Crocetin improves the quality of in vitro-produced bovine embryos: Implications for blastocyst development, cryotolerance, and apoptosis.

The aim of this work was to assess the effect of supplementation of bovine culture medium with the natural antioxidant crocetin on in vitro blastocyst development and quality. This was evaluated as cryotolerance, apoptosis index, and total cells number and allocation. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid medium, supplemented with 0, 1, 2.5, and 5 µM crocetin (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and the blastocysts were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO, and 0.5 M sucrose. Finally, blastocysts produced on Day 8 in the absence (control) and presence of 1 µM crocetin were used for terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and differential staining to evaluate, respectively, the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm (TE) lineages (experiment 2). Embryo development was higher in the 1 µM crocetin group compared to the control, both in terms of total embryo output (37.7 ± 4.2%, 52.9 ± 6.3%, 40.9 ± 7.6%, and 42.4 ± 8.7%, respectively, with 0, 1, 2.5, and 5 µM; P < 0.01) and grade 1 and 2 blastocysts (33.6 ± 4.9%, 46.1 ± 7.3%, 37.8 ± 7.9%, and 39.4 ± 7.9%, respectively, with 0, 1, 2.5, and 5 µM; P < 0.05). Moreover, the percentage of fast-developing embryos increased in 1 µM crocetin group compared to the control (23.4 ± 4.7%, 32.7 ± 6.6%, 27.2 ± 6.6%, and 30.1 ± 7.2%, respectively, with 0, 1, 2.5, and 5 µM; P < 0.05). In addition, the enrichment of culture medium with 1 µM crocetin improved embryo cryotolerance compared to the control, as indicated by higher hatching rates recorded after 48 hours postwarming culture (46.5% vs. 60.4%; P < 0.05). Furthermore, 1 µM crocetin decreased both the average number (9.9 ± 0.4 vs. 7.1 ± 0.3) and the percentage of apoptotic cells (7.1 ± 0.4 vs. 4.2 ± 0.2) in blastocysts compared to the control (P < 0.01). However, no differences were recorded in the average number of ICM, TE, and total cells between 1 µM crocetin and control groups. In conclusion, the enrichment of bovine culture medium with 1 µM crocetin increased both blastocyst yield and quality, as indicated by the improved chronology of embryo development, increased resistance to cryopreservation, and reduced incidence of apoptosis.

L-ergothioneine supplementation during culture improves quality of bovine in vitro-produced embryos.

The aim of this study was to evaluate whether supplementation of bovine culture medium with the natural antioxidant L-ergothioneine (LE), improves in vitro blastocyst development and quality, assessed as resistance to cryopreservation, total cells number, cellular differentiation, and apoptosis index. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid with 0, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM of LE (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On the basis of the results of this dose-response trial, the range of concentrations to test was reduced in experiment 2, in which presumptive zygotes were cultured with 0, 0.05 mM, and 0.1 mM of LE. On Day 7, embryo yields were assessed, and the blastocysts (BL) were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO and 0.5 M sucrose. Finally, BL produced on Day 8 in the absence (control) and presence of 0.1 mM LE were used for transferase-mediated dUTP nick end labeling and differential staining to evaluate, respectively the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm lineages (experiment 3). Despite similar blastocyst yields, supplementation of culture medium with 0.1 mM LE improved the cryotolerance of in vitro-produced (IVP) embryos compared to the control group, as indicated by higher (P < 0.05) hatching rates recorded after 48-hour post-warming culture (48.5%, 50.0%, and 63.8%, respectively with 0, 0.05, and 0.1 mM LE). Interestingly, when embryos were cultured in the presence of 0.1 mM LE, the percentage of BL with the most physiological ICM:total cells ratio (20%-40%) increased (85.1 vs. 66.0%, P < 0.05), confirming a beneficial effect on embryo quality. Furthermore, 0.1 mM LE decreased (P < 0.01) both the average number (4.3 ± 0.2 vs. 9.1 ± 0.3) and the proportion (3.6 ± 0.3 vs. 8.1 ± 0.5) of apoptotic cells in BL compared to the control. In conclusion, the enrichment of bovine culture medium with 0.1 mM LE improves embryo quality, as indicated by the improved cryotolerance, the lower apoptotic rate, and the higher percentage of BL with the most physiological ICM:total cells ratio.

Effect of resveratrol supplementation during culture on the quality and cryotolerance of bovine in vitro produced embryos.

The aim of the study was to evaluate whether resveratrol supplementation of bovine culture medium improves in vitro blastocyst development, embryo cryotolerance and cell numbers. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, zygotes were cultured in SOF medium, supplemented with 0 (control, n=439), 0.25µM (n=422), 0.5µM (n=447) and 1µM resveratrol (n=416). On Day 7 (IVF=Day 0) blastocysts were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide and 0.5M sucrose. Development rate, i.e. the percentage of embryos resuming development to reach a more advanced stage, and hatching rate were evaluated after 24 and 48h culture. Blastocysts cultured with (0.5µM) and without resveratrol underwent differential staining to count inner cell mass (ICM) and trophectoderm (TE) cells. Resveratrol during culture did not increase blastocyst yields (57.1, 57.7, 59.2 and 46.6%, respectively in 0, 0.25, 0.5 and 1µM resveratrol). However, 0.5µM resveratrol improved embryo cryotolerance compared to the control, as indicated by higher development rates (67.3% vs 50.3%, respectively; P<0.01) and hatching rates (58.9% vs 30.9%, respectively; P<0.01) recorded after 48h post-warming culture. Blastocysts produced in the control and in 0.5µM resveratrol groups had similar numbers of ICM (34.1 and 36.4, respectively), TE (88.1 and 85.3, respectively) and total (122.2 and 121.7, respectively) cells. In conclusion, low levels of resveratrol during in vitro culture improve the quality of IVP bovine embryos, as indicated by their increased resistance to cryopreservation.

Influence of oocyte donor on in vitro embryo production in buffalo.

The aim of this research was to estimate the variability between buffalo as oocyte donors. In Experiment 1, reproductive variables were retrospectively analyzed in buffalo (n=40) that underwent repeated ovum pick up (OPU), over 16 puncture sessions (PS). The follicular recruitment among individuals and the relationship between follicular population and oocyte production were evaluated. In Experiment 2, eight buffalo underwent OPU for 28 PS and the oocytes were processed separately to correlate follicular and oocyte population at the first PS to blastocyst (BL) production. In Experiment 1, the average number of total follicles (TFL), small follicles (SFL), cumulus-oocyte complexes (COC) and Grade A+B COC recorded in each 4-PS period had great repeatability (r=0.52, 0.54, 0.60 and 0.57, respectively). The average number of Grade A+B COC recovered during the subsequent 15 PS was positively correlated with the first PS number of TFL (r=0.60; P<0.001), SFL (r=0.68; P<0.001), COC (r=0.48; P<0.01) and Grade A+B COC (r=0.40; P<0.05). In Experiment 2, a large variability among animals was observed in blastocyst yields. When animals were grouped according to the BL yield, the greatest BL yield group had a greater (P<0.05) number of TFL (8.3 ± 0.9 compared with 5.6 ± 0.7) and SFL (7.3 ± 0.3 compared with 3.8 ± 0.7) at the first PS than the lesser BL yield group. The average number of BL produced over the subsequent sessions was correlated with the number of TFL (r=0.80; P<0.05) and COC (r=0.76; P<0.05) observed at the first PS. These results demonstrated a donor influence on the oocyte and BL production, suggesting a preliminary screening to select the donors with greater potential.

Health and economic impact of HPV 16 and 18 vaccination and cervical cancer screening in India.

Cervical cancer is a leading cause of cancer death among women in low-income countries, with approximately 25% of cases worldwide occurring in India. We estimated the potential health and economic impact of different cervical cancer prevention strategies. After empirically calibrating a cervical cancer model to country-specific epidemiologic data, we projected cancer incidence, life expectancy, and lifetime costs (I$2005), and calculated incremental cost-effectiveness ratios (I$/YLS) for the following strategies: pre-adolescent vaccination of girls before age 12, screening of women over age 30, and combined vaccination and screening. Screening differed by test (cytology, visual inspection, HPV DNA testing), number of clinical visits (1, 2 or 3), frequency (1 x , 2 x , 3 x per lifetime), and age range (35-45). Vaccine efficacy, coverage, and costs were varied in sensitivity analyses. Assuming 70% coverage, mean reduction in lifetime cancer risk was 44% (range, 28-57%) with HPV 16,18 vaccination alone, and 21-33% with screening three times per lifetime. Combining vaccination and screening three times per lifetime provided a mean reduction of 56% (vaccination plus 3-visit conventional cytology) to 63% (vaccination plus 2-visit HPV DNA testing). At a cost per vaccinated girl of I$10 (per dose cost of $2), pre-adolescent vaccination followed by screening three times per lifetime using either VIA or HPV DNA testing, would be considered cost-effective using the country's per capita gross domestic product (I$3452) as a threshold. In India, if high coverage of pre-adolescent girls with a low-cost HPV vaccine that provides long-term protection is achievable, vaccination followed by screening three times per lifetime is expected to reduce cancer deaths by half, and be cost-effective.