Factors affecting the survival of bisected sheep embryos in vivo

Two experiments were carried out to examine the effects of different factors on the survival of split sheep embryos. In Experiment 1, embryos collected on Day-6, Day-7 or Day-8 were bisected and transferred into recipient ewes in pairs. The proportions of Day-6, Day-7 and Day-8 demi-embryos developing to lambs were 26% (14 54 ), 30% (31 102 ) and 32% (24 74 ), respectively. Replacement of bisected late morula to expanded late blastocyst stage embryos into zonae did not affect their survival rate (P>0.5). The proportion of demi-embryos developing to lambs in recipients with two or more ovulations was higher (35%, 53 152 ) than in recipients with a single ovulation (21%, 16 78 ; P<0.05). In Experiment 2, Day-6 embryos were split with or without exposure to 0.25 M of sucrose and were transferred into recipients in pairs or singly. Exposure to 0.25 M of sucrose decreased the proportion of split embryos developing to lambs compared with that of the controls (31%, 22 70 vs 49%, 34 70 ; P<0.05). The effects of the number of demi-embryos transferred or the stage of development on the survival rate were not significant (P>0.05). The number of lambs born per original embryo was the highest when the embryos were split without exposure to sucrose and transferred into recipients singly (106%, 17 16 ).     

Development of reconstituted mouse embryos produced from bisected and electrofused pronuclear-stage embryos

The present study was designed to investigate the in vitro and in vivo development potential of reconstituted mouse embryos produced by bisection and electrofusion of pronuclear stage embryos (PN-E). Pronuclear-stage ICR and F1 (C57BL x CBA) strain mouse embryos were bisected manually with a fine glass needle under the dissecting microscope to produce karyoplasts (KP) and cytoplasts (CP). The KP of ICR PN-E and CP of F1 PN-E (KP: ICR + CP:F1) or the KP of F1 PN-E and CP of F1 PN-E (KP:F1 + CP:ICR) were attached using phytohemagglutinin-P (PHA-P) and then electrofused. High fusion rates of the KP and CP of PN-E were obtained (93.5%). The fused embryos were encapsulated in alginate gel and cultured for 72 or 96 hours. The cleavage rates of reconstituted embryos were also high (98.8%). Developmental rates to the blastocyst stage in vitro for the 96-hour culture of reconstituted embryos were 68.9% (KP:ICR + CP:F1) and 78.4% (KP:F1 + CP:ICR). Furthermore, the developmental ability of reconstituted embryos in vivo was investigated, and some live young were obtained (KP:ICR + CP:F1, 7.5% and KP:F1 + CP:ICR, 10.8%). In this study, it was confirmed that reconstituted embryos produced by bisection and electrofusion of pronuclear stage embryos were able to develop into blastocysts in vitro and into live young in vivo.     

Growth and reproduction of mice developed from bisected embryos

Embryos from three groups of mice, ICR (I), a synthetic (S), and an F(1) hybrid from C57BL males and S females (F), were used to examine effects of embryo bisection on subsequent viability, postnatal growth and reproduction. In two experiments, Group S females used as recipients received the following combinations of whole (W) and/or bisected (B) embryos: W(I) and W(F), W(I) and B(F), B(I) and W(F), and B(I) and B(F) in Experiment 1 and W(I) and W(S), W(I) and B(S), B(I) and W(S), and B(I) and B(S) in Experiment 2. Eight to 12 embryos of both types were transferred surgically to two horns of the uterus. Overall survival rate of whole embryos (32.6%) was significantly greater (P < 0.001) than that for demi-embryos (13.2%). The gestation period after transfer was significantly longer (P < 0.05) for demi-embryos (17.5d) than for whole embryos (16.4 d). Mice developed from demi and whole embryos were not different in mean body weight at birth and at 21, 42 and 63 d of age. Fertility and litter size at first parity of mice that developed from bisected embryos were comparable with those that developed from whole embryos. Hence, bisection decreased embryo viability, increased gestation period of recipients but did not affect postnatal growth and reproduction.     

Production of monozygotic twins following the transfer of bisected embryos in the goats

Embryos at the morula, blastocyst and hatched blastocyst stage were obtained from superovulated and naturally ovulated Japanese native goats. They were bisected into halves with a glass needle, and transferred immediately or after culture (for morula) to recipients. None of five does which received bisected morula became pregnant. Three of nine goats became pregnant after transfer of bisected hatched blastocysts, six of eleven recipients became pregnant. Four of them produced monozygotic twins and the remaining two produced singles. The present study demonstrated that the hatched blastocyst is suitable for bisection in the goat.     

Survival of frozen rabbit embryos

Preimplantation stage rabbit embryos were successfully frozen to ?196 °C. The effects of various cooling rates, warming rates, thawing procedures, dimethyl sulfoxide concentrations and developmental stages were examined to determine their effects on the survival of Dutch-Belted rabbit embryos. When these factors were optimized, 65 % of the frozen embryos developed to the blastocyst stage in vitro. Some of these embryos developed into fetuses upon reimplantation to a foster mother.     

The biopolitics of frozen embryos

The unresolved debate about frozen embryos has left open the discussion on "what to do with them". There are only three ways to deal with frozen embryos: 1) to leave them frozen indefinitely; 2) to defrost and discard them and 3) to use them for research. In this paper, we suggest that the application of current scientific knowledge, instead of inappropriately referring to ethical principles or to the concept of person, could help with the decision about what to do with hundreds of thousands of frozen embryos, thus bringing the sensitive debate on bioethical issues to shared practical solutions. We face a new individual only when a new functional copy of his genome is formed. In both natural and artificial animal and plant reproduction, this principle applies. This status occurs in humans at the 4-8 cell stage. Acknowledgement of this factual datum would allow advocates of all religious and ideological beliefs to defend their principles and to realign their positions to a setting within the boundaries of current scientific knowledge.     

Viability of frozen-thawed bovine embryos bisected in sucrose: A preliminary report

A method for producing identical twin calves is described in which Day 7 frozen-thawed bovine embryos in 12.5% sucrose solution were bisected using a fine microsurgical blade. The resulting bisected embryos were transferred nonsurgically to the uterine horn ipsilateral to the corpus luteum of synchronous recipients (+/-1 d), two bisected embryos per recipient. The pregnancy rate when both halves remained in the same zona pellucida was 50% (5 10 ); the pregnancy rate was 1 5 for morulae and 4 5 for blastocysts. The pregnancy rate for unfrozen morulae bisected in PBS and transferred without zona pellucida was 27% (4 15 ). The in vitro survival rate of embryos bisected in 12.5% sucrose when both halves remained in the original zona pellucida was 82% (18 22 ), which was higher than when embryos were bisected in PBS (53%, 9 17 ).     

Development of reconstituted mouse embryos produced from the cytoplast of bisected oocytes or pronuclear-stage embryos and single blastomeres of 2-cell stage embryos

The present study investigated the in vitro developmental potential of reconstituted mouse embryos produced from the cytoplast of pronuclear-stage embryos or oocytes and single blastomeres of 2-cell stage embryos by electrofusion. The cytoplast of pronuclear-stage embryos and oocytes were obtained by manual bisection with a fine glass needle under a dissecting microscope. The fusion rates of the reconstituted embryos produced from the cytoplast of oocytes and single blastomeres of 2-cell stage embryos (O-SB2: 38.1 and 41.5%) were significantly lower than those produced from the cytoplast of pronuclear-stage embryos and single blastomeres of 2-cell stage embryos (P-SB2: 91.2 and 97.6%; P<0.001). Reconstituted embryos were encapsulated in alginate gel and were cultured for 96 hours. Similarly, the cleavage and development rates to the blastocyst stage of O-SB2 (56.3, 61.2 and 2.0, 3.1%, respectively) were significantly lower than those of the P-SB2 (91.0, 91.2 and 18.6, 20.7%; respectively, P<0.05). The cleavage and development rates to the blastocyst stage (61.2 and 2.0%) of reconstituted embryos produced from single blastomeres of late 2-cell stage embryos and oocyte cytoplast improved after activation by ethanol treatment (76.1 and 21.7%). However, the use of single blastomeres of early 2-cell stage embryos as nuclear donors did not enhance the cleavage and development rates of the reconstituted embryos to the blastocyst stage.     

Cervical transfer of deep frozen cattle embryos

Cattle blastocysts were collected from 29 donors 7-8 days after estrus and frozen and stored in liquid nitrogen up to several months. Two procedures were used for freezing and thawing: After thawing, the embryos were cultured from 8 to 12 hours before transfer; 36% of the embryos continued normal development during culture; both procedures resulted in a high pregnancy rate (procedure A: 10 15 ; procedure B: 11 15 ) after single cervical transfer of the frozen thawed embryos which developed normaly in vitro . However the overall survival rate was low (25%) and varied between donors, indicating that progress must be made before the technique of freezing can be extended to applied conditions.     

Cultivation of frozen mouse bone marrow cells in agar.

The authors attempted to cultivate frozen mouse bone marrow cells in a semisolid medium. They demonstrated that the stem haematopoietic cells of frozen mouse bone marrow were capable of proliferation and of colony formation on agar. The much smaller number of colonies from frozen mouse bone marrow (about 80% fewer) compared with fresh marrow is evidence that part of the stem haematopoietic cell population retains proliferative capacity even after freezing.     

Prior frozen storage enhances the effect of edible coatings against Listeria monocytogenes on cold-smoked salmon during subsequent refrigerated storage

Aims: Listeria monocytogenes is a major safety concern for ready‐to‐eat foods. The overall objective of this study was to investigate whether prior frozen storage could enhance the efficacy of edible coatings against L. monocytogenes on cold‐smoked salmon during subsequent refrigerated storage. Methods and Results: A formulation consisting of sodium lactate (SL, 1·2–2·4%) and sodium diacetate (SD, 0·125–0·25%) or 2·5% Opti.Form (a commercial formulation of SL and SD) was incorporated into each of five edible coatings: alginate, κ‐carrageenan, pectin, gelatin and starch. The coatings were applied onto the surface of cold‐smoked salmon slices inoculated with L. monocytogenes at a level of 500 CFU cm^?2. In the first phase, the slices were first frozen at ?18°C for 6 days and stored at 22°C for 6 days. Alginate, gelatin and starch appeared to be the most effective carriers. In the second phase, cold‐smoked salmon slices were inoculated with L. monocytogenes, coated with alginate, gelatin or starch with or without the antimicrobials and stored frozen at ?18°C for 12 months. Every 2 months, samples were removed from the freezer and kept at 4°C for 30 days. Prior frozen storage at ?18°C substantially enhanced the antilisterial efficacy of the edible coatings with or without antimicrobials during the subsequent refrigerated storage. Conclusions: Plain coatings with ≥2 months frozen storage and antimicrobial edible coatings represent an effective intervention to inhibit the growth of L. monocytogenes on cold‐smoked salmon. Significance and Impact of the Study: This study demonstrates the effectiveness of the conjunct application of frozen storage and edible coatings to control the growth of L. monocytogenes to enhance the microbiological safety of cold‐smoked salmon.     

Genetic control of survival of frozen mouse embryos

Lines of mice selected for increased litter size (L+), increased body weight (W+), or randomly (K) were used to study genetic variation in embryo cryosurvival in response to standard cryopreservation protocols. A total of 60528-cell embryos from 400 females were used in two studies. In Study 1, embryos from L+, W+, and K were frozen by slow-cool and ultrarapid (direct-plunge) methods to evaluate effects of selection on cryosurvival and genotype X freezing method interaction. Post-thaw survival (PTS) was measured as percentage of recovered embryos developing in vitro to blastocyst per donor female. Nonfrozen control embryos developed similarly for each line. Within slow-cool freezing, lines differed (W+ greater than K, W+ = L+, L+ = K; p less than 0.05); no differences were observed within the ultrarapid freezing. However, line X method interaction effects on PTS were not significant. In Study 2, reciprocal crosses were made between L+ and K and between W+ and K. Hybrid and pure line embryos were frozen by slow-cooling. Control embryos developed similarly for all genotypes. Selection lines did not differ for overall PTS. However, hybrid embryos from L+ dams were superior to those from K dams (84 vs. 61%; p less than .001). No overall embryo heterosis was observed. Differences were not significant among embryo genotypes or treatments for cell number or in vivo survival. These results demonstrate significant correlated responses in embryo post-thaw cryosurvival due to selection, and implicate both maternal and embryonic genomes as controlling mouse embryo cryosurvival.     

Basic aspects of frozen storage of semen

Basic concepts of cryopreservation and the causes of cryoinjury are reviewed. The possible roles of cryoprotectants and additives are considered in the context of their putative interactions with the sperm plasma membrane. Modern approaches to the laboratory assessment of spermatozoa after freeze-thawing are also briefly discussed.     

The effect of freezer type, cryoprotectant, and processing methods on viability of frozen embryos

Two hundred forty excellent-quality blastocysts flushed from 53 superovulated Holstein heifers were frozen by 1 of 16 procedures in a 2 x 2 x 2 x 2 factorial design. The main effects included a simple, inexpensive, portable mechanical freezing unit instead of a programmable Liquid Nitrogen (LN) freezer for freezing bovine embryos, cryoprotective agents dimethyl sulfoxide (DMSO) and glycerol, addition rates of the cryoprotectants and freezing rates. Embryo viability was assessed morphologically and by fluorescein diacetate (FDA) evaluation. Neither the type of freezer, the cryoprotectant nor the rate of cryoprotectant addition affected embryo viability. Embryo survival after 12 h of incubation was higher (P < 0.05) using a conventional freezing rate than a two-step method (37.2 vs 16.5%). The correlation coefficient between viability evaluation methods (morphology vs FDA) was influenced by cryoprotectant and embryo processing methods and ranged from -0.13 to +0.70. This study indicates that more simplified embryo freezing equipment and handling procedures may provide protection equal to that of more complicated, expensive equipment and more time-consuming methods. Economical on-farm embryo freezing is feasible.     

Beneficial acclimation and the Bogert effect

Few studies have examined the extent to which phenotypic plasticity in a given trait might be influenced by behavioural responses to an environmental cue. Regulatory behaviour might eliminate environmental variation such that little selection for physiological change would take place. Here, to test this Bogert effect on acclimation, we use two life-stages of a kelp fly that inhabit the same habitat, but differ profoundly in their behaviour. We predicted that when denied opportunities for behavioural regulation, mobile, though brachypterous adults would show a performance advantage in most thermal environments following acclimation to their preferred temperature(s). By contrast, in the less mobile larvae, that have a broader thermal preference, beneficial acclimation would be more evident. Ordered factor anova with orthogonal polynomial contrasts revealed that adults recovered faster from chill coma following any one of six short-term temperature treatments if they had been acclimated at low temperature, whilst larvae showed beneficial acclimation.