Abstract
Assisted Reproductive Techniques (ARTs) can play an important role in rapid generation of cows with desired traits. The objectives were to measure ovaries and oocyte production, and to optimize in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) media for in vitro production of embryos of indigenous zebu cows in Bangladesh. The ovarian size was compared with respect to cyclic status of ovaries collected from slaughterhouses. The weight (3.3±1.1 gm), length (2.3±0.3 cm) and width (2.1±0.4 cm) were higher in ovaries with corpus luteum (CL) than in those with no CL (1.6±0.7 gm, 1.8±0.4 cm, 1.8±0.4 cm, respectively) (P<0.05). The number of follicles per ovary was similar in ovaries from pregnant cows (7.9±4.5), non pregnant cows (8.7±5.4) and cows with CL groups (8.6±4.9). There were more medium size follicles (2-8 mm diameter) in ovaries from non pregnant cows (5.6±3.8) than in ovaries from pregnant cows (4.0±2.7) (P<0.05). The number of retrieved cumulus oocytes complex (COCs) per ovary was higher in ovaries with no CL (4.3±2.6) than that of ovaries with pregnant cows (3.5±2.0) and in ovaries from non pregnant cows with CL (3.6±2.3) (P<0.05). The oocytes recovery rate was higher in ovaries with no CL (49.9%) than in ovaries with CL (40.7%) (P<0.05). More grade A (1.1±1.0) and grade B (1.2±0.7) COCS was retrieved from ovaries with no CL than from ovaries of pregnant cows (0.6±0.5 and 0.9±0.6) and non pregnant cows with CL (0.9±0.6 and 1.0±0.7) (P<0.05). Tissue culture medium (TCM) 199, modified synthetic oviductual fluid (mSOF) and Tyrode's albumin lactate pyruvate (TALP) medium were compared as IVM media. More oocytes matured in TCM 199 (74.5%) than in TALP (58.2%) media (P<0.05). The maturation rate of oocytes was higher in FSH (73.3%) supplemented TCM 199 than in gonadotrophin (FSH+LH) supplemented medium (60.2%) (P<0.05). The maturation rate of oocytes was similar when TCM 199 was supplementation with fetal bovine serum (FBS) (73.0%), oestrous cow serum (OCS) (71.1%) or bovine serum albumin (BSA) (62.5%) as protein. To determine an effective IVF medium, cleavage rate was compared with TCM 199, TALP and, Bracket and Oliphant (BO) medium. Cleavage rate was higher in oocytes fertilized in TALP (63.3%) than in TCM 199 (47.5%) (P<0.05). Cleavage rate was higher in oocytes fertilized by spermatozoa separated by percoll gradient method (62.3%) than by centrifugation (51.6%) (P<0.05). Cleavage rate of oocytes was higher when insemination was done with spermatozoa capacitated in TALP supplemented with heparin, and penicillamine, hypotaurine and epinephrine (PHE) (61.3%) compared to control (40.9%) (no capacitating agent) (P<0.05). Zygotes were cultured in SOF (commercial), mSOF (produced in laboratory) and potassium simplex optimization medium (KSOM) (produced in laboratory) supplemented with BSA. No difference in cleavage (50.5, 63.0 and 59.8%, respectively) and embryo development to 16-cell stage (1.9, 5.5 and 3.1%, respectively) was observed (P>0.05). When presumptive zygotes were cultured, more 16-cell embryos developed in mSOF medium supplemented with epidermal growth factor (EGF) and cysteamine (11.0%) than with EGF (2.5%) or cysteamine (5.3%) alone (P<0.05). Most 16-cell embryos developed, when IVC medium was supplemented with 100 μM cysteamine (14.5%) compared with control (no supplementation, 2.1%), 50 μM (8.9%) and 150 μM (10.5%) groups (P<0.05). The protocols for IVM, IVF and IVC were developed for 16-cell embryos in indigenous zebu cows. However, further study is necessary using IVF and IVC media to develop the transferable blastocysts.