226 HEAT SHOCK OF GERMINAL VESICLE-STAGE BOVINE OOCYTES REDUCES EMBRYO DEVELOPMENT

Abstract

Culture of germinal vesicle (GV)-stage oocytes at an elevated temperature occurring in heat-stressed dairy cattle reduced ability of oocytes to progress to metaphase II after resumption of meiosis (Payton RR et al., 2003 Biol. Reprod. 68, 343 abst). The objective of this study was to evaluate embryo development of oocytes heat-shocked at GV stage. To prevent cumulus-oocyte complexes from resuming meiosis after removal from follicles, oocytes were cultured in roscovitine (cell cycle inhibitor of p34^cdc2/cyclin B kinase;; 50 μM) for 24 h (McCann LM et al., 2000 Biol. Reprod. 64, 141 abst and Payton RR et al., 2003 Biol. Reprod. 68, 343 abst) showed that roscovitine is effective for maintaining >90% of oocytes at GV-stage in a reversible manner. Germinal vesicle-stage oocytes were 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. In addition, a group of COC were not cultured in roscovitine but placed in maturation medium (lab control). After a total of 24 h, COC were washed extensively of roscovitine and cultured for an additional 24 h in maturation medium. Oocytes presumed mature were fertilized with Percoll-prepared frozen-thawed semen. Presumptive zygotes were cultured in KSOM containing 1X nonessential amino acids in 5.5% CO₂, 7% O₂, and 87.5% N₂ at 38.5°C in humidified air. Cleavage and development to blastocyst were 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 (2000) after testing for normality. Use of roscovitine for maintaining oocytes at GV-stage for 24 h did not alter cleavage (80.5 and 73.4%; SEM = 5.8; lab and experimental controls), development to 8–16 cell (50.4 and 52.6%; SEM = 4.6; lab and experimental controls), or blastocyst (29.7 and 24.8%; SEM = 3.2; lab and experimental controls) stages. Culture of GV-stage oocytes at 41°C for up to 24 h did not increase lysis (8.0–11.1%; SEM = 2.7). Heat shock of GV-stage oocytes for as few as 6 h reduced the proportion developing to 8–16 cell stage after release from inhibitor (Table 1). When experimental control and HS 0–6 were pooled for comparison to HS 0–12, effects of heat shock for reducing development to blastocyst were noted (P < 0.005). Moreover, negative effects of heat shock for reducing developmental competence of GV-stage oocytes increased as duration of heat shock increased (linear contrast;; experimental control, HS 0–12, and HS 0–24; P < 0.04). Results indicate that a physiologically relevant elevated temperature for as few as 6 h compromises continued development of GV-stage oocytes. Seasonal depressions in fertility of heat-stressed cattle may be due in part to direct effects of elevated temperature on GV-stage oocytes.

Table 1