Introduction
Recent trends in climate change observed on the Korean peninsula showed a rate of change f 0.41℃ / 10 years from 1981 to 2010 and 0.5℃ / 10 years from 2001 to 2010, and scenarios using the regional climate model for the future climate forecast predicted the temperature increased by 4℃ and the precipitation increased by 17% for all regions of the Korean peninsula (Chung et al., 2004; Lee, 2015). According to the report, South Korea is transforming into a subtropical region, and the climatic changes can impact on productivity of livestock (Rojas-Downing et al., 2017). Moreover, the occurrences of heat waves defined as extreme weather conditions such as daily maximum temperature over 33℃ has increased in the 1990s and 2000s, and the hot events have been more intense in summer from June to August over southern provinces in South Korea (Shim et al., 2017).
Interestingly, our analysis of reproductive performance in Korean native cattle (Hanwoo) demonstrated that calving interval was increased 4.3% and pregnancy rate was deceased about up-to 2.8% year-on-year due to subfertility in summer season (Cho et al., 2016; Choi and Cho, 2016). In this study, we investigated association of heat waves in summer with developmental competency of oocyte in Korean native cattle, Hanwoo.
Material and Methods
All other chemicals used in this study were purchased from Sigma–Aldrich (St. Louis, MO, USA), unless otherwise indicated.
Statement of Animal Rights
All practices and procedures for this experiment were reviewed and approved by the Animal Ethics Committee of the Chungnam National University (CNU-00599).
Ovary collection and in vitro production of embryos
Ovaries were collected from a local slaughter house within 7 days after heat waves during summer, and at November, respectively. In vitro maturation, parthenogenetic activation, and embryo culture procedures have been described elsewhere (Kelly et al., 2010). The maturation and embryo development rates were assessed by extrusion of the first polar body and morphology such as blastocoel.
Reactive oxygen species (ROS) levels and mitochondrial distribution
The ROS levels were examined to determine whether heat stress induced by heat waves affect cytoplasmic competency of oocytes. ROS levels of oocytes were measured with Image-iT Live Green Reactive Oxygen Species Detection kit (Invitrogen, Eugene, OR, USA). To investigate mitochondrial distribution, matured oocytes were incubated with 100mM MitoTracker Red TM (Invitrogen) for 30 min and examined using a confocal laser-scanning microscope (Zeiss LSM 710 META; Jena, Germany). The fluorescent intensities of ROS level and the distribution of fluorescence signal intensities along a line drawn across MII oocytes were analysed using Image J (1.48v) software (https://imagej.nih.gov/ij/index.html).
Results and Discussion
There were no significant differences in nuclear maturation assessed by polar body extrusion between HS (heat stress) and Non-HS groups. The cleavage and blastocyst development was examined on day 2 and 7 after oocyte activation. 2-cell cleavage and blastocyst development were significantly lower in the HS groups, respectively (Table1).
We examined ROS and mitochondria distribution because mitochondria are essential for oocyte maturation and subsequent embryo development (Wang et al., 2009). We found a significant increase in the levels of ROS in the HS oocytes after IVM, compared to those in non-HS oocytes (Table 2). Mitochondria in non-HS oocyte were homogeneously distributed in the cytoplasm, however mitochondria were polarized in the HS oocyte, particularly in the pericortical cytoplasm (Fig. 1).
Fig. 1. Intercellular distribution of mitochondria in matured oocytes. a: Mitochondria are stained red(MitoTracker Red) Non-HS (Non-heat stressed condition; oocytes collected and matured in winter), HS (Heat stressed condition; oocytes collected following exposure of heat waves). Scale bar 50 μM. b: Intensities of stained mitochondria (along a line drawn across MII oocytes; white dot line in Fig 1. a) are measured using image J. ZP (zona pellucida).
Oocytes from summer season showed lower cleavage and blastocyst rates although nuclear maturation rates were not different from oocytes obtained in winter season. We therefore postulated that insufficient cytoplasmic maturation affect further developmental competency. Our findings that oocytes exposed to summer heat waves increased the production of ROS and had polarized peripheral distribution of mitochondria, rather than homogeneous cytoplasmic distribution, indicate that the lower developmental competency of bovine oocytes collected under heat waves is attributed to insufficient cytoplasmic maturation such as increased generation of ROS and dysfunction of mitochondria (Roth et al., 2008; Roth, 2015; Paes et al., 2016). Moreover, taurine type of Korea native cattle (Hanwoo) has more genetic diversities including genetic features of African taurine and indicine cattle breeds (less than 10%) (Lee et al., 2014; Makina et al., 2014; Sharma et al., 2016).
Conclusion
We supposed that Hanwoo might be less resistant to increased temperature and humidity as seen in Bos taurus cattle, compared to Bos indicus (Paula-Lopes et al., 2013; Silva et al., 2013). Taken together, our findings suggest that embryo transfer using cryopreserved embryos/oocytes obtained during non-heat stress season can improve pregnancy rates in the summer because HS adversely affects cytoplasmic maturation and subsequent embryonic development.
