Supplementing α-Linolenic acid in the in vitro maturation media improves nuclear maturation rate of oocytes and early embryonic development in the Nili Ravi buffalo

The present study was conducted to investigate the effect of omega-3 poly unsaturated fatty acid (PUFA), α-linolenic acid (ALA; 18:3 n-3) on the in vitro maturation (IVM) of buffalo oocytes and subsequent embryonic development. Buffalo cumulusoocyte complexes (COCs; n = 2282) were in vitro matured in TCM-199 (0.6% fatty acid free bovine serum albumin, 0.02 Units/ml FSH, 1 μg/ml 17-β-estradiol, 10 μg/ml epidermal growth factor, 50 μg/ml gentamicin) supplemented with 0 (control), 25, 50, 100, 150 or 300 μm ALA under an atmosphere of 5% CO2 in air at 38.5oC for 22-24 h. The matured oocytes were then fertilized in Tyrode’s Albumin Lactate Pyruvate (TALP) medium and cultured in synthetic oviductal fluid (SOF) medium. Concentrations up to 100 μm ALA improves (P ≤ 0.05) the cumulus expansion compared to control. Higher percentage of oocytes reaching MII stage was observed at 50 μm and 100 μm of ALA compared to control (P ≤ 0.05) . Concentrations of 150 and 300 μm ALA were detrimental both for cumulus expansion and nuclear maturation rate of buffalo oocytes. Moreover, supplementation with 100 μm ALA improved (P ≤ 0.05) cleavage rate compared to control and treatment with 50 and 100 μm ALA yielded significantly higher morulae compared to control. The results of present study indicate that the supplementation with 100 μm ALA to the IVM medium improves nuclear maturation rate of buffalo oocytes and subsequent early embryonic development.


Introduction
The Nili-Ravi buffalo is considered to be one of the best milk producers among other breeds of buffaloes in the world (Warriach et al., 2008).Although, this breed has the potential of producing more than 5000 L of milk/lactation (Bilal et al., 2006), the average milk yield is quite low, which raises the opportunities of genetic improvement through assisted reproductive technologies like artificial insemination, and multiple ovulation and embryo transfer (MOET).
However, the implementation of these technologies is hampered due to some inherent reproductive problems in buffalo such as fewer primordial follicles, which results in smaller number of recruitable follicles, high level of atresia, poor estrus behavior detection and a poor response to superovulation protocols (Madan, 1990).Recently, the emphasis has now been shifted towards the in vitro production (IVP) of buffalo embryos (Hansen, 2006) utilizing germ plasm of both male and female animals simultaneously (Barakat et al., 2012).
In vitro maturation (IVM) is a key step during which oocytes undergo all the necessary changes required for successful fertilization and further embryonic development (Wang et al., 1997).Therefore, the maturation medium is crucial, not only for the maturation of oocyte itself but also for its further competence after IVF (Bavister et al., 1992;Kharche et al., 2006).Previously, several studies have evaluated different types of media (Totey et al., 1993;Abdoon et al., 2001;Zicarelli et al., 2003), and its supplements including different sources of protein (Chauhan et al., 1998), hormones (Totey et al., 1993), thiol compounds (Zicarelli et al., 2005;Gasparrini et al., 2006), growth factors (Purohit et al., 2005) and antioxidants (Ullah et al., 2006) for in vitro maturation of buffalo oocytes with variable success rates and blastocyst production rate of no more than 10-20% (Kumar and Anand, 2012) which is relatively lower when compared to 30-40% blastocyst production rate in cattle (Rizos et al., 2008).
PUFAs must be provided by diet, since their in vivo synthesis is not possible due to absence of proper enzymes (Gurr et al., 2002).Change in the composition of dietary fatty acids not only modifies fatty acid composition in the blood plasma but also of the reproductive tissues including, follicular fluid, cumulus cells and the oocytes (Ferguson and Leese, 1999;Zeron et al., 2002;Bilby et al., 2006;Childs et al., 2008;Fouladi-Nashta et al., 2009;Wonnacott et al., 2010), which can directly influence the competence of oocytes for further development and/or fertility (Armstrong et al., 1990;Burke et al., 1997;Petit et al., 2001;Wonnacott et al., 2010).For instance, dietary fish oil supplementation has been shown to alter the specific n-3 and n-6 fatty acids in follicular fluid, cumulus cells and oocytes in both beef heifers (Childs et al., 2008) and ewes (Zeron et al., 2002;Wonnacott et al., 2010).Such changes in the fatty acid profile may improve oocyte maturation, which is essential for successful fertilization and further embryo development (Marei et al., 2009) Fatty acids supplemented into IVM medium yielded positive effects on oocyte maturation, fertilization, and embryonic development in the rat (Khandoker and Tsujii, 1999), goat, sheep (Veshkini et al., 2015) and cattle (Kim et al., 2001).
Alpha-linolenic acid (ALA; C18:3) is the dietary precursor for the long-chain omega-3 PUFAs (Brenna et al., 2009).It plays an important role in folliculogenesis, developmental competence of oocyte (Moallem et al., 2013), fertilization rate and embryo quality (Thangavelu et al., 2007).As ALA is produced by the ovarian follicles and the amount of ALA increases as the ovarian follicles enlarge (Veshkini et al., 2015); a role of specific unsaturated fatty acids like ALA may be speculated in the oocyte maturation and/or follicular growth.In fact, ALA has been shown to improve the fertility rate in both cattle and sheep by improving folliculogenesis and fertilization rate in vivo (Moallem et al., 2013).When supplemented in the in vitro maturation medium it was reported to regulate the molecular mechanism leading to increased number of MII stage oocytes and improved their subsequent development into early embryos in both cattle (Fouladi-Nashta et al., 2009;Marei et al., 2009) and sheep (Ghaffarilaleh et al., 2014).However, the role of ALA supplementation of IVM media for Nili Ravi buffalo oocytes has not been studied yet.In this study we hypothesized that the supplementation of ALA in IVM media improves the quality of in vitro matured buffalo oocytes and their subsequent development postfertilization.The main objective of the study was to evaluate the effects of ALA supplementation of the IVM media on the in vitro oocyte maturation and subsequent embryonic development in buffalo.

Collection of oocytes from buffalo ovaries
Buffalo ovaries (n = 2500) were collected from the local abattoir and within two hours of collection transferred to the laboratory in a thermos containing sterilized phosphate buffered saline (PBS) kept at 33-35°C.Fresh PBS was used to wash the ovaries immediately after arrival.Sterile disposable plastic syringe (10 ml) fitted with 18 gauge needle was used to aspire cumulus-oocyte complexes (COCs) from antral follicles (2-8 mm).Searching for COCs was done under stereomicroscope in PBS and was classified as grade A, B, C and D, on the basis of their cumulus investment and ooplasm homogeneity (Sabasthin et al., 2013).Grade A COCs had four or more than four compact cumulus layers with homogenous ooplasm, while grade B oocytes had two to three layers of compact cells with homogenous ooplasm.Grade C oocytes had only one layer of less compact cells with irregular ooplasm, while the oocytes having irregular dark ooplasm and highly expanded cumulus cells and denuded oocytes were considered as grade D (Sabasthin et al., 2013).COCs (n = 2282) with homogenous ooplasm and more than two compact layers of cumulus cells were selected for the experiments.

In vitro maturation (IVM) of oocytes
COCs were in vitro matured in 100 µl drops covered with sterile mineral oil for 24 h at 38.5ºC in 5% CO 2 in air with 95% humidity.All the media and culture dishes were equilibrated at 38°C in CO 2 incubator for at least 1-2 h before experiment.

Assessment of cumulus cell expansion
After 24 h of maturation, cumulus cell expansion was assessed by visual assessment using stereomicroscope as 1) not expanded (no expansion observed in cumulus cells), 2) partially expanded (expansion of outer layers of cumulus cells only), or 3) fully expanded (expansion of all layers of cumulus cells; Azam et al., 2017).

Oocyte staining and determination of stage of nuclear maturation
For determination of nuclear stage in meiosis, COCs were completely denuded.Denuded oocytes were washed twice in PBS and fixed overnight in acetoethanol.Oocytes were placed on a grease free glass slide and covered with a cover slip.Oocytes were slightly compressed onto the glass slide with a needle until drop containing oocytes touched the cover slip.The oocytes were then stained with 1% aceto-orcein and de-stained with aceto-glycerol as described by Azam et al. (2017).Oocyte's nuclear maturation was evaluated with a phase contrast microscope at 200X to 400X magnification.
Based on morphology, nuclear maturation was categorized as described by Azam et al. (2017).Oocyte nucleus with a nucleolus and filamentous chromatin is at germinal vesicle (GV) stage.Oocyte without nucleolus, nuclear membrane and shortening of chromosomes is at germinal vesicle breakdown (GVBD) stage.At MI stage chromosomes look as thick dots arranged at metaphase plate.The segregation of chromosomes start at anaphase-I and at Telophase-I, complete segregation of chromosomal sets occur.First polar body is released at M-II.

In vitro fertilization (IVF) of oocytes
Frozen semen was used for in vitro fertilization (IVF).Three 0.5 ml straws of cryopreserved buffalo semen were thawed in water bath at 37°C for 30 sec.Thawed semen was placed in a 15 ml conical tube.Spermatozoa with maximum motility were collected by swim up technique (Parrish et al., 1986).About 250 µl of thawed semen was deposited at the bottom of four 15 ml tubes containing 3 ml of pre warmed sperm wash medium (TALP: modified calcium-free Tyrode's Albumin Lactate Pyruvate with 6 mg/ml BSA fraction-V).Tubes were incubated at 45° angle for 30 min.Supernatant from each tube was removed and transferred into another 15 ml conical tube and centrifuged at 1600 rpm for 10 min.The pellet obtained after centrifugation of supernatant was assessed for sperm motility, and concentration was determined in an improved Neubauer counting chamber.Subsequently, sperm suspention was resuspended in pre-warmed fertilization TALP (supplemented with 0.1mM hypotaurine, 0.2mM penicillamine, 0.01mM epinephrine, 10 μg/ml heparin) to a final concentration of 2 x 10 6 cells ml −1 .
After 24 h of IVM, buffalo oocytes were washed in fertilization media and were placed in fertilization droplets (5 COCs/50 µl droplet) of pre warmed fertilization media under mineral oil.Oocytes and spermatozoa were co-incubated for 20 h at 38.5°C under 5% CO 2 with maximum humidity (Gasparrini et al., 2008).The day of fertilization was defined as day 0.

In vitro culture (IVC) of embryos
Following fertilization, presumptive zygotes were denuded by vigorous pipetting in PBS and transferred to 25 µl drops containing IVC media (SOF) supplemented with BME, MEM and 5% fetal calf serum (FCS).Embryo culture was performed at 38.5°C in a humidified incubator with 5% CO 2 in air.On day 2 the cleavage rate (number of oocytes cleaved/total COCs incubated × 100) was evaluated.Further developmental stages (4-8 cell stage, >8 cell stage and morula) were evaluated and recorded every other day.

Experiment 2
Oocytes (1082) were matured as in the experiment 1. Matured oocytes were fertilized and cultured.The experiment was replicated seven times.Embryonic development was assessed as cleaved, 4-8 cell stage, >8 cell stage and morula formation.

Statistical analysis
The data on the effect of different doses of ALA on COCs expansion, MII stage, cleavage rate and developmental stages (2 cell stage, 4-8 cell stage, >8 cell stage and morula) were analyzed by one-way analysis of variance (ANOVA) after square root transformation (Y = √X+0.5)and presented as Mean ± SEM.Duncan's multiple range test (DMRT) was used to compare the treatment means.Results were considered significant at P < 0.05.

Experiment 1
The data on the effect of ALA supplementation of maturation media on cumulus expansion are shown in Fig. 1 and 2. The addition of 25 μm ALA in maturation media did not show improvement (P > 0.05) in cumulus expansion compared to control.However, supplementation with 50 and 100 μm ALA resulted in increased rate of cumulus expansion (P ≤ 0.05 ) compared to control.The higher levels of ALA, i.e., 150 and 300 μm resulted in significant decrease (P < 0.05) in expansion of cumulus cells compared to other groups (Fig. 1 and 2).

Discussion
The fatty acid content of developing oocytes changes depending on the environment in which they develop (Wakefield et al., 2008).Changing the microenvironment of oocyte also changes intra-cytoplasmic concentration of lipids.Therefore, PUFAs in the microenvironment may replace saturated FAs in the oocyte cytoplasm and make the oocyte membrane comparatively more permeable for sperm entry and increase fertilization rate and developmental competence of the oocyte.Present study shows that supplementation of maturation media with 100 µm ALA increased the number of fully expanded oocytes.Supporting our current findings, a previous study (Marei et al., 2009) demonstrated that supplementation of maturation medium with ALA at a concentration of lower than 100 µm had no adverse effect on cumulus cell expansion in cattle.The deleterious effect of ALA at a concentration of 150 and 300 µm in the present study is consistent with the previous studies (Coyral-Castel et al., 2010;Ghaffarilaleh et al., 2014;Veshkini et al., 2015) that reported decreased viability of granulosa cells with higher concentration of ALA after 24 h of maturation.In the present study, high levels of ALA in maturation medium might have been outside the tolerance threshold of the cells, exerted toxic effect leading to a reduction in cell viability (Andrade et al., 2005).
Cumulus cells are important for keeping the oocytes under meiotic arrest, inducing meiotic resumption and supporting cytoplasmic maturation.These functions have been attributed to their gap junctions and their specific metabolizing capabilities (Tanghe et al., 2002).It is anticipated that cumulus cells mediate signals and regulate the synthesis of important cytoplasmic factors which support nuclear maturation (Albertini et al., 2001).However, it is not fully understood that expansion of cumulus cells is prerequisite for nuclear maturation or both processes are not inter-related.It has been reported in the ovine that cumulus cell expansion is prerequisite for nuclear maturation (Amini et al., 2016), at the same time no such connection has also been reported in the bovine (Marei et al., 2010;Khalil et al., 2013).
Meiotic competence of oocytes determines the developmental capacity for subsequent embryo development.In the present study, supplementation of maturation medium with 100 µm ALA increased the MII rate of buffalo oocytes.High doses of ALA in IVM medium inhibited maturation progression, manifested by high percentage of oocytes arrested at GVBD or MI stage and a concomitant decrease in percentage of oocytes that completed second meiotic division metaphase.Our results are in agreement with the results of Veshkini et al. (2015) who reported improved nuclear maturation in sheep oocytes at 100 µm ALA in maturation medium.The positive effect of ALA on oocyte's meiotic competence may be mediated directly by improvement of cytoplasmic maturation via the mitogen-activated protein kinase pathway and indirectly through PGE2 synthesis (Marei et al., 2009).The PGE2 is a key paracrine and/or autocrine regulator of cumulus cell functions and has been proposed to play a major role in oocyte nuclear maturation (Elvin et al., 2000).An elevated concentration of PGE2 positively stimulated the extent of cumulus expansion and nuclear maturation by enhancing the phosphorylation of MAPK1 and MAPK2 in both oocytes and cumulus cells through the elevation of cAMP levels (Marei et al., 2009).
In addition to the positive effects observed on cumulus expansion and oocyte nuclear maturation, improvement in all developmental stages of buffalo embryos was observed when oocytes were matured in the presence of 50-100 µm ALA in the maturation media.Similar to the findings of our study, an increased rate of embryonic development has been observed with supplementation of ALA in maturation media in the bovine (Marei et al., 2009), sheep (Ghaffarilaleh et al., 2014) and goat oocytes (Veshkini et al., 2015).Provision of ALA in maturation media might have improved the cellular processes during different phases of maturation, which in turn influenced the developmental competence of subsequent embryos (Krisher and Bavister, 1998).In another study on the bovine, addition of ALA to the culture medium did not improve the cleavage and blastocyst rates; however, the viability of the embryos was influenced in a positive manner by the presence of ALA in culture medium (Al Darwich et al., 2010).It is relevant to mention that the improvement in embryo development rate in present study can only be attributed to supplementation of ALA during oocyte maturation as maturation medium was serum free and there was no supplementation during in vitro embryo culture.These finding s suggest that ALA might have been more important to the oocytes if added to the maturation medium rather than to the culture medium after fertilization.
In the present study, ALA at higher concentrations (300 µm ALA) resulted in reduced number of cleaved embryos and no morula stage embryos.Since PUFAs are more susceptible to peroxidation than monounsaturated and saturated fatty acids, supplementation at higher levels might have increased the oxidative stress (Song et al., 2000;Gladine et al., 2007).Higher levels of ALA might have disturbed the balance of unsaturated and saturated fatty acids, so it might be the ratio of saturated vs unsaturated FAs that is more critical in the oocyte's microenvironment rather than a certain concentration of the unsaturated FAs per se.Therefore, one may speculate that the higher concentrations of ALA in our study might have generated oxidative stress after oxidative phosphorylation of ALA (Wakefield et al., 2008) which might have compromised the developmental competence of these oocytes and be responsible for the observed reduction of embryonic growth.Further studies may be suggested to investigate whether antioxidant supplementation of ALA-supplemented IVM medium could abrogate negative effects as observed for higher concentrations of ALA.In conclusion, the results of the present study have shown that supplementation of in vitro maturation media with 1167 ALA at 100 µm improves the in vitro maturation of buffalo oocytes that in turn improves early embryonic development.

Figure 1 .
Figure 1.Representative pictures of COCs (A: fully expanded, B: partially expanded, C: not expanded) after 24 h of maturation.

Figure 2 .
Figure 2. Cumulus expansion rate of buffalo COCs after 24 h of maturation in IVM medium supplemented with different concentrations of ALA.Data are shown as Mean percentage ± SEM.Bars with different superscripts differ significantly (P < 0.05).0 (Control), 25 (supplementation with 25 µm ALA in the maturation media), 50 (supplementation with 50 µm ALA in the maturation media), 100 (supplementation with 100 µm ALA in the maturation media), 150 (supplementation with 150 µm ALA in the maturation media), 300 (supplementation with 300 µm ALA in the maturation media).

Figure 3 .
Figure 3. Nuclear maturation of buffalo oocytes after 24 h of maturation in IVM medium supplemented with different concentrations of ALA.Data are shown as Mean percentage ± SEM.Bars with different superscripts differ significantly (P < 0.05).GV: germinal vesicle, GVBD: germinal vesicle breakdown, MI: metaphase I, MII: metaphase II, Deg: degenerated.

Table 1 .
Effect of supplementation of IVM medium with different concentrations of ALA on the cleavage rate and subsequent embryo development of buffalo oocytes after in vitro fertilization.