EFFICIENCY OF FATTY ACID-FREE DEFINED SYSTEM FOR IN VITRO MATURATION OF BUFFALO OOCYTES.
The evaluation of medium for in vitro maturation of buffalo oocytes has progressed towards more defined conditions for the studies evaluating effect of specific components in the medium. Therefore, aim of the present study was to evaluate the effect of fatty acid free-bovine serum albumin (FAF-BSA) alone or supplemented with epidermal growth factor (EGF) in the in vitro maturation medium on in vitro maturation of oocytes and subsequent embryo development. The cumulus oocyte complexes (COCs) were collected from ovaries (900) of slaughtered animals by aspirating follicles, and incubated in maturation medium for 24 h. Three types of maturation media (MM) supplemented with 1) 10% fetal calf serum (FCS), 2) 6 mg/ml FAF-BSA or 3) 6 mg/ml FAF-BSA + 10.0 ng/mL epidermal growth factor (EGF) were evaluated. After maturation, Tyrode's albumin lactate pyruvate (TALP) and synthetic oviductal fluid (SOF) media were used for fertilization and culture environments, respectively.
The percentage of COCs with expanded cumulus was higher (P 0.05) was observed in meiotic resumption and nuclear maturation rate in all three types of maturation media. Percentage of embryos crossing 8-16 cell stage and reaching up to 32 cell stage (morula) after IVC was lower (P 0.05) with development in MM supplemented with FCS. In conclusion, serum free defined system having FAF-BSA+EGF was comparable to the MM containing FCS and can be used to replace serum supplemented media for the in vitro maturation of buffalo oocytes and further embryo development.
Key words: In vitro maturation, serum free medium, defined medium, epidermal growth factor.
Buffalo represents an integral part of the agricultural economy in Pakistan contributing high quality animal protein, both milk and meat. However, the production of milk in buffalo is lower than its potential (Bilal et al., 2006). The production potential can be improved by genetic selection of superior animals using assisted reproductive technologies. Success rate of the reproductive biotechnologies viz., artificial insemination, super ovulation and embryo transfer is limited in buffalo as compared to cattle, due to few primordial follicles, smaller number of recruitable follicles, high level of follicle atresia, poor estrus detection and poor response to superovulation (Madan, 1990). Therefore, the emphasis has now been shifted towards in vitro production of high quality buffalo embryos (IVEP) for research, biotechnology and commercial settings (Hansen, 2006) utilizing germ plasm of both male and female animals simultaneously (Barakat et al., 2012).
During IVEP, in vitro maturation is a crucial step for the generation of quality oocytes capable of being fertilized and developed into normal embryos (Wang et al., 1997). The process of meiotic maturation and acquisition of developmental competence determines the ability of the oocyte to undergo successful fertilization, cleavage, and embryonic development. The culture conditions in which oocytes develop are critical; especially the composition of media affects nuclear and cytoplasmic maturation of oocytes and ultimately the early embryonic development (Gordon, 2003). It is relevant to mention that the maturation medium and its protein supplements play an important role in oocyte maturation and in vitro development following IVF (Bavister et al., 1992). Serum is commonly used constituent of maturation media that promote in vitro maturation of oocytes and post cleavage development of embryos (Kim et al., 2001).
It contains essential components like proteins, fatty acids, vitamins, trace elements, hormones, and growth factors (Harper and Brackett, 1993). Although serum has an inevitable ability to support oocyte maturation and embryo development in vitro, there are certain disadvantages. Due to its complex and undefined nature, it may contain varying concentrations of unknown molecules and batch to batch variation exists in its activity (Takagi et al., 1991). Furthermore, being blood derivative, serum can induce contamination and infection of oocytes and gametes (Freshney, 2000). Therefore, recently, research efforts have focused at replacement of serum with alternative compounds including polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA), chicken egg albumin, BSA-V, purified BSA, essentially fatty acid-free BSA (Ali and Sirard, 2002; Collado et al., 2014) particularly in studies evaluating effect of specific components in the media (Korhonen et al., 2010).
Bovine serum albumin (BSA) is an alternative to serum and is a relatively pure fraction than serum (Bavister, 1995). The constituents of BSA include albumin, estradiol, low molecular weight substrates and growth factors (Mingoti et al., 2002). Essentially fatty acid-free bovine serum albumin (FAF-BSA) is another form of BSA that can also replace serum as protein source to obtain more defined IVM environment (Ali and Sirard, 2002). It is pertinent to mention that undefined source of lipids in maturation media results in accumulation of lipid droplets in in-vitro produced bovine embryos that increase their sensitivity to cryopreservation (Rizos et al., 2003). However, the factors that induce lipid accumulation are removed during purification (Yotsushima et al., 2004) and had not been identified in fatty acid free-BSA. The requirement of a fatty acid free defined media is further intensified if the effect of certain fatty acids is to be investigated.
Along with the merits of fatty acid free defined media, there is also a drawback in its use. It has been investigated in bovine that embryo yield is compromised in culture medium supplemented with FAF-BSA compared to serum supplemented group (Korhonen et al., 2010). It is therefore needed to reinforce the fatty acid free defined media to acquire full advantage during IVM of oocytes. It is known that growth factors present in the serum contribute towards increased proportion of matured oocytes and embryos developed to blastocyst stage (Chauhan et al., 1999; Kumar and Purohit, 2004; Sadeesh et al., 2014). Among these, epidermal growth factor (EGF) is reported to resume meiosis in various mammalian species (Gall et al., 2004). It was therefore hypothesized that serum in the in vitro maturation media can be replaced with FAF-BSA if fortified with epidermal growth factor.
The objective of the present study was to evaluate the effect of replacing serum with fatty-acid free BSA (FAF-BSA) alone or with epidermal growth factor (EGF) on in vitro maturation of oocytes and subsequent development of the embryos.
MATERIALS AND METHODS
Unless otherwise stated, all reagents were purchased from Sigma.
Collection of ovaries: During breeding season, buffalo ovaries were collected from slaughtered animals and transferred to the IVF laboratory, Buffalo Research Institute (BRI), Pattoki, within two hours in sterilized phosphate buffered saline (PBS) kept at 33-35degC. Fresh PBS was used to wash the ovaries immediately after arrival. A total of 900 ovaries were collected in five replicates of the study.
Collection of oocytes: Sterile disposable plastic syringe (10 ml) fitted with 18 gauge needle was used for aspiration of follicular fluid with immature cumulus-oocyte complexes (COCs) from 2-8 mm follicles. Searching for COCs in the follicular fluid was done in a 35 mm petri dish under stereomicroscope and collected. The COCs were classified as grade A, B, C and D, on the basis of their cumulus investment and ooplasm homogeneity (Sabasthin et al., 2013). Only type A and B COCs were selected for IVM.
In vitro Maturation (IVM): Selected COCs were washed twice in PBS (37oC) and twice in pre-equilibrated maturation medium (MM: TCM-199 supplemented with 0.02 IU/mL FSH, 1 u g/mL estradiol-17b (E2), 50 u g/mL gentamicin and 1) 10% fetal calf serum (FCS), 2) 6 mg/ml FAF-BSA or 3) 6 mg/ml FAF-BSA + 10.0 ng/mL epidermal growth factor (EGF). The washed COCs were randomly allocated into 100 ul of respective culture droplets covered with sterile mineral oil and matured in vitro in an incubator at 38.5degC under 5% CO2 in air with 95% relative humidity for 24 hours. All the media and culture dishes were equilibrated at 38 degC in CO2 incubator for at least 1-2 h before experiment. Data were collected in five independent repeats.
Assessment of cumulus cell expansion: After 24 hours of maturation, cumulus cell expansion was assessed by visual assessment using stereomicroscope as 1) not expanded 2) Partially expanded or 3) Fully expanded (Kobayashi et al., 1994). Criterion is shown in Plate 1. Data on cumulus cells expansion of oocytes after 24 hours of maturation, from five (5) replicates are pooled and presented.
Nuclear maturation assessment: For determination of nuclear stage in meiosis, COCs were completely denuded, stained with 1% aceto-orcein and examined for GV (Germinal vesicle), GVBD (Germinal vesicle break down), MI (Metaphase I) and MII (Metaphase II) stage under a phase contrast microscope (Yadav et al., 1997). Criterion is shown in Plate 2. Data on nuclear stage of oocytes after 24 hours of maturation, from five (5) replicates are pooled and presented.
In vitro fertilization (IVF): Motile spermatozoa were separated by swim up technique (Parrish et al., 1986) in Tyrode's albumin lactate pyruvate (TALP) medium. Oocytes were washed in fertilization media and were placed in the 50 ul droplet of pre warmed fertilization media under mineral oil with final sperm concentration of 2x106 mL-1. The oocyte and spermatozoa were incubated at 38.5 degC under 5% CO2 with 95% humidity for 20 h (Gasparrini et al., 2008).
In vitro Embryo Culture (IVC): After 20 h of fertilization, cumulus cells were removed by vigorous pipetting in PBS. After one time washing with PBS and two times with culture media; synthetic oviductal fluid medium (SOF), presumptive zygotes were transferred to 25 u l of culture droplet under mineral oil in four-well dish and cultured at 38.5degC in a humidified incubator with 5% CO2 in air.
Cleavage Rate and Early embryo development: On day 2 of development (Day 0 = day of insemination) the cleavage rate (number of oocytes cleaved/total COCs incubated x 100) was observed. The culture was continued and further developmental stages were recorded every other day.
Statistical Analysis: Differences in cumulus expansion, nuclear maturation of oocytes after 24 h after maturation and further developmental stages were analyzed by one-way analysis of variance (ANOVA) at 5% level of significance. Least Significant Difference (LSD) test was used to compare treatment means.
Cumulus Expansion: Data on cumulus mass expansion of the oocytes matured in different media are presented in Table 1. Higher (P < 0.05) number of COCs with fully expanded cumulus mass was recorded in IVM medium supplemented with serum compared to medium supplemented with FAF-BSA. However, the difference between IVM medium supplemented with serum and the medium with FAF-BSA+EGF (10ng/mL) was non-significant. Percentage of COCs with partial cumulus expansion remained similar among treatment groups, while COCs with non-expanded cumulus mass were lower (P 0.05) among experimental groups.
Early Embryonic Development: Data on development of embryos after maturation of oocytes in the media supplemented with FCS, FAF-BSA or FAF-BSA + EGF are given in Table 3. The cleavage rate and percentage of embryos reaching 4-8 cell stage after IVC were similar (P > 0.05) among treatment groups. However, number of embryos crossing 4-8 cell block and reaching 8-16 cell stage were lower (P 0.05) trend was seen for embryos reaching 32 cell stage (Morula).
Table 1. Cumulus expansion rate of buffalo oocytes after 24 hours of maturation in IVM medium supplemented with FCS, FAF-BSA or FAF-BSA+EGF.
Treatments###Cumulus Expansion (Mean percentage+-SEM)
###N###Fully Expanded###Partially Expanded###Not Expanded
###N (%)###N (%)###N (%)
FCS###129###88 (68.21+-1.74)a###30 (23.25+-1.36)a###11 (8.50+-0.86)b
FAF-BSA###129###70 (54.26+-1.26)b###34 (26.35+-0.67)a###25 (19.37+-1.10)a
FAF-BSA+EGF###129###84 (65.11+-2.04)###31 (24.03+-0.79)a###14 (10.85+-1.41)b
Table 2. Nuclear maturation status of buffalo oocytes after 24 hours of maturation in IVM medium supplemented with serum, FAF-BSA or FAF-BSA+ EGF.
Treatments###Nuclear Stage in Meiosis (Mean percentage+-SEM)
###N (%)###N (%)###N (%)###N (%)
FCS###129###3 (2.32+-0.94)###28 (21.71+-1.25)###19 (14.73+-0.83)###79 (61.24+-1.71)
FAF- BSA###129###2 (1.55+-0.92)###29 (22.48+-1.59)###21 (16.28+-0.93)###77 (59.68+-1.26)
FAF- BSA+EGF###128###2 (1.56+-0.92)###29 (22.65+-1.17)###20 (15.62+-1.18)###77 (60.15+-1.57)
Table 3. Effect of supplementation of IVM medium with FCS, FAF-BSA and FAF-BSA+EGF on the cleavage rate and subsequent development of buffalo oocytes in vitro.
Treatments###Developmental stages (Mean percentage+-SEM)
###N###Cleaved###4-8 cell embryos###8-16 cell embryos###32 cell embryos (Morula)
###N (%)###N (%)###N (%)###N (%)
FCS###129###75 (58.1+-1.58)a###60 (46.37+-3.52)a###35 (27.12+-1.24)a###23 (17.79+-0.74)a
FAF- BSA###129###69 (53.48+-1.12)a###53 (41.01+-1.43)a###22 (17.05+-0.93)b###10 (7.70+-1.24)b
FAF- BSA+EGF###128###71 (55.04+-0.49)###56 (43.69+-1.93)###30 (23.41+-1.64)###18 (14.06+-0.94)ab
To obtain serum free IVM medium, bovine serum albumin (BSA) is an alternate source for oocyte maturation (Wydooghe et al., 2014). In present study, BSA alone in the IVM medium could not initiate proper protein synthesis for embryo development compared to serum supplemented IVM medium. Therefore, EGF was supplemented along with BSA in the maturation media. It is relevant to mention that oocyte utilizes EGF as a source of protein and it also alters the pattern of proteins neosynthesis during IVM (Lonergan et al., 1996). Receptors for EGF are reported on cumulus cells of oocytes that are the major site of action of the growth factors for regulation of oocyte maturation (Downs et al., 1988).
In the present study, when FAF-BSA was used to replace fetal calf serum, the cumulus expansion was compromised during IVM. These results are in line with previous studies that reported reduced cumulus cell expansion when serum was substituted with bovine serum albumin (Cetica et al., 1999). However, with the addition of EGF, cumulus expansion was improved compared to FAF-BSA alone and was comparable with expansion obtained in MM supplemented with FCS. This improvement in cumulus expansion of the oocytes matured in the media having FAF-BSA+EGF may be attributed to the reinforcement of FAF-BSA alone treatment group with epidermal growth factor. The present study is in line with the previous reports that buffalo COCs respond well towards EGF and resulted in improved meiotic resumption, increased proportion of matured oocytes and embryos developed to blastocyst stage (Chauhan et al., 1999; Kumar and Purohit, 2004; Sadeesh et al., 2014).
In the present study, the nuclear maturation rate was not compromised when serum was substituted with FAF-BSA alone or supplemented with EGF, although there was significant lower cumulus expansion when serum was substituted with FAF-BSA in IVM medium. Similar results have been reported by Saeki et al. (1991) in bovine where the nuclear maturation rate of oocytes did not differ among media supplemented with variable protein sources (FCS or PVP). The results of nuclear maturation in our study were independent of the cumulus expansion rate and it has previously been reported that the rate of oocyte nuclear maturation or fertilization does not seem to depend on the extent of cumulus expansion (Sirard et al., 1988).
The developmental competence of an oocyte is determined by its nuclear and cytoplasmic maturation rate in vitro. During maturation period, transcription factors, mRNAs and proteins are stored, which are necessary for maturation, fertilization and the beginning of early embryonic development (Ferreira et al., 2009). In the current study, percentages of embryos crossing 8-16 cell stage and reaching up to 32 cell stage (morula) after IVC was lower (P < 0.05) in MM supplemented with FAF-BSA compared to development in MM supplemented with FCS, however with addition of EGF, embryo development was improved, although it was statistically non significant and was comparable to the development in MM supplemented with FCS. Compromised nature of FAF-BSA alone experimental group was improved (non-significant) by reinforcing it with EGF, which might have enhanced the oocyte maturation through changing the pattern of protein synthesis, as has been reported in cattle (Park et al., 1999; Lonergan et al., 1996).
Conclusion: In conclusion, serum free defined system having FAF-BSA+EGF was comparable to the MM containing FCS and can be used to replace serum supplemented media for the in vitro maturation of buffalo oocytes and further embryo development.
Acknowledgements: The authors thank to Higher Education Commission, Pakistan for financial support under 5000 PhD indigenous fellowship scheme.
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