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J. Ali and J. N. Shelton

The aim of the experiments described here was to investigate cryopreservation of day-6 sheep embryos by vitrification methods in which the preliminary procedures can be performed at room temperature using VS1 (5.5 mol ethylene glycol l−1 and 2.5 mol glycerol l−1), VS11 (6.0 mol ethylene glycol l−1 and 1.8 mol glycerol l−1) and VS14 (5.5 mol ethylene glycol l−1 and 1.0 mol sucrose l−1). None of the day-6 sheep embryos vitrified with VS1 survived. Day-6 sheep embryos with the exception of blastocysts were vitrified with VS11 with no loss of viability in vitro. The viability of transferred day-6 embryos vitrified with VS11 was however extremely poor. Osmotic damage was avoided by initially exposing the embryos to one of four dilutions (20%, 30%, 40% and 50%) of VS11 for 5 min at 25°C and then vitrifying with the undiluted VS11. The highest survival (88.2%) in vitro was obtained when embryos were exposed to 30% VS11 before vitrification with the undiluted VS11. Survival of transferred embryos exposed to 30% VS11 and then vitrified with undiluted VS11 was 55% (16 of 29) for morulae and 62% (18 of 29) for blastocysts. The pregnancy rate for recipients that received two vitrified sheep embryos of these developmental stages per ewe was 79% (22 of 28). In a small study performed with VS14 the survival of day-6 sheep embryos vitrified with VS14 (in one-step) was 100% in vitro and 50% after transfer.

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J. Ali and J. N. Shelton

Three vitrification solutions (VS) namely VS1 (5.5 mol ethylene glycol l−1 and 2.5 mol glycerol l−1), VS11 (6.0 mol ethylene glycol l−1 and 1.8 mol glycerol l−1) and VS14 (5.5 mol ethylene glycol l−1 and 1 mol sucrose l−1) were tested for cryopreservation by vitrification of all developmental stages of mouse preimplantation embryos. In these experiments all preparative work was at room temperature (25°C). VS1 was toxic to embryos at and earlier than the eight-cell stage, whereas VS11 was toxic to the four-cell and earlier stages. VS14 was the least toxic VS. All three VS resulted in good viability of vitrified Swiss Outbred day-4 embryos (morulae, early blastocysts and blastocysts) in vitro and vitrification with VS14 resulted in no loss of viability in all preimplantation stage Swiss Outbred embryos except one-cell embryos. One-cell F1 embryos were vitrified successfully with VS14 and VS1. The minimal equilibration time essential for successful vitrification of embryos suggests that concentration of the intracellular solutes by dehydration has a major role in establishing conditions conducive to intracellular vitrification. Studies in vitro suggested that sucrose dilution was not necessary in the removal of cryoprotectant from vitrified eight-cell and day-4 mouse embryos but, in contrast, development of vitrified day-4 embryos in vivo was better when the VS was diluted with sucrose. When VS1 or VS11 was removed from vitrified embryos with sucrose the number of live fetuses obtained after transfer to surrogates did not differ from the number obtained from untreated embryos. Vitrification was not teratogenic; all mice that developed from vitrified embryos appeared normal and later reproduced normally. The present study demonstrated that vitrification can be routinely used to cryopreserve mouse embryos with no loss of viability.

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J. Ali and J. N. Shelton

A series of experiments was performed to determine the concentrations at which ten cryoprotectants singly and in pairs would vitrify on plunging into liquid nitrogen and remain vitreous when warmed by plunging into a water bath at 25°C. From these tests eight solutions (VS) were selected for testing of toxicity to mouse morulae in vitro. One of these (VS1) was modified as a further five VS of which one (VS11) was tested for toxicity to all stages of mouse embryos and to sheep compacted morulae. The concentrations at which the cryoprotectants vitrified on cooling were: butylene glycol, 3.0 mol l−1; propylene glycol, 4.0 mol l−1; dimethyl sulfoxide (DMSO) and glycerol 5.0 mol l−1; ethylene glycol, 6.5 mol l−1. None of these, at the highest concentration tested, remained vitreous during warming. Methanol and the high molecular weight polymers, dextran, Ficoll, polyethylene glycol and polyvinylpyrrolidone, did not vitrify at the concentrations tested. Toxicity studies showed the order of increasing toxicity to be ethylene glycol, methanol, DMSO, glycerol, propylene glycol and butylene glycol. Of the mixtures composed of two cryoprotectants, those containing ethylene glycol and glycerol were the least toxic at vitrifying concentrations. VS11 (6.0 mol ethylene glycol l−1 and 1.8 mol glycerol l−1) was well tolerated by mouse morulae, less well by eight- and one-cell embryos and poorly by two-cell embryos. Dilution of the VS11 from mouse embryos by exposure to 1.0 mol sucrose l−1 for 10 min did not enhance their survival. VS14 (5.5 mol ethylene glycol l−1 and 1.0 mol sucrose l−1) was a good vitrifying mixture and was non-toxic to mouse embryos when they were exposed for up to 30 min. The survival of sheep compacted morulae in vitro was not affected by exposure to VS11 for up to 20 min when dilution of the VS11 was done by exposure to 1.0 mol sucrose l−1 for 10 min. Without sucrose dilution, exposure to VS11 for 10 min was detrimental to embryo survival.

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A. Széll and J. N. Shelton

Summary. The time requirements for permeation by glycerol and dehydration by sucrose before rapid freezing of Day-3 mouse embryos by direct transfer to − 180°C were studied. When the embryos were equilibrated in 2·0, 3·0, or 4·0 m-glycerol + 0·25 m-sucrose for 2·5 to 40 min, the post-thaw viability increased (P < 0·001) with the length of equilibration period at 4°C. At 20°C the volume of embryos increased with the duration of equilibration up to 20 min (P < 0·001), but the post-thaw viability was not affected.

The effect of equilibration in glycerol–sucrose was determined at 20°C for embryos which were previously permeated by glycerol, dehydrated by sucrose or left in PBS + 5% FCS. The survival of previously permeated embryos was not affected by equilibration for 1–16 min in glycerol–sucrose. The maximum survival rate was attained after shorter equilibration in glycerol–sucrose for embryos without pretreatment (4 min) than for those previously dehydrated (8 min).

It is concluded that increases in the intracellular glycerol level are beneficial for the viability of rapidly frozen mouse embryos and previous or concommitant exposure to sucrose unfavourably affects glycerol permeation.

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A. Széll and J. N. Shelton

Summary. The toxic effects of sucrose and the conditions of in-straw glycerol removal after freezing and thawing were studied using Day-3 mouse embryos. At 20°C, exposure to ≤1·0 m-sucrose for periods up to 30 min had no adverse effects on freshly collected embryos. At 25 and 36°C, however, ≥ 1·0 m-sucrose significantly reduced the developmental potential (P < 0·001).

In the freezing experiments the embryos were placed in 0·5 ml straws containing 40 μl freezing medium separated by an air bubble from 440 μl sucrose solution. The straws were frozen rapidly in the vapour about 1 cm above the surface of liquid nitrogen. The post-thaw viability was substantially better after sucrose dilution at 20°C than at 36°C. Mixing the freezing medium with the sucrose diluent immediately after thawing further improved the rate of survival relative to mixing just before freezing (P < 0·001). The best survival was obtained when the freezing medium contained 3·0 M-glycerol + 0·25 m-sucrose; it was mixed with the diluent after thawing and the glycerol was removed at 20°C. Under such conditions the sucrose concentration in the diluent had no significant effect on the rate of development (0·5 m, 69%; 1·0 m, 73%; 1·5 m, 64%).

The results show that during sucrose dilution the temperature should be strictly controlled and suggest that intracellular and extracellular concentrations of glycerol are important in the cryoprotection of embryos.

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András Széll and J. N. Shelton

Summary. The relative volume of Day-3 mouse embryos changed as a linear function of the reciprocal of osmolarity of non-permeating solutes after 10 min exposure to sucrose and glycerol—sucrose solutions at 20°C. The slope of the linear regression line was less in glycerol—sucrose than in sucrose solutions because glycerol permeation caused re-expansion.

Before freezing by direct transfer to − 180°C the embryos were placed into glycerol-sucrose in 1-step (1-step equilibration) or first into glycerol and then into glycerol-sucrose (2-step equilibration). Using 2-step equilibration the post-thaw survival rate was substantially higher at 3·0 and 4·0 m-glycerol levels and less dependent on changes in the sucrose concentration within the range of 0·125 to 1·0 M than with 1-step equilibration. Under optimal conditions 90–95% of rapidly frozen embryos developed to blastocysts in vitro and 30% into live young in vivo.

It is suggested that the cryoprotective role of glycerol is due to its ability to reduce osmotic pressure differences between the extra and intracellular spaces during rapid freezing of embryos.

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J. N. SHELTON and N. W. MOORE

Summary.

When compared in ewes at three dose-rates horse anterior pituitary extract (hap) produced a different ovarian response to that of pregnant mare serum gonadotrophin (pmsg). Although there was no difference in number of ovulations, there was a greater number of persistent follicles after pmsg. Because persistent follicles are associated with a decreased percentage of fertilized ova, hap is more suited than pmsg to the production of large numbers of fertilized sheep ova.

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N. W. MOORE and J. N. SHELTON

Summary.

A trial of factorial design is described, in which was studied the response of the cyclic ewe to a horse anterior pituitary extract (hap) Three total-dose levels of hap (60, 90 and 135 mg) were administered over 2 or 3 days commencing on the 12th day of the oestrous cycle. The total dose was given as either two or three equal daily injections, or four or six equal twice-daily injections.

Highly significant (P<0·001) linear dose-response relationships were observed in the ovulatory response to both 2- and 3-day treatments. Treatment for 3 days was significantly better than treatment for 2 days in the proportion of ewes ovulating (P<0·05), ovulation rates (P<0·001) and fertilization of eggs shed (P< 0·001).

For both 2- and 3-day treatments the ovarian responses varied with the time elapsing between the final hap injection and the onset of oestrus. Maximum responses were observed in ewes in oestrus 24 to 48 hr after the cessation of treatment. Fertilization in ewes treated for 2 days, but not in those treated for 3 days, also varied with the time elapsing between the cessation of treatment and onset of oestrus.

The results showed hap to be a most effective preparation for the induction of multiple ovulation in the ewe.

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J. N. SHELTON and N. W. MOORE

Summary.

Control of the onset of oestrus in ewes by the daily intramuscular injection of progesterone had no effect on the survival of fertilized eggs transferred at the rate of two per ewe to the Fallopian tubes or the uterus. More ewes lambed and more lambs were born following transfer of eight (or more) cell eggs to the uterus than after transfer of eggs of less than eight cells to the Fallopian tubes.

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N. W. MOORE and J. N. SHELTON

Summary.

A factorial experiment is described in which fertilized sheep eggs were transferred at a rate of two per animal to 162 recipient ewes all of which were allowed to go to term.

The factors included and the results obtained were:

  1. Degree of synchronization between donor and recipient. Transfers were made to recipients in oestrus 48 hr before to 48 hr after their respective donors. Highly significant effects were observed in the number of ewes which lambed (P<0·01) and number of eggs surviving to parturition (P<0·001), with maximum results in ewes in oestrus 12 hr before to 12 hr after their respective donors.
  2. Age of eggs. Eggs were collected 48 to 60, 60 to 72 and 72 to 84 hr after the donors were first observed in oestrus. There was a significant increase in the number of ewes which lambed (P<0·02) and lambs born (P<0·01) with increase in age of the transferred eggs.
  3. Site of transfer. Transfers were made to the Fallopian tubes or uterine horns. Tubal transfers were significantly more successful than uterine transfers in the number of ewes which lambed (P<0·05) and number of lambs born (P<0·01).

There were no significant interactions.