Masters Theses

Date of Award

8-2003

Degree Type

Thesis

Degree Name

Master of Science

Major

Animal Science

Major Professor

J. Lannett Edwards

Committee Members

Neal Schrick, Arnold Saxton

Abstract

The overall objective was to evaluate susceptibility of germinal vesicle (GV)-stage bovine oocytes to elevated temperature. Because GV-stage oocytes spontaneously resume meiosis (begin to mature) after removal from ovarian follicles, roscovitine (inhibitor of p34cdc2 /cyclin B kinase) was utilized to prevent breakdown of the GV (resumption of meiosis). In the first experiment, oocytes were maintained at GV-stage after removal from 3-8 mm follicles by culturing in 50 μM roscovitine for 24 h. Previous efforts have shown that culture of oocytes in roscovitine is effective for maintaining >90% at GV-stage with effects being reversible. In addition, a group of oocytes were not cultured in roscovitine but placed in maturation medium containing gonodotropins (lab control) to assess any potential negative effects of roscovitine on development. Germinal vesicle-stage oocytes were cultured at 38.5 C for 24 h (Control) or 41 C for 12 h followed by 38.5 C for 12 h (HS). Thereafter, oocytes were washed of roscovitine, matured for 24 h, and fertilized. Oocytes were evaluated after culture in roscovitine, after maturation, or 18-20 h after adding sperm. Specific experimental endpoints included hardening of zona pellucidae, cortical granule type, nuclear stage, and assessment of fertilization. Most data were analyzed using Chi-Square. Culture of GV-stage oocytes at 41°C did not alter zona pellucida hardening at any time point (185.6 to 231.8 s). Resumption of meiosis was prevented by roscovitine (93.9 and 96.1 % of oocytes had an intact GV for Control and HS). Culture at 41°C reduced ability of GV-stage oocytes to undergo nuclear maturation after removal from roscovitine (91.9 versus 83.6% at metaphase II for Control and HS; P<0.06). Also, culture at 41 °C increased oocytes having type Ill cortical granules (6.9 versus 20.5% for Control and HS; P<0.05). However, increased type Ill cortical granules in HS oocytes were not evident after maturation (68.3 and 65.0% for Control and HS). Penetration (72.1 and 71.9%), number of sperm within each oocyte (1.6 and 1.8), pronuclear formation (70.7 and 71.6%), monospermic (67.4 and 58.0%) and putative embryos (48.4 and 41.8%) were similar for Control and HS, respectively. In the second experiment, oocytes were maintained at G\/-stage using 50 μM roscovitine and cultured at 38.5 °C for 24 h (experimental control) or 41.0°C as follows: HS 0-6 (41°C for 6 h, 38.5°C for 18 h), HS 0-12 (41°C for 12 h, 38.5°C for 12 h), HS 12-24 (38.5°C for 12 h, 41°C for 12 h), HS 18-24 (38.5°C for 18 h, 41°C for 6 h), or HS 0-24 (41°C for 24 h) in 5.5% CO2 and humidified air. A lab control was included to assess any potential negative effects of roscovitine on development. After 24 h, oocytes were washed of roscovitine, matured for 24 h, and fertilized. Ability of putative zygotes to cleave and develop to blastocyst was recorded on days 3 and 8 post-insemination, respectively. Data were collected in 7 replicates and analyzed as an incomplete block using mixed models of SAS (2001) after testing for normality. In addition, contrasts were performed to examine the linear relationship between the combination of experimental control and HS 0-6 versus HS 0-12 and the relationship between experimental control, HS 0-12, and HS 0-24. Culture of GV-stage oocytes in roscovitine did not affect cleavage (80.5 and 73.4% for lab control and experimental control, respectively; P>0.40; SEM=5.8), development to 8-16 cell stage (50.4 and 52.6% for lab control and experimental control, respectively; P>0.75; SEM=4.6), or development to blastocyst (29.7 and 24.8% of cleaved for lab control and experimental control, respectively; P>0.30; SEM=3.2). Culture of GV-stage oocytes at 41°C for up to 24 h did not increase proportion of oocytes that lysed (8.0 to 11.1%; P>0.95; SEM=2.7). After maturation and fertilization, a greater proportion of oocytes heat-shocked at GV-stage for as little as 6 h arrested at the 4-cell stage with a concomitant decrease in 8-16 cell embryos. When compared to control, blastocysts were decreased only for HS 0-24. When experimental control and HS 0-6 were pooled and compared to HS 0-12, culture of GV-stage oocytes at 41°C for 12 h reduced development to blastocyst (P<0.006). A linear contrast between experimental control, HS 0-12, and HS 0-24 revealed a linear decrease in development to blastocyst as duration of heat shock increased (P<0.04). Number of nuclei in blastocysts did not differ. In summary, exposure of GV-stage oocytes to a physiologically relevant heat shock compromised subsequent development. Seasonal depressions in fertility of heat-stressed cattle may be due in part to direct effects of elevated temperature on reducing developmental competence of GV-stage oocytes.

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