Impact of ethanol and heat stress-dependent effect of ultra-diluted Arnica montana 6 cH on in vitro embryo production in cattle.

This study evaluated the effect of adding ultra-diluted and dynamized Arnica montana 6 cH, and its vehicle (0.3% ethanol) to the in vitro maturation (IVM) medium, in the absence (experiment 1) or presence (experiment 2) of heat stress (HS), on bovine oocyte maturation and in vitro embryo production (IVEP). In experiment 1 (n = 902 cumulus oocyte complexes, COCs), the treatments were 1) IVM medium (Control treatment), 2) IVM medium + 0.3% ethanol, and 3) IVM medium + Arnica montana 6 cH. In experiment 2 (n = 1064 COCs), the treatments were 1) IVM medium without HS, 2) IVM medium under HS, 3) IVM medium + ethanol under HS, and 4) IVM medium + Arnica montana under HS. In the absence of HS (experiment 1), the addition of Arnica montana to the IVM medium had a deleterious effect on the IVEP (cleavage and blastocyst rates) and the total cell number/blastocysts. On the other hand, ethanol (0.3%) increased IVEP in relation to the Control and Arnica montana treatments. However, in the presence of HS during IVM (experiment 2), the addition of ethanol or Arnica montana increased IVEP when compared to the HS treatment alone, and the Arnica montana treatment resulted in greater total cell number/blastocysts compared to the other treatments. In conclusion, this study showed for the first time that the negative or positive effect of Arnica montana 6 cH on IVEP depends on the culture condition (i.e., absence or presence of HS during IVM). On the other hand, ethanol showed beneficial and consistent results on IVEP regardless of exposure to HS.

Influence of cooling rate on the ability of frozen-thawed sperm to bind to heterologous zona pellucida, as assessed by competitive in vitro binding assays in the ocelot (Leopardus pardalis) and tigrina (Leopardus tigrinus).

We evaluated the influence of two cooling rates (from 25 to 5 degrees C) on post-thaw function of frozen sperm in ocelots (Leopardus pardalis; n=3 males) and tigrinas (Leopardus tigrinus; n=4 males). Seven normospermic (>70% normal sperm) electroejaculates from each species were diluted with a 4% glycerol freezing medium, divided into two aliquots, and assigned to one of two cooling rates: fast or slow (0.7 or 0.16 degrees C/min, respectively). Sperm motility index (SMI) and percentage of sperm with an intact acrosome were assessed before freezing and after thawing, and the ability of sperm to bind to the zona pellucida of IVM domestic cat oocytes were assessed in a competitive in vitro sperm-binding assay. Regardless of the cooling rate, frozen-thawed sperm from both species exhibited a SMI of 50; approximately 20 and approximately 32% of post-thaw sperm had an intact acrosome in ocelots and tigrinas, respectively (P<0.05). The mean (+/-S.E.M.) number of sperm bound per oocyte was higher for fast-cooled (8.5+/-1.3) than slow-cooled (2.5+/-0.3; P<0.01) ocelot sperm. In contrast, more tigrina sperm bound to domestic cat oocytes when cooled slowly versus quickly (5.8+/-0.9 versus 2.7+/-0.4, P<0.05). In conclusion, cryopreservation decreased sperm function in both species, and the oocyte-binding assay was the most efficient method to detect functional differences in post-thaw sperm.