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

BackgroundMost 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.MethodsIn 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.ResultsIn 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).ConclusionsThe 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.

The association of follicular fluid volume with human oolemma stretchability during intracytoplasmic sperm injection

“…The present data show that the FF volume was correlated with follicular diameter, which is consistent with the findings of Arashiro et al [ 22 ]. We focused on the relationship between follicle shape and follicle diameter under two-dimensional transvaginal ultrasonography.…”

Characterization of blood flow and the effects of exogenous estradiol benzoate on residual follicles formed after ultrasound-guided transvaginal follicle aspiration in cattle

“…At D9, the largest follicle present on the ovaries, presumptive dominant follicle, was measured and aspirated by TVFA, with the same ultrasound device, except that a micro-convex 7.5 MHz transducer was used for TVFA, which was performed as previously described [ 15 ]. A conventional TVFA system was adapted for individual recovery of follicular fluid (FF) from targeted follicles into 1.5 mL tubes [ 5 ]. Follicular fluid was then centrifuged at 600 × g for 10 min to remove cells and cumulus-oocyte complexes and the supernatant was stored at -20 o C until radioimmunoassay (RIA) analysis.…”

“…This technique is less invasive than other approaches as laparotomy or laparoscopy and so can be used repeatedly in the same oocyte donor with low risk of sequels [ 2 ] or disturbance of animal welfare [ 3 ]. Moreover, TVFA permits the recovery of follicular fluid and granulosa cell samples for studies on the physiology of the intrafollicular microenvironment [ 4 , 5 ].…”

Methods to study ovarian function in monovulatory species using the cow as a model

“…Furthermore, oocytes, follicular fluid and granulosa cells can be collected from individual follicles under epidural anesthesia using an adapted ovum pick up system, without the need to euthanize animals (Beg et al, 2001;Arashiro et al, 2013;Sanchez et al, 2014). Based on our experience, IFI is an excellent method to manipulate ligand/receptor signaling in a follicle of known developmental status without interfering with such signals in other follicles or tissues.…”