Vitrification yields higher cryo-survival rate than slow freezing in biopsied bovine in vitro produced blastocysts.

Vitrification and slow freezing are the two commonly used embryo cryopreservation methods. In most studies, vitrification of intact embryos has proven superior in several respects, including cell and embryo survival and pregnancy rate. However, there is a lack of data for comparing these two methods in in vitro produced (IVP) bovine blastocysts, which have been subjected to the retrieval of trophectoderm (TE) biopsy. Day 7 IVP blastocysts were pooled and randomized into four groups: 1) non-biopsy (NB), 2) biopsy (B), 3) biopsy-vitrification (BV), 4) biopsy-slow freeze (BSF). The blastocysts in the B, BV, and BSF groups were subjected to TE biopsy. For the B group, this was followed by 5 hours (h) incubation and subsequent scoring of the biopsy-survival (re-expansion) rate before processing for further analyses. For the BV and BSF groups, the biopsy procedure was followed by 2 h incubation, allowing for a quick re-expansion, after which the blastocysts were subjected to vitrification and slow freezing, respectively. After warming and thawing, respectively, they were then incubated for 5 h followed by scoring the cryo-survival (re-expansion) rates before processing for further analyses. These included quantification of ICM and TE cells, cleaved caspase-3- and TUNEL-positive cells, quantitative PCR on cellular stress markers (SOD1 and PRDX1), and ultrastructural analysis. The biopsy-survival rate in the B group was 94% (307/326). The cryo-survival rate in BV (86%, 138/161) was higher than that in BSF (57%, 81/142; P < 0.001). No differences were noted between the average ICM, TE, and total cell numbers of the groups. The percentages of cleaved caspase-3-positive cells were higher in BV vs. NB (P < 0.05), in BSF vs. NB (P < 0.001), and in BSF vs. B (P < 0.001). The percentages of TUNEL-positive cells were higher in BV vs. NB (P < 0.05) and in BSF vs. NB (P < 0.001). The levels of mRNA abundance for SOD1 and PRDX1 in B, BV, and BSF were not different from that in NB. The ultrastructural analysis of blastocysts in the BV and BSF groups showed distension of extracellular spaces and appearance of intracellular vacuoles in the ICM, distension of mitochondria, and disorganization of mitochondrial cristae in both ICM and TE, and weakened tight junctions between adjacent TE cells. In summary, our findings demonstrate that vitrification yields a higher cryo-survival rate than slow freezing in biopsied bovine IVP blastocysts. However, biopsy-vitrification and biopsy-slow-freeze values are comparable in terms of ICM, TE, and total blastocyst cell numbers, as well as cleaved caspase-3- and TUNEL-positive cell rates. Moreover, biopsy and cryopreservation performed alone had no effect on ICM, TE, total blastocyst cell numbers, or TUNEL-positive cell rates. Biopsy and vitrification performed alone had no effect on the cleaved caspase-3 positive cell rates, whereas slow freezing resulted in an increased rate. Furthermore, double traumatization with a combination of biopsy and cryopreservation, either vitrification or slow freezing, resulted in increased rates of cleaved caspase-3- and TUNEL-positive cells.

Inhibition of Small-Conductance Calcium-Activated Potassium Current (I K,Ca) Leads to Differential Atrial Electrophysiological Effects in a Horse Model of Persistent Atrial Fibrillation.

BACKGROUND: Small-conductance Ca2+-activated K+ (KCa2) channels have been proposed as a possible atrial-selective target to pharmacologically terminate atrial fibrillation (AF) and to maintain sinus rhythm. However, it has been hypothesized that the importance of the KCa2 current-and thereby the efficacy of small-conductance Ca2+-activated K+ current (I K,Ca) inhibition-might be negatively related to AF duration and the extent of AF-induced remodeling. EXPERIMENTAL APPROACH AND METHODS: To address the hypothesis of the efficacy of I K,Ca inhibition being dependent on AF duration, the anti-arrhythmic properties of the I K,Ca inhibitor NS8593 (5 mg/kg) and its influence on atrial conduction were studied using epicardial high-density contact mapping in horses with persistent AF. Eleven Standardbred mares with tachypacing-induced persistent AF (42 ± 5 days of AF) were studied in an open-chest experiment. Unipolar AF electrograms were recorded and isochronal high-density maps analyzed to allow for the reconstruction of wave patterns and changes in electrophysiological parameters, such as atrial conduction velocity and AF cycle length. Atrial anti-arrhythmic properties and adverse effects of NS8593 on ventricular electrophysiology were evaluated by continuous surface ECG monitoring. RESULTS: I K,Ca inhibition by NS8593 administered intravenously had divergent effects on right and left AF complexity and propagation properties in this equine model of persistent AF. Despite global prolongation of AF cycle length, a slowing of conduction in the right atrium led to increased anisotropy and electrical dissociation, thus increasing AF complexity. In contrast, there was no significant change in AF complexity in the LA, and cardioversion of AF was not achieved. CONCLUSIONS: Intra-atrial heterogeneity in response to I K,Ca inhibition by NS8593 was observed. The investigated dose of NS8593 increased the AF cycle length but was not sufficient to induce cardioversion. In terms of propagation properties during AF, I K,Ca inhibition by NS8593 led to divergent effects in the right and left atrium. This divergent behavior may have impeded the cardioversion success.

Effect of selective IK,ACh inhibition by XAF-1407 in an equine model of tachypacing-induced persistent atrial fibrillation.

BACKGROUND AND PURPOSE: Inhibition of the G-protein gated ACh-activated inward rectifier potassium current, IK,ACh may be an effective atrial selective treatment strategy for atrial fibrillation (AF). Therefore, the anti-arrhythmic and electrophysiological properties of a novel putatively potent and highly specific IK,ACh inhibitor, XAF-1407 (3-methyl-1-[5-phenyl-4-[4-(2-pyrrolidin-1-ylethoxymethyl)-1-piperidyl]thieno[2,3-d]pyrimidin-6-yl]azetidin-3-ol), were characterised for the first time in vitro and investigated in horses with persistent AF. EXPERIMENTAL APPROACH: The pharmacological ion channel profile of XAF-1407 was investigated using cell lines expressing relevant ion channels. In addition, eleven horses were implanted with implantable cardioverter defibrillators enabling atrial tachypacing into self-sustained AF. The electrophysiological effects of XAF-1407 were investigated after serial cardioversions over a period of 1 month. Cardioversion success, drug-induced changes of atrial tissue refractoriness, and ventricular electrophysiology were assessed at baseline (day 0) and days 3, 5, 11, 17, and 29 after AF induction. KEY RESULTS: XAF-1407 potently and selectively inhibited Kir 3.1/3.4 and Kir 3.4/3.4, underlying the IK,ACh current. XAF-1407 treatment in horses prolonged atrial effective refractory period as well as decreased atrial fibrillatory rate significantly (~20%) and successfully cardioverted AF, although with a decreasing efficacy over time. XAF-1407 shortened atrioventricular-nodal refractoriness, without effect on QRS duration. QTc prolongation (4%) within 15 min of drug infusion was observed, however, without any evidence of ventricular arrhythmia. CONCLUSION AND IMPLICATIONS: XAF-1407 efficiently cardioverted sustained tachypacing-induced AF of short duration in horses without notable side effects. This supports IK,ACh inhibition as a potentially safe treatment of paroxysmal AF in horses, suggesting potential clinical value for other species including humans.

Effect of induced chronic atrial fibrillation on exercise performance in Standardbred trotters.

BACKGROUND: Atrial fibrillation (AF) is the most common arrhythmia affecting performance in horses. However, no previous studies have quantified the performance reduction in horses suffering from AF. OBJECTIVES: To quantify the effect of AF on maximum velocity (Vmax ), maximum heart rate (HRmax ), heart rate recovery (T100 ), hematologic parameters and development of abnormal QRS complexes. ANIMALS: Nine Standardbred trotters. METHODS: Two-arm controlled trial. Six horses had AF induced by means of a pacemaker and 3 served as sham-operated controls. All horses were subjected to an exercise test to fatigue before (SET1) and after (SET2) 2 months of AF or sham. The Vmax and HRmax were assessed using a linear mixed normal model. Abnormal QRS complexes were counted manually on surface ECGs. RESULTS: Atrial fibrillation resulted in a 1.56 m/sec decrease in Vmax (P < .0001). In the AF group, HRmax ± SD increased from 226 ± 11 bpm at SET1 to 311 ± 27 bpm at SET 2. The AF group had higher HRmax at SET2 compared with controls (P < .0001), whereas no difference between the control and AF groups was observed at SET1 (P = .96). Several episodes of wide complex tachycardia were observed during exercise in 3 of the AF horses during SET2. CONCLUSIONS AND CLINICAL IMPORTANCE: Atrial fibrillation resulted in a significant reduction in performance, an increase in HR and development of abnormal QRS complexes during exercise, which may be a risk factor for collapse or sudden cardiac death.