117 Supplementation of IVF medium with nerve growth factor improved bovine embryonic cleavage rates during summer months

Abstract

Nerve growth factor-β (NGF), a protein originally associated with regulation of neuron development, has been found to play a role in the reproductive system of mammals. Previous research showed that administration of NGF to cows resulted in enhanced conceptus development. Although these effects were speculated to be a result of improved corpus luteum function, whether NGF could act directly on the embryo remained undetermined. Therefore, the direct effects of NGF on fertilization and embryo development warrant investigation to see whether it can be used as a novel tool to improve cleavage and blastocyst rates when producing embryos via IVF during periods of suboptimal oocyte quality, such as with heat stress. The objective of this study was to explore how supplementation of NGF, purified from bull seminal plasma, during IVF may directly affect embryo development in oocytes harvested in the summer. Abattoir-derived bovine ovaries were used for recovery of cumulus-oocyte complexes (COCs) over eight replicates through May and June. On Day −1, COCs were collected and matured for 20 h in oocyte maturation medium incubated at 38.5°C. On Day 0, matured oocytes were added to a solution of IVF-Tyrode's albumin lactate pyruvate (TALP) and either phosphate-buffered saline (PBS; control) or 100 ng mL⁻¹ NGF. Pooled frozen-thawed semen from two different bulls per replicate were added to the IVF solutions and incubated with COCs for 20 h at 38.5°C in a humidified atmosphere of 5% CO₂. On Day 1, zygotes were washed in HEPES-TALP, and cumulus cells were removed using 1% hyaluronidase. The zygotes were plated in synthetic oviductal fluid (SOF-BE2) culture medium and incubated at 38.5°C in a tri-gas chamber (5% CO₂, 5% O₂, and balanced N₂). Cleavage rates were recorded at 24 and 48 h, calculated by dividing the number of cleaved embryos by the total zygote count. Embryos were incubated until Day 8, when the rate of blastocysts was assessed. This study found that the treatment of IVF medium with NGF increased the cleavage rate of embryos after 48 h (Control: 59%; NGF: 66%; P = 0.04) and the hatched blastocyst percentage per oocyte on Day 8 (Control: 6.7%; NGF: 9.4%; P = 0.01). The treatment did not affect the percentage of blastocysts per cleaved embryos (Control: 21%; NGF: 22%; P = 0.16) or the hatched blastocyst rate at Day 8 (Control: 53%; NGF: 55%; P = 0.67). These results show that NGF can act directly on the oocyte during fertilization to alter subsequent development, specifically through increased embryonic cleavage rates. Further studies are needed to assess different dosages of NGF in order to mitigate the detrimental effects of heat stress on oocyte competence for use in IVF. Follow-up studies using a whole-animal model are needed to understand the clinical relevance of these findings in the ability of embryos to promote maternal recognition of pregnancy.