The specific conditions of the embryo culture can affect blastocyst quality and cell counts. However, in vitro embryo development can be affected by such oxidative stress sources as reactive oxygen species that can damage embryo development. Antioxidants act as free radical scavengers and protect cells. The addition of antioxidants to the embryo culture medium has been shown to improve in vitro embryo development .
In a previous study examining the effect of boron-supplemented feed on the embryonic development of mice, doses of 0.04, 2.05 and 11.8 µg B/g-diet were administered, and the negative effects on the in vitro development of embryos collected from those fed 0.04 µg B/g-diet  were established. In the present study, as the first of its kind in literature, low doses (10− 2, 10− 3, 10− 4 and 10− 5 ppm) of BA were added to the culture medium to understand the positive effects on embryo development and its mechanism.
Ince et al. found that feeding rats boron via a gavage tube (0.04 and 2.05 g) for 14 days improved gene expression in the early embryonic period after conception (HEX, NANOG, and OCT-3/4) and improved the fetal development of the rats . Similarly, our study found NANOG levels to significantly promote embryo development when compared to the control group.
In contrast, a recent study reported that the addition of 20–250 µM/L BA as an antioxidant to in vitro cultures did not promote bovine embryo development in vitro . Nonetheless, B (0.3-09 g) supplemented to the quality of Angora buck sperma after freezing and thawing and some positive effects on spermatological parameters after thawing . Another study, in which B (0.25, 0.5 and 1 mm) was added to thawed ram sperm parameters, adverse effects on sperm parameters and gene expressions level of antioxidants . It was noted in the present study that embryo development is positively affected at much lower doses (up to 10− 5), and that the use of in vitro methods is important for the dose determination of newly used antioxidants or similar growth factors.
Erhui et al. established that 0.4 and 40 ppm B doses positively affected the proliferation of spleen lymphocytes. A study examining the effects of rat Sertoli cells cultured with different B doses (0, 0.25, 0.5, 1.0, 5.0, 10.0, 40.0 and 80.0 mmol) on in vitro cytotoxicity, apoptosis and cell cycle reported a positive effect on proliferation for only 0.5 mmol B . The B boron and BA used in the present study had a greater effect on cell growth, particularly at low doses.
Yalcin et al., in their study investigating the effects of BA on cell viability of mouse TM3 Leydig cells, observed no cytotoxicity after exposure to BA in varying concentrations (0.5, 1, 5, 10, 50, 100, 500 and 1000 µM) for 24 hours . The present study observed that boric acid (10− 2, 10− 3 10− 4 and 10− 5 ppm) significantly improved viable cell growth.
In fertilized trout eggs fed B in quantities ranging from 2.2 to 90.6 mmol/L, B was found to promote development in a dose-dependent manner . Similarly, our study showed that BA promoted embryo development depending on the dose. Adult Xenopus laevis were fed a diet containing 45, 310 and 1850 microg B/kg B (-B), and abnormal developmental disorders were observed in the high-dose groups (310 and 1850 microg B/kg), while the low dose group (45 micrograms) had normal reproductive system development after 120 days . The present study, considering the effect of BA on embryo development, observed a negative effect especially in high doses in MTT, while the effect was positive in low-dose groups, and therefore, dose escalation was performed.
Recent studies have investigated the antioxidant properties of BA under in vivo and in vitro conditions. Various doses of BA were administered to Galleria mellonella larvae as a supplementary diet, leading to increased SOD activity at 156 and 620 ppm doses, while high doses of 1250 and 2500 ppm led to decreased antioxidant levels and increased larval and pupal mortality . A previous study investigating the ability of BA to protect against aflatoxin b1 toxicity in human blood found that 2 ppm BA reduced the oxidative stress caused by aflatoxin by increasing the levels of such antioxidants as SOD, CAT and GSH-Px . Furthermore, another study investigating the effects of different doses of BA injected into the egg on the bursa of Fabricius and spleen found that low boric acid doses (1000 ppm) caused led to the involution of the bursa of Fabricius, and indirectly increased the plasma cell counts in the spleen . In another study assessing the immune and antioxidant status and the growth performance of lambs fed diets with or without sufficient Ca + showed that 40 ppm BA increased the total antioxidant activity and SOD1 gene expression . In the present study, in which embryo development after the addition of BA to in vitro mouse embryo cultures was monitored, a dose-dependent increase in TAS, NANOG, SOD1, SOD2, GPX1 and GPX4 levels was established at 0.001 ppm BA, and this increase in antioxidant levels suggests a decrease in the oxidative stress factors in the embryo culture and an increase in embryo development rates and quality.
In conclusion, the use of low-dose BA (0.00001) the better positively affects embryo development. The findings of the present study can be used for the development of a model for use in veterinary and human medicine. Embryo production is especially important in farm animals (cattle, sheep). Increasing the production of healthy embryos through the use of BA would contribute to the improvement and development of livestock. Future original researches exploring the effects of boric acid-supplemented culture media on in vitro and in vivo embryo development together in studies of such processes as embryo cryopreservation, somatic cell nuclear transfer (SCNT), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) would further contribute to literature.