57 THE EFFECT OF VITRIFICATION FOR SHEEP EMBRYO VIABILITY

This work evaluated different methods for sheep embryo cryopreservation by vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). The vitrification method was applied according to the method described by Vajta et al. Both treatments used a vitrification solution (VS) containing 20% ethylene glycol, 20% dimethylsulfoxide (Me2SO), 0.5 mol L–1 sucrose in Dulbecco's phosphate buffered saline (DPBS) with 10% BSA. The super-cooled LN facilitates heat transmission between LN and the cryosolution interface, and this is efficient for bovine semen and blastocyst cryoconservation (Arav et al. 2002). By surgical flushing 25 super-stimulated ewes, 109 transferable morulae were harvested; 35 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 36) or SCURV (n = 38), respectively, thawed or warmed, and transferred to recipients. Embryos were vitrified using the HSV Kit. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by HSV Kit, which was immediately submerged into and stored in LN. Warming was done by placing the narrow end of the straw into DPBS + 0.25 M sucrose for 5 min. Embryos were then transferred into DPBS + 0.125 M sucrose for 3 min and finally to DPBS until transfer. The SCURV morulae were then exposed to 50 and 100% VS at 37°C for 2 min and 30 s, respectively. Embryos after saturation in VS were transferred on a surface of a nylon loop (volume 20 μL, diameter 0.5 mm) and using negative pressure of LN in the chamber for freezing with the VIT-Master. Thawing vitrified embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 M and 0.125 M with expositions 2 and 3 min accordingly. After thawing embryos, only good-quality embryos were transferred. Statistical analyses were performed with Student's t-test. The lambing rate following transfer of fresh, frozen-thawed vitrification and SCURV methods were 18, 12, 14 lambs accordingly. No statistical difference was found for the percentage of does lambing following transfer thawed after vitrification (33.4 ± 5.2a%) and SCURV methods (36.8 ± 6.3b%). The survival rate following transfer of fresh embryos (51.4 ± 4.8c) was higher and in line with previous findings using VS. Differences were statistically significant (ac,bc P < 0.05). Importantly, our data suggest that the HSV Kit can be used to produce viable morulae for implantation as the SCURV, and to as vitrification method. Although further work on the developmental competence of embryos cryopreserved with the SCURV method are needed, these data suggest that with SCURV a faster freeze rate and lower level of cryoprotectants is able to minimize ice crystal formation and should be further evaluated as a routine mechanism for cryopreserving sheep embryos.

120 SUCCESSFUL LAMBING AFTER SUPER-COOLING ULTRARAPID VITRIFICATION SHEEP MORULAE

The aim of this work was to establish alternative methods for sheep morulae cryopreservation by using vitrification by open pulled straw (OPS) methods and super-cooling ultra-rapid vitrification (SCURV). Both treatments used a vitrification solution (VS) of 20% (3.6 mol L-1) ethylene glycol (EG), 20% (2.4 mol L-1) dimethylsulfoxide (DMSO), 0.5 mol L-1 sucrose in DPBS with 10% BSA in both methods. In our experiment we used the Vit-Master™ (MTG, Germany). The super-cooled LN facilitates heat transmission between LN and the cryosolution interface, and this is efficient for bovine semen and blastocyst cryoconservation (Arav et al. 2002). By surgical flushing of 24 super stimulated ewes 121 transferrable morulae were harvested; 30 morulae were transferred fresh to synchronised recipients and the others were cryopreserved by OPS (n = 49) or SCURV (n = 42), respectively thawed or warmed, and transferred to recipients. Embryos were vitrified using the OPS method. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by touching a 1-μL drop with the straw, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the straw into DPBS + 0.25M sucrose for 5 min. Embryos were then transferred into DPBS + 0.125 M sucrose for 3 min and finally to DPBS until transfer. The SCURV morulae were then exposed to 50 and 100% VS at 37°C for 2 min and 30 s, respectively. Embryos after saturation VS have been transferred by on a surface of a nylon loop (volume 20 μL, diameter 0.5 mm) and using negative pressure temperature of liquid nitrogen in the chamber for freezing with the VIT-Master. Thawing vitrified embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 and 0.125 with expositions of 2 and 3 min, accordingly. After embryos were thawed, only good quality embryos were transferred. Importantly, our data suggest that by using the SCURV method, the toxic elements contained in the cryopreservation solution can be reduced while maintaining a similar ability to produce viable morulae for implantation as the OPS method. Although further work on the developmental competence of embryos cryopreserved with the SCURV method are needed, these data suggest that the faster freeze rate and lower levels of cryoprotectants of SCURV are able to minimize ice crystal formation and should be further evaluated as a routine mechanism for cryopreserving sheep morulae. Table 1.Effect vitrification and ultra-rapid super-cooling vitrification on the viability and lambing of sheep morulae

Regulation of canine jejunal transit

The aim of this study was to determine what factors influence the direction of movement of canine jejunal chyme. In four dogs, pacing electrodes were implanted near each end of a 50-cm jejunal Vella loop, while recording electrodes and intraluminal pressure catheters were spaced along the loop. After recovery, the loop was perfused from either the proximal stoma (forward flow) or the distal stoma (reversed flow), and effluent was collected from the nonperfused stoma. The pacesetter potentials were paced electrically in a forward (aborad) or a reverse (orad) direction. During control conditions (forward flow-forward pacing), the mean transit time of liquids was 2.6 +/- 0.1 min (mean +/- SE) and the static volume of the loop was 8.8 +/- 0.3 ml. Reversing both direction of flow and direction of pacesetter potential propagation slowed transit (4.4 +/- 0.4 min; P less than 0.05) and increased loop volume (16.0 +/- 1.0 ml; P less than 0.01). Reversing flow with forward pacing resulted in even slower transit (7.5 +/- 1.2 min; P less than 0.05), maintained a large volume (33 +/- 10 ml), and increased basal pressure in the loop from -1.2 +/- 1.7 (control) to 2.3 +/- 1.6 mmHg (P less than 0.05). These observations suggest that the direction of propagation of jejunal pacesetter potentials influences the direction of movement of jejunal chyme but that other factors have a role as well.

A Study of the Decay Phase of an X-Ray Flare on Algol

We have re-analyzed the X-ray flare on Algol which was observed with EXOSAT (White et al. (1986)). The common practice of estimating loop volume and length from the decay time of the flare is discussed extensively. We show that during the decay phase of the flare both scaling laws for coronal loops are valid. This implies a unique determination of loop volume and length and allows a check whether additional heating occurs in the decay phase of a flare.

Maturation of diluting capacity in loop of Henle of rat superficial nephrons

The postnatal development of renal diluting capacity was studied by free-flow micropuncture and by microdissection of single superficial loops of Henle in rats kept on a high salt diet. Total renal filtration rate, sodium absorption, total solute excretion, and systemic arterial pressure were monitored during single nephron evaluation at two maturational stages (12–15 days and 27–35 days postnatal). Nephron filtration rates were identical when measured in early distal and late proximal segments of the same nephron. Absolute fluid absorption between these sites increased by a factor of 2.5, whereas loop fractional absorption of the volume load changed from 38.1 +/- 6.9 to 49.8 +/- 4.8% (SD), while proximal volume delivery to the loop increased from 4.33 +/- 1.12 to 7.14 +/- 0.65 nl/min. Simultaneously, the osmolarity of early distal fluid (8.8% of distal length) decreased significantly from 284 + 19.8 to 180.9 +/- 18.2 mosmol/liter during maturation. This study suggests that the ability of the loop of Henle to generate hypotonic fluid is attained only gradually during ontogeny. Volume absorption in the loop of Henle increases disproportionately to the loop volume load.

Physical Factors Influencing Fluid Reabsorption from Henle's Loop

Our objective was to produce reductions in the luminal volume of Henle's loop and increases in linear flow velocity through the loop. We did this in a recollection micropuncture study by collecting fluid with and without suction from early distal tubules. With suction, transit time of fast green dye through the loop decreased by 34%, calculated loop volume decreased by 28%, and fractional water reabsorption fell from 73.6 to 70.3% (p < 0.025) in water diuretic rats. Absolute water reabsorption did not decrease significantly. In urea–saline diuretic rats transit time decreased 25%, calculated loop volume decreased 22%, fractional reabsorption fell from 59.0 to 51.7% (p < 0.001), and absolute reabsorption decreased by 2.3 nl/min(p < 0.025). Single nephron glomerular filtration rate, distal tubular sodium concentration, and osmolality were unaffected. The less pronounced effect of collection with suction in water diuretic rats may be related to the lower medullary fluid osmolality, which was 338 ± 9(S.E.)mOsmol/kg as compared to497 ± 35 in urea–saline diuretic rats. Collecting fluid with suction from late proximal tubules did not alter glomerular filtration rate or fractional water reabsorption. Stumpe et al. ((1970) J. Clin. Invest. 49, 1200–1212) noted an inverse correlation between fluid reabsorption from Henle's loop and flow velocity in rats with hypertension or congestive heart failure. One can reproduce this correlation by artificially altering the transmural pressure gradient in the loop.