Towards 'in vitro' produced germline stem cells in the bovine

Bovine spermatogonial stem cells (SSCs) have potential to be used in advanced reproductive technologies such as testis cell transplantation, where identification and purification of large numbers of SSCs is required. There are at least two possible sources of SSCs: isolation from the testis, or in vitro differentiation from pluripotent stem cells. The long-term goal of this thesis was to work towards the generation of SSCs from bovine somatic cells using induced pluripotent stem (iPS) cell technology. In order to do so, it was first important to characterise molecular markers expressed by bovine SSCs to allow for their identification in culture, and secondly to explore the feasibility of producing bovine iPS cells. In order to achieve the first goal, a screening platform was developed based on comparative analysis of gene expression levels in SSC enriched and depleted cell populations. Expression of established testis cell markers was used to confirm the validity of the screening platform. This method was then used to examine expression of candidate spermatogonial markers in the bovine testis. STRA8, KIT, GFRA1, CLDN8, DDX6 and NAP1L4 were shown to be putative markers for bovine spermatogonia. Further analysis of CLDN8 showed expression by both a subset of spermatogonia and a subset of Sertoli cells, leading to the hypothesis that CLDN8 plays a role in the maintenance of SSCs in the SSC niche. Reprogramming of bovine somatic cells was undertaken by introducing canonical reprogramming factors through a lentiviral vector. Initial experiments found that the reprogramming protocol was sufficient to produce cells exhibiting stem cell-like characteristics. Analysis of these cells indicated partial reprogramming had been achieved. A number of small molecules were then tested for their ability to enhance the success of cell reprogramming. A combination of three small molecules was found to accelerate the kinetics of the reprogramming process and also promoted further reprogramming where cells could differentiate to the three different germ layers. Further research is now required to define the optimal culture conditions for the maintenance and expansion of bovine pluripotent cells in long term culture, and to test whether they can be differentiated towards the germ line

The case for pre-parturient selenium and iodine supplementation of ewes for improving lamb survival

Lamb survival is an issue of high relevance to the Australian sheep industry, as lamb survival rates have direct bearing on overall reproductive performance of the sheep, and also constitute a main concern from an animal welfare perspective (Mellor and Stafford 2004). Both genetic and management factors play an important role in this complex issue (Hinch and Brien 2014). Ewe nutrition is of particular relevance as the intrauterine growth conditions prepare the lamb for the crucial transition to life outside the uterus. Effects of body condition of the ewe during various stage of the pregnancy have been investigated in detail, yet much less is known about the critical role of micronutrient provision to the ewe. Although several risk factors for Se and I subclinical deficiencies exist in Australia, determining micronutrient status in sheep or pasture is not straightforward

The case for pre-parturient selenium and iodine supplementation of ewes for improving lamb survival

Lamb survival is an issue of high relevance to the Australian sheep industry, as lamb-survival rates have direct bearing on overall reproductive performance of the sheep, and also constitute a main concern from an animal welfare perspective (Mellor and Stafford 2004). Both genetic and management factors play an important role in this complex issue (Hinch and Brien 2014). Ewe nutrition is of particular relevance as the intrauterine growth conditions prepare the lamb for the crucial transition to life outside the uterus. Effects of body condition of the ewe during various stages of the pregnancy have been investigated in detail, yet much less is known about the critical role of micronutrient provision to the ewe. Although several risk factors for selenium (Se) and iodine (I) subclinical deficiencies exist for sheep on pasture in Australia, determining micronutrient status in sheep or pasture is not straightforward. Several studies have separately found effects of Se and I supplementation on lamb survival. Studies investigating the interaction of Se and I supplementation have been few but results have demonstrated an interaction between Se and I. With increased twinning rates as a result of increased selection of numbers of lambs weaned, nutritional demands during pregnancy across flocks are increasing, and effects of micronutrients on lamb health and survival have greater impact. New opportunities in nutritional research are encouraging new studies into the effects of Se and I supplementation on lamb survival

The effects of mild prenatal stress during mid- and late-pregnancy, on lamb vigour traits

The datasets include data gathered on both pregnant ewes and newborn lambs.
 In pregnant ewes we collected, health checks and blood samples which were analysed for glucose and lactate before and after the stress treatments.
 In newborn lambs we collected data relating to vigour traits such as body dimensions, reactivity during a behaviour test and body surface temperature using IRT during a cold challenge

Lamb survival, a challenge for the decades

Lamb survival to weaning is the major profit driver in the Australian sheep industry, and equally an important issue for animal welfare and ethical livestock production. Research aimed at improving lamb survival and reproductive efficiency remains consistently one of the highest- ranking priorities identified by industry consultation groups. The extensive body of knowledge generated by research efforts across Australia have led to comprehensive management guidelines and extension programs to support their uptake. Nevertheless, reported rates of loss in many regions remains higher than acceptable, despite lamb loss rates in general improving under improved management strategies. One of the biggest challenges is to break down this complex phenotype into measurable meaningful component traits. As for any complex phenotype, a combination of improved management and genetic selection may be most likely to lead to success but for either application, phenotypes must be less dependent on uncontrollable environmental influences than counts of losses. Dystocia has been conclusively shown to be one of the most important causes of lamb loss and development of approaches to measure incidence of dystocia will aid the development of new phenotypes to facilitate genetic and management strategies to improve lamb survival

Investigations of Methods for Non-Viral Generation of Bovine Cells to Pluripotency, With a View on Potential Use in Reproductive Technologies

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

The effects of mild prenatal stress during mid- and late-pregnancy, on lamb vigour traits

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

Using Pen-Side Measurable Blood Parameters to Predict or Identify Dystocic Lambing Events

Dystocia is the greatest contributor to neonatal lamb mortality in Australia and poses significant welfare and economic concerns worldwide. In this study, we set out to investigate whether pen-side analysis technology could be employed to detect blood parameters predictive of dystocic labour events in sheep. In a pilot trial, we collected and analysed blood samples in pen-side assays for glucose, lactate, pH, pCO2, pO2, base excess, HCO3, TCO2, sO2, lactate, sodium, potassium, chloride, calcium, urea nitrogen, creatinine, haematocrit, haemoglobin and anion gap. From the pilot data, we identified creatinine, TCO2, chloride and calcium as potentially useful markers. To develop a time course and to establish variability of the selected blood parameters, a time series of samples was collected from 12 ewes, from mid-gestation to 48 h after birth. For the main trial, blood samples were collected at mid- and late gestation for glucose determination and for the full set of blood parameters at three time points before, at and after birth. Possible predictors of lambing difficulty were chloride, haematocrit and haemoglobin, sampled one week before birth; creatinine, sampled at birth; and blood pH and base excess after birth. In conclusion, we found that pen-side analysis of blood markers showed promise in identifying dystocic lambing events

Physiological and Behavioural Patterns Associated with Prolonged Lambing Events in Sheep

Dystocia is one of the most important issues faced by sheep producers nationally and internationally. In Australia specifically, dystocia directly and indirectly accounts for 50-75% of all neonatal lamb mortalities. Similar figures have been reported in flocks in New Zealand and the UK. Dystocia has been the subject of research for decades" however, the outcome studied is almost always 'number of lambs dead', therefore excluding dystocic events that did not result in death. This thesis is comprised of a series of experimental manuscripts investigating dystocia and its effect on maternal behaviour pre- and post-birth" assessing on-animal accelerometers to identify differences in eutocic and dystocic ewe activity pre-birth" and using pen-side blood analysis techniques to identify maternal blood markers that may be indicative of a dystocic event before and after birth. Dystocia has a marked effect on the behaviour of the ewe in the hours before birth. Dystocic ewes are significantly more active overall – identified by accelerometer data alongside traditional observation and annotation methods – but reach the peak in their activity 3 h before birth, whereas eutocic ewes reach the peak in activity at birth. There is also a significant difference in ewe behaviour after birth, with dystocic ewes performing fewer maternal care behaviours and more avoidance behaviours. Finally, there are multiple blood markers seen to be different in eutocic and dystocic ewes before and immediately after birth" however they are cohort-dependent.Dystocia is a complex and multifaceted problem, and requires a multifaceted approach in order to appropriately address the underlying issues and develop practical detection and prevention strategies

Claudin-8 expression in Sertoli cells and putative spermatogonial stem cells in the bovine testis

Adhesion molecules are expressed by both adult and embryonic stem cells, with different classes of adhesion molecules involved in cell-membrane and intercellular contacts. In this study the expression of the adhesion molecule claudin-8 (CLDN8), a tight-junction protein, was investigated as a potential marker for undifferentiated spermatogonia in the bovine testis. We found that CLDN8 was expressed by both spermatogonia and a subset of Sertoli cells in the bovine testis. We also showed co-expression of GFRα1 in testis cells with CLDN8 and with 'Dolichos biflorus' agglutinin-fluorescein isothiocyanate (DBA-FITC) staining. We observed co-enrichment of spermatogonia and CLDN8-expressing Sertoli cells in DBA-FITC-assisted magnetic-activated cell sorting (MACS), an observation supported by results from fluorescence-activated cell sorting analysis, which showed CLDN8-expressing cells were over-represented in the MACS-positive cell fraction, leading to the hypothesis that CLDN8 may play a role in the spermatogonial stem-cell niche