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Determination of an Effective Media and Its Hormone and Protein Supplementation 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 follicles. The oocytes were cultured in the 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 an 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 the TCM medium than that of the TALP medium (58.2±6.2). To determine an effective hormone supplementation for maturation medium, the oocytes were cultured 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 the 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 was 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 conclusion, TCM supplemented with either FBS, OCS, or BSA as protein and FSH as hormone may be used as a medium for IVM of oocytes of indigenous zebu cows.

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

Location: 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

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. All the chemicals, reagents and media constituents were purchased from Sigma-Aldrich Inc., St Louis, USA. Media and reagents were prepared using a standard protocol and under aseptic conditions. All media were filtered using a 0.22µm pore size filter (Durapure^® membrane filter, Carrigtwohill, Ireland) and culture medium was routinely equilibrated in an 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. The ovaries of indigenous zebu cows were collected from a local slaughterhouse 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). 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). 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. 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 the medium in an incubator at 38.5°C with 5% CO₂ in humidified air for 24 hrs. The culture drops were examined under the stereomicroscope for expansion of cumulus cells after 24 hrs of culture in the maturation media. Presumptive maturation was confirmed by the degree of cumulus expansion. To examine the presence of the first polar body extrusion, the COCs were denuded by using a 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 the presence of polar body under an inverted microscope (Leica DM IRE2, Germany) with the help of a mouth controlled pipette. 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 pyruvate (TALP) medium supplemented with 10% fetal bovine serum (FBS), 5µg/ml follicle-stimulating hormone (FSH) and 1µg/ml oestradiol (OE2). Each experiment was repeated 10 times across the days. To determine an effective hormone supplementation in the 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 8 times across the days. 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 8 times across the days. The data were recorded in a Microsoft Excel spreadsheet and descriptive statistics was performed. The maturation rates were expressed as mean ± SD and a 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

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Achievement/Findings:

In conclusion, the 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 a protein source and FSH may be used as a hormone supplement in a basic medium for optimum IVM rate of indigenous Zebu cow’s oocytes in Bangladesh.

Knowledge Management: Journal