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.

Department of Veterinary Sciences Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague 165 00 Prague Czech Republic

Department Physiology International Excellence Campus for Higher Education and Research Campus Mare Nostrum and Institute for Biomedical Research of Murcia University of Murcia 30071 Murcia Spain

Laboratory of Reproductive Biology Institute of Biotechnology of the Czech Academy of Sciences BIOCEV 252 50 Vestec Czech Republic

Zobrazit více v PubMed

Sakkas D., Ramalingam M., Garrido N., Barratt C.L.R. Sperm selection in natural conception: What can we learn from Mother Nature to improve assisted reproduction outcomes? Hum. Reprod. Update. 2015;21:711–726. doi: 10.1093/humupd/dmv042. PubMed DOI PMC

Luongo C., González-Brusi L., Cots-Rodríguez P., Izquierdo-Rico M.J., Avilés M., García-Vázquez F.A. Sperm proteome after interaction with reproductive fluids in porcine: From the ejaculation to the fertilization site. Int. J. Mol. Sci. 2020;21:6060. doi: 10.3390/ijms21176060. PubMed DOI PMC

Matás C., Vieira L., García-Vázquez F.A., Avilés-López K., López-Úbeda R., Carvajal J.A., Gadea J. Effects of centrifugation through three different discontinuous Percoll gradients on boar sperm function. Anim. Reprod. Sci. 2011;127:62–72. doi: 10.1016/j.anireprosci.2011.06.009. PubMed DOI

Matás C., Sansegundo M., Ruiz S., García-Vázquez F.A., Gadea J., Romar R., Coy P. Sperm treatment affects capacitation parameters and penetration ability of ejaculated and epididymal boar spermatozoa. Theriogenology. 2010;74:1327–1340. doi: 10.1016/j.theriogenology.2010.06.002. PubMed DOI

Ferraz M.A.M.M., Henning H.H.W., Stout T.A.E., Vos P.L.A.M., Gadella B.M. Designing 3-Dimensional In Vitro Oviduct Culture Systems to Study Mammalian Fertilization and Embryo Production. Ann. Biomed. Eng. 2017;45:1731–1744. doi: 10.1007/s10439-016-1760-x. PubMed DOI PMC

Romar R., Cánovas S., Matás C., Gadea J., Coy P. Pig in vitro fertilization: Where are we and where do we go? Theriogenology. 2019;137:113–121. doi: 10.1016/j.theriogenology.2019.05.045. PubMed DOI

Alvarez J.G., Lasso J.L., Blasco L., Nuñez R.C., Heyner S., Caballero P.P., Storey B.T. Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime. Hum. Reprod. 1993;8:1087–1092. doi: 10.1093/oxfordjournals.humrep.a138198. PubMed DOI

Amiri I., Ghorbani M., Heshmati S. Comparison of the DNA fragmentation and the sperm parameters after processing by the density gradient and the swim up methods. J. Clin. Diagn. Res. 2012;6:1451–1453. doi: 10.7860/JCDR/2012/4198.2530. PubMed DOI PMC

Parrish J.J., Krogenaes A., Susko-Parrish J.L. Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development. Theriogenology. 1995;44:859–869. doi: 10.1016/0093-691X(95)00271-9. PubMed DOI

Arias M.E., Andara K., Briones E., Felmer R. Bovine sperm separation by Swim-up and density gradients (Percoll and BoviPure): Effect on sperm quality, function and gene expression. Reprod. Biol. 2017;17:126–132. doi: 10.1016/j.repbio.2017.03.002. PubMed DOI

Clarke R.N., Johnson L.A. Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro. Gamete Res. 1987;16:193–204. doi: 10.1002/mrd.1120160302. PubMed DOI

Holt W.V., Hernandez M., Warrell L., Satake N. The long and the short of sperm selection in vitro and in vivo: Swim-up techniques select for the longer and faster swimming mammalian sperm. J. Evol. Biol. 2010;23:598–608. doi: 10.1111/j.1420-9101.2010.01935.x. PubMed DOI

Cánovas S., Ivanova E., Romar R., García-Martínez S., Soriano-Úbeda C., García-Vázquez F.A., Saadeh H., Andrews S., Kelsey G., Coy P. DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids. Elife. 2017;6:e23670. doi: 10.7554/eLife.23670. PubMed DOI PMC

García-Martínez S., Sánchez Hurtado M.A., Gutiérrez H., Sánchez Margallo F.M., Romar R., Latorre R., Coy P., López Albors O. Mimicking physiological O2 tension in the female reproductive tract improves assisted reproduction outcomes in pig. MHR Basic Sci. Reprod. Med. 2018;24:260–270. doi: 10.1093/molehr/gay008. PubMed DOI

García-Martínez S., Latorre R., Sánchez-Hurtado M.A., Sánchez-Margallo F.M., Bernabò N., Romar R., López-Albors O., Coy P. Mimicking the temperature gradient between the sow’s oviduct and uterus improves in vitro embryo culture output. Mol. Hum. Reprod. 2020;26:748–759. doi: 10.1093/molehr/gaaa053. PubMed DOI

Hamze J.G., Jiménez-Movilla M., Romar R. Sperm-Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus-Oocyte Complex. Curr. Protoc. Toxicol. 2020;86 doi: 10.1002/cptx.100. PubMed DOI

Coy P., Gadea J., Romar R., Matás C., García E. Effect of in vitro fertilization medium on the acrosome reaction, cortical reaction, zona pellucida hardening and in vitro development in pigs. Reproduction. 2002;124:279–288. doi: 10.1530/rep.0.1240279. PubMed DOI

Romar R., Funahashi H., Coy P. In vitro fertilization in pigs: New molecules and protocols to consider in the forthcoming years. Theriogenology. 2016;85:125–134. doi: 10.1016/j.theriogenology.2015.07.017. PubMed DOI

Acuña O.S., Avilés M., López-Úbeda R., Guillén-Martínez A., Soriano-Úbeda C., Torrecillas A., Coy P., Izquierdo-Rico M.J. Differential gene expression in porcine oviduct during the oestrous cycle. Reprod. Fertil. Dev. 2017;29:2387–2399. doi: 10.1071/RD16457. PubMed DOI

Küçük N., Lopes J.S., Soriano-Úbeda C., Hidalgo C.O., Romar R., Gadea J. Effect of oviductal fluid on bull sperm functionality and fertility under non-capacitating and capacitating incubation conditions. Theriogenology. 2020;158:406–415. doi: 10.1016/j.theriogenology.2020.09.035. PubMed DOI

Kim N.H., Day B.N., Lim J.G., Lee H.T., Chung K.S. Effects of oviductal fluid and heparin on fertility and characteristics of porcine spermatozoa. Zygote. 1997;5:61–65. doi: 10.1017/S0967199400003567. PubMed DOI

McCauley T.C., Buhi W.C., Wu G.M., Mao J., Caamaño J., Didion B.A., Day B.N. Oviduct-Specific Glycoprotein Modulates Sperm-Zona Binding and Improves Efficiency of Porcine Fertilization In Vitro1. Biol. Reprod. 2003;69:828–834. doi: 10.1095/biolreprod.103.016444. PubMed DOI

Coy P., Lloyd R., Romar R., Satake N., Matas C., Gadea J., Holt W.V. Effects of porcine pre-ovulatory oviductal fluid on boar sperm function. Theriogenology. 2010;74:632–642. doi: 10.1016/j.theriogenology.2010.03.005. PubMed DOI

Kumaresan A., Johannisson A., Humblot P., Bergqvist A.-S. Oviductal fluid modulates the dynamics of tyrosine phosphorylation in cryopreserved boar spermatozoa during capacitation. Mol. Reprod. Dev. 2012;79:525–540. doi: 10.1002/mrd.22058. PubMed DOI

Kumaresan A., González R., Johannisson A., Berqvist A.S. Dynamic quantification of intracellular calcium and protein tyrosine phosphorylation in cryopreserved boar spermatozoa during short-time incubation with oviductal fluid. Theriogenology. 2014;82:1145–1153. doi: 10.1016/j.theriogenology.2014.07.029. PubMed DOI

Zapata-Carmona H., Soriano-Úbeda C., París-Oller E., Matás C. Periovulatory oviductal fluid decreases sperm protein kinase A activity, tyrosine phosphorylation, and in vitro fertilization in pig. Andrology. 2020;8:756–768. doi: 10.1111/andr.12751. PubMed DOI

Gadea J., Sellés E., Marco M.A., Coy P., Matás C., Romar R., Ruiz S. Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders. Theriogenology. 2004;62:690–701. doi: 10.1016/j.theriogenology.2003.11.013. PubMed DOI

González-Abreu D., García-Martínez S., Fernández-Espín V., Romar R., Gadea J. Incubation of boar spermatozoa in viscous media by addition of methylcellulose improves sperm quality and penetration rates during in vitro fertilization. Theriogenology. 2017;92:14–23. doi: 10.1016/j.theriogenology.2017.01.016. PubMed DOI

Yeste M., Fernández-Novell J.M., Ramió-Lluch L., Estrada E., Rocha L.G., Cebrián-Pérez J.A., Muiño-Blanco T., Concha I.I., Ramírez A., Rodríguez-Gil J.E. Intracellular calcium movements of boar spermatozoa during “in vitro” capacitation and subsequent acrosome exocytosis follow a multiple-storage place, extracellular calcium-dependent model. Andrology. 2015;3:729–747. doi: 10.1111/andr.12054. PubMed DOI

Petters R.M., Wells K.D. Culture of pig embryos. Biosci. Proc. 1993;48:61–73. doi: 10.1530/biosciprocs.14.005. PubMed DOI

Lopes J.S., Canha-Gouveia A., París-Oller E., Coy P. Supplementation of bovine follicular fluid during in vitro maturation increases oocyte cumulus expansion, blastocyst developmental kinetics, and blastocyst cell number. Theriogenology. 2018;126 doi: 10.1016/j.theriogenology.2018.12.010. PubMed DOI

Abaigar T., Holt W.V., Harrison R.A.P., Del Barrio G. Sperm subpopulations in Boar (Sus scrofa) and Gazelle (Gazella dama mhorr) semen as revealed by pattern analysis of computer-assisted motility assessments. Biol. Reprod. 1999;60:32–41. doi: 10.1095/biolreprod60.1.32. PubMed DOI

Ballester L., Romero-Aguirregomezcorta J., Soriano-Úbeda C., Matás C., Romar R., Coy P. Timing of oviductal fluid collection, steroid concentrations, and sperm preservation method affect porcine in vitro fertilization efficiency. Fertil. Steril. 2014;102:1762–1768. doi: 10.1016/j.fertnstert.2014.08.009. PubMed DOI

Coy P., Canovas S., Mondejar I., Saavedra M.D., Romar R., Grullon L., Matas C., Aviles M. Oviduct-specific glycoprotein and heparin modulate sperm-zona pellucida interaction during fertilization and contribute to the control of polyspermy. Proc. Natl. Acad. Sci. USA. 2008;105:15809–15814. doi: 10.1073/pnas.0804422105. PubMed DOI PMC

Kim S.K., Jee B.C., Kim S.H. Histone methylation and acetylation in ejaculated human sperm: Effects of swim-up and smoking. Fertil. Steril. 2015;103:1425–1431. doi: 10.1016/j.fertnstert.2015.03.007. PubMed DOI

Magdanz V., Boryshpolets S., Ridzewski C., Eckel B., Reinhardt K. The motility-based swim-up technique separates bull sperm based on differences in metabolic rates and tail length. PLoS ONE. 2019;14 doi: 10.1371/journal.pone.0223576. PubMed DOI PMC

Park C.H., Lee S.G., Choi D.H., Lee C.K. A modified swim-up method reduces polyspermy during in vitro fertilization of porcine oocytes. Anim. Reprod. Sci. 2009;115:169–181. doi: 10.1016/j.anireprosci.2008.12.004. PubMed DOI

Boerke A., Tsai P.S., Garcia-Gil N., Brewis I.A., Gadella B.M. Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: Functional relationship with zona binding and the zona-induced acrosome reaction. Theriogenology. 2008;70:1188–1196. doi: 10.1016/j.theriogenology.2008.06.021. PubMed DOI

Espinosa F., López-González I., Muñoz-Garay C., Felix R., De la Vega-Beltrán J.L., Kopf G.S., Visconti P.E., Darszon A. Dual regulation of the T-type Ca(2+) current by serum albumin and beta-estradiol in mammalian spermatogenic cells. FEBS Lett. 2000;475:251–256. doi: 10.1016/S0014-5793(00)01688-4. PubMed DOI

Alcântara-Neto A.S., Fernandez-Rufete M., Corbin E., Tsikis G., Uzbekov R., Garanina A.S., Coy P., Almiñana C., Mermillod P. Oviduct fluid extracellular vesicles regulate polyspermy during porcine invitro fertilisation. Reprod. Fertil. Dev. 2020;32 doi: 10.1071/RD19058. PubMed DOI

Navarro-Serna S., Hachem A., Canha-Gouveia A., Hanbashi A., Garrappa G., Lopes J.S., Paris-Oller E., Bermejo-Álvarez P., Matas C., Romar R., et al. Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination. Cris. J. 2021;4:1–16. doi: 10.1089/crispr.2020.0078. PubMed DOI

Functional characterization and phenotyping of RAB2A and Lactadherin/MFGE8 as boar sperm zona pellucida binding proteins

Time to revise: impact of methodology on boar sperm capacitation in vitro via phosphotyrosine patterns