title: Timing of oviductal fluid collection, steroid concentrations, and sperm preservation method affect porcine in vitro fertilization efficiency.
abstract: OBJECTIVE
  To determine optimal conditions for the inclusion of oviductal fluid (OF) in IVF protocols.
  
  
  METHODS
  Experimental prospective study.
  
  
  METHODS
  Mammalian reproduction research laboratory.
  
  
  METHODS
  Oviducts and ovaries from porcine females were collected at a slaughterhouse. A total of 30 oviducts and 1,285 oocytes were used. Boar-ejaculated spermatozoa were also used.
  
  
  METHODS
  In vitro-matured porcine oocytes were preincubated with OF collected from animals before or after ovulation and later fertilized in vitro. Zona pellucida digestion time in oocytes after preincubation in OF was assessed. Concentrations of E2 and P4 in OF were measured. IVF was performed, including within the culture media the E2 and P4 concentrations found in the preovulatory OF. The effect of preovulatory OF on IVF efficiency was compared between fresh and frozen-thawed spermatozoa.
  
  
  METHODS
  E2 and P4 concentrations in OF; penetration and monospermy rates; number of spermatozoa within the ooplasm and on the zona pellucida after IVF under different experimental conditions; zona pellucida resistance to protease digestion.
  
  
  RESULTS
  Preincubation of oocytes in OF collected before ovulation enhances IVF efficiency in the pig compared with OF collected after ovulation (29.58 ± 3.84 vs. 11.03 ± 2.69). When frozen-thawed spermatozoa are used for the IVF of these OF-treated oocytes, their fertilization ability increases compared with fresh semen. OF collected before and after ovulation shows significantly different concentrations of E2 (99.00 ± 8.72 vs. <10 pg/mL) and P4 (2.53 ± 0.66 vs. 12.27 ± 2.33 ng/mL), respectively. Addition of E2 and P4 at concentrations similar to those in the OF before ovulation partially simulates the effect of the fluid on IVF outcome.
  
  
  CONCLUSIONS
  Preincubation of oocytes in OF collected before ovulation is a suitable protocol for increasing the efficiency of IVF with fresh semen in the pig model and could be a useful tool to increase the fertilization ability of frozen-thawed spermatozoa in other species. E2 concentrations in preovulatory OF are higher than those reported in blood serum at the same phase of the estrous cycle.
authors.affiliation: Department of Physiology, Veterinary Faculty, University of Murcia, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum) and Institute for Biomedical Research of Murcia, Murcia, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Ballester
authors.affiliation: Department of Physiology, Veterinary Faculty, University of Murcia, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum) and Institute for Biomedical Research of Murcia, Murcia, Spain.
authors.forename: Jon
authors.initials: J
authors.lastname: Romero-Aguirregomezcorta
authors.affiliation: Department of Physiology, Veterinary Faculty, University of Murcia, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum) and Institute for Biomedical Research of Murcia, Murcia, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Soriano-Úbeda
authors.affiliation: Department of Physiology, Veterinary Faculty, University of Murcia, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum) and Institute for Biomedical Research of Murcia, Murcia, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Matás
authors.affiliation: Department of Physiology, Veterinary Faculty, University of Murcia, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum) and Institute for Biomedical Research of Murcia, Murcia, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: Romar
authors.affiliation: Department of Physiology, Veterinary Faculty, University of Murcia, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum) and Institute for Biomedical Research of Murcia, Murcia, Spain. Electronic address: pcoy@um.es.
authors.forename: Pilar
authors.initials: P
authors.lastname: Coy
mesh_terms: D000818:Animals; D001826:Body Fluids / Q000737:chemistry*; D004958:Estradiol / Q000378:metabolism; D005187:Fallopian Tubes / Q000378:metabolism*; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary*; D005615:Freezing; D008297:Male; D011374:Progesterone / Q000378:metabolism; D011446:Prospective Studies; D013094:Spermatozoa; D013552:Swine; D013997:Time Factors
chemical_list: D011374:Progesterone; D004958:Estradiol
keywords: Oviductal fluid; frozen-thawed spermatozoa; in vitro fertilization; steroids