The effect of leukaemia inhibitory factor (LIF) on bovine embryo development in vitro : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Animal Science at Massey University
The aim of the study was to investigate the effect of Leukaemia Inhibitory Factor (LIF) either during in vitro maturation (IVM) or in vitro culture (IVC) on bovine embryo development. Three main experiments were conducted using oocytes aspirated from 2-8 mm diameter follicles collected from cows slaughtered at local abattoirs, Hamilton. The oocytes were matured in a modified TCM-199 containing 10 µ/ml of FSH and LH, and 1 µg/ml E2, fertilised in TALP and cultured in SOF/AA/BSA. Experiment 1 examined the effect of LIF (0, 500, 1000 or 2000 U/ml) and various time periods of IVM (18, 22 or 28 h), in a 4 × 3 factorial design on oocyte maturation. Following maturation, oocytes were stripped out of cumulus cells, then denuded oocytes were stained in 1% lacmoid for determination of maturation stage while the cumulus cells were examined for the incidence of apoptosis by observation of DNA fragmentation using gel electrophoresis procedures. Experiment 2 comprised two parts, (a) the effect of LIF (0, 500, 1000 or 2000 U/ml) at 24 h IVM in a randomised block design on in vitro development of embryos, (b) comparison of 20 vs 24 h IVM in the presence of LIF (0, 500, 1000 or 2000 U/ml) in a 2 × 4 factorial experiment on embryo development. In the two studies, the proportion of bovine oocytes that cleaved and developed to blastocyst stage was recorded. In addition, cell numbers of blastocysts after Giemsa staining were counted. Experiment 3 examined the effect of LIF during IVM (0 vs 1000 U/ml) or IVC (0, 500, 1000 or 2000 U/ml) in a 2 × 4 factorial design on development of embryos. The incidence of cleavage and blastocyst development and cell numbers of blastocysts were recorded. In addition, blastocysts were further categorised into early, expanded and hatched blastocyst stages and cell numbers of blastocyst inner cell mass (ICM) and trophectoderm (TE) after differential staining with Hoechst 33342 and propidium iodide were determined. In Experiment One, an interaction of LIF concentration and duration of IVM was not observed for the proportion of immature oocytes reaching metaphase II (P>0.05). The presence of LIF (500, 1000 or 2000 U/ml) increased the proportion of oocytes at metaphase II at 18 h (50%, 52% or 58%, respectively, compared to without LIF= 27%), indication that LIF may accelerate the maturation process in vitro. Supplementation of LIF during IVM did not affect the incidence of apoptosis of the cumulus cells. In Experiment Two, compared to 24 h IVM in the presence of LIF, 20 h IVM significantly increased blastocyst rates (Σ blastocysts : Σ cleaved, P<0.05). Cell numbers of blastocysts were not different from oocytes matured for 20 or 24 h in the presence of LIF (P>0.05), however the data show that treatment groups of 20 h IVM in LIF resulted in higher cell numbers of blastocysts than achieved by 24 h IVM. In Experiment Three, there was a correlation between LIF during IVM and LIF during IVC in the proportion of blastocysts (P<0.05). This finding shows that the proportion of blastocysts decreased when oocytes were matured in the absence of LIF and cultured in LIF. In contrast, more blastocysts developed when the oocytes were matured and then cultured in media containing LIF. There was no effect of addition of LIF during IVM and IVC for cell numbers of blastocysts (P>0.05). However, blastocysts derived from oocytes matured without LIF had significantly increased cell numbers (121 cells) compared to those matured in 1000 U/ml LIF (109 cells, P<0.05). Supplementation of LIF both during IVM and IVC did not affect the proportion of blastocyst stages (P>0.05). However, a concentration of 2000 U/ml LIF during IVC accelerated blastocyst development with more blastocysts hatching (60%, P<0.05). Cell numbers of inner cell mass (ICM), trophectoderm (TE), and the proportion of ICM were not affected by supplementation of LIF during IVM or IVC (P>0.05). A concentration of 1000 U/ml LEF during IVC resulted in higher cell numbers of ICM (P<0.05). This study suggests that LIF of 500, 1000 or 2000 U/ml increased the proportion of metaphase II bovine oocytes and even reduced the time course of IVM. Supplementation of LIF during IVM may suppress the incidence of apoptosis of the cumulus cells. IVM for 20 h in the presence of LIF resulted in a higher number of blastocysts and 1000 U/ml LIF during IVM and culture in LIF increased the proportion of blastocysts. A higher concentration of LIF is required for reaching the hatched blastocyst stage. A level of 1000 U/ml LIF during IVC promoted higher cell numbers of ICM.