Bovine pre-implantation embryos are sensitive to high temperatures. The hyperthermia resulting from maternal heat stress leads to an increased loss of early stage embryos. The effects of hyperthermia appear to be more pronounced in the zygote than blastocyst stages. There is also evidence to suggest that B. indicus embryos show better resistance to heat shock as compared to B. taurus embryos. The mechanism of thermotolerance in B. indicus embryos is unknown and it is also unclear if the thermotolerance of these embryos is a result of maternal or paternal contributions. There is also evidence to suggest that male embryos survive heat shock better than female embryos. This research therefore aimed 1. To examine the effect of heat shock on day-7 bovine embryos 2. To study the influence of maternal and paternal genotype on the embryo's ability to survive up to 48 hours post heat shock treatment 3. To study the expression of HSP70 in heat-shocked and non-heat shocked embryos and compare B. indicus and B. taurus embryos. 4. To compare the effects of heat shock on rapidly developing (day-6 morulae) and slowly developing (day-7 morulae) embryos to heat shock treatments 5. To determine the effect of heat shock on the sex ratio of the surviving embryos The work described in this thesis used an in vitro protocol for heat shock studies on embryos, based on observed in vivo rectal temperature fluctuations in heat stressed cows. The first study examined the effects of heat shock on embryos at the morula or blastocyst stage on day 7 of in vitro culture. The embryos were produced in vitro from oocytes collected from ovaries of either pure breed B. indicus or B. taurus and fertilised by frozen semen straws of either B. indicus or B. taurus bulls. Day-7 morulae or blastocysts were used for the study. The embryos were subjected to heat shock (HS) (41.5°C) or non-heat shock (NHS) (39°C) temperatures. Embryo survival and development 48 hours (48 h) post treatment were recorded. The data were analysed using binary logistic regression using logit link function to determine the Odds ratio (OR) and 95% confidence limits for individual factors. The variables used as factors in the analysis were replicate, heat shock, maternal genotype (B. indicus vs B. taurus), paternal genotype (B. indicus vs B. taurus) and stage at which heat shock was applied (morula or blastocyst). The outcome variables used for analysis were the number of viable embryos from the total treated and the proportion reaching expanded or hatched blastocyst stage, and the proportion of male embryos. Heat shock significantly reduced the probability of embryo survival by more than half (OR = 0.47; P ≤ 0.001) and reduced the probability of the embryo's progression to the expanded or hatched blastocyst stage by almost half (OR = 0.58; P = 0.005) as compared to NHS embryos. The probability of survival of embryos with B. indicus paternal genotype (confounded by the use of a single sire) was double (OR = 2.00; P = 0.002) that of embryos with B. taurus paternal genotype. The heat shock x paternal B. indicus genotype interaction was found to be non-significant. Maternal B. indicus genotype showed no influence on thermotolerance. Nevertheless, the probability of progression to expanded blastocysts for embryos with maternal B. indicus genotypes was double (OR = 2.05; P = 0.030) that of embryos with maternal B. taurus genotype. Comparison of day-7 morulae with day-7 blastocysts showed that the probability of day-7 blastocysts surviving until 48 h after treatment was almost two and half times greater (OR = 2.39; P ≤ 0.001). The probability of survival of a blastocyst with paternal B. indicus genotype was almost twice (OR = 1.95; P = 0.041) when compared to a blastocyst with B. taurus paternal genotype. The interaction term of heat shock × blastocyst stage was found to be significant (OR = 2.70; P = 0.038) indicating that blastocyst stage embryos were resistant to heat shock. The study showed a trend towards survival of more male embryos (61% males vs 39% females) under heat shock conditions (OR = 1.64; P = 0.070) indicating that male embryos may be resistant to heat shock. The second study was designed to detect the induced form of HSP70 in heat shocked and non-heat shocked day-7 bovine embryos and to compare the patterns between B. indicus embryos and B. taurus embryos. Comparison of confocal images showed that staining for HSP70 was present in most embryos whether heat shocked or not and found to be concentrated in the nuclei and cytoplasm. Heat shock appears to have increased HSP70 staining intensity in both the nucleus and the cytoplasm, suggesting increased expression of HSP70 after heat shock. The same general staining patterns were seen in heat shocked and non-heat shocked embryos of B. indicus and B. taurus embryos. In the third experiment, embryos were examined for the effects of heat shock on day-7 or on day-6 of their in vitro culture. The data were analysed to study the effects of heat shock, stage at heat shock (day-7 blastocysts vs day-7 morulae) and day of heat shock (day-7 vs day-6) on embryo survival and progression to expanded or hatched blastocysts 48 h post treatment. We found that heat shock reduced the probability of survival by more than half (OR = 0.40; P = 0.004). Probability of survival of day-7 embryos (confounded by rapidly developing blastocysts and slow developing morulae) was less than half (OR = 0.36; P = 0.008), that of day-6 embryos. The data were then analysed to study the effects of heat shock on day-7 blastocysts and day-7 morulae for embryo survival post treatment. Heat shock negatively affected embryo survival (OR = 0.35; P = 0.007). The probability of day-7 blastocysts surviving HS and NHS was more than two and a half times greater (OR = 2.71; P = 0.008) than that of day-7 morulae. No interaction of heat shock and blastocyst stage was noticed. Subsequently the effects of heat shock and the effects of the day of heat shock on morula stage embryos were examined. Overall, heat shock reduced the probability of survival (OR = 0.35; P = 0.009) and the probability of embryo development to the expanded or hatched blastocysts (OR = 0.35; P ≤ 0.001), 48 h post treatment. The probability of day-6 morulae surviving HS and NHS was almost three times higher (OR = 2.81; P = 0.007) than that of day-7 morulae. The interaction of stage of development with heat shock was not significant. We concluded that an embryo that was capable of developing to the blastocyst stage by day-7 or morula stage by day-6 had better survival and higher probability of progressing to expanded or hatched stage when compared to an embryo that had slow development (day-7 morulae). The study showed a significantly different effect of heat shock on the survival of male and female embryos (63% males vs 36% females, OR = 1.79; P = 0.014). In our final study, embryos were produced from three different bulls each of B. indicus or B. taurus genotype. Heat shock effects consistently and negatively affected survival of embryos (OR = 0.29; P ≤ 0.001) and their ability to progress to the expanded or hatched blastocysts (OR = 0.42; P ≤ 0.001). Embryos with paternal B. indicus genotype showed no advantage of survival over embryos with paternal B. taurus genotype embryos. When the day-7 blastocysts were compared with the day-7 morulae, the probability of day-7 blastocysts surviving HS or NHS was more than double (OR = 2.23; P = 0.009) that of day-7 morulae. In this study the effect of survival of the interaction term of heat shock treatment with the blastocyst stage was significant and negative (OR = 0.49; P = 0.040). However the interaction term of blastocyst stage x HS for an embryos ability to progress to expanded or hatched blastocyst stage was positive (OR = 2.00; P = 0.052), indicating that the embryos that did survive heat shock were capable of continuing their developmental progress. Although more male embryos (55% male vs 45% female) survived heat shock, the effect for this study was non-significant. In summary, the studies described in this thesis successfully tested a new in vitro heat shock protocol based on in vivo temperature changes experienced by heat stressed cows. Heat shock was consistent in negatively affecting embryo survival. Heat shock also negatively affected the embryo's progression to the expanded or hatched blastocyst stage by 48 h post treatment. We observed variable cytoplasmic and nuclear staining for HSP70 in day-7 blastocysts of both B. indicus and B. taurus. This distribution did not change dramatically after heat shock, but staining appeared to be more intense in heat shocked embryos, suggesting that there was increased expression of HSP70 after heat shock. No difference in this pattern was observed between B. indicus and B. taurus embryos. The speed of development of an embryo until being subjected to either heat shock or non heat shock temperatures affected the probability of survival and further development. However, the interaction term of day-7 blastocyst stage and heat shock was inconsistent, meaning that the rate of development appeared to specifically protect against heat shock in some studies while in the other studies, the apparent protective effect extended to control as well as heat shocked embryos. Our study found no significant influence of B. indicus paternal genotype on embryo survival. Our study showed evidence of B. indicus maternal genotype influence on the embryo's ability to progress to expanded blastocysts but not on embryo survival. The study found a clear trend towards an increased proportion of male embryos surviving heat shock conditions. In some studies this was significant, and in others it was not, but the trend was always in the same direction. This suggests that male embryos are more resistant to the effects of heat shock in vitro.
| Identifer | oai:union.ndltd.org:ADTP/289576 |
| Creators | Naik, Veena |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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