Replacement of Albumin by Preovulatory Oviductal Fluid in Swim-Up Sperm Preparation Method Modifies Boar Sperm Parameters and Improves In Vitro Penetration of Oocytes

Abstract

More suitable and efficient methods to protect gametes from external harmful effects during in vitro handling can be achieved by adding preovulatory porcine oviductal fluid (pOF) to in vitro culture media. The objective of this study was to assess the swim-up procedure's suitability as a sperm selection method using a medium supplemented with 1mg/mL BSA, 1% preovulatory pOF (v/v), 1% v/v pOF plus 1mg/mL BSA, and 5mg/mL BSA. After selection, various sperm parameters were studied, such as sperm recovery rate, sperm morphology, motility (by CASA), vitality, acrosome status and intracellular calcium (by flow cytometry) and ability to penetrate oocytes in vitro. Around 2% of sperm were recovered after swim-up, and the replacement of BSA by pOF showed a beneficial reduction of motility parameters calcium concentration, resulting in an increased penetration rate. The combination of albumin and oviductal fluid in the medium did not improve the sperm parameters results, whereas a high concentration of BSA increased sperm morphological abnormalities, motility, and acrosome damage, with a reduction of calcium concentration and penetration rate. In conclusion, the replacement of albumin by preovulatory oviductal fluid in the swim-up sperm preparation method modifies boar sperm parameters and improves the in vitro penetration of oocytes.

Keywords: albumin; oviductal fluid; porcine; sperm selection; swim-up.

Figure 1

Schematic of the workflow procedure. Refrigerated ejaculated boar spermatozoa were centrifuged (Before swim-up group; SU) and then subjected to swim-up selection method in a medium supplemented with different protein combinations of bovine serum albumin (BSA group) and late follicular phase-porcine oviductal fluid (pOF): 1 mg/mL BSA (1BSA), 1% v/v pOF (1pOF group), 1 mg/mL BSA + 1% v/v pOF (1BSA-1pOF group) and 5 mg/mL BSA (5BSA group). Samples were incubated for 20 min at 38 °C with the tube inclinated 45° for swim-up selection. After selection, recovery rate, morphology, motility parameters, sperm viability, acrosome integrity, intracellular calcium concentrations and fertilization results after IVF in TALP medium supplemented with 3 mg/mL BSA were evaluated. S/O: number of spermatozoa per penetrated oocyte; S/ZP: number of spermatozoa bound to the zona pellucida of penetrated oocytes; PNM: male pronucleus formation rate.

Figure 2

Motion parameters in ejaculated boar spermatozoa selected by swim-up method with different protein supplementation: 1 mg/mL BSA (1BSA), 1% v/v pOF (1pOF), 1 mg/mL BSA + 1% v/v pOF (1BSA-1pOF) and 5 mg/mL BSA (5BSA). Data were evaluated by computer-assisted semen analysis (CASA) and represented as mean ± SEM. ^a–c Values for each parameter are significantly different (p-value < 0.05). VSL; straight-line velocity; VAP, average path velocity.