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&degC) or non-heat shock (NHS) (39&degC) 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 embryo’s 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.

A large proportion of the beef industry in northern Australia is composed of tropically adapted cattle, due to their propensity for tick and drought resistance. It has been suggested that a large conical cervix is responsible for decreased fertility in these cattle where a conical cervix is defined as having a caudal to cranial cervical diameter difference of greater than 15 mm. Studies were conducted in order to ascertain the incidence of conical cervix and the heritability of cervical diameter in tropically adapted beef cattle in Queensland. An initial study was conducted on 246 Santa Gertrudis females from four properties. Caudal and cranial cervical measurements were taken with transrectal B-mode ultrasound using the Aloka SSD_500 scanner and a 5 MHz linear probe. Analysis of data was by AS REML, including the effects of sire, parity and property. Sire was included as a random effect, whereas parity and property were included in the analysis as fixed effects. Sire effects were significant. A sire model was used to calculate a heritability estimate of 0.60 ± 0.27 for uterine cervical diameter. An analysis of variance showed that parity had a significant effect on cervical diameter, with cervical diameter increasing as parity level increased. Property had no effect on cervical diameter A more detailed study was conducted on 850 cattle from five properties. Breeds represented were Santa Gertrudis, Brahman, Brahman Cross, Belmont Red and Belmond Red Cross. Caudal and cranial cervical diameter, sire, parity and breed were recorded for each animal. Once again, sire effects were significant resulting in a heritability estimate of 0.46 ± 0.15. An analysis of variance showed parity effects to be significant (α=0.05) while property and breed effects did not influence cervical diameter. However property and breed were heavily confounded due to the practice of many stud breeders of running one breed per property. Breed and parity were also heavily confounded as Santa Gertrudis breed cattle were the only cattle available that had already produced one or more calves. In order to examine the relationship between cervical diameter and liveweight, a subset of 568 cows from the above group also had their weight in kilograms recorded. Animals were chosen for this study based on the availability of scales at each property. A Pearson correlation test resulted in a correlation coefficient of 0.043 between uterine cervical diameter and animal liveweight. This demonstrates that there is a negligible linear relationship between cervical diameter and liveweight. The relationship between cervix diameter and calving outcome was not investigated in this study. Although calving records were obtained for a number of animals in this study, many variables affecting calving outcome could not be accounted for such as disease outbreaks, artificial insemination technique and other management factors. Previous studies have suggested a relationship between cervical diameter and infertility but the present study was unable to draw conclusions regarding this. Although this study has shown that uterine cervical diameter is a moderately to highly heritable trait, it is not recommended that producers cull animals based on their uterine cervical dimensions at this stage, as the relationship between uterine cervical diameter and fertility is still poorly understood.

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&degC) or non-heat shock (NHS) (39&degC) 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 embryo’s 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.

No description available.

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&degC) or non-heat shock (NHS) (39&degC) 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 embryo’s 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.

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&degC) or non-heat shock (NHS) (39&degC) 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 embryo’s 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.

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&degC) or non-heat shock (NHS) (39&degC) 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 embryo’s 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.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Net feed intake or residual feed intake is the feed intake of an animal after adjustment for its average weight and weight gain while on the feed test. High net feed efficiency (NFE) animals have a low net feed intake, so the aim is to select animals that have high net feed efficiency in order to reduce the 70% expenditure for feed costs. Thus far, very few studies have been undertaken on beef cattle to identify genetic markers for NFE and to understand the molecular genetics of feed intake regulation and energy balance. Therefore, in an attempt to identify genes and metabolic pathways controlling feed efficiency in beef cattle, three different experimental approaches were taken herein: a) linkage and linkage disequilibrium quantitative trait loci (QTL) mapping for net feed intake in Lirnousin x Jersey animals, b) mitochondrial oxidative phosphorylation enzyme assays in high and low NFE cattle, and c) 2-dimensional fluorescent gel electrophoresis (DIGE) proteomics analysis of mitochondrial proteins. For the cattle QTL mapping, the results from a previous trial were utilized. In the trial, a double back-cross design was employed using two extremely divergent Bos taurus breeds [Jersey (J) dairy breed and Limousin (L) beef breed]. These breeds known to differ in many traits including carcass composition, fat colour, body size, and meat tenderness. Three first cross (F1=X) sires were mated to pure Jersey or pure Limousin cows, creating in total 366 XJ and XL backcross progeny (range 120-156 progeny per sire). The amount of feed consumed each day during the 70-100 day test was recorded electronically for each animal. Feed intake data were processed by calculating the least-square means for each animal over the test period. The data for net feed intake were analysed using a QTL half-sib interval-mapping model. The interval linkage analysis of whole genome detected six suggestive QTL (BTA 1, 6, 8, 9, 16, and 20) segregating for NFE. Of these 6 QTL, 4 NFE QTL (BTA 1, 6,16, and 20) were homeologous to QTL for NFE observed in fullsib F2 families of mouse selection lines (Fenton 2004). After the NFE data were reanalysed for outliers, a QTL on BTA 11 was re-ranked and placed in the top 4 NFE QTL in terms of size of effect and statistical support, whereas the OTL on BTA 6 and BTA 16 had less support. Since the QTL on BTA 9 was not independent of growth, only 4 QTL (BTA 1, 8, 11 and 20) were targeted for further study herein. These NFE QTL were cross-validated in Angus NFE selection line animals in collaboration with Department of Primary Industries (DPI), Victoria by microsatellite linkage mapping. Two of the QTL on BTA 8 and 20 were confirmed and three other minor QTL on BTA 1, 11, and 20 were detected in the Angus animals. Based on this background information, a comparative genome mapping study was undertaken to identify candidate genes. Using the human and bovine genome Ensembl databases, 205 NFE candidate genes underlying the 4 major QTL regions (BTA 1, 8, 11, and 20) were identified and 61 were sequenced in the mapping F1 Limousin x Jersey mapping sires. In these 61 genes, 308 SNPs were discovered, of which 27 were potentially functional SNPs changing the amino acids. 84 SNPs were selected for genotyping and used for fine mapping the 4 QTL and for SNP association studies with NFE. From the positions of the analyses, the 4 NFE QTL were refined and 27 candidate SNPs in 20 genes showed strong association with NFE in the Limousin x Jersey animals. A ParAllele whole genome scan with a bovine 10K SNP chip was also performed on a subset of the Angus NFE selection line animals by DPI Victoria. 100 ParAllele SNPs had significant association with NFE in the Angus selection line animals. These ParAllele SNPs were tested in the Limousin x Jersey animals and 16 ParAllele SNPs were significantly associated with NFE. Four of these SNPs were located in the NFE QTL on BTA 1, 11 and 20. Based on the candidate genes underlying the 4 NFE QTL, 8 potential metabolic pathways contributing to NFE were identified. These metabolic pathways included mitochondrial oxidative phosphorylation and glucose turnover. Therefore, to determine if these specific pathways are indeed involved in net feed efficiency, oxidative phosphorylation enzyme assays and mitochondrial protein profiling were conducted on progeny from the Angus Trangie NFE selection line animals. Liver and skeletal muscle samples were obtained from extreme high and low NFE animals with an average phenotypic difference of 3 kg net feed intake per day. Using these liver and muscle samples, mitochondria were prepared and assessed. The mitochondrial preparations were assayed for enzyme activity of 3 complexes (Complex I, II and IV) involved in oxidative phosphorylation. The enzyme activities were measured spectrophotometrically and analysed by regression analysis. The activity of the liver mitochondrial Complex I was found to be significantly decreased in the high NFE animals compared to the low NFE animals (p<O.0001). The Complex II and IV activities were increased in the high NFE cattle, but the differences were not statistically significant. Using the mitochondrial preparations, 2-D polyacrylamide gel electrophoresis differential gel electrophoresis (2-D PAGE DIGE) was used to generate a mitochondrial protein profile for the high and low NFE Angus cattle. An ontological analysis based on the differentially expressed proteins (>1.5 fold difference) in the high vs. low NFE cattle unambiguously identified a total of 27 proteins in 6 physiologically different groups. The mitochondria proteomics results also confirmed the involvement of oxidative phosphorylation in net feed intake regulation. Eleven oxidative phosphorylation complex subunit proteins were found to be differentially expressed between the high and low NFE animals. Other differentially expressed proteins included six stress-related proteins, seven energy production and glucose turnover proteins, two protein turnover and nitrogen balance enzymes, and two proteins involved in mitochondrial DNA and protein biosynthesis. Four of the differentially expressed proteins were found in the NFE QTL regions. The results of these experiments provide a better understanding of the relationship between variation in feed efficiency and cellular energy production mechanisms in beef cattle. The proteomics and mitochondrial enzyme assay results suggest that energy metabolism and homeostasis may not be an efficient process in low NFE cattle. Lastly, a set of candidate SNPs are now available for the further validation as markers for selection of NFE in cattle breeding programs. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1349183 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

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&degC) or non-heat shock (NHS) (39&degC) 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 embryo’s 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.

A large proportion of the beef industry in northern Australia is composed of tropically adapted cattle, due to their propensity for tick and drought resistance. It has been suggested that a large conical cervix is responsible for decreased fertility in these cattle where a conical cervix is defined as having a caudal to cranial cervical diameter difference of greater than 15 mm. Studies were conducted in order to ascertain the incidence of conical cervix and the heritability of cervical diameter in tropically adapted beef cattle in Queensland. An initial study was conducted on 246 Santa Gertrudis females from four properties. Caudal and cranial cervical measurements were taken with transrectal B-mode ultrasound using the Aloka SSD_500 scanner and a 5 MHz linear probe. Analysis of data was by AS REML, including the effects of sire, parity and property. Sire was included as a random effect, whereas parity and property were included in the analysis as fixed effects. Sire effects were significant. A sire model was used to calculate a heritability estimate of 0.60 ± 0.27 for uterine cervical diameter. An analysis of variance showed that parity had a significant effect on cervical diameter, with cervical diameter increasing as parity level increased. Property had no effect on cervical diameter A more detailed study was conducted on 850 cattle from five properties. Breeds represented were Santa Gertrudis, Brahman, Brahman Cross, Belmont Red and Belmond Red Cross. Caudal and cranial cervical diameter, sire, parity and breed were recorded for each animal. Once again, sire effects were significant resulting in a heritability estimate of 0.46 ± 0.15. An analysis of variance showed parity effects to be significant (α=0.05) while property and breed effects did not influence cervical diameter. However property and breed were heavily confounded due to the practice of many stud breeders of running one breed per property. Breed and parity were also heavily confounded as Santa Gertrudis breed cattle were the only cattle available that had already produced one or more calves. In order to examine the relationship between cervical diameter and liveweight, a subset of 568 cows from the above group also had their weight in kilograms recorded. Animals were chosen for this study based on the availability of scales at each property. A Pearson correlation test resulted in a correlation coefficient of 0.043 between uterine cervical diameter and animal liveweight. This demonstrates that there is a negligible linear relationship between cervical diameter and liveweight. The relationship between cervix diameter and calving outcome was not investigated in this study. Although calving records were obtained for a number of animals in this study, many variables affecting calving outcome could not be accounted for such as disease outbreaks, artificial insemination technique and other management factors. Previous studies have suggested a relationship between cervical diameter and infertility but the present study was unable to draw conclusions regarding this. Although this study has shown that uterine cervical diameter is a moderately to highly heritable trait, it is not recommended that producers cull animals based on their uterine cervical dimensions at this stage, as the relationship between uterine cervical diameter and fertility is still poorly understood.