Cryopreservation of Porcine Embryos: RESULTS
Experiment 1 Table 1 summarizes the results of cryopreserving IVP embryos vitrified at the expanded blastocyst stage, where about 40% of the IVP blastocysts survived vitrification (Fig. 2a), although their average cell number was lower compared to that of the nonvitrified blastocysts (38.4 ± 5.1 vs. 61.9 ± 5.8, P < 0.05). Note that survival rate and embryo quality of IVP embryos was significantly improved (41.2% vs. 83.3%; 38.4 ± 5.1 vs. 51.8 ± 4.2, P < 0.05) when cryopreserved after delipation. In fact, the survival rate of vitrified delipated blastocysts was nearly the same as that of nonvitrified controls (intact blastocysts, 88.0%; delipated nonvitrified blastocysts, 92.9%). Cell number of the delipated blastocysts after vitrification was also not significantly different compared to that of nonvitrified controls. As shown in Figure 2b, many of the delipated blastocysts tended to herniate from the zona pellcidae in culture after thawing, indicating that decline of embryo quality was minimized when IVP blastocysts were cryopreserved after de-lipation.
Experiment 2 Table 2 summarizes the results of cryopreserving IVP embryos at the 4-cell stage. Most embryos vitrified without delipation degenerated within 24 h after thawing (Fig. 3a), having a very low developmental rate to blastocysts (6/62, 9.7%). By contrast, delipation of embryos prior to vitrification significantly improved post-thaw development (32/ 89, 36.0%, P < 0.05). Most embryos vitrified after delipation herniated from the zona pellucidae as shown in Figure 3b in culture after thawing. canadian neighbor pharmacy viagra
Experiment 3 As shown in Table 3, survival of IVP morulae vitrified after trypsin treatment and centrifugation (tryp/centr; 52/ 63, 82.5%; Fig. 4, b and c) was equal to that of the deli-pated morulae (46/56, 82.1%; Fig. 4e). These survival rates were as high as those of the nonvitrified control embryos (Fig. 4a).
Centrifugation without trypsin treatment was also shown to render the IVP morulae cryotolerant (survival, 24/44, 54.5%; Fig. 4d), although their survival rate was significantly lower than that of the tryp/centr and delipated groups (P < 0.05). In contrast, only a few (3/35, 8.6%) of the intact morulae survived vitrification (Fig. 4f).
TABLE 2. Survival of parthenogenetic porcine IVP embryos after vitrification at the 4-cell stage.
|Delipatio||n Vitrification+||No. (%)Examined62||of embryosDeveloped to blastocysts6 (9.7)a||No. of cells in blastocysts (mean ± SEM)66.5 ± 6.7a|
|+||+||89||32 (36.0)b||36.7 ± 3.8b|
|+||–||50||29 (58.0)c||38.5 ± 3.0b|
|–||–||53||33 (62.3)c||58.5 ± 5.2a|
a‘b‘c Values with different superscripts within columns differ significantly (P< 0.05).
FIG. 1. Polarization of the cytoplasmic lipid droplets after centrifugation of a parthenogenetic porcine IVP morula. A morula-stage embryo centrifuged with (a) and without (b) trypsin treatment. Note that complete separation of lipid droplets from blastomeres could be achieved (a). Scale bar = 50 ^m.
FIG. 2. Survival of parthenogenetic porcine IVP blastocysts after vitrification. Day 8 blastocysts developed from nondelipated (a) and delipated (b) embryos after vitrification. Photographs were taken 48 h after thawing. Scale bar = 150 ^m.
TABLE 3. Survival of parthenogenetic porcine IVP morulae vitrified after delipation by a noninvasive method.
* Embryos were delipated by micromanipulation.
a,b,c Values with different superscripts within columns differ significantly (P < 0.05).
FIG. 3. Survival of parthenogenetic porcine IVP embryos vitrified at the 4-cell stage. Most of the nondelipated embryos
(a) degenerated by 24 h after vitrification, whereas embryos vitrified after delipation
(b) developed to blastocysts 120 h after thawing. Scale bar = 150 ^m.
FIG. 4. Development of parthenogenetic porcine IVP morulae vitrified after delipation by various methods. All the photographs except for (b) were taken 48 h after thawing (Day 6). a) Intact control nonvitrified embryos that developed to expanded blastocysts. b) Day 4 morulae centrifuged after trypsin treatment. c) Embryos vitrified after trypsin treatment and centrifugation. d) Embryos vitrified after centrifugation without trypsin treatment. e) Delipated morulae that developed to blastocysts after vitrification. f) Control vitrified morulae. Scale bar = 150 ^m.