Small Blastocysts Research Articles

P-258 Not too big, not too small: euploid blastocysts that lead to live birth have an optimal surface area, diameter and optimal pace of development

Abstract Study question Does embryo diameter and surface area at tsB and tB as determined by AI predict live birth (LB) potential amongst euploid blastocysts? Summary answer Euploid blastocysts with optimal diameter (tsB:120-129; tB:136-140μm) and surface area (tsB:11771-13577; tB:15000-15643μm2) are more likely to lead to LB than larger or smaller blastocysts. What is known already Current embryology consensus favours larger blastocysts based on Gardner grading, associating expansion with higher quality. Human assessment of embryos is subjective, and it is unknown whether this dogma holds when assessing pre-expansion blastocyst stages. The impact of blastocyst surface area and diameter at tSB and tB; and pace of development on LB remains unclear. This may help clarify whether large blastocysts are associated with accelerated embryo growth and abnormal metabolic processes. The study’s objective was to use AI to quantitatively determine the optimal embryo surface area and diameter of euploid blastocysts that lead to live birth. Study design, size, duration Retrospective study including 97 time-lapse videos of euploid embryos with known live birth outcomes from January to December 2021 Participants/materials, setting, methods Embryo diameter and surface area were measured at start of blastulation (tSB) and at time of blastulation (tB) by an AI model (CHLOE-EQ, Fairtility Ltd). Live birth (LB) outcomes were manually annotated by embryologists. An Optimal embryo surface area and diameter were determined by the highest live birth rate and the suboptimal range was determine by a decreased Live birth rate. A comparison between the optimal and suboptimal live birth rate was measured using chi-square. Main results and the role of chance Moderately sized embryos were found to have higher LB outcomes. At tSB, optimal surface area was 11771-13577 um2, while optimal embryo diameter was 120-129μm. LB rate for optimal embryo surface area was higher than for suboptimal [Optimal 80% (20/25) vs Suboptimal 55% (40/72), p < 0.001]. In addition, optimal embryo diameter had higher LB rates than embryos with suboptimal diameter [80% (16/20) vs 52% (31/60), p < 0.05). At tB, optimal embryo area was 15000-15643μm2 and optimal embryo diameter 136-140μm. LB rate for optimal embryo surface area was higher than for suboptimal 82% (14/17) vs 49% (36/73), p < 0.05]. Optimal embryo diameter had higher LB rates than embryos with suboptimal diameter (77% 20/26 vs 47% 30/64, p < 0.05). Embryos with an optimal surface area and diameter were slower than bigger suboptimal embryos: t2 (26±2 vs 25±2), t8 (59±8 vs 53±6), t9 + (71±6 vs 67±6), tM (82±7 vs 77±8), tSB (99±6 vs 93±6), tB (107±7 vs 100±6), tEB (137±2 vs 122±12); t8-t5 (12±10 vs 6±5), t8-t2 (33±8 vs 28±5), tEB-tSB (34±2 vs 23±9), p < 0.05. Optimal vs small suboptimal embryos were faster in t9 + (71±6 vs 76±4), tEB (121±11 vs 137±2, tEB-tSB (20±10 vs 34±1). But were slower in t8-t5 (12±10 vs 5±6), t8-t2 (33±8 vs 28±5), p < 0.05. Limitations, reasons for caution This is the first time a quantitative embryo surface area and diameter has been studied in its correlation with live birth. Generalisation requires the replication across diverse centers with varying demographic profiles to validate findings and establish broader applicability within the context of assisted reproductive technologies. Wider implications of the findings This data-driven approach can support the embryo selection process, correlating quantitative parameters with live birth outcomes, thereby refining the precision of the decision-making in IVF. This level of quantitative data of embryo surface area and diameter is only accessible through AI tools. Trial registration number IRB-007CITY-FL21.08.2023

P-302 Correlation of blastocyst shrinkage-pattern during vitrification and post-warming embryo performance

Abstract Study question Can blastocyst shrinkage-pattern during vitrification be predictive of post-warming survival and reproductive potential? Summary answer Blastocysts assuming a specific shrinkage-pattern during vitrification are liable to lower post-warming morphology and, potentially, degeneration. What is known already Blastocyst vitrification is a highly standardized technique. However, there are still possible factors than can compromise the outcome of the technique which should be minimized. For example, the degree of blastocoel expansion before vitrification may negatively influence embryo survival post-warming. There are studies which demonstrate that expanded blastocysts exhibit lower survival rate because of their higher blastocoel volume and amount of water, which is more prone to the detrimental ice crystal formation during vitrification. Moreover, shrinkage of expanded blastocysts during vitrification differs as some of them shrink in a uniform, spherical way while others shrink in a non-canonical way Study design, size, duration A retrospective study was conducted at IAKENTRO fertility clinic, Thessaloniki. A total of 2167 blastocysts vitrified between November 2018-December 2022, out of which 532 blastocysts warmed during the same period, were included in the present study. No biopsied or PGT-a tested embryos were included. Embryo vitrification was performed following the same protocol during the whole period of the study. For the clinical outcomes, 178 single-embryo transfers and 123 double-embryo transfers were analyzed Participants/materials, setting, methods During blastocyst exposure to the vitrification solution two shrinkage patterns were detected, allocating embryos into Group-1 for blastocysts presenting shrinkage without any detachment of the trophectoderm cells from the zona pellucida, leading to a ZP reforming which eventually resembled a “deflated” ball and into Group-2 for blastocysts that collapsed in a uniform way, having their TE cells fully detached from the ZP, which remained spherical throughout the exposure to the vitrification solution Main results and the role of chance In the total cohort of 2167 assessed blastocysts, 437 were allocated in Group-1 and 1730 in Group-2, which can be translated as 20,2% event occurrence. The Group-1 blastocysts were mostly of expansion degree 4 (76,7%) and degree 3 (23,3%), according to the Gardner system, while no smaller blastocysts were assigned in this group. As primary outcome was examined post-warming embryo survival with blastocysts in Group-1 presenting significantly higher degeneration rate (22/187, 11,7%) respect to blastocysts in Group-2 (7/345, 2%) (χ2 test, p < 0.0001, OR = 5,798, 95%CI = 2,511 to 14,55). Moreover, in order to assess any potential decline in embryo morphology post-warming, we corresponded Gardner’s system evaluation to a numerical score and we calculated the integrity in morphology before and after vitrification. Before vitrification, embryos’ morphology score was similar in both groups (74,8% and 73,1% for Group-1 and Group-2, respectively) while, after warming, Group-1 blastocysts exhibited significantly lower integrity of their morphology (86,52% integrity in score) respect to Group-2 blastocysts (93,82% integrity in score) (t-test, p < 0,0001). However, when analyzing PR and CPR no significant differences were observed between the two groups Limitations, reasons for caution The retrospective aspect of the study is per se a general limitation. Moreover, given that the analysis of the data was not limited to freeze-all cases, the embryos chosen for the fresh embryo-transfers, which means those with the highest implantation potential, were excluded from the study’s cohort Wider implications of the findings Our results show that blastocysts resembling a “deflated” ball during vitrification have compromised post-warming survival. Given that this phenomenon is exclusively observed in expanded blastocysts, whose ZP is thin and TE cells are stretched, identifying the expansion grade over which is happening could be beneficial in clinical practice Trial registration number Not applicable

Effects of single or serial embryo splitting on the development and morphokinetics of in vitro produced bovine embryos

Embryo splitting can be used in cattle in vitro production (IVP) to improve embryo availability and to increase selection intensity. Despite this widespread utility, a comparative investigation of the viability of IVP embryos split at Day 2 (2-cell stage), Day 3 (8-cell stage), and blastocyst stage has not been undertaken. Similarly, the suitability of splitting Day 3 embryos with atypical numbers of blastomeres, and the feasibility of serial-splitting cleavage stage embryos, have not been investigated in cattle. Here, we demonstrate that the strategy most likely to produce the greatest output of viable embryos is the splitting of Day 3 embryos into four parts, regardless of whether embryos with exactly eight cells or an atypical number of blastomeres are used. This approach was found to produce 1.8 blastocysts per zygote on average compared to just 0.4 blastocysts per zygote for non-split controls. Single-splitting was also found to be superior to serial-splitting which, whilst feasible, impaired embryo viability as judged by cell number at day 7 post-insemination. Interestingly, zygotes (≥2 cells) split once on either Day 2 or Day 3 post-insemination, whilst resulting in smaller blastocysts than control embryos, displayed higher cell counts than expected at the blastocyst stage, suggesting a compensatory mechanism might be at play. Indeed, time-lapse imagery revealed that zygotes split at 2-cells reached the compact morula and expanded blastocyst stages earlier than either those split at Day 3 or non-split controls. Developmental events between splits originating from the same progenitor appeared well synchronized only up to the third cleavage division.

Vitrification of in vitro-produced and in vivo-recovered equine blastocysts in a clinical program

There is a clinical demand for cryopreservation of both in vivo-recovered and in vitro-produced (IVP) equine embryos. We previously reported successful vitrification of expanded equine blastocysts in fine-diameter microloader pipette tips (MPTs) after blastocoel collapse, in a research setting. Here, we report the results of clinical application of the MPT vitrification technique for both in vivo-recovered and IVP blastocysts. In vivo-recovered blastocysts were obtained by referring veterinarians on Days 6 to 8 after ovulation, and shipped 1 to 10 hours to the laboratory before vitrification. IVP blastocysts (<300 μm in diameter) were produced by intracytoplasmic sperm injection and in vitro embryo culture. All vitrified-warmed embryos were shipped (0.5–12 hours) for transfer to recipient mares. In experiment 1, 47 IVP embryos from our clinical intracytoplasmic sperm injection program were vitrified using the MPT and transferred. The rates of initial pregnancy (59%) and foaling (45%) were equivalent to those for 52 IVP embryos from the same mare aspiration sessions and shipped for the same duration but transferred fresh (75% and 45%, respectively). The pregnancy and foaling rates for in vivo-recovered embryos were 76 and 71%, respectively for 17 small blastocysts (<300 μm in diameter), and 55 and 45%, respectively for 11 large blastocysts (303–608 μm in diameter, collapsed before vitrification; P > 0.1). In experiment 2, the MPT was cut lengthwise to form an open vitrification device, designated “Sujo”. Research IVP blastocysts were vitrified at 1, 2, or 3 embryos per Sujo (n = 34 embryos), or singly on a commercial open device (Cryolock; n = 11). After warming, 97% and 91% of embryos, respectively, grew in culture. Similarly, culture of two in vivo-recovered large blastocysts after collapse and vitrification on Sujos both resulted in embryo growth. However, transfer of four in vivo-recovered expanded blastocysts after collapse, vitrification on Sujos, and warming resulted in only one foal. These data indicate that vitrification of equine IVP embryos and small in vivo-recovered embryos is efficient under clinical conditions. Collapse and vitrification of in vivo-recovered large blastocysts in MPT under our clinical conditions resulted in a 45% foaling rate. While numbers are low, use of an open vitrification system did not appear to improve results for these embryos.

Female bovine blastocysts are more prone to apoptosis than male ones

Female and male embryos show differences in gene expression and metabolism from the onset of their genome. Those differences are affected by environmental factors. The objective of the study was to compare the apoptotic rates of in vitro–produced female and male bovine blastocysts cultured in different conditions. Day 7 blastocysts obtained after IVF with sex-sorted semen and culture in two synthetic oviductal fluid–based media (containing fetal calf serum [FCS] or BSA, insulin, transferrin, and selenium) were simultaneously evaluated for two markers of apoptosis after 3D reconstruction from confocal images: active caspase 3 by immunofluorescence and DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling. Higher levels of apoptotic cells were observed in female embryos whatever the culture condition but with a more pronounced difference in FCS medium. This result was confirmed using the unsexed semen of two bulls. The sex effect on apoptosis was detected in both the inner cell mass and the trophectoderm but was dependent on the embryonic size. In conclusion, this study reported that female bovine blastocysts are more prone to apoptosis than male ones but that culture in FCS exacerbates the differences in apoptosis between sexes, especially in small blastocysts.

Effect of bovine blastocyst size at embryo transfer on day 7 on conceptus length on day 14: Can supplementary progesterone rescue small embryos?

Conceptus size on Day 14 after multiple embryo transfer of Day 7 in vitro–produced blastocysts varies greatly within animal. One explanation for this variation may be related to blastocyst cell number at the time of transfer. The aim of this study was to examine the effect of Day 7 blastocyst cell number on Day 14 conceptus size and to examine the effect of progesterone (P4) supplementation on embryo development after the transfer of Day 7 blastocysts containing a low total cell number. The estrous cycles of crossbred beef heifers were synchronized using an 8-day progesterone (P4)–releasing intravaginal device (PRID) with the administration of a prostaglandin F2α analog on the day before device removal. Only those heifers recorded in standing estrus (Day 0) were used. Heifers were randomly assigned to one of four treatment groups: (1) control: large blastocysts (high total cell number), (2) control: small blastocysts (low total cell number), (3) small blastocysts plus a single intramuscular injection of 3000 IU human chorionic gonadotropin (hCG) on Day 2 after estrus, or (4) small blastocysts plus insertion of a vaginal P4 insert (PRID, 1.55 g P4) between Days 3 and 5 after estrus. In vitro–produced blastocysts were transferred to each heifer on Day 7 (n = 10 blastocysts per heifer), and conceptuses were recovered at slaughter on Day 14. Daily blood samples were collected from Day 0 to 14 to measure serum P4 concentrations. Data were analyzed using the PROC MIXED procedure of SAS. Total cell number on Day 7 was significantly lower in small versus large blastocysts (72.4 ± 3.93 vs. 144.8 ± 3.90, P < 0.05). Conceptus recovery rate was 53.8% overall (140 of 260) and was highest in the large blastocyst group (68.3%, 41 of 60) compared with the other groups (45.7%–55.0%). Concentrations of serum P4 were similar in the two unmanipulated recipient groups but were significantly elevated (P < 0.05) by Day 8 in the hCG-treated heifers and on Days 4 and 5 in the PRID group (P < 0.003). In the absence of supplemental P4, Day 14 conceptuses resulting from the transfer of small blastocysts (2.48 ± 0.54 mm) were smaller than those from large blastocysts (3.32 ± 0.52 mm). Administration of hCG on Day 2 approximately doubled conceptus length on Day 14 (4.94 ± 1.15 mm; P < 0.05), whereas insertion of a PRID from Days 3 to 5 increased conceptus length approximately fivefold (13.09 ± 2.11 mm; P < 0.05) compared with controls. In conclusion, results indicate that supplemental P4 is capable of “rescuing” poor-quality blastocysts, presumably via the now well-described actions on the endometrium and consequent effects on uterine lumen fluid composition.

76 RELATIONSHIP BETWEEN BLASTOCYST CELL NUMBER AT EMBRYO TRANSFER AND CONCEPTUS ELONGATION ON DAY 14: CAN SUPPLEMENTARY PROGESTERONE RESCUE SMALL EMBRYOS?

Conceptus size on Day 14 following multiple embryo transfer of Day 7 in vitro-produced blastocysts varies greatly within animal. One explanation for this variation may be related to blastocyst cell number at the time of transfer. The aim of this study was to examine the effect of Day 7 blastocyst cell number on Day 14 conceptus size and to examine the effect of progesterone (P4) supplementation on embryo development following the transfer of Day 7 blastocysts containing a low total cell number. The oestrous cycles of crossbred beef heifers were synchronised using an 8-day CIDR (1.38 g of P4) treatment with administration of a prostaglandin F2α analogue (Estrumate™, Merck Animal Health, Summit, NJ, USA) on the day before CIDR removal. Only those heifers recorded in standing oestrus (Day 0) were used. Heifers were randomly assigned to 1 of 4 treatment groups: (1) Control: large blastocysts (high total cell number), (2) Control: small blastocysts (low total cell number), (3) small blastocysts plus a single intramuscular injection of 3000 IU of hCG (Chorulon®, Merck Animal Health) on Day 2 after oestrus, or (4) small blastocysts plus insertion of a vaginal progesterone insert (PRID, 1.55 g of P4) between Days 3 to 5 after oestrus. In vitro-produced blastocysts were transferred to each heifer on Day 7 (n = 10 blastocysts per heifer) and conceptuses were recovered at slaughter on Day 14. A sample of blastocysts was fixed and stained using Hoechst 33342 to determine the mean total cell number per group. Daily blood samples were collected from Day 0 to 14 to measure serum P4 concentrations. Data were analysed using the PROC MIXED procedure of SAS (SAS Institute Inc. Cary, NC, USA). Total cell number on Day 7 was significantly lower in small compared with large blastocysts (72.4 ± 3.93 v. 144.8 ± 3.90; P &lt; 0.05). Conceptus recovery rate was 53.8% overall (140/260) and was highest in the large blastocyst group (68.3%, 41/60) compared with the other groups (45.7–55.0%). Concentrations of serum P4 were similar in the 2 unmanipulated recipient groups but were significantly elevated (P &lt; 0.05) by Day 8 in hCG-treated heifers and on Days 4 and 5 in the PRID group (P &lt; 0.003). In the absence of supplemental P4, Day 14 conceptuses resulting from the transfer of small blastocysts (2.48 ± 0.54 mm) were smaller than those from large blastocysts (3.32 ± 0.52 mm). Administration of hCG on Day 2 approximately doubled conceptus length on Day 14 (4.94 ± 1.15 mm), whereas insertion of a PRID from Day 3 to 5 increased conceptus length ~5-fold (13.09 ± 2.11 mm; P &lt; 0.05) compared with controls. In conclusion, results indicate that supplemental P4 is capable of “rescuing” poor quality blastocysts, presumably via the now well-described actions on the endometrium and consequent effects on uterine lumen fluid composition. Supported by Science Foundation Ireland (07/SRC/B1156).

In-vitro Developmental Potential of Single Blastomeres Derived from Day 3 Discarded Human Embryos

Objective To investigate whether isolated blastomeres of discarded human embryos could develop into blastocysts cultured in vitro without zona pellucida. Methods Total discarded 60 embryos, which were not suitable for transplanting or freezing, were collected from 21 patients. Of 60 embryos, 10, 8, 24, 12, and 4 embryos were at 2-, 3-, 4-, 5- and 6-cell stage, respectively. These embryos were split by 0.5% protease combined with mechanical method. The resulting single blastomere was cultured individually, evaluated daily and counted blastocyst development. Pluripotency of inner cell mass (ICM) of the blastocyst was analyzed by the expression of alkaline phosphatase(AKP). Results A total of 229 single blastomeres were isolated from 60 embryos. Defining the day when the embryos were split as the first day (d 1). The majority of the blastomere-derived embryos followed the normal pattern of development with compaction on d 3 and cavitationon d 4 and developed into small blastocysts on d 5. Rates of division, compaction, cavity and expansion of single blastomeres from 4-cell embryos were higher than those in other groups (P Conclusion Some of the blastomeres from discarded human embryo are flexible and able to develop into blastocysts, the potential was related to their donor embryos closely. They seem to follow development pattern of their donor embryos. The blastomere-derived blastocysts have smaller size and fewer cells compared with regular in vitro cultured human embryos. However, AKP expression showed ICM cells with pluripotency. We believe that the value of single blastomere of discarded embryos will be further confirmed in the future.

The four blastomeres of a 4-cell stage human embryo are able to develop individually into blastocysts with inner cell mass and trophectoderm

Early mammalian blastomeres are thought to be flexible and totipotent allowing the embryo to overcome perturbations in its organization during preimplantation development. In the past, experiments using single blastomeres from 2-, 4- and 8-cell stage mammalian embryos have provided evidence that at least some of the isolated cells can develop into healthy fertile animals and therefore are totipotent. We investigated whether isolated blastomeres of human 4-cell stage embryos could develop in vitro into blastocysts with trophectoderm (TE) and inner cell mass (ICM). Six 4-cell stage human embryos were split and the four blastomeres were cultured individually. The expression of NANOG, a marker for ICM cells, was analysed by immunocytochemistry. The majority of the blastomere-derived embryos followed the normal pattern of development with compaction on Day 4 and cavitation on Day 5 and developed into small blastocysts with TE and ICM on Day 6 (n = 12). The four cells of one embryo were individually capable of developing into blastocysts with TE and ICM, and NANOG was expressed in the ICM. Although based on a small number of embryos, we conclude that the blastomeres of a 4-cell stage human embryo are flexible and able to develop into blastocysts with ICM and TE.

Vitrification of zona-free rabbit expanded or hatching blastocysts: a possible model for human blastocysts.

The purpose of this study was to test the effectiveness of one two-step (A) and two one-step (B1 and B2) vitrification procedures on denuded expanded or hatching rabbit blastocysts held in standard sealed plastic straws as a possible model for human blastocysts. The effect of blastocyst size was also studied on the basis of three size categories (I: diameter <200 micro m; II: diameter 200-299 micro m; III: diameter >/==" BORDER="0">300 micro m). Rabbit expanded or hatching blastocysts were vitrified at day 4 or 5. Before vitrification, the zona pellucida was removed using acidic phosphate buffered saline. For the two-step procedure, prior to vitrification, blastocysts were pre- equilibrated in a solution containing 10% dimethyl sulphoxide (DMSO) and 10% ethylene glycol (EG) for 1 min. Different final vitrification solutions were compared: 20% DMSO and 20% EG with (A and B1) or without (B2) 0.5 mol/l sucrose. Of 198 vitrified blastocysts, 181 (91%) survived, regardless of the vitrification procedure applied. Vitrification procedure A showed significantly higher re-expansion (88%), attachment (86%) and trophectoderm outgrowth (80%) rates than the two one-step vitrification procedures, B1 and B2 (46 and 21%, 20 and 33%, and 18 and 23%, respectively). After warming, blastocysts of greater size (II and III) showed significantly higher attachment (54 and 64%) and trophectoderm outgrowth (44 and 58%) rates than smaller blastocysts (I, attachment: 29%; trophectoderm outgrowth: 25%). These result demonstrate that denuded expanded or hatching rabbit blastocysts of greater size can be satisfactorily vitrified by use of a two-step procedure. The similarity of vitrification solutions used in humans could make it feasible to test such a procedure on human denuded blastocysts of different sizes.

Ultrastructure of the embryonic stem cells of the 8-day pig blastocyst before and after in vitro manipulation: development of junctional apparatus and the lethal effects of PBS mediated cell-cell dissociation.

Ultrastructural examination of 8-day hatched pig blastocysts (large and small), their cultured inner cell mass (ICM), and cultured epiblast tissue (embryonic stem cells) was undertaken to assess the development of epiblast cell junctions and cytoskeletal elements. In small blastocysts, epiblast cells had no desmosomes or tight junction (TJ) connections and few organized microfilament bundles, whereas in large blastocysts the epiblast cells were connected by TJ and desmosomes with associated microfilaments. ICM isolation by immunodissection damaged the endoderm cells beneath the trophectoderm cells but did not appear to damage the epiblast cells or their associated endoderm cells. Epiblast cells in cultured ICMs were similar in character to those in the intact large blastocyst except that perinuclear microfilaments were observed. Isolated pig epiblasts, cultured for approximately 36 hr on STO feeder layers, formed a monolayer whose cells were connected by TJ, adherens junctions and desmosomes with prominent microfilament bundles running parallel to the apical cytoplasmic membranes. Perinuclear microfilaments were a consistent feature in the approximately 36 hr cultured epiblast cells. A feature characteristic of differentiation into notochordal cells, i.e., a solitary cilium, was also observed in the cultured epiblast. Exposure of the cultured epiblast cells to Ca(++)-Mg(++)-free phosphate buffered saline (PBS) for 5-10 min resulted in extensive cell blebbing and lysis. The results may indicate that pig epiblast cells could be more easily dissociated from early blastocysts ( approximately 400 microm in diameter) if immunodissection damage to the ICM can be avoided. It may be difficult, however, to establish them as embryonic stem cell lines because the cultured pig epiblast cells were easily lysed by standard cell-cell dissociation methods.

Timing of the first cleavage post-insemination affects cryosurvival of in vitro-produced bovine blastocysts.

The time of the first cleavage of bovine zygotes during in vitro culture can affect the rate of development and cell number of the blastocysts. The aim of this work was to study the effect of the timing of first cleavage on the cryosurvival of the resulting blastocysts. Following standard IVM and IVF, zygotes were cultured in modified synthetic oviduct fluid (SOF), with 10% fetal calf serum (FCS) added 48 hr post insemination, in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2. Embryos which cleaved by 24, 27, 30, 33, or 36 hr after insemination (IVF) were harvested and further cultured to the blastocyst stage (day 7 or day 8 post IVF). All developing blastocysts on days 7 and 8 were classified into three groups and were cryopreserved by vitrification. Group A consisted of blastocysts (<150 microm, small blastocysts); group B consisted of expanded or hatching blastocysts (>150 microm, large blastocysts); and group C consisted of morphologically poor quality blastocysts. The vitrification solution consisted of 6.5 M glycerol and 6% bovine serum albumin in PBS (VS3a). Thawed embryos were cultured further and survival was defined as the re-expansion and maintenance of the blastocoel over 24, 48, and 72 hr, respectively. Overall survival and hatching at 72 hr post-thawing was higher in blastocysts formed by day 7 than those formed by day 8 (60% vs. 40% survival; 63% vs. 45% hatching). Large blastocysts from day-7 and day-8 groups survived significantly better than small or poor quality blastocysts (76% vs. 63% and 31%; 72% vs. 30% and 26%, respectively; P < 0.05). Day-7 blastocysts from the 27- and 30-hr cleavage groups survived significantly better than those from the 36-hr group (63% and 66% vs. 25%, P < 0.05). Day-8 blastocysts from later cleaved (30 hr) zygotes had a higher survival than the 27-hr cleavage groups (52% vs. 26%, P < 0.05). These results indicate that the day of blastocyst appearance, developmental stage, and timing of the first cleavage post-insemination can influence the cryosurvival of bovine blastocysts following vitrification.

Removal of cytoplasm from one-celled mouse embryos induces early blastocyst formation.

It has been recognized for several decades that the number of cleavage divisions which precede blastocyst formation in the mammalian embryo is rigorously fixed, such that removal of cells from the embryo, or augmentation of cell number by embryo aggregation, does not affect the timing of blastulation. Instead, embryos manipulated so as to reduce cell number form small blastocysts with fewer numbers of cells, while aggregate embryos form giant blastocysts. This tight control of the number of cleavage divisions ensures that the timing of blastocyst formation corresponds to the period of uterine receptivity for implantation. As yet, no experimental manipulation has succeeded in altering control of the number of cleavage divisions prior to blastulation, and as a consequence, the biological basis for the control mechanism is entirely obscure. We report here that removal of cytoplasm from one-celled mouse embryos does not alter the rate of cleavage, but does induce precocious formation of small blastocysts. These findings suggest that the early embryo "counts" cleavage divisions by measuring the size of its blastomeres, and that experimental reduction of cell size disturbs the counting mechanism and leads to abnormally early blastulation.

The sex ratio of bovine embryos produced in vitro in serum-free oviduct cell-conditioned medium is not altered

The sex ratio of bovine blastocysts produced in vitro in serum-free oviduct cell-conditioned medium was investigated. Bovine embryos reaching the blastocyst stage were removed from culture medium on Days 6,7, 8 and 9 and were identified as small, mid-sized or expanded blastocysts. One third (29/91) of the blastocysts appeared on Day 6. Twelve from them were small blastocysts (5 males), 7 were mid-sized blastocysts (4 males) and 10 were expanded blastocysts (5 males). On Day 7, 33 blastocysts were obtained: 8 small (5 males), 9 mid-sized (3 males) and 16 expanded (13 males) blastocysts. Finally, on Days 8 and 9, 29 blastocysts were obtained: 12 small (9 males), 9 mid-sized (6 males) and 8 (3 males) expanded blastocysts. Sexing of the 91 blastocysts was performed by using an original polymerase chain reaction (PCR) protocol generating discreet internal control signals from both female and male samples and Y-specific smears from the male samples. Proportions of male embryos on Days 6, 7 and on Days 8+9 were 48, 64 and 62%, respectively. These values did not differ significantly among days and did not differ from 50%. Fifty-nine percent of small blastocysts, 52% of mid-sized blastocyst and 62% of expanded blastocysts were male. No difference between these values or with respect to 50% could be observed. These results show that bovine blastocysts produced in serum-free oviduct cell-conditioned medium do not have an altered sex ratio.

Large equine blastocysts are damaged by vitrification procedures

Viability following vitrification of equine blastocysts with different sizes was investigated in vitro. Twenty-four blastocysts were classified into three groups according to their diameters (< 200 microns, 200-300 microns and > 300 microns; n = 8 each). The solution used for vitrification was defined as EFS and contained 40% ethylene glycol, 18% Ficoll and 0.3 M sucrose in modified-phosphate-buffered saline (m-PBS). During pretreatment with 20% ethylene glycol in m-PBS for 20 min, the larger blastocysts responded to the osmotic pressure caused by 20% ethylene glycol more slowly than the smaller blastocysts. Single blastocysts were loaded into the EFS in 0.25-mL straws, left to stand for 1 min and vitrified in nitrogen vapour. After thawing for 20 s in water (20 degrees C), a fractured zona pellucida or capsule was seen in: 1 of 8 blastocysts < 200 microns in diameter; 1 of 8 blastocysts 200-300 microns in diameter; and 2 of 8 blastocysts > 300 microns in diameter. When the blastocysts were cultured for 48 h in TCM199 supplemented with 10% fetal bovine serum at 37 degrees C in 5% CO2 in air, 7 of 8 (88%) blastocysts < 200 microns in diameter and 6 of 8 (75%) blastocysts 200-300 microns in diameter developed with re-expansion of the blastocoele. However, the developmental ability of blastocysts > 300 microns in diameter (2 of 8, 25%) was significantly lower than that of blastocysts < 200 microns in diameter (P < 0.05).

Embryogenesis recapitulates oogenesis in swine.

Events during oogenesis can affect embryogenesis so dramatically that oocytes can be identified that are progenitors of embryos which would probably die if they remained in the host pig, but would live if appropriately transferred to another female. This review goes backward from embryonic to oocyte development, first discussing how subtle differences between littermate embryos can result in the death of some embryos and then relating the causes of those differences to events during follicular maturation. Embryonic development is not uniform in swine. The larger blastocysts within a litter synthesize estradiol sooner than their less developed contemporaries. Estradiol advances uterine secretions to the benefit of the more developed blastocysts, but results in an asynchronous and hostile environment for the less developed blastocysts. Through a series of experiments, the pattern of oocyte and follicular development was found to be one of the sources of subsequent disparity among blastocysts. In pigs mated before ovulation, the first oocytes released at ovulation were the first fertilized and became the more developed blastocysts 12 days later. Inversely, the later ovulated oocytes were the last to be fertilized and became the smaller blastocysts. These smaller blastocysts can develop normally, but because of estrogenic advancement of uterine secretions, they will preferentially die.

Survival of small and large littermate blastocysts in swine after synchronous and asynchronous transfer procedures

Thirty-two crossbred sows were assigned to synchronous and asynchronous embryo transfer procedures to determine if, within a litter, small blastocysts were as viable as large blastocysts. Synchronous embryo transfers were established when donors and recipients displayed the onset of estrus (Day 0) within 6 h of each other. Asynchronous transfers were established when recipients displayed the onset of estrus 18 to 24 h after that of donors. An equal number (four or five) of the smallest and largest diameter blastocysts, from a Day 7 donor, were transferred to separate uterine horns of a Day 7 (synchronous) or a Day 6 (asynchronous) recipient. Each recipient's uterine horns were ligated at the external bifurcation to prevent transuterine embryonic migration. The percentage of blastocysts surviving was determined 300 h (12.5 d) after donors exhibited estrus. Small as well as large Day 7 blastocysts survived following asynchronos transfer to a Day 6 recipient. However, fewer (P<0.01) small blastocysts survived synchronous transfer than large blastocysts. These data suggested that small blastocysts were lost due to asynchrony with the uterine environment; however, when transferred to a less advanced environment, small blastocysts were equally viable as large blastocysts.

Fertilization and early embryonic development in androstenedione-immunized Merino ewes

Ewes were immunized against androstenedione (Fecundin) and assigned to be mated 14 days (179 ewes Group C) or 25 days (174 ewes Group B) after a booster immunization with Fecundin. The anti-androstenedione titres at these times were 6790 and 3240 respectively (P less than 0.01). The remaining 169 ewes were untreated controls (Group A). Ewes were mated to entire rams (12 rams to 180 ewes) at their second oestrus after synchronization of oestrus. Immunization against androstenedione caused a shortening of the time from sponge removal to mating (Day 0) and a decrease in the percentage of ewes mated by the rams. Also, ovulation rate was increased after immunization (P less than 0.01), being 1.42, 2.16 and 1.93 for Groups A, C and B respectively. Egg recovery rates on Day 2 were lower in immunized ewes and there was some indication that fertilization rates were lowered. On Day 13 after mating a higher proportion of blastocysts was recovered from ewes in Group A than from those in Groups B and C. Immunization resulted in lower fertilization rates and smaller blastocysts with lower mitotic indexes (P less than 0.01). At Days 24-32 of pregnancy fetal weight was lower in the immunized ewes. At all sampling stages, the proportion of ewes pregnant (fertility) was lowered in immunized ewes. The results of the present study show that significant reproductive wastage occurs in androstenedione-immunized Merino ewes, with lower rates of embryo recovery and delayed embryonic development being found in comparison to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The Marsupial Blastocyst - a Study of the Blastocysts in the Hill Collection

Blastocysts in the Hill Collection from Trichosurus vulpecula, Pefrogale penicillata, Macropus ruficollis (= M. rufogriseus), Macropus parma, Onychogalea fraenata, Bettongia gaimardi, Perameles obesula (=lsoodon obesulus), Perameles nasuta, Dasyurus viverrinus, Didelphis aurita (=D. marsupialis) and Didelphis virginiana were examined. They ranged from incomplete unilaminar blastocysts to late bilaminar blastocysts. The mode of formation of the unilaminar blastocyst appeared to be influenced by the presence or absence of the yolk mass. A unilaminar blastocyst lined by uniform protoderm cells occurred in a wide variety of marsupials. Differentiation of the unilaminar blastocyst into embryonic and extra-embryonic areas occurred at different stages of development. In macropodids and Didelphis it was found in small blastocysts soon after blastocyst completion. In dasyurids, Perameles and some other groups it was found in larger blastocysts, at least four cell generations after blastocyst completion. The first histological signs of differentiation of the unilaminar blastocyst into embryonic and extra-embryonic areas varied between different marsupials. In Didelphis, enlarged endoderm mother cells developed from the protoderm cells of one hemisphere. The protoderm cells of this hemisphere later differentiated as embryonic ectoderm and the endoderm mother cells gave rise to the primary endoderm. In D. viverrinus, bandicoots and T. vulpecula, the protoderm cells of one hemisphere differentiated simultaneously into cuboidal embryonic ectoderm and endoderm mother cells. In P. penicillata, M. ruficollis and M. parma the protoderm cells of one hemisphere proliferated to form a multilayered embryonic area which later differentiated into embryonic ectoderm and primary endoderm.

The developmental capacity of blastomeres from 4- and 8-cell sheep embryos

ABSTRACT The developmental capacity of (a) single blastomeres of 4-cell embryos, (b) pairs of blastomeres of 8-cell embryos and (c) single blastomeres of 8-cell embryos was studied. The blastomeres were inserted into evacuated foreign zonae pellucidae, embedded in agar, transferred to ligated oviducts of dioestrous ewes, and recovered when their total age was to days. At lecovery the majority of embryos belonging to categories (a) and (b) had developed into small blastocysts, whereas most embryos of category (c) were vesicular forms with no clearly observable inner cell mass. The viability of embryos which had continued their development during culture in the sheep oviduct was tested by their transfer to the uterine horns of recipient ewes which had come into oestrus on the same day as the respective embryo donors. Three lambs were produced after transfer of eight embryos of category (a) deriving from two parent embryos. These three lambs derived from a single 4-cell embryo. Nine lambs were produced after transfer of 16 embryos of category (b) deriving from four parent embryos. Four of these lambs derived from a single 8-cell embryo. When ewes which had come into oestrus one or two days after the respective embryo donors were used as recipients for 16 embryos of category (a) and 16 embryos of category (b) one and five lambs, respectively, were produced. Finally, two lambs were produced after transfer of 31 embryos of category (c). These lambs derived from two different parent embryos. The principal conclusions drawn from the study are that at least some individual blastomeres from 4- and 8-cell sheep embryos can give rise to an entire conceptus, and that the viability of embryos produced by blastomere separation depends more on the degree of reduction in cell number than on the stage of development at which separation is performed.