No description available.

No description available.

Thesis (Ph.D.)--Aberdeen University, 2009. / Title from web page (viewed on July 20, 2009). Includes bibliographical references.

<p>    </p> <p>In honey bees, male fertility is directly involved in colony fitness. Males not only provide fifty percent of colony genetics, but they also provide material benefits involved with female fertility. Recent evidence suggests there is tremendous variation in drone fertility. Of drones which are sexually mature, as few as one in ten may be able to produce enough sperm to successfully inseminate a queen. If drones are not producing healthy sperm, their mates will not produce healthy colonies. Despite this, we have very little understanding of precisely how variation in drone reproductive quality is generated. Specifically, we know comparatively little about the role genetics plays in shaping drone reproductive traits. This standing genetic variation can contribute to phenotypic variation observed among honey bee stocks and contribute to the success (or failure) of colonies. <strong>The major goal of this thesis is to identify the major mechanisms and genes driving genotypic and phenotypic variation in honey bee males. </strong>First, we used a population genetics approach to estimate the evolutionary impact of haploid selection <strong>(Chapter 2) </strong>and variation in sexual selection <strong>(Chapter 3) </strong>on male genes. In <strong>Chapter 2</strong>, we found genes expressed by males had an increased genetic diversity, rate of adaptation, and more efficient purifying selection than non-haploid selected female-expression genes. This suggests haploid and sexually- expressed genes experience increases in the rate of molecular evolution. In <strong>Chapter 3</strong>, we found selection on sperm length shifted over evolutionary time within corbiculate bees, resulting in different strengths of sexual selection. As selection for sperm length intensified in male bees, there was rapid evolution of expression patterns and gene sequences associated with male-biased genes. Second, we used a quantitative genetics approach to connect genetic variation to the trait components of fitness <strong>(Chapter 4)</strong>. We identified key genes connected to honey bee male sperm and maturity traits. These genes also appear to have different rates of evolution. Overall, we combined both population and quantitative genetic approaches to provide comprehensive insights into the evolution of honey bee male genotypes and phenotypes. This powerful approach allowed me to identify the genetic and mechanistic underpinnings driving variation in fitness-related traits. This information can be used to identify candidate genes associated with honey bee male fitness. </p>

In this investigation energy demands from offspring were varied for the MF1 mouse after litter sizes were manipulated and set to 5 pups/litter or 16 pups/litter by cross-fostering to examine the effects on immediate lactation and subsequent reproductive performance. Pups reared in manipulated large litters grew slower and were weaned lighter (half the size of pups reared in small litters) than pups reared in manipulated small litters.  Mortality of pups was high in the large litters but pups did not die in small litters. The results of a subsequent reproduction that followed the first lactation suggested an apparent cost for the female group that previously reared pups in large litters.  This is because in the subsequent reproduction, the group raised litters that were significantly (P&lt;0.05) 12.7g less in mass than litters raised by females that previously reared pups in small litters.  This apparent cost, however, did not appear to have severe negative impact on the general reproductive performance because data showed comparable litter sizes and masses at birth, growth rates of pups, and the number and masses of pups weaned between this group and females that previously reared pups in small litters. At reproductive age, female offspring previously weaned from large litters weighted significantly (P&lt;0.05) 5.8g less than female offspring previously weaned from small litters.  During their first breeding attempt or episode, female offspring previously weaned from large litters did not catch-up in body mass with those previously weaned from small litters.  In spite of this, the two groups ingested similar quantities of food prior to mating and during lactation.  The groups did not differ significantly (P&gt;0.05) in efficiencies of energy utilization, milk energy output and daily energy expenditure.  Consequently, they showed comparable breeding success. Different age groups of male and female offspring previously weaned from manipulated small (5 pups/litter) or large litters (16 pups/litter) and unmanipulated normal litters (10 pups/litter) varied significantly in body mass and did not show compensatory growth at the time they were examined for susceptibility to obesity and glucose intolerance.  A batch of six months old male offspring previously weaned from litters whose mothers were fed a particular macronutrient diet during lactation did not differ significantly in body mass when they were also examined for susceptibility to obesity.  All groups and sexes showed a tendency to develop obesity when they were challenged with a diet that contained up to 45% fat by calories.  Development of obesity did not appear to depend on  a particular litter size in which the mice were previously reared and weaned or whether the mice were previously suckled by mothers that were fed a particular macronutrient diet.  However, the development of obesity exhibited sexual dimorphism with the male offspring showing a higher tendency to develop obesity than female offspring.

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.

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.