Our objective was to determine if replacing glucose with fructose would decrease cytoplasmic lipid accumulation during culture of embryos with or without regulators of metabolism. In vitro-produced bovine zygotes were cultured 60 hr in chemically defined medium-1 (CDM-1) plus 0.5% BSA and 0.5 mM fructose or glucose in Experiment 1, and glucose in Experiment 2. In both experiments, 8-cell embryos were next cultured 135 hr in CDM-2 plus 2 mM fructose or glucose in factorial combination with five treatments: (Experiment 1: control, 10% fetal calf serum (FCS), 0.3 microM phenazine ethosulfate (PES), 30 microM dinitrophenol (DNP), and PES + DNP), and (Experiment 2: control, PES, PES + DNP, and 1 and 3 microg/ml cerulenin (C1 and C3)). Day 7.5 blastocysts were stained with Sudan Black B to quantify cytoplasmic lipid droplets as small (SD, <2 microm), medium (MD, 2-6 microm), or large (LD, >6 microm). Blastocyst rates per oocyte were 22% (Experiment 1) and 15% (Experiment 2) higher (P < 0.05) for fructose than glucose. For Experiment 1, numbers of MD were lower for PES, DNP, and PES + DNP than control and FCS (P < 0.05). LD were lower for PES and DNP than control, and higher for FCS than all other treatments (P < 0.05). For Experiment 2, MD were lower (P < 0.05) for PES, and PES + DNP than C1, C3, and control. For LD, PES was lower (P < 0.05) than control, C1, and C3, but not different from PES + DNP. The only effect of hexose on lipids was that fructose resulted in fewer MD (P < 0.01) in Experiment 2. In conclusion, fructose produced more blastocysts than glucose, and PES reduced lipid accumulation.

This study investigated effects of hexoses, fetal calf serum (FCS), and phenazine ethosulfate (PES) during the culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. The basal, control medium was chemically defined (CDM) plus 0.5% fatty acid-free BSA. In vitro-produced bovine zygotes were cultured in CDM-1 with 0.5 mM glucose; after 60 hr, 8-cell embryos were cultured 4.5 days in CDM-2. The 8-cell embryos were randomly allocated to a 2 x 3 x 2 x 3 factorial experimental design with two energy substrates (2 mM glucose or fructose); three additives (0.3 microM PES, 10% FCS, and control); two cryopreservation methods using no animal products (conventional slow freezing or vitrification); and semen from three bulls with two replicates for each bull. A total of 1,107 blastocysts were produced. Fructose resulted in 13% more blastocysts per oocyte than glucose (37.2% vs. 32.9%), and per 8-cell embryo (51.3% vs. 45.3%; P < 0.01). No differences were found for additives (P > 0.1) control, FCS, or PES for blastocysts per oocyte or per 8-cell embryo. There was a significant interaction (P < 0.05) between additives and hexoses for blastocyst production; although trends were similar, the benefit of fructose compared to glucose was greater for controls than for FCS or PES. Culture of embryos with PES, which reduces cytoplasmic lipid content, improved cryotolerance of bovine embryos; post-cryopreservation survival of blastocysts averaged over vitrification and slow freezing (between which there was no difference) was 91.9%, 84.9%, and 60.2% of unfrozen controls (P < 0.01) for PES, control, and FCS groups, respectively.

The objective of these experiments was to study bovine in vitro fertilization (IVF) conditions for blastocyst production using non-sexed sperm (Experiment 1) and sexed sperm (Experiment 2). For Experiment 1, in vitro-matured oocytes (N=707) were allocated to a 2 × 3 × 4 factorial design: time of co-incubation of gametes for fertilization (4 and 18 h), sperm dose (1, 0.33, and 0.11 × 10(6) frozen-thawed sperm/ml, and sperm source (four bulls). Pronuclear status was evaluated for a subset. Experiment 2 (N=2155 oocytes) was a 2 × 3 × 2 × 6 factorial design: sex of sperm (X and Y), sperm dose (1, 0.33, and 0.11 × 10(6) frozen-thawed sperm/ml), and sperm-sorting pressures (40 and 50 psi), replicated with sperm of six bulls. Presumptive zygotes were cultured 60 h in chemically defined medium-1 (CDM-1), and for 114 h in CDM-2. For Experiment 1, pronuclear formation, cleavage and blastocysts rates were greater for 1, and 0.33 × 10(6) than 0.11 × 10(6) sperm/ml (72 and 62 vs 42%; 89 and 81 vs 58%; and 21 and 17 vs 9%, respectively; all p<0.01); polyspermy was greater for 1, than 0.33 and 0.11 × 10(6) sperm concentrations (24 vs 2 and 0%; p<0.01). There were greater main effects (p<0.01) of pronuclear formation (69 vs 48%), polyspermy (13 vs 4%), and cleavage (63 vs 54%), at 18 than at 4 h of co-incubation of gametes (all p<0.01). For Experiment 2, cleavage and blastocyst rates were greater for 1 × 10(6) sperm/ml vs 0.33 and 0.11 (69%, 47%, and 30% cleavage and 30%, 14%, and 8% blastocysts) and 40 vs 50 psi (54% and 44% cleavage and 18% and 15% blastocysts) (p<0.01). A marked bull by fertilization sperm dose interaction was found for cleavage (p<0.05). The main conclusion was that the optimal sperm concentration for cleavage and producing blastocysts via IVF with sexed sperm was considerably higher and more variable among bulls than for unsexed sperm.

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