Experimental groups.

<p>Twenty New Zealand rabbits were initially distributed at random in three groups: NCR, HCR, OO at 5 months of age (corresponding to experimental time (et) = 0 months). After 4 months, NCR continued with normal diet (ND) and HCR was split in two subgroups: HCR and ½ HCR. HCR continued with experimental diet 1 (ED1) and the last group was fed with ED2. Four months later (et = 8 m) ½ HCR was divided again in two (subgroup II): ½ HCR (fed with ED2) and ½ HCR+½ OO (fed with ED3). m = months, n = number of experimental animals. More details in the text.</p

General characteristics of fresh rabbit semen samples (mean ± SEM).

*<p>p<0.05, n = 25.</p

Effect of dietary treatment on sperm membrane cholesterol.

<p>Fluorescence micrographs showing cholesterol content in plasma membrane of ejaculated rabbit spermatozoa detected by filipin probe. Images correspond to filipin-stained sperm cells (×630) from NCR (A), OO (B) HCR (C), ½ HCR (D) and ½ HCR+½ OO (E). Compare the strong signal detected in HCR (C) with the one from the ½ HCR+½ OO (E). The experiment was performed at least three times with 20 sperm from each animal.</p

Effect of diet on blood cholesterol.

<p>Bars represent the mean ± SEM of plasma cholesterol concentration from NCR, OO, HCR, ½ HCR and ½ HCR+½ OO during the last 3 months of experiment. n = 45 samples. *** indicates significantly different from NCR (p<0.001).</p

Impact of diet on sperm functionality: membrane response to hypo-osmotic swelling test.

<p>Bars represent the percentage (mean ± SEM) of spermatozoa swollen from NCR (black bar), OO, HCR, ½ HCR and ½ HCR+½ OO (white bars). The experiment was performed at least three times with each animal. n = 25 samples. *** = significantly different from NCR (p<0.001).</p

Impact of diet on sperm functionality: sperm capacitation and acrosome reaction.

<p>Protein tyrosine phosphorylation (A) and acrosomal exocytosis index (B) of rabbit spermatozoa. A: phospho-Y proteins from control (NCR), HCR and ½ HCR+½ OO showed different patterns ranging from one band (non-capacitated, culture medium without BSA (−), approximately 60 kDa) to many bands (capacitated with BSA (+), from over 20 to 100 kDa). Notice that capacitated sperms from HCR decreased the p-Y protein pattern compared with NCR, and ½ HCR+½ OO shows a p-Y pattern resembling control (NCR) conditions. The experiment was performed at least three times and representative blot is shown. B: Bars represent AR index of spermatozoa from NCR, ½ HCR, ½ HCR+½ OO and OO after 10 µM progesterone incubation. AR index corresponds to normalized data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052386#s2" target="_blank">Materials and Methods</a>). n = 25 samples. *** = significantly different from NCR (p<0.001).</p