrast, and the supravital test was completed with eosin/nigrosin staining. For DGC, 20 × 106 cells were used in a gradient of 90% and 45% percoll. MACS used 10 × 106 cells with 20 μL of nanoparticles attached to annexin V, and filtered through the MiniMACS magnetic separation column. Membrane integrity was assessed with SYBR-14/IP and mitochondrial potential with JC-1 by flow cytometry. Processing sperm by MACDGC, was more effective in obtaining samples with high quality sperm, verified by the total of abnormalities in the samples: 35.04 ± 2.29%, 21.50 ± 1.47%, 17.30 ± 1.10%, 30.68 ± 1.94% and 10.50 ± 1.46%, respectively for fresh, DGC, MAC, MACPOOR, and MACDGC. The subpopulation of non-apoptotic sperm had a high number of live cells (82.65%), membrane integrity (56.60%) and mitochondrial potential (83.98%) (p < 0.05). These findings suggest that this nanotechnological method, that uses nanoparticles, is efficient in the production of high-quality semen samples for assisted reproduction procedures in cattle.

Keywords

Acknowledgement

Dr. Paula A. Navarro, Human Reproduction Division, Department of Gynecology and Obstetrics, Ribeirao Preto School for the temporary assignment of MiniMACS equipment. Alta Genetics, Uberaba, MG for the facilities and animals used in the research. This research did not receive any specific scholarship from public, commercial or non-profit sectors. This study was financed in part (english review service) by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior - Brasil (CAPES) - Finance Code 001.

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