Many researchers have investigated conditions required to improve in vitro maturation (IVM) and subsequent development of cattle oocytes [1,2]. In this respect various supplements to culture media have been examined including serum [3], follicular fluid, co-culture [4], growth factors and/or gonadotropic hormones [2]. However, IVM of bovine oocytes is not only related to the constituents of the culture medium but also to the incubation temperature [5]. A basic variable in the cellular environment of in vitro cultures is temperature [4]. Conventional in vitro bovine embryo production technology including oocyte maturation, fertilization and embryo culture is performed at 38.5°C or 39°C which represents core body temperature of cattle [6]. However, Leese et al [5] reported that preovulatory follicles have a temperature of approximately 1.5°C to 2°C less than that of the ovarian stroma or core body temperature in cattle, but this fact is rarely or never taken into account in in vitro embryo production. Moreover, McEvoy et al [7] suggested that present in vitro oocyte maturation technology is far from the in vivo reality due to incubation temperature employed. Also, Hunter et al [8] suggested that in vivo formation of meiotic and cytoplasmic maturation of oocyte, which occur at low temperature, should be considered in in-vitro studies. These phenomena lead to the suggestion that a temperature lower than core body temperature of cattle may provide better oogenesis or maturation of the bovine oocytes when cultured in vitro, and therefore, may result in an improvement in the subsequent development after fertilization.

Intra-cellular ATP production is realized as a result of oxidative metabolism, but also reactive oxygen species (ROS) are produced as undesired by-products of the process [9]. The amount of ROS production can vary depending on the rate of oxidative metabolism, and higher metabolic activity is associated with increased amounts of ROS production [10] that may cause cellular damage such as cellular enzymes inactivation, DNA damage and cell membrane lipid peroxidation in embryonic cells [5]. Generation of ROS is an unavoidable result of oxidative reactions, but various preservative scavengers such as superoxide dismutase, catalase and glutathione remove radical species before they can cause significant damage in in vitro embryo production systems. However, it would be possible to achieve a lower production of ROS with reduced metabolic activity and/or oxidative metabolism rate. In that case, culture media may require comparatively less or no supplementation with an antioxidant system against the ROS. Therefore, if environmental conditions of the in vitro culture of oocytes do not allow an increase in the rate of embryo metabolism, it may be possible to eliminate or reduce supplementations of the culture media with antioxidants. To this effect Leese [11] has put forward the ‘Quiet Embryo Hypothesis’, which proposed that low (quiet) embryo metabolism would better serve the embryo viability than active metabolism. This leads to the notion that the oocyte and the early embryo may function better at a lower temperature which is also representative of the in vivo preovulatory follicle environment [5]. Therefore, in this study we tested the hypothesis that exposure to lower temperature (36.5°C) representative of the in vivo thermal conditions of the bovine preovulatory follicles, improves the maturation and subsequent post-fertilization development of the bovine oocytes. The main aim was to compare the effects of low (36.5°C) and conventional (38.5°C) culture temperatures on the oocyte maturation parameters and the embryonic development including the cell numbers, diameter and blastocyst quality.

All chemicals and media used in this study were purchased from Sigma-Aldrich, Turkey, except where otherwise indicated.

In the present study an attempt was made to carry out IVM of bovine oocytes at low temperature of 36.5°C, which is considered to be the temperature of preovulatory follicles and at which supposedly in vivo oocyte maturation occurs. The results for the Grade A oocytes showed that cumulus expansion rate, first polar body formation and the proportion of oocytes reaching the M II stage were similar between the tested two maturation temperatures (36.5°C or 38.5°C). This suggests that low incubation temperature of 36.5°C during the maturation process does not alter the progress of bovine nuclear maturation. Similarly, previous studies have shown that low incubation temperature during IVM did not affect the rate of cumulus expansion [15,16], first polar body formation [15–17] and proportion of oocytes that reached to M II stage [4,6,15, 16]. Ye et al [4] have reported that addition of follicular fluid to the maturation media did not alter nuclear maturation level of porcine oocytes matured at 35.5°C, compared to counterparts matured at 39.0°C and 37.0°C. Moreover, supplementation of estrous cow serum into the maturation media did not affect nuclear maturation level of bovine oocytes cultured at 37°C and 39°C [18]. Overall, these studies suggest that serum may protect oocytes from possible negative effects, if any, of low incubation temperatures. It has been suggested previously [4] that, lower follicular ambient temperature is advantageous to complete oocyte maturation or development, within the follicular microenvironment. However, our results for Grade B oocytes have shown that low incubation temperature decreased the proportion of oocytes that reached M II or first polar body formed. These results suggest that lower temperatures do have negative impact on the maturation process which in our study was not obvious in the case of Grade A oocytes but was present in the case of comparatively vulnerable Grade B oocytes probably due to less cumulus cell layers which are known to assist in the nuclear maturation of mammalian oocytes [19].

As expected, embryos from Grade A oocytes had better developmental competence following IVF. Although cleavage rates of embryos obtained from oocytes matured at 36.5°C were similar to those from matured oocytes at 38.5°C, the proportion of embryos in the morula stage were higher for Grade A oocytes matured at 38.5°C. However, this difference disappeared at the blastocyst stage. Shi et al [6] have similarly shown that neither 37.0°C nor 38.5°C incubation temperatures during lVM had any significant effect on embryonic development until development to the blastocyst stage. Ravindranatha et al [16] reported that embryos from buffalo oocytes maturated at four different temperatures had similar development to 8 to 16 cells and blastocysts. Antioxidant supplementation, as expected, increased the ratio of embryos in the blastocyst stage, irrespective of maturation temperature whether the oocytes were maturated at 36.5°C or at 38.5°C. Similar results on the effect of antioxidant supplementation have been informed by Luvoni et al [9] and Uysal et al [20].

The viability of the pre-implant embryo depends on a well-organized formation of ICM cells [21]. Ganeshan et al [21] reported that in vitro environmental or culture conditions have primarily major effects at the developmental potential of the ICM cell lineage rather than the TE cell lineage. Previous studies reported that in vitro produced blastocysts had less ICM cells than in vivo counterparts [22,23]. Van Soom et al [13] and Kuran et al [24] reported that lower ICM or overall cell numbers of in vitro derived blastocysts are related to poor embryo quality. In the present study, incubation temperatures during IVM did not influence overall, ICM and TE cell numbers, but blastocysts obtained from Grade A oocytes matured at 36.5°C and cultured in antioxidants supplemented media, had more ICM cells. This could be a sign of an improvement in antioxidant defense system and low culture temperature during maturation had strengthened the antioxidant defense system in the morula and blastocyst stage of embryo development and may increase the quality of embryos. Additionally, this improvement may be caused by mimicking of the in vivo Graaf follicle environmental conditions. On the contrary, blastocysts, which were obtained from good quality oocytes matured at 38.5°C and cultured in antioxidant supplemented media, had more TE cells. These results indicate that bovine oocytes maturation in conventional culture temperature with antioxidant supplementations in embryo culture have improved subsequent development of embryonic TE cells from balancing the metabolic activity. Because, Hewitson and Leese [25] showed that TE cells consume more oxygen and nutrients due to having more mitochondria than the ICM cells. Also, ICM cells have more glycolytic metabolism than those of the TE in mouse embryo [25]. This low metabolic activity may reflect pluripotency of ICM cells [26]. Unfortunately, observations regarding to the embryo transfer and metabolic activity were not recorded in the present study. However future studies may help to interpret whether low maturation temperature had any effect on ICM cell metabolic activity and success rate of embryo transfer in cattle.

In the present study, we have observed that the incubation temperatures during maturation did not influence blastocyst diameter, but supplementation of antioxidants during embryo culture increased both overall cell number and diameter of blastocysts. In vitro produced blastocysts with a smaller diameter are known to have some anomalies in mitotic, osmotic or secretory activity of blastomeres [27]. Thus, these results of the present study may indicate that supplementation of antioxidants in embryo culture media may improve cell proliferation or the osmotic/secretory activity of the blastomeres in blastocyst rather than reflecting the culture temperatures during maturation stage.

In conclusion, the results of the present study demonstrated that bovine oocytes can complete maturation process at 36.5°C culture temperature and they exhibit similar embryonic development following IVF as the oocytes matured at conventional (38.5°C) culture temperature. Low culture temperature during IVM could be useful to bovine in vitro embryo production programs. Future studies are required to investigate the effect of low incubation temperature during maturation on the metabolic activity, gene expression, DNA methylation and antioxidant capacity of both oocytes and embryos.