Effects of vitrification of cumulus-enclosed porcine oocytes at the germinal vesicle stage on cumulus expansion, nuclear progression and cytoplasmic maturation.

Although offspring have been produced from porcine oocytes vitrified at the germinal vesicle (GV) stage, the rate of embryo development remains low. In the present study, nuclear morphology and progression, cumulus expansion, transzonal projections (TZPs), ATP and glutathione (GSH) levels were compared between vitrified cumulus-oocyte complexes (COCs) and control COCs (no cryoprotectant treatment and no cooling), as well as a toxicity control (no cooling). Vitrification was performed with 17.5% (v/v) ethylene glycol and 17.5% (v/v) propylene glycol. Vitrification at the GV stage caused premature meiotic progression, reflected by earlier GV breakdown and untimely attainment of the MII stage. However, cytoplasmic maturation, investigated by measurement of ATP and GSH levels, as well as cumulus expansion, proceeded normally despite detectable damage to TZPs in vitrified COCs. Moreover, treatment with cryoprotectants caused fragmentation of nucleolus precursor bodies and morphological changes in F-actin from which oocytes were able to recover during subsequent IVM culture. Reduced developmental competence may be explained by premature nuclear maturation leading to oocyte aging, although other mechanisms, such as initiation of apoptosis and reduction of cytoplasmic mRNA, can also be considered. Further research will be required to clarify the presence and effects of these phenomena during the vitrification of immature COCs.

Selection of porcine oocytes in vitro through brilliant cresyl blue staining in distinct incubation media.

Staining with brilliant cresyl blue (BCB) may be used for oocyte selection, but BCB staining itself and the most commonly used selection medium (DMPBS) may compromise the development of porcine oocytes in vitro. This study evaluated DNA fragmentation, nuclear maturation, the area of migration of cortical granules (CG) and embryo development for stained (BCB+) and unstained (BCB-) oocytes incubated in DMPBS and in a modified medium (ReproPel) tested for the first time. Unexposed (UN), BCB+ and BCB- oocytes were incubated composing six groups: DMPBS/UN; DMPBS/BCB+; DMPBS/BCB-; ReproPel/UN; ReproPel/BCB+; and ReproPel/BCB-. There were more BCB+ oocytes in ReproPel than in DMPBS (P < 0.05). The DNA fragmentation was evaluated for oocytes in DMPBS/BCB+, DMPBS/BCB-, ReproPel/BCB+, ReproPel/BCB- and in porcine follicular fluid (control). The frequency of oocytes with no DNA fragmentation was greatest (64.6%) in DMPBS/BCB+ and lowest in ReproPel/BCB+ and ReproPel/BCB- (26.8 and 34.1%, respectively) (P < 0.05). Nuclear maturation rates were greater (P < 0.05) for DMPBS/BCB+ (63.1%), ReproPel/UN (55.1%) and ReproPel/BCB+ (50.2%) than for DMPBS/UN (40.8%) and ReproPel/BCB- (35.5%). The area of CG was greater (P < 0.05) for ReproPel/BCB- (80.7%) and DMPBS/UN (77.6%) than for ReproPel/UN (34.7%). Cleavage rates for DMPBS/BCB+ and ReproPel/BCB+ were greater than for DMPBS/UN (P < 0.05). Blastocyst development rates were greatest (P < 0.05) for ReproPel/UN and ReproPel/BCB+. In both media, BCB staining was apparently unable to select competent oocytes, which likely occurred due to toxicity. Despite the similar nuclear maturation and area of CG compared with DMPBS, oocytes selected in ReproPel presented impaired DNA integrity.

In vitro assays for vesper mice (Calomys laucha) sperm using heterologous substrates from nonrodent species.

Small vesper mice (Calomys laucha) may be considered as an animal model for in vitro fertilization studies, but limited data about in vitro evaluations of their sperm quality and fertility are available. The in vitro penetration (IVP) assay is used to estimate potential sperm fertility for many mammal species, but it still requires reduction in cost and labor. This study tested improvements in the IVP assay for C. laucha sperm using swine oocytes and perivitelline layers (PVL) of chicken eggs as substrates, and evaluated associations among C. laucha sperm quality, IVP, and in vivo fertility after natural mating. In the IVP assay, gametes coincubation was carried out flat-bottomed wells with M2, in water bath at 37°C for 2 hr. C. laucha sperm presented motility, normal morphology, membrane integrity, and acrosome integrity equal to 90.6 ± 5.6, 90.2 ± 6.6, 88.7 ± 9.6, and 90.5 ± 11.5%, respectively. The IVP rate was 39.8% in swine oocytes and 87.5% in the inner PVL. Considering in vivo fertility as the gold standard, the IVP assay in swine oocytes presented a sensitivity of 16.0% and specificity of 83.3%. The sensitivity of the IVP assay in the inner PVL was 84.0%, but the specificity was not determined because there were no true negative results. Sperm membrane integrity was correlated with parturition after natural mating (r = 0.38, P<0.01) and litter size (r = 0.54; P<0.0002).The IVP assay using swine oocytes as substrates can be performed in nearly 2 hr without gametes' coincubation in CO(2).