Procedure fo r De termination o f Volume Changes o f Morula when Exposed to VS

The procedures employed for the determination of volume changes in the day-4 mouse and day-6 sheep compacted morulae were similar with minor differences. The volume changes after exposure to VS 11 in the mouse embryos were calculated at 0, 5, 10, 15 and 20 minutes at 25°C and in the sheep they were calculated at 0, 3, 5, 10, 15 and 20 minutes also at 25°C. The holding pipette used for manipulating the sheep morulae was larger than that used for handling mouse morulae (see Chapter 2, section 2.2.8.2.).

Micromanipulation of embryos for volume studies was performed with a Leitz Diavert (Leitz, Germany) inverted microscope and Leitz manipulators. The holding pipette described in section 2.2.8.2. was filled with liquid paraffin and fitted to the paraffin filled plastic instrument tubing attached to a Hamilton micro-syringe on the left side of the microscope. The holding pipette was then inserted in the manipulator instrument holder. The paraffin filled probe (small pipette; see Chapter 2, section 2.2.8.2.) was then similarly fitted to the paraffin filled plastic tubing attached to a Hamilton micro-syringe on the right side of the microscope and then inserted in the other manipulator instrument holder. All microscopic observations

were continually recorded by a video camera connected to a recorder and a monitor screen.

A transparent plastic plate was placed on the microscope stage and a drop of Hepes buffered medium (HWM, if mouse morula; or HSOF, if sheep morula) was placed on the plastic plate in the centre of the field of vision. One freshly collected healthy morula (either mouse or sheep) was introduced into the drop of medium. The embryo was quickly brought into focus and video recording was commenced. The morula was then held firmly but without too much suction with the holding pipette. The probe was brought from the opposite side and made to just touch the morula with minimal pressure. This stabilized the morula during subsequent manipulation. The medium on the plate was gently siphoned off withan embryohandling capillary pipette without dislodging the embryo from the holding pipette. Immediately after removal of the medium, VS11 was flooded on to the spot where the morula was being held. The entire events under the microscope were continously recorded and monitored. The amount of VS flooded on to the plate was 50 times more than the original volume of the medium so that the dilution of VS by the medium remaining on the plate was negligible. With the aid of the holding pipette and probe the morula was positioned at various angles so that changes in its shape due to the osmotic challenge by the VS could be recorded from all sides. The recordings continued over a period of 20 minutes.

9.3.1.2. Procedure for the Calculation of Volume of Morula Before and After Exposure to VS 11

For measurement of the volume while in isosmotic medium it was assumed the embryo was spherical. After immersion in VS 11 dehydration of the embryo accompanied by a reduction in volume and alteration in shape was almost immediate. The new shape was observed and recorded by using the holding pipette and probe to adjust the attitude of the embryo. In its most stable position the embryo appeared round with reduced diameter. However it was clearly not spherical and when held in its least stable attitude the appearance resembled a tennis ball which had been partially evacuated. One side had become concave and the other remained convex or was slightly flattened. Calculation of the volume of the altered embryos required the use of the appropriate models in solid geometry. Two models were used. The prefered model for each j embryo was selected by the appearance of the embryo. The models are shown in Figs. 9.3 and 9.4. In the first, Fig.9.3 the volume of the embryo was obtained by subtracting the volume of a

segment of a sphere from the volume of a segment of another sphere. The volume of such a segment is obtained by the formula given in Fig. 9.3. In the second model, Fig.9.4 the volume of the embryo was obtained as the difference between the volumes of two hemispheres.

The measurements were made with aid of an image analyzer (Image-1/AT, USA) on images selected from the video recording for each chosen time interval. Each image was viewed on five separate occasions. On each occasion each measurement was made six times and the means of these six measurements were used in the formulae given in Figs. 9.3 and 9.4. Thus there were five calculations of the volume of each embryo, and each was expressed as a percentage of the volume of the embryo when in isosmotic medium.

Statistical analysis was performed by Chi-square test. 9.4. Results

9.4.1. Experiment 9.1: Volume Changes in Mouse Compacted Morulae when Exposed to VS 11 for 0 to 20 Minutes at 25°C

The relative volumes of each mouse embryo are shown in Table 9.1 and are illustrated in Fig. 9.1. Statistical analyses of the relative volumes of the three mouse morulae showed that there was a difference between embryos (pcO.OOl) and between durations of exposure, and there was a significant interaction between embryos and durations of exposure (p<0.001).

At the first measurement after 5 minutes of exposure to VS the relative volumes ranged from 20.3% to 26.1. At 20 minutes the range increased to 25.2% to 50.5%. Further examination of the data showed that one embryo continually increased in relative volume between 5 and 20 minutes of exposure. The remaining two embryos increased in relative volume up to 15 minutes and then declined.

9.4.2. Experiment 9.2: Volume Changes in Sheep Compacted Morulae when Exposed to VS 11 for 0 to 20 Minutes at 25°C

The relative volumes of each sheep embryo are shown in Table 9.2 and Fig.9.2. There was a significant difference between embryos (p<0.001) and durations of exposure and a significant interaction between embryos and durations (p<0.001).

S C O

CD

m

e

Fig.9.1: Relative volumes (percent) of individual day-4 mouse morulae when challenged with VS11

Percent relative 50 -- volume 40 ..

O VA) O V")

Duration of exposure

Fig.9.2: Relative volumes (percent) of individual day-6 sheep morulae when challenged with VS11

100 « 70 - - 60 - - Percent relative 50 -- volume 40 .. 30 - - i o - - Duration of exposure

Fig. 9.3. Volume of segment of a sphere (between two parallel planes) Volume of segment V Jlh (3r2 + 3r3 + h )2 2 2 = 0.52359878h (3r2 + 3 r3 + h 2) Volume of embryo

Vol. of embryo (Shaded) =

Vol. of segment of a sphere (striped & shaded) - Vol. of unshaded (striped) segment

PLATE 9.1

The following are prints of images as shown by image analyzer. These prints and drawings complement Fig 9.3

TOP LEFT:Untreated fresh day-4 mouse morula

TOP RIGHT: The same morula described above

superimposed with a dotted circle and diameter "d"

MIDDLE LEFT:Day-4 mouse morula after exposure to VS shown at its most stable position. It assumes a shape akin to that of a evacuated ball

MIDDLE RIGHT:The same morula described above

superimposed with 2 dotted outer and inner circles and outer and inner diameters "dj" and "d2 indicating the outer and inner circumference of the embryo after it assumed the deflated appearance

BOTTOM LEFT:Day-4 mouse morula after exposure to VS shown at its least stable position showing the deflated appearance.

BOTTOM RIGHT:The same morula described above

superimposed with 2 dotted outer and inner circles and outer diameter "dj" and inner diameter "&2

Fig. 9.4. Volume of Sphere

Volume of embryo exposed to VS

~ Volume of treated embryo (shaded) = (V2 Vol. of larger sphere) -

(V2 Vol. of smaller sphere)

Key:

r = radius of untreated embryo d = diameter of untreated embryo

dj= diameter of larger sphere of treated embryo d2 = diameter of smaller sphere of treated embryo

PLATE 9.2

The following are prints of images as shown by image analyzer. These prints and drawings complement Fig 9.4

TOP LEFT:Untreated fresh day-6 sheep morula

TOP RIGHT: The same morula described above superimposed with a dotted circle and diameter "d"

MIDDLE L£FT:Day-6 sheep morula after exposure to VS shown at its most stable position. It assumes a shape akin to that of a evacuated ball

MIDDLE RIGHT:The same morula described above superimposed with 2 dotted outer and inner circles and outer and inner diameters "dj" and "d2" indicating the

outer and inner circumference of the embryo after it assumed the deflated appearance

BOTTOM LEFT:Dny-6 sheep morula after exposure to VS shown at its least stable position showing the deflated appearance.

BOTTOM RIGHT:The same morula described above superimposed with 2 dotted outer and inner circles and outer diameter "dj" and inner diameter "d2"

The data show that no two embryo responded in the same manner. For the three embryos, Al, A3 and A4, the highest relative volume was recorded at 10 minutes, 15 minutes and 20 minutes respectively. For all three embryos the relative volumes at 3 minutes ranged from 15.7% to 30% and at 20 minutes from 31.4 to 49.1%.

9.5. Discussion

The volume changes of mouse and sheep morulae exposed to VS 11 were characterized by an initial instantaneous shrinkage followed by a gradual re­ expansion similar to that reported in glycerol solution by Jackowski et al. (1980) and in glycerol-sucrose solutions by Szell and Shelton, (1986b).

The embryos exposed to VS 11 underwent a sudden drastic decrease in volume which resulted in gross alterations to shape. Although the shapes had some quite constant features there was some variation between embryos and this necessitated the application of two different formulae for the calculation of volume. Further, because of the irregularities in the solid geometry of individual embryos, the calculated volumes can be considered as approximations only. Neverthless observations on trends remain valid. It is clear also that when embryos are exposed to solutions of high molarity it is not valid to calculate their volume on the assumption that they remain spherical in shape. This may account for the deviation from the predicted shrinkage in 1.0M sucrose reported by Szell and Shelton (1987).

The relative volume of mouse and sheep morulae after exposure to VS11 for 3 to 5 minutes respectively ranged from 20.3% to 26.1% and 15.7% and 30%. Because of technical difficulty no earlier volumes were recorded; however, in view of the estimate (Leibo, 1986) that 80% to 85% of the volume of ova consists of water, it is apparent that a very high degree of dehydration was induced immediately after exposure to VS11. As embryos were frozen (Chapter 7 and 8) after 1 to 3 minutes of exposure to VS11 it is obvious that vitrification was induced when intracellular water and VS 11 contents were very low. It was not possible in these embryos to ascertain the intracellular concentrations of VS 11 but obviously in combination with the high concentration of cell solutes it was sufficient to permit freezing with little or no formation of ice (vitrification).

Two of the mouse embyros showed a decrease in relative volume after 20 minutes of exposure to VS 11 and two sheep morulae after 15 minutes and 20 minutes of exposure. This was probably related to cellular damage to the embryos and would

accord with the findings of Chapter 5 and 6 that VS11 is toxic to embryos after 20 minutes of exposure

Complete equilibration of embryos with VS is not only unnecessary for cryopreservation by vitrification but the long exposure necessary to achieve complete equilibration is lethal. This time related toxicity is due to the prolonged osmotic stress because of the slow rate of permeation by VS 11 and /or chemical toxicity as discussed in Chapter 5.