Neonatal lamb vigour has been identified as an important trait for the subsequent survival of the lamb. To date, time to perform critical early lamb behaviours, up to time to suckle, has been the most common method of assessing lamb vigour. The aim of this thesis was to identify and evaluate other methods of assessing vigour and assessing it at a time when the ewe would be moving from the birth site because this time is critical for the maintenance of contact between the ewe and the lamb. Improving the ease of assessing lamb vigour may also provide a practical tool for improving lamb survival through genetic selection on lamb vigour traits. A modified barrier test and the physiological response of lambs to a cold stress between four and six hours of age were used as novel methods for assessing lamb vigour. Cold stress was induced a number of ways including via noradrenaline injections, the use of an ice vest and simulation of cold weather conditions using fans and cold water. Responses to cold stress were measured using changes in rectal temperature collected continuously over the treatment period, the behavioural responses of the lamb during the cold challenge and their performance in the modified barrier test following the cold challenge. Differences in vigour due to variation in late pregnancy nutrition, breed and sire within breed were also assessed. Sires were selected from the Sheep CRCs information nucleus flock (INF) and correlations between the INF sire vigour score and time to suckle were determined. It was found that sub maintenance nutrition in late pregnancy had no effect on lamb vigour as measured by time to suckle. Sire breed did not account for differences in lamb vigour however within breed sire differences were found. There were high correlations between INF vigour score and time to suckle and rectal temperature at 10 minutes of age (0.77, -0.67 and -0.71 respectively). It was concluded that a modified barrier test could be used to assess lamb vigour after the lamb has suckled to provide information on the capacity of the lamb to respond to the ewe at a time when she may be moving from the birth site. The use of lamb vigour scoring as used in the INF may be a useful indirect selection criterion for improving lamb survival as it appears to be providing information on critical neonatal behaviours.

Two experiments were conducted on commercial layer hens to study the effects of dietary supplementation with two levels of 25-hydroxycholecalciferol (25(OH)D₃) and two levels of calcium pidolate (with or without 25(OH)D₃) on production performance and egg quality. Experiment 1 was conducted using 90 Hy-Line Brown layer hens from 19 to 80 weeks of age and commenced in November 2012. Birds were divided into three groups of 30 hens as follows: group A (control group) fed with normal commercial layer mash feed, and groups B and C (treated groups) fed with normal commercial layer mash feed plus 0.5 g of 25(OH)D3 [premix (68.9 μg 25(OH)D₃)] per kg of feed and 1 g of 25(OH)D₃ [premix (137.8 μg 25(OH)D₃)] per kg of feed, respectively. Experiment 2 was conducted using 147 Lohmann Brown layer hens from 21 to 80 weeks of age and commenced in March 2014.

Shearing during pregnancy has been shown to increase lamb birthweight but the underlying mechanisms remain unknown.
This thesis aims to examine the effects of prenatal husbandry-like and cold stressors during mid- (pregnancy day 85–95) and late-pregnancy (pregnancy day 120–130) on lamb vigour traits. The long-term effects of prenatal mild stress on lamb vigour traits were assessed using lamb growth and body dimensions, neonatal behaviour and reactivity after a cold challenge and the lamb’s thermoregulatory abilities when exposed to cold. Maternal glucose response to the stressors and lamb metabolic responses were also examined.
It was hypothesized that increased birthweights were a result of a maternal stress response to the husbandry-like stressors involved with shearing, particularly handling, and/or the subsequent cold exposure of shearing. It was also hypothesized that this maternal stress response would impact on lamb vigour traits such as behaviour and thermoregulation. This thesis examined the effects of a range of mild stressors commonly experienced by ewes during husbandry procedures during mid-pregnancy (pregnancy day 85–95) or during late-pregnancy (pregnancy day 120–130) such as yarding, handling, shearing, transport as well as cold exposure. The effects of these stressors on lamb vigour traits were assessed using a separation test for reactivity, before and after a 1 hr separation and cold challenge and thermal imaging during the cold challenge to determine the ability of the lamb to maintain body surface temperature.
Shearing during mid-pregnancy increased lamb body dimensions and weight at birth while other stressors did not, which suggests that the causal mechanisms are something other than psychological and cold stress. Cold exposure during both mid- and late-pregnancy (cold exposure and/or cold exposure resulting from shearing) negatively impacted the reactivity levels of newborn lambs by increasing their latency to bleat, stand and return to ewe during a separation test. Cold exposure combined with acute stressors in keeping with husbandry procedures resulted in improved body surface temperature maintenance in newborn lambs when exposed to cold. The presence of effects of mild stressors for both stages of pregnancy on all three lamb vigour traits tested, suggests a number of alternate mechanisms are triggered by maternal-pregnancy stress impacting foetal development. Understanding how to modulate these effects could allow flock management to be modified to improve lamb survival.

Spermatogonial stem cells (SSCs) from livestock species have the potential to be used for reproductive technologies and for the production of transgenic animals. The isolation of a relatively pure population of SSCs from livestock species has proven difficult; however, the recent advances in the production of induced pluripotent stem (iPS) cells may provide an alternative source of SSCs through the differentiation of iPS cells toward the germline. The general aim of this thesis was to produce bovine iPS cells that may be differentiated toward the germ line for potential use in reproductive technologies such as germ cell transplantation. Due to difficulties in isolating a pure population of bovine SSCs, methods to improve the enrichment of these cells are of interest in order to improve the success of bovine germ cell transplantation. A number of different methods to enrich bovine spermatogonia were trialled to determine which of the method for enrichment was most effective. The combination of enrichment by differential plating followed by separation of cells by discontinuous Percoll gradient centrifugation, was found to isolate the most enriched population of un-differentiated spermatogonia.

This thesis explores various methods to optimise breeding programs that use female reproductive technologies and genomic selection. Simulation studies have shown that female reproductive technologies such as multiple ovulation and embryo transfer (MOET) and juvenile in vitro embryo production and transfer (JIVET) can increase rates of genetic gain through increased female selection intensity and decreased generation interval. Furthermore the use of genomic selection has facilitated better selection decisions to be made on younger selection candidates that may not have phenotypic measurements. When combining genomic selection with reproductive technologies the rate of genetic gain could be further accelerated. However intensive use of the best females in breeding programs can also increase the rate of inbreeding to unsustainable levels. This thesis aimed to stochastically simulate breeding programs where reproductive and genomic technologies are optimally implemented while maintaining a sustainable increase of inbreeding. The impacts of using reproductive technologies and/or genomic selection were evaluated for breeding programs across species. Furthermore, the thesis investigated a cost-benefit analysis of using reproductive technologies which led to a further study that optimized the use of reproductive technologies that considered their costs as well as future co-ancestry during selection.

The successful breeding of ewes before the age of 12 months presents an opportunity to improve flock reproduction efficiency and increase returns for sheep producers. However, uptake of this practice is currently low in Australia and New Zealand. A contributing factor to this is that reproductive performance at 1 year of age is lower and more variable than in older ewes; between flocks and also from year to year. Whilst previous research has explored what factors contribute to successful reproduction at 1 year of age, there is no universally accepted measure of puberty and sexual maturity in sheep nor is there an accurate phenotypic predictor of yearling reproductive success. Although established genetic correlations between reproduction and production traits exist, reproduction has previously been analysed as a repeated records trait across parities, rather than treating first parity as a separate trait. Model studies show that genomic information offers the opportunity to select animals more accurately at younger ages. However the implications of a lower and more variable fertility rate of ewes mated prior to 1 year of age has been largely ignored. The aim of this thesis was to evaluate the impact of mating ewes prior to 1 year of age on flock genetic progress and to quantify the relationship between potential pubertal traits, yearling reproduction traits, reproduction traits at later parities and other key production traits.

Nine experiments were performed to investigate how the synthesis and/or deposition of protoporphyrin IX (PP IX) into eggshell is influenced in brown-egg laying hens. The findings obtained in the first experiment (Chapter 2) showed that flock age and production system affected overall eggshell and egg quality. Egg weight was significantly higher in cage eggs, while albumen height was significantly higher in barn eggs. The mammillary layer ultrastructural variables showed no clear relationship with production system and flock age. Cuticle cover (ΔE*ab) was significantly higher in barn eggs compared with free range and cage eggs and was significantly higher in eggs from the 44 week old flock than for 64 and 73 week old flocks. In 1 gram of eggshell with and without cuticle, there was more PP IX in cage eggs followed by free range and barn eggs. The findings in experiment 2 (Chapter 3) indicate that eggs laid earlier in the day had deeper brown eggshell colour compared with the eggs laid later in the day. Egg position in a clutch had a clearer effect on eggshell quality in long clutches, as compared with medium and short clutches. The findings of the third experiment (Chapter 4) showed that different infectious bronchitis virus (IBV) strains affected the level of PP IX in eggshell differently. In unvaccinated laying hens, the mean PP IX per gram of shell was significantly higher on day 1 post-infection (p.i.) compared to day 7, after which PP IX increased with day p.i. In unvaccinated and vaccinated laying hens, PP IX decreased with increased day p.i. until day 12. The effect on loss of shell colour was more marked in the T strain infected group followed by N1/88, Vic S and A3 strains. Experiment four (Chapter 5) investigated reference gene stability in the shell gland in relation to time-points (time post-oviposition) of eggshell formation and nicarbazin treatment. The two most stable reference genes selected, HPRT1 and HMBS, were used for the normalisation of gene expression data obtained in experiment five (Chapter 6). The findings in experiment five showed that mitochondria per cell did not vary significantly with different time-points of eggshell formation and nicarbazin treatment. Genes involved in the synthesis of PP IX were regulated differentially in relation to time-points of eggshell formation. Feeding nicarbazin caused down-regulation of the ALAS1 gene that resulted in lower production of PP IX appearing in the shell gland tissue and eggshell. Experiment six (Chapter 7) investigated reference gene stability in the shell gland and spleen of infectious bronchitis virus (IBV) infected hens. The two most stable reference genes selected, TBP and YWHAZ, were used for the normalisation of gene expression data obtained in experiments seven and eight (Chapters 8 and 9). The RNA-sequencing findings in experiment seven showed that there were no differentially expressed genes (DEGs) involved in the mucosal immune system and eggshell formation in the shell gland between IBV challenged and control laying hens. However, there were 1608 and 1806 DEGs at 5 and 15 hrs time-points of eggshell formation, respectively. The Gene Ontology (GO) terms and functional gene analysis showed that the DEGs at 5 hr post-oviposition were mainly involved in ion transport and synthetic activities, while the DEGs at 15 hr were involved in energy metabolism and secretory activities, reflecting the peak stage of eggshell formation. The findings in experiment eight (Chapter 9) showed that IBV T infection significantly lowered mitochondrial count per cell in the shell gland region of the oviduct but not in the magnum or isthmus. The expression levels of nuclear DNA encoded genes involved in mitochondrial biogenesis and fission showed no clear correlation with mitochondrial count and were not significantly different between the control and challenged samples. The expression levels of all the genes except for PGC-1α were significantly affected by the time-points of eggshell formation. Experiment nine (Chapter 10) attempted to localize ALAS1, ALAD and FECH enzymes in shell gland tissue collected at different time-points (time post-oviposition) and in response to nicarbazin treatment. The findings showed that these antibodies did not recognise their respective proteins, possibly due to their amino acid sequences being derived from humans. PP IX fluorescence was not detected in Zenker Formol fixed tissue sections, stained or unstained, taken at different time-points and with or without nicarbazin treatment. Future work is suggested using chicken specific antibodies to localise cells involved in PP IX synthesis. Taken together, the findings in the current study broaden the understanding of factors affecting the synthesis and deposition of PP IX into eggshells.

The viability and survival of the neonate lamb relies on its ability to communicate and maintain a strong attachment with its dam. To date there has been little concise information available about the role of the lamb's behaviour, and in particular the importance of acoustic cues, in this relationship as greater attention has been focused on maternal attributes important in facilitating the maternal-young bond. In human and rodent neonates, acoustic features of the distress vocalisation are used as indices of neurological deficit and integrity both at birth and in infant acoustic cry analysis. The aim of this thesis was to investigate potential behavioural indicators of lamb vigour, with a particular focus on vocal behaviour, within the first 12 hours of life. Such measures could provide valuable information for development of reproductive breeding objectives, and provide clarity regarding the role of the lamb in failed maternal-young interactions. Delayed vocalisation initiation in response to a separation stimulus was found to be associated with poor vigour-related behaviour reflecting the capacity of the lamb to reunite and follow the dam over 12 hours postpartum. Vocalisation delay was also associated with risk factors related to poor lamb survival including longer parturition duration, male sex, first parity, heavier birth weight and sire-related conformational attributes likely to result in a more difficult birth. Blood assay markers reflecting fetal distress including poor blood oxygenation, and elevated plasma glucose and lactate levels sampled at birth were also demonstrated to be correlated with vocalisation latency. These associations were concluded to reflect impacts on the lamb's neurological system rather than genetic influences because of evidence provided by within-litter comparisons, and to demonstrate neuroregenerative processes over a 12 hour measurement period. An analysis of lamb distress signals modelled on acoustic cry analysis of the human neonate was also undertaken to compare vocalisation characteristics of lambs with delayed responses to those with rapid responses indicating vigour. Signal features of delayed response lambs were more likely to demonstrate acoustic parameters reflecting glottal instability, lower amplitude and reduced repetition rate. These lambs were more likely to emit inefficient or inappropriate signals in the context of isolation. A significantly higher fundamental frequency, an indicator of pathology in the human infant, was not clearly demonstrated to be associated with compromised lambs in this study. It was also found in a two-choice test, where sheep dams were required to demonstrate a preference for signals of their own co-twins, that ewes preferred acoustic signals of lambs correlated with rapid vocalisation response, higher pitch and greater signal stability. The results indicate that delayed vocalisation responsiveness and other acoustic measures are associated with fetal compromise in the neonate lamb, as shown in the human and rodent models. It was concluded that delayed vocal initiation is a marker for poor postnatal outcome characterised by diminished responsiveness to a distress condition. This research has important implications for understanding failed maternal-young relationships and the consequences for survival in mammalian neonates.