Comparison of Two In vitro Maturation Media in Their Efficiency for Production of Competent Alpaca Oocytes

Abstract

Oocyte maturation is the most critical process in in vitro culture, since the oocyte acquires competence for future processes, which involve the resumption of meiosis, epigenetic regulation, polyspermy block, activation of the zygote, exchange of protamines to histones, cleavage, cell polarization, embryonic stem cell proliferation, and implantation. Therefore, the objective of this study was to evaluate the efficiency of two different in vitro maturation media in the production of competent alpaca oocytes matured in vitro. The competency evaluated by morphometry and cytoplasmic and nuclear staining techniques. Alpaca ovaries (n=44) were sourced from slaughterhouse, transported to laboratory in thermoses with saline solution at 33-36ºC and subjected to follicular aspiration and/or dissection. A total of 212 oocytes were obtained. The oocytes were selected, randomized and distributed in one of the two experimental groups: Maturation medium 1 (MM1) and Maturation medium 2 (MM2) with three replicates per experiment. In vitro maturation was carried out in a portable incubator for 40h under 5% CO2 conditions (produced by effervescent granules) and 38.5ºC (by thermostatic bath). Nuclear maturation was assessed using acetate-orcein staining, cytoplasmic maturation using Brilliant Cresyl Blue (BCB) staining and morphometry using ImageJ Software. Nuclear maturation was classified according to the meiotic stage. Cytoplasmic maturation was classified by the presence (BCB+) or absence (BCB-) of BCB staining. Morphometry evaluated the measurements of the diameter and thickness of the zona pellucida and perivitelline space of the oocyte. Nuclear maturation rate was evaluated in Metaphase, observing no significant differences (P=0.088) between MM1 (46.08 ± 5.90 %) and MM2 (54.65 ± 3.00%) in this parameter. The cytoplasmic maturation rate of MM1 (67.78 ± 4.50%) was significantly lower compared to MM2 (77.61 ± 3.58) % (P=0.014). For the diameter of the oocyte, MM1 (133.42 ± 14.56 μm) and MM2 (136.14 ± 15.2 μm) were no different (P=0.397), same for the thickness of the zona pellucida, MM1 (16.84 ± 3.96 μm) and MM2 (16.73 ± 4.98 μm) were no different (P=0.919). For the perivitelline space area, MM1 (11018.91 ± 2465.40 μm2 ) and MM2 (8686.18 ± 3016.88 μm2 ) were significantly different (P=0.0005). In conclusion, the present study shows that the MM2 medium results are better in cytoplasmic maturation and the area of the perivitelline space. The other parameters (nuclear maturation, diameter of the oocyte and thickness of the zona pellucida) were no different between the two maturation media evaluated.