Male weight problems, which often co-presents with micronutrient deficiencies, is associated with sub-fertility. sperm intracellular reactive oxygen species (ROS) concentrations and 8OHdG lesions, which resulted in reduced 8OHdG lesions in the male pronucleus, increased 2-cell cleavage rates, and partial restoration of fetal weight similar to controls. Sub-fertility associated with male obesity may be restored with very short-term micronutrient supplementation that targets the timing of the transit of sperm through the epididymis, which is the developmental windows where sperm are the most susceptible to oxidative damage. = 48) were randomly assigned in the pre-intervention period to either a control diet (CD) (= 24) or a high fat diet (HFD) (= 24) (SF12-012 and SF13-109 respectively; Specialty Feeds, Perth, Australia; Table 1) for 10 weeks. We have previously shown that this time on the diet initiates increased adiposity and perturbed sperm function in those animals allocated to the HFD [9,15,41]. After this initial diet phase mice were allocated to one of four diets for the intervention period, whereby mice initially fed a CD either (i) continued a CD, or were fed a (ii) CD supplemented with micronutrients (CD + S; SF12-013; Specialty Feeds, Table 1), and mice in the beginning fed a HFD either (iii) continued a HFD or were fed a (iv) HFD supplemented micronutrients (HFD + S; SF13-110; Specialty Feeds, Table 1) MLN8054 novel inhibtior for an additional 12 MLN8054 novel inhibtior days. We added micronutrients to the diets that are known to be reduced in obese individuals [34,35], MLN8054 novel inhibtior are important for sperm function [42,43,44,45] and have been shown to improve sperm oxidative damage after oral administration in sub fertile men . The 12-day duration was used to cover the duration of epididymal transit by sperm in mice (~9.5 days) . Animals were housed individually with ad libitum access to food and water. Individual body weights were recorded weekly. The use and care of all mice were approved by the Animal Ethics Committee of the University or college of Adelaide (M-165-13) and were handled in accordance with the Australian Code of Practices for the Care and Use of Animals for Scientific Purposes. Table 1 Composition of diets. (peanut) agglutinin (Lectin PNA; Molecular Probes, Eugene, USA) as previously explained . Sperm were incubated in G-IVF + 10% HSA for 1 h in 6% CO2 and 5% O2 at 37 C, washed in PBS, and incubated in Lectin PNA Alexa 594 antibody (1:100) in PBS for 45 min. Samples were stained with Hoechst to identify sperm nuclei. A minimum of 200 sperm were counted per sample and were classified as capacitated, non-capacitated, or acrosome reacted. 2.7. Sperm Binding to the Zona Pellucida of MII Oocyte and Fertilisation Rates To assess sperm binding to the Rabbit Polyclonal to SEMA4A zona pellucida, mature cumulus-enclosed oocytes (COCs) were collected from 4C5-week aged CBAF1 female mice 12C13 h following ovulation induction by an IP injection of pregnant mares serum gonadotrophin (PMSG; Folligon; Intervet, Bendigo East, Australia) and hCG (Pregnyl; Organon, Australia) administered 48 h apart . COCs were placed in 80 L drops of G-IVF + 10% HSA in 6% CO2 and 5% O2 at 37 C. After a 1-h incubation of the sperm, COCs were inseminated with 1 104/mL of motile sperm and co-incubated at 6% CO2 and 5% O2 at 37 C for 4 h. At 4 h post-insemination, sperm binding was assessed by counting the number of sperm bound to the zona pellucida of each MII oocyte as previously explained . Following MLN8054 novel inhibtior this, zygotes were transferred to G1.3 media (Vitrolife, Goteberg, Sweden) and progression to the 2-cell embryo was assessed 24 h post co-incubation of sperm and eggs. Fertilization rates MLN8054 novel inhibtior were assessed as the total quantity of COCs that cleaved to the 2-cell embryo. 2.8. Sperm Intracellular ROS Concentrations (DCFDA) Intracellular reactive.