title: In vivo collection of follicular fluid and granulosa cells from individual follicles of different diameters in cattle by an adapted ovum pick-up system.
abstract: BACKGROUND
  Most studies on granulosa cell (GC) function in cattle have been performed using GC and follicular fluid (FF) samples collected from slaughterhouse ovaries. Using this approach, the follicular developmental stage and functional status are unknown and indirectly inferred, limiting data interpretation. Ultrasound-guided follicle aspiration has previously been used to recover GC or FF samples, but this was mostly carried out in large follicles or pools of small follicles, without recording the efficiency of recovery. The present study was aimed at adapting and evaluating an ovum pick-up (OPU) system for the in vivo recovery of FF and GC from individual follicles of different diameters.
  
  
  METHODS
  In the first trial, the losses of fluid inside the tubing system were calculated using a conventional or an adapted-OPU system. Blood plasma volumes equivalent to the amount of FF in follicles of different diameters were aspirated using a conventional OPU Teflon circuit. The OPU system was then adapted by connecting 0.25 mL straws to the circuit. A second trial evaluated the efficiency of FF recovery in vivo. Follicles ranging from 4.0 to 16.8 mm in diameter were aspirated individually using the conventional or adapted-OPU systems. A third trial assessed the in vivo recovery of GC and the subsequent amount of RNA obtained from the follicles of different diameters from Holstein and Gir cattle.
  
  
  RESULTS
  In Trial I, the plasma recovery efficiency was similar (P > 0.05) for the volumes expected for 12 and 10 mm follicles, but decreased (P < 0.05) for smaller follicles (45.7+/-4.0%, 12.4+/-4.3% and 0.0+/-0.0% for 8, 6, and 4 mm follicles, respectively). Using the adaptation, the losses intrinsic to the aspiration system were similar for all follicle diameters. In Trial II, the expected and recovered volumes of FF were correlated (r = 0.89) and the efficiency of recovery was similar among follicles <12 mm, while larger follicles had a progressive increase in FF losses that was not related to the tubing system. In Trial III, the number of GC and amount of RNA obtained were not affected (P > 0.05) by follicle size, but differed according to breed (615,054+/-58,122 vs 458,095+/-36,407 for Holstein and Gir, respectively; P < 0.05).
  
  
  CONCLUSIONS
  The adapted-OPU system can be successfully used for the in vivo collection of FF and GC from follicles of different diameters. This will enable further endocrine, cellular, and gene expression analyses.
authors.affiliation: Embrapa, Juiz de Fora, MG 36038-330, Brazil.
authors.forename: Eduardo K N
authors.initials: EK
authors.lastname: Arashiro
authors.forename: Miller P
authors.initials: MP
authors.lastname: Palhao
authors.forename: Sabine
authors.initials: S
authors.lastname: Wohlres-Viana
authors.forename: Luiz G B
authors.initials: LG
authors.lastname: Siqueira
authors.forename: Luiz S A
authors.initials: LS
authors.lastname: Camargo
authors.forename: Marc
authors.initials: M
authors.lastname: Henry
authors.forename: Joao H M
authors.initials: JH
authors.lastname: Viana
mesh_terms: D000818:Animals; D002417:Cattle; D005260:Female; D015571:Follicular Fluid / Q000378:metabolism*; D006107:Granulosa Cells / Q000166:cytology*; D054315:Oocyte Retrieval / Q000295:instrumentation / Q000379:methods*; D006080:Ovarian Follicle / Q000166:cytology* / Q000378:metabolism; D010063:Ovum / Q000166:cytology*; D015203:Reproducibility of Results
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