Effects of Gestational Nutrition on Post-Natal Fertility in Sheep: A thesis submitted for the degree of Doctor of Philosophy at the University of Otago, Dunedin, New Zealand

Changes to feed availability resulting from global climate change have the potential to exacerbate fertility issues already facing the NZ livestock industry. Appropriate feeding levels during gestation is gaining more attention as a number of studies have illustrated that underfeeding during gestation can have negative impacts on the fertility of female offspring. However, the mechanisms underlying this relationship remain obscure. Therefore, the aims of this study were to firstly establish a model in sheep whereby restricted gestational nutrition influenced fertility of the female offspring. A second aim was to identify potential mechanisms underlying the relationship between restricted gestational nutrition and postnatal fertility. Ewes were provided with either a maintenance diet, or a 0.6 of maintenance diet for the first 55 days of gestation. Thereafter, all ewes were fed ad-lib for the remainder of gestation. Fetuses were collected at days 55 and 75 of gestation to examine fetal ovarian development using stereology, and RNAseq was used to examine gene expression. Steroid profiles were generated from both maternal and fetal (day 75 only) plasma samples. Female offspring were monitored from birth until 19 months of age. From these offspring, the time of onset of puberty was recorded, indicators of fertility (ovulation rate and antral follicle counts) were assessed at 8 and 19 months of age, and key hormone profiles were generated at 19 months of age. Surprisingly, female offspring at 19 months of age, but not 8 months of age, showed increases in key indicators of fertility: ovulation rate (p < 0.05) and antral follicle count (AFC, p < 0.01). Additionally, these animals showed an increase in plasma progesterone concentrations (p < 0.05) indicative of increased embryo survival. Changes to the pattern of FSH secretion (p < 0.05) were also observed. Fetal ovaries exposed to restricted nutrition contained more germ cells at day 75 but not at day 55 of gestation (p < 0.01). RNAseq identified 69 sequences differentially expressed in fetal ovaries at day 55, and 145 sequences at day 75. Fold changes observed at day 75 were less than those observed at day 55. Amongst differentially expressed genes, germ cell specific genes were prominent at both ages. Prominent Gene Ontology categories at both ages were ion transport and protease inhibitors. Pathways identified as affected using IPA analysis included some related to the metabolism of arginine to nitric oxide and citrulline, LXR/RXR and FXR/RXR activation, quantity of germ cells, GADD45 signalling, and acute phase response signalling. Taken together, the data supports increased indicators of fertility in female offspring whose dams were exposed to restricted nutrition during gestation. The observed differences appear to originate within the ovary. The results are consistent with the concept that it is not the restricted nutrition alone, but the change in nutrition from restricted to ad-lib which may be generating the observed changes in the offspring. Further, the data offers insights into potential mechanisms underlying the phenotype observed with both nitric oxide and protease inhibitors being possible candidates for involvement. The results open new avenues of research to either address current fertility issues in livestock, or to improve livestock fertility through manipulating gestational nutrition.