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Determination of an Effective Media and Its Hormone and Protein Supplemenntation for in Vitro Maturation of Oocytes of Indigenous Zebu Cows

M. M. Rahman
Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

S. M. N. Morshed
Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

N. S. Juyeana
Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. M. U. Bhuiyan
Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

In vitro maturation (IVM) of oocytes is the first important step for successful in vitro embryo production of any mammalian species. The objectives of the present study were to determine an effective basic medium and its hormone and protein supplementation for IVM of oocytes of indigenous zebu cows. The oocytes were derived from ovaries of locally slaughtered cows after aspiration of follicle. The oocytes were cultured in medium for 24 hrs at 38.5ºC with 5% CO2 in humidified air for maturation. The maturation of oocytes was evaluated by examining the presence of first polar body extrusion in denuded oocytes under inverted microscope. To determine an effective basic medium, the oocytes were cultured in fetal bovine serum (FBS) supplemented tissue culture medium (TCM), modified synthetic oviduct fluid (mSOF) and Tyrodes albumin lactate pyruvate (TALP) medium. The maturation rate was significantly higher (74±4.2) in TCM medium than that of TALP medium (58.2±6.2). To determine an effective hormone supplementation for maturation medium, the oocytes were cultured in either in follicle stimulating hormone (FSH) or gonadotrophin supplemented TCM. The maturation rate of oocytes was significantly (p>0.05) higher (73.3±4.0) in FSH supplemented medium than that of gonadotrophin supplemented counterpart (60.2±6.6). To determine an effective protein supplementation, the oocytes were cultured in FBS, oestrus cow serum (OCS) and bovine serum albumin (BSA) supplemented TCM 199. The maturation rate of oocytes were 73.0±5.9, 71.1±2.8, and 62.5±9.4 in medium supplemented with FBS, OCS and BSA respectively (p>0.05). In conclusions, TCM supplemented with either FBS, OCS or BSA as protein and FSH as hormone may be used as medium for IVM of oocytes of indigenous zebu cows.

Keywords: Hormone, IVM, Media, Oocytes, Protein, Zebu cows

Location: The Community-based Dairy Veterinary Foundation (CDVF) Laboratory, Department of Surgery and Obstetrics, Bangladesh Agricultural University, Mymensingh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Cow, Hormone

Objectives:

To select an efficient basic medium including supplementation of suitable hormone and protein in it for optimal rate IVM of oocytes of indigenous Zebu cows in Bangladesh.

Methodology:

The study was carried out at the Community-based Dairy Veterinary Foundation (CDVF) Laboratory, Department of Surgery and Obstetrics, Bangladesh Agricultural University, Mymensingh.
Chemicals and media: All the chemicals, reagents and media constituents were purchased from Sigma-Aldrich Inc., St Louis, USA. Media and reagents were prepared using standard protocol and under aseptic condition. All media were filtered using 0.22µm pore size filter (Durapure® membrane filter, Carrigtwohill, Ireland) and culture medium was routinely equilibrated in incubator (VS-9000C, Vision Scientific Co. Ltd. South Korea) at 38.5°C with 5% CO2 in humidified air for at least 2 hrs before use. Collection of ovaries: The ovaries of indigenous zebu cows were collected from local slaughter house after slaughtering within 2 hrs and carried to the laboratory in a thermo flask containing warm normal saline (37°C, 0.9% sodium chloride solution, w/v). Collection of oocytes: In the laboratory, the ovaries were rinsed three times with warm (37°C) normal saline. The follicular fluid of 2 to 8 mm diameter follicles was aspirated using an 18 gauge needle (TERUMO®, Beijing, China) fitted with a 10 ml disposable plastic syringe (JMI Syringes and Medical Devices Ltd ®, Chauddagram, Comilla, Bangladesh). Selection of oocytes for culture: The aspirated follicular fluid was transferred in a 60 mm petridish (Greiner bio-one, Frickenhausen, Germany) and left for 5 minutes for sedimentation. The retrieved follicular aspirate was diluted with HEPES-buffered tissue culture medium (TCM) 199 supplemented with bovine serum albumin (BSA). Oocytes were selected under a stereomicroscope (LABOMED, USA). The cumulus-oocyte-complexes (COCs) with more than 3 compact cumulus cell layers and homogenous ooplasm were selected for maturation. The COCS were washed three times in washing medium followed by once washing in maturation medium. Culture of oocytes for maturation: Four 50µl drops of maturation medium were prepared in 35mm petridish (FALCON, Becton Dickinson Labware, USA) and covered with embryo tested mineral oil. For IVM, 8-10 COCs were cultured in each drop of medium in incubator at 38.5°C with 5% CO2 in humidified air for 24 hrs. Evaluation of oocytes for maturation:The culture drops were examined under the stereo microscope for expansion of cumulus cell after 24 hrs of culture in the maturation media. Presumptive maturation was confirmed by the degree of cumulus expansion. To examine the presence of first polar body extrusion, the COCs were denuded by using denuding agent (3% sodium citrate, w/v in HEPES buffered TCM 199 medium) and pipetting. After pipetting, the denuded oocytes were kept in 10µl drops of HEPES buffered TCM 199 and examined for presence of polar body under inverted microscope (Leica DM IRE2, Germany) with the help of a mouth controlled pipette. Experimental design: Experiment 1-To determine an effective basal maturation medium, the COCs were cultured in TCM 199 (TCM, Earle’s salts with sodium bicarbonate), modified synthetic oviductal fluid (mSOF) medium and Tyrodes albumin lactate pyrovate (TALP) medium supplemented with 10% fetal bovine serum (FBS), 5µg/ml follicle stimulating hormone (FSH) and 1µg/ml oestradiol (OE2). Each experiment was repeated for 10 times across the days. Experiment 2 - To determine an effective hormone supplementation in maturation medium, the oocytes were cultured in TCM 199 supplemented with 5µg/ml FSH or 10 IU/ml Gonadotrophin, 10% FBS and 1µg/ml OE2. Each experiment was repeated for 8 times across the days. Experiment 3 - To determine an effective protein supplementation in maturation medium, the COCs were cultured in TCM 199 supplemented with 10% FBS, 10% OCS (Oestrous Cow Serum) or 3% BSA (Bovine serum albumin) and 5µg/ml FSH and 1µg/ml OE2. Each experiment was repeated for 8 times across the days. Statistical analysis: The data were recorded in Microsoft Excel spread sheet and descriptive statistics was performed. The maturation rates were expressed as mean ± SD and Z test was done to determine the significant effect of different treatments on the maturation rate using SPSS Version 20. The difference between groups was considered significant when P-value was < 0.05.

Source of Information: Bangl. J. Vet. Med. (2018). 16(1): 45–51 ISSN: 1729-7893 (Print), 2308-0922 (Online)

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Funding Source:

1.

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Total Budget (Tk.) :

Achievement/Findings:

The objectives of the present study were to select an effective basic medium and its effective hormone and protein supplementation for IVM of oocytes of indigenous zebu cows. A total of 2340 oocytes were collected from 585 ovaries and the mean number of oocytes collection from each ovary was 4.00. The present mean number of retrieved oocytes per ovary is higher than that of previous study (Morshed et al., 2014; Singha et al., 2015; Choudhury et al., 2017) and lower than that of an earlier study (Talukder et al., 2008). The reason for variations in oocyte retrieval rate among studies may be due to variation in skillness of follicle aspirators. Moreover, seasons of oocytes retrieval and cyclic status of cows may influence the oocyte retrieval rate from ovaries (Dode and Adona, 2001). Additionally, an increase in FSH level in blood may influence the number of oocytes retrieved (Fortune, 1994). Further, nutrition and temperature may influence the gonadotrophin concentrations and affect the population of follicles and number of oocytes retrieved (Zeitoun et al., 1996).In the present study, the maturation rate of oocytes did not vary in OCS supplemented TCM 199 than that of FBS or BSA supplemented counterpart (P>0.05). Contrasting to the present finding, Lu and Gordon (1987) reported that OCS had a significant and marked effect on oocytes maturation compared to FBS supplementation. Further, similar result to earlier report has been demonstrated elsewhere (Schellander et al., 1990). There are different sources for supplemented sera such as FBS (Kobayeshi et al., 1994; Nandi, 1998; Mahmoud and Nawito, 2005; Das et al., 2006; Talukder et al., 2008), OCS (Schellander et al., 1990; Singha et al., 2015), steer serum (Roy et al., 1968; Nandi et al., 2001) and superovulated cow serum (Boediono et al., 1994). Although, there are reports to use different sera sources for supplementation in maturation medium, comparison was made within FBS, OCS and BSA only in the present study. Because, FBS, OCS and BSA are the most widely used protein supplementation for maturation culture of bovine oocytes in the world. In conclusions, selection of a suitable oocytes culture medium is crucial for optimal results in bovine IVM/IVF. For IVM, both TCM and mSOF may be used as a basic medium supplemented with FBS, OCS or BSA as protein source and FSH may be used as a hormone supplement in basic medium for optimum IVM rate of indigenous Zebu cow’s oocytes in Bangladesh.

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