Definition of protocols for cryopreservation and three-dimensional in vitro culture of prepubertal goat testicular tissue after histomorphological, ultrastructural, and functional analysis.

This study aims to define the best method (slow freezing or vitrification) and fragment size (1, 5, or 9 mm³) for prepubertal goat testis cryopreservation, as well as to evaluate testicular morphological integrity after cryopreservation and in vitro culture (IVC). Initially (experiment I), 1, 5, or 9 mm³ testis fragments were cryopreserved by slow freezing using a Mr. Frosty container with 20% Dimethylsulfoxide (DMSO) or vitrified using the Ovarian Tissue Cryosystem (OTC) device, (Equilibration solution - ES: 10% DMSO and 10% ethylene glycol - EG; Vitrification solution - VS: 20% DMSO and 20% EG) and then subjected to morphological analysis, type I and III collagen quantification and gene expression (Oct4, C-kit, Bax, and Bcl-2). Subsequently, (experiment II), fresh or cryopreserved by slow freezing testis fragments were cultured in vitro and submitted to morphological analysis by scanning electron microscopy. The data from the experiment I revealed fewer morphological alterations in 1 and 5 mm³ fragments after vitrification and slow freezing, respectively. The percentage of type I collagen fibers in 5 and 9 mm³ frozen was higher than in fresh or vitrified fragments. For type III collagen, fresh or frozen fragments of 1 and 5 mm3 showed a higher percentage than fragments of 9 mm3. Gene expression for Oct4 and C-kit after slow freezing or vitrification in the 5 mm3 fragments was lower than that observed in the fresh fragments. The Bax:Bcl-2 ratio in the 1 and 9 mm³ fragments was lower than in the 5 mm³ fragments for fresh fragments or after freezing. In experiment II, fragments cultured in vitro, previously frozen or not, showed more morphological alterations than fresh or frozen fragments. We concluded that slow freezing of 5 mm³ fragments was the best protocol for cryopreserving prepubertal goat testis and although the results of IVC are encouraging, it still needs improvement to restore testicular function after cryopreservation.

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.

Contractility changes of the right and left ventricular muscle after chronic myocardial infarction

Contractility changes and adaptive responses resulting from acute left ventricular (LV) myocardial infarction are not restricted to the LV myocardium. The reduced LV function increases the right ventricular (RV) pressure load and neurohumoral factors, activated by the infarction episode, might have pan-cardiac effects. In the present study we investigated the mechanical activity of RV and LV isolated papillary muscles from 30-day infarcted male Wistar, 3-4-months old rat hearts. LV myocardial infarction was produced by ligature of the descending anterior branches of the left coronary artery (INF group). Control animals were submitted to sham surgery (SO group). Both groups were studied 30 days after the infarction procedure. Post-infarction hypertrophy was evaluated by measuring the cell diameters in the nuclear region. Contractility changes were analyzed by determining the isometric force (F) and the rate of force development (dF/dt) of papillary muscles from LV and RV. The effects of variations in extracellular Ca2+ concentrations (0.6, 1.25, 2.5 and 3.75 mM)were determined on twitches and tetanic contractures obtained during caffeine perfusion (2.5 mM) and were used to assess changes at the contractile protein level. The activity of the sarcoplasmic reticulum was evaluated by using the post-rest potentiation phenomenon. Hypertrophy occurred in both ventricles after infarction, with the RV chamber showing a pressure overload pattern while LV myocytes developed a volume overload pattern. F and dF/dt of LV papillary muscles decreased after infarction but did not change in the RV preparations. Positive inotropic changes obtained with increasing Ca2+ concentrations and the development of tetanic tension were reduced after infarction only in LV papillary muscles. The relative potentiation of post-rest contractions was only affected in the LV myocardium showing a decrease after infarction. These results suggest that different adaptive changes occur in the LV and RV myocardium after infarction. While the RV myocardium maintains its contractility the LV myocardium displays a depressed mechanical activity problably due to changes at the contractile mechinery level and to alterations in the Ca2+ handling process.