Modifications of semen quality related to ejaculation frequency is one of the most important and neglected factors from the standpoint of artificial insemination or sperm competition. New Zealand white rabbits (Oryctolagus cuniculus) offer an advantageous experimental model because they have characteristic sexual behavior, they present rapid ejaculation after a single intromission, they have a very short interval between successive ejaculations, and semen can be easily collected. The authors studied the modifications on sperm quality (semen volume, sperm concentration, sperm motility) produced by 14 consecutive ejaculations recovered every 15 min using stimulus females and an artificial vagina. Bucks were exposed every 15 min to a sexually receptive female. After each ejaculation the female was removed and reintroduced 15 min later. Sperm concentration showed a clear biphasic conduct. The amount of spermatozoa per milliliter decreased rapidly until ejaculate number 6, showed a highly significant increase in ejaculates 7-9, and decreased to nil in the last 2 ejaculates. Total number of ejaculated spermatozoa was 557 x 10(6), 76% of which were recovered from the first 4 ejaculates. Ejaculate volume also showed a biphasic conduct. In the first ejaculates the volume decreased linearly until ejaculate number 6, showed a significant increase in ejaculates 7-10, and then decreased. The total semen volume recovered during the experiment was 2.44 mL, 40% of which (0.98 mL) was recovered from the first 2 ejaculates. Individual motility in the first 6 ejaculates was preferentially progressive (60% of the sperms) and turned to random or in situ from the seventh ejaculate up. The proportion of spermatozoa with cytoplasmic droplets increased from ejaculates 6 and 7 up. The results seem to reflect an acceleration of semen transport through the epididymis when the demands for spermatozoa increase.

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Maternal age dependency of gestation time in hamster and in other mammals is a well demonstrated fact. We have recently shown that adult nulliparous and multiparous hamster females show significant asynchrony and retard on early embryo development (from two blastomeres to morula stages) when compared with nulliparous young females. The number of cell-cell adhesions between blastomeres in early embryo development has been reported to be a good indication of the ability of embryos to cleave and develop. In this work we studied, by indirect immunofluorescence, the presence and distribution of E-cadherin in 4-cell embryos obtained from nulliparous young (NYF), nulliparous adult (NAF) and multiparous adult (MAF) hamster females. Distribution and intensity of fluorescence was observed and registered using confocal microscopy. Staining intensities for E-cadherin were quantified by computed densitometry in the free membrane regions, in the cytoplasm region and in the cell-cell adhesion zones of each embryo. E-Cadherin in all the studied zones was significantly higher (p < 0.01) in NYF. Cadherin concentration in the intercellular membranes was always statistically higher (p < 0.05) than in the free membrane regions. An appreciable concentration of E-cadherin was found in the cytoplasm of the 4-cell embryos obtained from the three groups of females, but was significantly higher in NYF. No statistical differences were observed in any of the parameters studied between NAF and MAF. Our results seem to indicate that changes in the reproductive behavior related to age and/or multiparity may be correlated with changes in the processes related to intercellular adhesions during early cleavage.

Studies have demonstrated that the early pre-implantation embryo is very sensitive to effects of heat stress in vitro. Heat stress reduces the total cell number in blastocysts and increases apoptosis in blastomeres. Insulin-like growth factor-1 (IGF-1) has been widely studied as a thermoprotective agent for its anti-apoptotic actions. Addition of IGF-1 to the culture medium decreases the effects of heat stress on blastocysts but has no effects on 2-cell embryos. Molecular mechanisms by which IGF-1 decreases apoptosis involve activation of the PI3K/Akt pathway. It is also known that adherens junctions contribute to PI3K/AKT activation mediated by the transmembrane glycoprotein E-cadherin, which is involved in Ca2+-dependent cell-cell adhesion. Within 2- to 8-cell embryos, E-cadherin is mainly inactive and has cytoplasmic localization. 6-Dimethylaminopurine (6-DMAP) induces premature cell flattening and E-cadherin redistribution to adhesion sites in 4-cell embryos. The aim of this study was to induce E-cadherin redistribution in 4-cell hamster embryos and evaluate the thermoprotective function of IGF-1 in these embryos. Four-cell embryos were incubated in the presence of 6-DMAP to induce E-cadherin redistribution to adhesion sites and cultured for 24 h under conditions of heat stress and compared with controls without 6-DMAP. Culture medium was supplemented with IGF-1. At the end of culture, developmental stage and rate of apoptosis were determined and analysed by ANOVA using the General Linear Model (GLM) of SAS (SAS Institute Inc., Cary, NC, USA) procedure with statistical significance at P < 0.05. E-Cadherin redistribution induced by 6-DMAP increased development to the 6-cell stage after 24 h (63.57% v. 38.81%, respectively; P < 0.05) and reduced apoptosis (25% v. 33%, respectively; P < 0.05) under heat-stress conditions. In conclusion, we hypothesise a role for E-cadherin-mediated cell flattening in promoting IGF-1-mediated thermoprotection in pre-compact 4-cell hamster embryos. Further studies are required to confirm this link.