Abstract: The present research was undertaken to study the effect of a saturated fatty acid-Palmitic Acid (PA), an unsaturated fatty acid-Oleic Acid (OA) and also to investigate the efficacy of Oleic acid on prevention of detrimental effect of saturated fatty acids on oocyte development in ewes (Ovis aries). Sheep oocytes were matured in vitro in the presence of different concentration of palmitic acid (0, 20, 40 and 60 μM) in experiment 1 and oleic acid (0, 40, 60 and 80 μM) in experiment 2 for 24 h. Oocytes were then in vitro inseminated and evaluated for cleavage rates after 42 h post insemination and presumptive zygotes were evaluated for morulae/blastocyst stages after 7-8 days. In experiment 3, oocyte were matured in vitro in presence of, (a) Palmitic acid (60 μM)+oleic acid (40 μM) in oocyte maturation medium and (b) Oleic acid (40 μM)+stearic acid (30 μM) in medium for 24 h. Palmitic acid, was found to impair the maturation, viability, cleavage and embryo production rates at the level 60 μM in ewes. In contrast, oleic acid improved the oocyte development in vitro. Oleic acid also compensated for the unfavorable effects of palmitic and stearic acid on oocyte development.
INTRODUCTION
During postpartum period high yielding animals face metabolic stress condition which leads to Negative Energy Balance (NEB) because of energy loss by high production which cannot be reimburse by energy intake (Van Knegsel et al., 2005) and follicular fluid (Leroy et al., 2008a). This change and increase free fatty acid concentration affects oocyte quality which leads to impair fertility (Leroy et al., 2008b). Energy deficit feeding is accompanied by lipolysis and is typically featured by high Non-Esterified Fatty Acid (NEFA) in blend with low glucose concentrations in serum (Leroy et al., 2004). These NEFAs may have an effect on follicular growth and fertility by acting unswervingly on the oocyte and on other cell types within the growing follicle. Insufficient energy supply results in poor reproductive performance, which includes a delay in the onset of oestrous cycles during the post-partum period and a reduction in oocyte quality (Snijders et al., 2000; Butler, 2003). These reproductive alterations result in early embryonic loss and low conception rates (Lucy, 2001).
We observed that oleic acid, palmitic acid and stearic acid are the three predominant free fatty acids both in serum and in follicular in our laboratory (Nandi et al., 2013). The mean value of palmitic and oleic acid in sheep follicular fluid was found to be 17-24 and 34-43 μM, respectively (Nandi et al., 2013). Physiological NEFA concentrations of stearic acid, palmitic acid, oleic acid are 25, 50 and 75 μm, respectively (Van Hoeck et al., 2011). Bovine blastocyst production rates could be lowered when in vitro produced embryos were exposed to stearic acid, palmitic acid, oleic acid in concentration of 75, 150, 200 μm, respectively (Van Hoeck et al., 2011; Alves et al., 2015). We also demonstrated in our laboratory that stearic acid, a saturated fatty acid inhibited the ovine oocyte development in vitro at 30 μM level (Farman et al., 2015). The present study was undertaken to study the effect of different concentration of a saturated fatty acid, palmitic acid (PA: 0, 20, 40 and 60 μM) and an unsaturated fatty acid, oleic acid (OA: 0, 40, 60 and 80 μM) on in vitro oocyte development of ewes (Ovis aries). We also investigated the efficacy of oleic acid on prevention of detrimental effects of saturated fatty acids on oocyte competence.
MATERIALS AND METHODS
The ovary and testis collection, oocyte recovery, in vitro maturation, viability evaluation, in vitro fertilization and in vitro culture of embryos were as described earlier (Farman et al., 2015). Briefly, sheep ovaries were collected from a local slaughter house, Bangalore and they were brought to the laboratory within 2 h in a thermo flask containing warm 0.9% normal saline. Oocytes were recovered from ovaries using follicle aspiration technique. Oocytes were graded by morphological appearance of the cumulus cells investments and homogeneity of ooplasm under zoom stereomicroscope. Oocytes with a minimum of three layers of cumulus cells were selected for studies.
The oocytes were matured in groups of 8-12 in 50 μL maturation media and incubated under a humidified atmosphere of 5% CO2 in air for 24 h at 38.5°C. The control oocyte maturation medium consisted of TCM-199+FBS (10%)+FSH (10 μg mL–1)+Gentamicin (50 μg mL–1). Maturation of oocytes was assessed on the basis of cumulus cell expansion and first polar body extrusion after 24 h of incubation as described earlier (Nandi et al., 2002). For in vitro insemination, sperm cells were added to the fertilization medium to a final concentration of 2-3×106 sperm cells mL–1. The embryo culture medium consisted of TCM-199+FBS (10%)+Gentamicin (50 μg mL–1). After 40-42 h after inseminating the oocytes, the presumptive zygotes were evaluated under stereo zoom microscope at 110×magnification for evidence of cleavage. Results were recorded in terms of cleavage rate (percentage of oocytes inseminated and that were cleaved to 2 cell stage). The cleaved embryos were further cultured for 7 days for production of morulae/blastocytes.
Experimental designs
Experiment 1: To study the effect of a saturated (palmitic acid) and an unsaturated fatty acid (oleic acid) on in vitro development of ovine oocytes: Viable ovine oocytes were cultured at 38.5°C with 5% CO2 in air in the presence of palmitic acid (0, 20, 40, 60 μM) in oocyte maturation medium for 24 h. The viability and maturation rates were examined. The matured oocytes were in vitro inseminated and the fertilization and cleavage rates were examined.
Experiment 2: To study the effect of an unsaturated fatty acid (oleic acid) on in vitro development of ovine oocytes: Viable ovine oocytes were cultured at 38.5°C with 5% CO2 in air in the presence of oleic acid (0, 40, 60, 80 μM) in oocyte maturation medium for 24 h. The viability, maturation, cleavage and embryo production rates were examined as described in experiment 1.
Experiment 3: To examine the effect of oleic acid on prevention of detrimental effect of saturated fatty acids on oocyte development: The levels of palmitic acid and oleic acid were chosen for this experiment was 60 and 40 μM as these concentration caused significant detrimental and beneficial effects respectively in the experiment 1 and 2 of the present study. The level of stearic acid chosen for this experiment was 30 μM as this concentration was found to be detrimental for oocyte maturation in an earlier study in our laboratory (Farman et al., 2015). Viable ovine oocytes were cultured at 38.5°C with 5% CO2 in air in the presence of (a) Control maturation medium, (b) Palmitic acid (60 μM)+oleic acid (40 μM) in oocyte maturation medium and (c) Oleic acid (40 μM)+stearic acid (30 μM) in medium for 24 h. The viability, maturation, cleavage and embryo production rates were examined as described in experiment 1.
Statistical analysis: The maturation rates, fertilization rates and embryos yield were analysed by ANOVA followed by Tukey’s multiple comparison tests (percentage values were transformed to arcsine values before analysis). The statistical package of Graph Pad Prism, San Diego, USA was used for analyzing the data. A value of p<0.05 was considered statistically significant.
RESULTS AND DISCUSSION
Experiment 1: The effect of different concentration of palmitic and oleic acid on in vitro maturation, viability, cleavage and morulae/blastocyst formation is presented in Fig. 1. Exposure of oocytes to 60 μM concentration of palmitic acid in oocyte maturation medium significantly decreased maturation and cleavage rates compared to those observed in oocytes culture in media containing 0 μM (control group), 20 and 40 μM concentration of palmitic acid. The viability rates were not significantly affected in oocytes cultured in media containing 0, 20, 40 and 60 μM palmitic acid concentrations. No significant changes in the morulae/blastocyst yield were observed in oocytes cultured in 0, 20 and 40 μM palmitic acid. However, the morulae/blastocyst yield was significantly decreased in oocytes cultured in 60 μM group compared to lower concentration groups.
Experiment 2: The effect of different concentration of oleic and oleic acid on in vitro maturation, viability, cleavage and morulae/blastocyst formation is presented in Fig. 2. Exposure of oocytes to 40 and 60 μM concentration of oleic acid in oocyte maturation medium significantly increased maturation, cleavage and embryo production rates compared to lower control groups. No significant changes in maturation, cleavage and embryo production rates were observed between 40 and 60 μM levels. Increasing further to 80 μM levels reduced the maturation, cleavage and morulae/blastocyst yield compare with 40 and 60 μM but no significant changes were observed when compared with control group.
| Fig. 1: | Effect of palmitic acid on oocyte development |
| Fig. 2: | Effect of oleic acid on oocyte development |
| Fig. 3: | Effect of oleic acid on prevention of detrimental effect of saturated fatty acids on oocyte development |
No significant change was observed in viability rates in all level tested.
Experiment 3: No significant difference was observed in combined dose of PA+OA and OA+SA in terms of maturation, viability cleavage and morulae/blastocyst yield in comparison with control (Fig. 3).
High NEFA concentrations arised from up-regulated lipolysis had been occupied as a key factor in the association between metabolic imbalances, cellular dysfunction and related pathologies (Van Hoeck et al., 2011). The high circulating NEFA levels, associated with NEB were reflected in the follicular fluid of dominant follicles in ruminants early postpartum (Roth et al., 2001; Leroy et al., 2008a). The saturated long chain fatty acids in particular (such as: palmitic and stearic acid) aggravated an inhibition of maturation rate, leading to comparatively low fertilization, cleavage and blastocyst formation rates. In addition, it had been shown that palmitic acid induces apoptotic changes in the cumulus cells (Roth et al., 2001; Leroy et al., 2008b), which in turn sway oocyte maturation and perhaps embryo development in a negative way.
The reduction in blastocysts formed indicated that NEFA-exposure during oocyte development had a significant negative impact on post-genome activated development as well as on the pattern of gene transcription. It was generally recognized that saturated NEFA, such as palmitic acid (C16:0) and stearic acid (C18:0), could directly exert negative effects on cell viability (Lu et al., 2003).
Medium with elevated concentrations of PA showed a negative effect on the progression of meiosis. The subsequent fertilization and cleavage rates and blastocyst formation were significantly reduced. OA had no effect on any on the outcome of the variables, which confirms that maturation and fertilization proceeded normally (Rizos et al., 2002) which in agreement with our findings. The reduced fertilization rate and hampered in vitro development were most likely carry-over effects of the delayed or blocked maturation. The PA had a negative effect on rate of blastocyst formation yield. Exposing oocytes to higher NEFA concentrations hamper oocyte development, along with these its impact on the physiology of the resultant blastocytes (Van Hoeck et al., 2011; Van Hoeck et al., 2013a). Embryos arised from fertilized, NEFA-exposed oocytes have a significantly lower cell number, increased apoptotic cell index, abnormal transcriptional behavior, tainted amino acid turnover and misrepresented metabolism, with exacting allusion to glucose intolerance and reduced oxidative activity (Van Hoeck et al., 2011; Van Hoeck et al., 2013b). All these are indicators of lower embryo quality and viability . The NEFA cytotoxicity lead to certain modification in somatic cells like modification in cell membrane phospholipids, elevated nitric oxide production and increased ceramide concentration, which leads to apoptosis (Lu et al., 2003; Van Hoeck et al., 2013a).
Palmitic acid (16:0) is the second most copious in bovine follicular fluid (Leroy et al., 2005). It had been reported that saturated fatty acids were readily taken up by bovine oocytes (Adamiak et al., 2006; McKeegan and Sturmey, 2011) while, bovine oocyte maturation regressed in the presence of Palmitic Acids (PA) and significantly reduced fertilization, cleavage and blastocyst rates (Leroy et al., 2005). In contrast, for cat oocyte maturation and its metabolism is significantly increased in in vivo when compared with in vitro matured oocytes in presence of palmitate (McKeegan and Sturmey, 2011). Exposure of premature embryos to high concentrations of palmitic acid lead to deviant metabolism, augmented apoptosis (mainly due to smash up by Reactive Oxygen Species (ROS) and lengthy metabolic perturbation leaving the organism predisposed to diabetes and obesity (McKeegan and Sturmey, 2011). The mechanism of such a nongenomic hereditary condition had not yet been defined, although epigenetic modification was a likely candidate (Chason et al., 2011).
Oleic acid (18:1) was found in elevated concentration in bovine oocytes and follicular, oviducal and uterine fluids (Tsujii et al., 2001). Quinn and Whittingham (1982) reported that exogenous oleic and palmitic acids inhibited fertilization of mouse embryos but promote blastocyst formation and hatching when added to 8-cell embryos. Palmitic had a dose-dependent inhibitory effect on oocyte developmental competence. Oleic acid, in contrast caused an improvement of oocyte developmental capability. Likewise, the unfavorable effect of palmitic and stearic acid was found to be counteracted by oleic acid in our study.
CONCLUSION
Palmitic acid caused reduction in oocyte development at a very high dose. Oleic acid was beneficial to oocyte development at the physiological levels. Oleic acid also compensated for the unfavorable effects of palmitic and stearic acid. This imply that not only the concentration, but more prominently the ratio of saturated and unsaturated fatty acid in follicular fluid affect the developmental competence of the oocyte.
ACKNOWLEDGMENTS
We are grateful to the Director, NIANP, Bangalore for providing necessary facility to carry out the research work. We also like to thank to Mr. Gyan Prakash for his technical assistance. Financial help from Department of Biotechnology, Government of India is gratefully acknowledged.