The newly discovered oocyte-derived growth factor BMP15, like GDF9, is a member of the transforming growth factor-£] (TGF-£]) superfamily. To our knowledge, however, the expression and function of BMP15 in ovarian tissues has not yet been studied in the pig. We asked whether the relative abundance (RA) of BMP15 mRNA changes in oocytes and follicular cells during pre-ovulatory period or culture of cumulus-oocyte-complexs (COCs) in vitro. Denuded oocytes, cumulus cells and COCs were isolated from growing and pre-ovulatory follicles. Total RNA was extracted from the cells, and reverse transcriptase polymerase chain (RT-PCR) was carried out using specific oligo-nucleotide primers. Expansion of COCs was firstly observed at 18 h, when this period we found BMP15 mRNA expression obviously. But when we culture of denuded oocytes instead of COCs, BMP15 mRNA didn¡¦t express throughout the period. We also detected GDF9 and BMP15 mRNA in pig embryonic stage and in differentiation stage of pig stem cell. GDF9 mRNA level continued to express after fertilization, but BMP15 mRNA didn¡¦t appear. We also added BMP15 antibody against Expanding of COCs. The present results support the concept that BMP15 is a key mediator of oocyte-enabled cumulus expansion in pig.

Thesis (M. Sc. (Animal Production)) -- University of Limpopo, 2015 / The objectives of this study were: 1) To determine the effect of oocyte retrieval techniques (slicing and aspiration) on the quality and quantity of cattle oocytes, 2) To evaluate the effect of different concentrations of hormones on the maturational rate of cattle oocytes selected by brilliant cresyl blue staining, 3) To evaluate fertilization rate and cleavage/embryonic development of oocytes with or without cumulus cells, and 4) To compare the effect of fresh and frozen thawed semen on the fertilization rate of cattle oocytes. In Experiment 1: oocytes were recovered from abattoir derived ovaries using slicing and aspiration. The recovered oocytes were exposed for 90 minutes to 26μM of brilliant cresyl blue (BCB) stain and classified according to the colour of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). There was no difference (P>0.05) in the quality of oocytes recovered using slicing (60.7 %) or aspiration (53.7 %) techniques. In experiment 2: The BCB selected and the non-selected immature oocytes were randomly allocated into medium 199 + 10 % fetal bovine serum (FBS) maturation media. The media was supplemented with three different concentrations of hormones as treatments (T). The T1 (0.5 μg/ml of follicle stimulating hormone (FSH), 5mg/ml of luteinizing hormone (LH) and 2 μg/ml of estradiol (E2) as the control group. Then, T2 (1 μg/ml of FSH, 6 mg/ml of LH and 2.5 μg/ml of E2) and T3 (1.5 μg/ml of FSH, 7 mg/ml of LH and 4.5 μg/ml of E2). Maturation rate of oocytes was determined by the protrusion of the first polar bodies 24 hours following in vitro maturation. Treatment 2 yielded higher (P<0.05) maturation rate for both BCB+ (65.6 %) and without BCB (60.3 %) oocytes with T1 giving lower (P<0.05) maturation rate for BCB+ (22 %) and without BCB (16 %) oocytes. However, BCB- oocytes had lower (P<0.05) polar body extrusion (3.03 %, 8.1 % and 2.2 %) for T1, T2 and T3, respectively. In Experiment 3: one group of the presumptive zygotes was denuded of cumulus cells and the other group was cultured with cumulus cells. The presumptive zygotes were in vitro cultured in SOF-BSA and changed to SOF-FBS after 48 hours. High fertilization/cleavage rate was observed in oocytes cultured with cumulus cells (29.0 %) compared to the denuded oocytes (20.0 %) for 2-4 cells stage. Day 7 blastocysts were more (P<0.05) on oocytes cultured with cumulus cells (32 %) compared to denuded oocytes (13 %). In experiment 4: The matured oocytes were fertilized using fresh and frozen thawed semen. The oocytes fertilized with frozen thawed semen obtained a better number of 2-4 cell cleavage (23 %) when compared to fresh semen (19 %). Oocytes that were fertilized with frozen thawed semen also obtained higher morula (13 %) and blastocyst (8 %) compared to fresh semen with morula (3.4 %) and blastocyst (2 %). In conclusion, immature oocytes that were exposed to BCB+ and cultured in M199 supplemented with 10 % FBS, 0.5 μg/ml of FSH, 5 mg/ml of LH and 2 μg/ml of E2 had a higher (P<0.05) number of matured oocytes (extrusion of first polar body) compared to those that were not exposed to BCB (no BCB). Oocytes that were cultured with cumulus cells yielded a higher (P<0.05) number of cleaved embryos compared to the denuded oocytes. Slicing yielded a higher (P<0.05) number of oocytes, however the quality of oocytes recovered was similar compared to those recovered by the aspiration technique (P>0.05). Oocytes fertilized with frozen thawed semen yielded higher (P<0.05) number of 2-4 cell, morula and blastocyst when compared with oocytes that were fertilized using fresh semen. Keywords: ovaries, oocytes, slicing, aspiration, COCs, BCB, polar body and cattle

Thesis (M. Sc. (Animal Production)) -- University of Limpopo / The objectives of this study were: 1) To determine the effect of oocyte retrieval techniques (slicing and aspiration) on the quality and quantity of cattle oocytes, 2) To evaluate the effect of different concentrations of hormones on the maturational rate of cattle oocytes selected by brilliant cresyl blue staining, 3) To evaluate fertilization rate and cleavage/embryonic development of oocytes with or without cumulus cells, and 4) To compare the effect of fresh and frozen thawed semen on the fertilization rate of cattle oocytes. In Experiment 1: oocytes were recovered from abattoir derived ovaries using slicing and aspiration. The recovered oocytes were exposed for 90 minutes to 26μM of brilliant cresyl blue (BCB) stain and classified according to the colour of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). There was no difference (P>0.05) in the quality of oocytes recovered using slicing (60.7 %) or aspiration (53.7 %) techniques. In experiment 2: The BCB selected and the non-selected immature oocytes were randomly allocated into medium 199 + 10 % fetal bovine serum (FBS) maturation media. The media was supplemented with three different concentrations of hormones as treatments (T). The T1 (0.5 μg/ml of follicle stimulating hormone (FSH), 5mg/ml of luteinizing hormone (LH) and 2 μg/ml of estradiol (E2) as the control group. Then, T2 (1 μg/ml of FSH, 6 mg/ml of LH and 2.5 μg/ml of E2) and T3 (1.5 μg/ml of FSH, 7 mg/ml of LH and 4.5 μg/ml of E2). Maturation rate of oocytes was determined by the protrusion of the first polar bodies 24 hours following in vitro maturation. Treatment 2 yielded higher (P<0.05) maturation rate for both BCB+ (65.6 %) and without BCB (60.3 %) oocytes with T1 giving lower (P<0.05) maturation rate for BCB+ (22 %) and without BCB (16 %) oocytes. However, BCB- oocytes had lower (P<0.05) polar body extrusion (3.03 %, 8.1 % and 2.2 %) for T1, T2 and T3, respectively. In Experiment 3: one group of the presumptive zygotes was denuded of cumulus cells and the other group was cultured with cumulus cells. The presumptive zygotes were in vitro cultured in SOF-BSA and changed to SOF-FBS after 48 hours. High fertilization/cleavage rate was observed in oocytes cultured with cumulus cells (29.0 %) compared to the denuded oocytes (20.0 %) for 2-4 cells stage. Day 7 blastocysts were more (P<0.05) on oocytes cultured with cumulus cells (32 %) compared to denuded oocytes (13 %). In experiment 4: The matured oocytes were fertilized using fresh and frozen thawed semen. The oocytes fertilized with frozen thawed semen obtained a better number of 2-4 cell cleavage (23 %) when compared to fresh semen (19 %). Oocytes that were fertilized with frozen thawed semen also obtained higher morula (13 %) and blastocyst (8 %) compared to fresh semen with morula (3.4 %) and blastocyst (2 %). In conclusion, immature oocytes that were exposed to BCB+ and cultured in M199 supplemented with 10 % FBS, 0.5 μg/ml of FSH, 5 mg/ml of LH and 2 μg/ml of E2 had a higher (P<0.05) number of matured oocytes (extrusion of first polar body) compared to those that were not exposed to BCB (no BCB). Oocytes that were cultured with cumulus cells yielded a higher (P<0.05) number of cleaved embryos compared to the denuded oocytes. Slicing yielded a higher (P<0.05) number of oocytes, however the quality of oocytes recovered was similar compared to those recovered by the aspiration technique (P>0.05). Oocytes fertilized with frozen thawed semen yielded higher (P<0.05) number of 2-4 cell, morula and blastocyst when compared with oocytes that were fertilized using fresh semen. Keywords: ovaries, oocytes, slicing, aspiration, COCs, BCB, polar body and cattle

Thesis (M. Sc. (Animal Production)) -- University of Limpopo, 2015 / The objectives of this study were: 1) To determine the effect of oocyte retrieval techniques (slicing and aspiration) on the quality and quantity of cattle oocytes, 2) To evaluate the effect of different concentrations of hormones on the maturational rate of cattle oocytes selected by brilliant cresyl blue staining, 3) To evaluate fertilization rate and cleavage/embryonic development of oocytes with or without cumulus cells, and 4) To compare the effect of fresh and frozen thawed semen on the fertilization rate of cattle oocytes. In Experiment 1: oocytes were recovered from abattoir derived ovaries using slicing and aspiration. The recovered oocytes were exposed for 90 minutes to 26μM of brilliant cresyl blue (BCB) stain and classified according to the colour of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). There was no difference (P>0.05) in the quality of oocytes recovered using slicing (60.7 %) or aspiration (53.7 %) techniques. In experiment 2: The BCB selected and the non-selected immature oocytes were randomly allocated into medium 199 + 10 % fetal bovine serum (FBS) maturation media. The media was supplemented with three different concentrations of hormones as treatments (T). The T1 (0.5 μg/ml of follicle stimulating hormone (FSH), 5mg/ml of luteinizing hormone (LH) and 2 μg/ml of estradiol (E2) as the control group. Then, T2 (1 μg/ml of FSH, 6 mg/ml of LH and 2.5 μg/ml of E2) and T3 (1.5 μg/ml of FSH, 7 mg/ml of LH and 4.5 μg/ml of E2). Maturation rate of oocytes was determined by the protrusion of the first polar bodies 24 hours following in vitro maturation. Treatment 2 yielded higher (P<0.05) maturation rate for both BCB+ (65.6 %) and without BCB (60.3 %) oocytes with T1 giving lower (P<0.05) maturation rate for BCB+ (22 %) and without BCB (16 %) oocytes. However, BCB- oocytes had lower (P<0.05) polar body extrusion (3.03 %, 8.1 % and 2.2 %) for T1, T2 and T3, respectively. In Experiment 3: one group of the presumptive zygotes was denuded of cumulus cells and the other group was cultured with cumulus cells. The presumptive zygotes were in vitro cultured in SOF-BSA and changed to SOF-FBS after 48 hours. High fertilization/cleavage rate was observed in oocytes cultured with cumulus cells (29.0 %) compared to the denuded oocytes (20.0 %) for 2-4 cells stage. Day 7 blastocysts were more (P<0.05) on oocytes cultured with cumulus cells (32 %) compared to denuded oocytes (13 %). In experiment 4: The matured oocytes were fertilized using fresh and frozen thawed semen. The oocytes fertilized with frozen thawed semen obtained a better number of 2-4 cell cleavage (23 %) when compared to fresh semen (19 %). Oocytes that were fertilized with frozen thawed semen also obtained higher morula (13 %) and blastocyst (8 %) compared to fresh semen with morula (3.4 %) and blastocyst (2 %). In conclusion, immature oocytes that were exposed to BCB+ and cultured in M199 supplemented with 10 % FBS, 0.5 μg/ml of FSH, 5 mg/ml of LH and 2 μg/ml of E2 had a higher (P<0.05) number of matured oocytes (extrusion of first polar body) compared to those that were not exposed to BCB (no BCB). Oocytes that were cultured with cumulus cells yielded a higher (P<0.05) number of cleaved embryos compared to the denuded oocytes. Slicing yielded a higher (P<0.05) number of oocytes, however the quality of oocytes recovered was similar compared to those recovered by the aspiration technique (P>0.05). Oocytes fertilized with frozen thawed semen yielded higher (P<0.05) number of 2-4 cell, morula and blastocyst when compared with oocytes that were fertilized using fresh semen. Keywords: ovaries, oocytes, slicing, aspiration, COCs, BCB, polar body and cattle